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Gas Chromatography

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GAS CHROMATOGRAPHY

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Page 1: Gas Chromatography,  GC ppt

Gas Chromatography

Gas Chromatography Presented By -

Mr Shaise Jacob

Faculty

Nirmala College of Pharmacy

Muvattupuzha Kerala

India

Email ndash jacobshaisegmailcom

What is Gas ChromatographyWhat is Gas Chromatography

bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)

GAS CHROMATOGRAPHY

Separation of gaseous amp volatile substances Simple amp efficient in regard to separation

GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography

Gas rarr MP

Solid Liquid rarr SP

GSC not used because of limited no of SP

GSC principle is ADSORPTION

GLC principle is PARTITION

Sample to be separated is converted into vapour

And mixed with gaseous MP

Component more soluble in the SP rarr travels slower

Component less soluble in the SP rarr travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by GC 1VOLATILITY

2THERMOSTABILITY

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 2: Gas Chromatography,  GC ppt

Gas Chromatography Presented By -

Mr Shaise Jacob

Faculty

Nirmala College of Pharmacy

Muvattupuzha Kerala

India

Email ndash jacobshaisegmailcom

What is Gas ChromatographyWhat is Gas Chromatography

bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)

GAS CHROMATOGRAPHY

Separation of gaseous amp volatile substances Simple amp efficient in regard to separation

GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography

Gas rarr MP

Solid Liquid rarr SP

GSC not used because of limited no of SP

GSC principle is ADSORPTION

GLC principle is PARTITION

Sample to be separated is converted into vapour

And mixed with gaseous MP

Component more soluble in the SP rarr travels slower

Component less soluble in the SP rarr travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by GC 1VOLATILITY

2THERMOSTABILITY

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 3: Gas Chromatography,  GC ppt

What is Gas ChromatographyWhat is Gas Chromatography

bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)

GAS CHROMATOGRAPHY

Separation of gaseous amp volatile substances Simple amp efficient in regard to separation

GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography

Gas rarr MP

Solid Liquid rarr SP

GSC not used because of limited no of SP

GSC principle is ADSORPTION

GLC principle is PARTITION

Sample to be separated is converted into vapour

And mixed with gaseous MP

Component more soluble in the SP rarr travels slower

Component less soluble in the SP rarr travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by GC 1VOLATILITY

2THERMOSTABILITY

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 4: Gas Chromatography,  GC ppt

GAS CHROMATOGRAPHY

Separation of gaseous amp volatile substances Simple amp efficient in regard to separation

GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography

Gas rarr MP

Solid Liquid rarr SP

GSC not used because of limited no of SP

GSC principle is ADSORPTION

GLC principle is PARTITION

Sample to be separated is converted into vapour

And mixed with gaseous MP

Component more soluble in the SP rarr travels slower

Component less soluble in the SP rarr travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by GC 1VOLATILITY

2THERMOSTABILITY

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 5: Gas Chromatography,  GC ppt

Sample to be separated is converted into vapour

And mixed with gaseous MP

Component more soluble in the SP rarr travels slower

Component less soluble in the SP rarr travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by GC 1VOLATILITY

2THERMOSTABILITY

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 6: Gas Chromatography,  GC ppt

What is Gas ChromatographyWhat is Gas Chromatography

bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 7: Gas Chromatography,  GC ppt

The Next Generation in Gas Chromatography

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 8: Gas Chromatography,  GC ppt

How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 9: Gas Chromatography,  GC ppt

Chromatographic SeparationChromatographic Separation

ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid

within a columnwithin a column

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 10: Gas Chromatography,  GC ppt

Chromatographic SeparationChromatographic Separation

bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are

uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times

ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn

ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 11: Gas Chromatography,  GC ppt

Chromatographic Separation Chromatographic Separation (continued)(continued)

ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture

ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 12: Gas Chromatography,  GC ppt

Chromatographic AnalysisChromatographic Analysis

ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks

ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks

ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 13: Gas Chromatography,  GC ppt

Peaks and DataPeaks and Data

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 14: Gas Chromatography,  GC ppt

PRACTICAL REQUIREMENTS

bull Carrier gas

bull Flow regulators amp Flow meters

bull Injection devices

bull Columns

bull Temperature control devices

bull Detectors

bull Recorders amp Integrators

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 15: Gas Chromatography,  GC ppt

CARRIER GAS

raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated

compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 16: Gas Chromatography,  GC ppt

Requirements of a carrier gas

InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 17: Gas Chromatography,  GC ppt

Flow regulators amp Flow meters deliver the gas with uniform pressureflow

rate flow meters- Rota meter amp Soap bubble

flow meter

Rota meterplaced before column inlet

it has a glass tube with a float held on to a spring

the level of the float is determined by the flow rate of carrier gas

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 18: Gas Chromatography,  GC ppt

Soap Bubble Meter

loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 19: Gas Chromatography,  GC ppt

Injection Devices

Gases can be introduced into the column by valve devices

liquids can be injected through loop or septum devices

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 20: Gas Chromatography,  GC ppt

COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile

COLUMNS can be classified Depending on its use

1 Analytical column

1-15 meters length amp 3-6 mm dm

2 Preparative column

3-6 meters length 6-9mm dm

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 21: Gas Chromatography,  GC ppt

Depending on its nature

1Packed column columns are available in a packed manner

SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip

2Open tubular or Capillary column or Golay column

Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 22: Gas Chromatography,  GC ppt

3SCOT columns (Support coated open tubular column

Improved version of Golay Capillary columns have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column

Then coated with a thin film of liquid phase

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 23: Gas Chromatography,  GC ppt

Columns

bull Packed

bull Capillary

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 24: Gas Chromatography,  GC ppt

Equilibration of the column

Before introduction of the sample Column is attached to instrument amp

desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing

carrier gas for 24 hours

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 25: Gas Chromatography,  GC ppt

Temperature Control DevicesPreheaters convert sample into its vapour

form present along with injecting devices

Thermostatically controlled oven

temperature maintenance in a column is highly essential for efficient separation

Two types of operationsIsothermal programming-Linear programming- this method is

efficient for separation of complex mixtures

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 26: Gas Chromatography,  GC ppt

Temperature Control

bull Isothermal bull Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation - Oven

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 27: Gas Chromatography,  GC ppt

DETECTORSHeart of the apparatus

The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by

temperature flow ratehellip Non destructive Simple amp inexpensive

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 28: Gas Chromatography,  GC ppt

Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered

1Thermal Conductivity Detector(Katharometer Hot Wire Detector)

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 29: Gas Chromatography,  GC ppt

Thermal Conductivity Basics

When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal

The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas

Flo

w

Flo

w

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 30: Gas Chromatography,  GC ppt

When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column

The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded

Thermal Conductivity Detector

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 31: Gas Chromatography,  GC ppt

1048698 Measures heat loss from a hot filament ndash

1048698 filament heated to const Tbull when only carrier gas flows heat loss to

metal block is constant filament T remains constant

bull when an analyte species flows past the filament generally thermal conductivity goes

down T of filament will rise (resistance of the filament will rise)

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 32: Gas Chromatography,  GC ppt

Relative Thermal Conductivity

CompoundRelative Thermal

Conductivity

Carbon Tetrachloride 005

Benzene 011

Hexane 012

Argon 012

Methanol 013

Nitrogen 017

Helium 100

Hydrogen 128

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 33: Gas Chromatography,  GC ppt

Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive

Disadvantages Low sensitivityAffected by fluctuations in temperature and

flow rateBiological samples cannot be analyzed

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 34: Gas Chromatography,  GC ppt

Flame Ionization Detector

Destructive detector The effluent from the column is mixed with H

amp air and ignited Organic compounds burning in the flame

produce ions and electrons which can conduct electricity through the flame

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to electric current

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 35: Gas Chromatography,  GC ppt

FIDs are mass sensitive rather than conc sensitive

ADVANTAGESbull microg quantities of the solute can be

detectedbull Stablebull Responds to most of the organic

compoundsbull Linearity is excellent

bull DA destroy the sample

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 36: Gas Chromatography,  GC ppt

FID

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 37: Gas Chromatography,  GC ppt

Argon ionization detector Depends on the excitation of argon atoms to a

metastable state by using radioactive energy

Argonrarr irradiation Argon + e- rarrcollision Metastable

Argonrarr collision of sub rarr Ionization rarruarrCurrent

ADVANTAGES

1Responds to organic compounds

2High sensitivity

DISADVANTAGES

1Response is not absolute

2Linearity is poor

3 Sensitivity is affected by water

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 38: Gas Chromatography,  GC ppt

ELECTRON CAPTURE DETECTOR The detector consists of a cavity

that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 39: Gas Chromatography,  GC ppt

bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases

bull The detector selective for compounds with atoms of high electron affinity

bull This detector is frequently used in the analysis of chlorinated compounds

bull eg ndash pesticides polychlorinated biphenyls

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 40: Gas Chromatography,  GC ppt

ADVANTAGEHighly sensitive

DISADVANTAGEUsed only for compounds with electron

affinity

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 41: Gas Chromatography,  GC ppt

RECORDERS amp INTEGRATORS

Record the baseline and all the peaks obtained

INTEGRATORSRecord the individual peaks with Rt heighthellip

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 42: Gas Chromatography,  GC ppt

Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector

They are 2 types Precolumn derivatisation

Components are converted to volatile amp thermo stable derivative

Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 43: Gas Chromatography,  GC ppt

Post column derivatisation

Improve response shown by detector Components ionization affinity towards

electrons is increased

Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar

components like esters ethershellip

Techniques 1 use more polar liquid SP

2 Increasing amt of liquid phase

3Pretreatment of solid support to remove active sites

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 44: Gas Chromatography,  GC ppt

Parameters used in GC

Retention time (Rt)

It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds

(or) It is the time required for 50 of a component to be eluted from a column

Retention volume (Vr)

It is the volume of carrier gas which is required to elute 50 of the component from the column

Retention volume = Retention time ˣ Flow rate

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 45: Gas Chromatography,  GC ppt

Separation factor (S)

Ratio of partition co-efficient of the two components to be separated

If more difference in partition co-efficient bw two compounds the peaks are far apart amp S

Is more If partition co-efficient of two compounds are similar then peaks are closer

Resolution (R)

The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

It is the measure of both column amp solvent efficiencies

R= 2d

W1+W2

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 46: Gas Chromatography,  GC ppt

Retention time

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 47: Gas Chromatography,  GC ppt

Separation factor

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 48: Gas Chromatography,  GC ppt

Resolution

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 49: Gas Chromatography,  GC ppt

Resolution

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 50: Gas Chromatography,  GC ppt

THEORETICAL PLATE

An imaginary unit of the column where equilibrium has been established between SP amp MP

It can also be called as a functional unit of the column

HETP ndash Height Equivalent to a Theoretical Plate

Efficiency of a column is expressed by the number of theoretical plates in the column or HETP

If HETP is less the column is uarr efficient If HETP is more the column is darr efficient

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 51: Gas Chromatography,  GC ppt

HETP= L (length of the column)

N (no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion depends on flow rate

C = Effect of mass transferdepends on flow rate

u = Flow rate

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 52: Gas Chromatography,  GC ppt

Efficiency ( No of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

N = no of theoretical plates

Rt = retention time

W = peak width at baseThe no of theoretical plates is high the

column is highly efficientFor GC the value of 600 meter

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 53: Gas Chromatography,  GC ppt

Asymmetry Factor

Chromatographic peak should be symmetrical about its centre

If peak is not symmetrical- shows Fronting or Tailing

FRONTING

Due to saturation of SP amp can be avoided by using less quantity of sample

TAILING

Due to more active adsorption sites amp can be eliminated by support pretreatment

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 54: Gas Chromatography,  GC ppt

Asymmetry factor (095-105) can be calculated by using the formula AF=ba

b amp a calculated at 5 or 10 of the peak height

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 55: Gas Chromatography,  GC ppt

ADVANTAGES OF GC

Very high resolution power complex mixtures can be resolved into its components by this method

Very high sensitivity with TCD detect down to 100 ppm

It is a micro method small sample size is required

Fast analysis is possible gas as moving phase- rapid equilibrium

Relatively good precision amp accuracyQualitative amp quantitative analysis is possible

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 56: Gas Chromatography,  GC ppt

Gas Chromatography vials caps

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 57: Gas Chromatography,  GC ppt

Chromatographic AnalysisChromatographic Analysis

ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks

ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks

ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 58: Gas Chromatography,  GC ppt

Applications of GC

bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities

1 Qualitative analysis

Rt amp RV are used for the identification amp separation

2 Checking the purity of a compound

Compare the chromatogram of the std amp that of the sample

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 59: Gas Chromatography,  GC ppt

3 Quantitative analysis

It is necessary to measure the peak area or peak height of each component

4 used for analysis of drugs amp their metabolites

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 60: Gas Chromatography,  GC ppt

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Dete

ctor

Resp

onse

Retention Time

14

16

18

Pea

k A

rea

Sample Concentration (mgml)

2

4

6

8

10

05 10 15 20 25 30

The content of C fatty acids =C

C + C + C

= the content of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 61: Gas Chromatography,  GC ppt

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane16 min = RT

Res

pons

e

Unknown compound may be Hexane

16 min = RT

Retention Time on Carbowax-20 (min)

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 62: Gas Chromatography,  GC ppt

Res

p on s

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 40 min on SE-30

Retention TimesRetention Times

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 63: Gas Chromatography,  GC ppt

Advantages of Gas Chromatography

bull Very good separation

bull Time (analysis is short)

bull Small sample is needed - l

bull Good detection system

bull Quantitatively analyzed

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 64: Gas Chromatography,  GC ppt

How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works

ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column

ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas

ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column

  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85
Page 65: Gas Chromatography,  GC ppt
  • Gas Chromatography
  • Slide 2
  • What is Gas Chromatography
  • GAS CHROMATOGRAPHY
  • Slide 5
  • Slide 6
  • Slide 7
  • The Next Generation in Gas Chromatography
  • How a Gas Chromatography Machine Works
  • Chromatographic Separation
  • Chromatographic Separation
  • Chromatographic Separation (continued)
  • Chromatographic Analysis
  • Peaks and Data
  • Slide 15
  • Slide 16
  • Slide 17
  • Slide 18
  • Slide 19
  • Slide 20
  • PRACTICAL REQUIREMENTS
  • CARRIER GAS
  • Requirements of a carrier gas
  • Flow regulators amp Flow meters
  • Slide 25
  • Soap Bubble Meter
  • Injection Devices
  • COLUMNS
  • Depending on its nature
  • 3SCOT columns (Support coated open tubular column
  • Columns
  • Slide 32
  • Slide 33
  • Equilibration of the column
  • Temperature Control Devices
  • Temperature Control
  • DETECTORS
  • 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
  • Thermal Conductivity Basics
  • Slide 40
  • Slide 41
  • Slide 42
  • Relative Thermal Conductivity
  • Advantages of Katharometer
  • Flame Ionization Detector
  • Slide 46
  • FID
  • Slide 48
  • Argon ionization detector
  • Slide 50
  • Slide 51
  • Slide 52
  • Slide 53
  • Slide 54
  • Slide 55
  • RECORDERS amp INTEGRATORS
  • Derivatisation of sample
  • Post column derivatisation
  • Parameters used in GC
  • Slide 60
  • Retention time
  • Slide 62
  • Separation factor
  • Resolution
  • Slide 65
  • THEORETICAL PLATE
  • Slide 67
  • Efficiency ( No of Theoretical plates)
  • Slide 69
  • Slide 70
  • Asymmetry Factor
  • Slide 72
  • Slide 73
  • Slide 74
  • ADVANTAGES OF GC
  • Gas Chromatography vials caps
  • Slide 77
  • Applications of GC
  • Slide 79
  • Slide 80
  • Slide 81
  • Slide 84
  • Slide 85