methods in biochemistry and...
TRANSCRIPT
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http://facultymembers.sbu.ac.ir/mirzajani/courses/
Chromatography
1. HPLC
2. GC
Methods in Biochemistry and Biophysics
• Molecular Binding
Covalent non-Covalent
Polar non-Polar Hydrogen bond
• Molecules interactions
Aromatic resonance
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Bio molecules features
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Solvent Polarity • What is Polarity?
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Solvents can be broadly classified into two categories: polar and non-polar. Generally, the dielectric constant of the solvent provides a rough measure of a solvent's polarity. The strong polarity of water is indicated, at
20 °C, by a dielectric constant of 10..
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Solvent Chemical formula
Boiling point
[8]
Dielectric constant
[9]
Density
Dipole moment
Non-polar solvents
Pentane CH3-CH2-CH2-CH2-CH3 36 °C 1.84 0.626 g/ml 0.00 D
Cyclopentane C5H10 40 °C 1.97 0.751 g/ml 0.00 D
Hexane
CH3-CH2-CH2-CH2-CH2-CH3
69 °C 1.88 0.655 g/ml 0.00 D
Cyclohexane C6H12 81 °C 2.02 0.779 g/ml 0.00 D
Benzene C6H6 80 °C 2.3 0.879 g/ml 0.00 D
Toluene C6H5-CH3 111 °C 2.38 0.867 g/ml 0.36 D
1,4-Dioxane /-CH2-CH2-O-CH2-CH2-O-\ 101 °C 2.3 1.033 g/ml 0.45 D
Chloroform CHCl3 61 °C 4.81 1.498 g/ml 1.04 D
Diethyl ether CH3CH2-O-CH2-CH3 35 °C 4.3 0.713 g/ml 1.15 D
Polar aprotic solvents
Dichloromethane (DCM) CH2Cl2 40 °C 9.1 1.3266
g/ml 1.60 D
Tetrahydrofuran (THF) /-CH2-CH2-O-CH2-CH2-\ 66 °C 7.5 0.886 g/ml 1.75 D
Ethyl acetate CH3-C(=O)-O-CH2-CH3 77 °C 6.02 0.894 g/ml 1.78 D
Acetone CH3-C(=O)-CH3 56 °C 21 0.786 g/ml 2.88 D
Dimethylformamide (DMF)
H-C(=O)N(CH3)2 153 °C 38 0.944 g/ml 3.82 D
Acetonitrile (MeCN) CH3-C≡N 82 °C 37.5 0.786 g/ml 3.92 D
Dimethyl sulfoxide(DMSO)
CH3-S(=O)-CH3 189 °C 46.7 1.092 g/ml 3.96 D
Propylene carbonate C4H6O3 240 °C 64.0 1.205 g/ml 4.9 D
Polar protic solvents
Formic acid H-C(=O)OH 101 °C 58 1.21 g/ml 1.41 D
n-Butanol CH3-CH2-CH2-CH2-OH 118 °C 18 0.810 g/ml 1.63 D
Isopropanol (IPA) CH3-CH(-OH)-CH3 82 °C 18 0.785 g/ml 1.66 D
n-Propanol CH3-CH2-CH2-OH 97 °C 20 0.803 g/ml 1.68 D
Ethanol CH3-CH2-OH 79 °C 24.55 0.789 g/ml 1.69 D
Methanol CH3-OH 65 °C 33 0.791 g/ml 1.70 D
Acetic acid CH3-C(=O)OH 118 °C 6.2 1.049 g/ml 1.74 D
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Solvent Chemical formula
Boiling point
[8]
Dielectric constant
[9]
Density
Dipole moment
Non-polar solvents
Pentane CH3-CH2-CH2-CH2-CH3 36 °C 1.84 0.626 g/ml 0.00 D
Cyclopentane C5H10 40 °C 1.97 0.751 g/ml 0.00 D
Hexane
CH3-CH2-CH2-CH2-CH2-CH3
69 °C 1.88 0.655 g/ml 0.00 D
Cyclohexane C6H12 81 °C 2.02 0.779 g/ml 0.00 D
Benzene C6H6 80 °C 2.3 0.879 g/ml 0.00 D
Toluene C6H5-CH3 111 °C 2.38 0.867 g/ml 0.36 D
1,4-Dioxane /-CH2-CH2-O-CH2-CH2-O-\ 101 °C 2.3 1.033 g/ml 0.45 D
Chloroform CHCl3 61 °C 4.81 1.498 g/ml 1.04 D
Diethyl ether CH3CH2-O-CH2-CH3 35 °C 4.3 0.713 g/ml 1.15 D
Polar aprotic solvents
Dichloromethane (DCM) CH2Cl2 40 °C 9.1 1.3266
g/ml 1.60 D
Tetrahydrofuran (THF) /-CH2-CH2-O-CH2-CH2-\ 66 °C 7.5 0.886 g/ml 1.75 D
Ethyl acetate CH3-C(=O)-O-CH2-CH3 77 °C 6.02 0.894 g/ml 1.78 D
Acetone CH3-C(=O)-CH3 56 °C 21 0.786 g/ml 2.88 D
Dimethylformamide (DMF)
H-C(=O)N(CH3)2 153 °C 38 0.944 g/ml 3.82 D
Acetonitrile (MeCN) CH3-C≡N 82 °C 37.5 0.786 g/ml 3.92 D
Dimethyl sulfoxide(DMSO)
CH3-S(=O)-CH3 189 °C 46.7 1.092 g/ml 3.96 D
Propylene carbonate C4H6O3 240 °C 64.0 1.205 g/ml 4.9 D
Polar protic solvents
Formic acid H-C(=O)OH 101 °C 58 1.21 g/ml 1.41 D
n-Butanol CH3-CH2-CH2-CH2-OH 118 °C 18 0.810 g/ml 1.63 D
Isopropanol (IPA) CH3-CH(-OH)-CH3 82 °C 18 0.785 g/ml 1.66 D
n-Propanol CH3-CH2-CH2-OH 97 °C 20 0.803 g/ml 1.68 D
Ethanol CH3-CH2-OH 79 °C 24.55 0.789 g/ml 1.69 D
Methanol CH3-OH 65 °C 33 0.791 g/ml 1.70 D
Acetic acid CH3-C(=O)OH 118 °C 6.2 1.049 g/ml 1.74 D
pH
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Buffer
1. What is Buffer?
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A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. If the pH is expected to decrease during the experiment, choose a buffer with a pKa slightly below the working pH. Conversely, if the pH is expected to increase during the experiment, select a buffer with a pKa slightly above the working pH.
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The History of Chromatography
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Liquid chromatography (LC) was the first type of chromatography to be discovered and, in the form of liquid-solid chromatography (LSC) was originally used in the late 1890s by the Russian botanist, Tswett to separate and isolate various plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography (color writing) for this type of separation.
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Theory of Chromatography 1. Separation means….
2. Mechanisms of separations
3. Definition of retention
4. Van Demeter equation
5. Definition of resolution
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Separation means…
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B
A
C A
A
B
C
C
C Separation
C C C C
B B
A A A
Qualitative analysis
What are components A, B and C ?
Quantitative analysis
What is the concentration of
components A, B and C ?
A B
C
Chromatogram containing three peaks Qualitative analysis (identification) and Quantitative analysis (determination) Can be performed using the information contained in the chromatogram
Chromatography : Method Chromatogram : Results Chromatograph : Instrument
A B C
Column
Packing material
↓ ↓ ↓ ↓ Mobile phase (solvent)
Chromatogram
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Inlet Separation line Compounds Sample/Solvent outlet
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Definition of retention
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Typical in RP/HPLC
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Mechanisms of separations
Superpositioning of partitioning
Gel-Filtration (Size-Exclusion)
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Mechanisms of separations
• Electrostatic interactions
• Hydrogen bindings
±
±
±
1. Actual type of stationary phase
2. Mobile phase or buffer condition
3. Type and concentration of salt
4. pH
pH pH
• Ion Exchange
• Affinity
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Definition of retention
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We have a line of stores (chance for molecular interactions) and costumers (a molecules want to have interaction)…
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van Deemter equation
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H= A + B/u + Cu
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Terms of …
• Resolution:
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Terms of …
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van Deemter equation
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Equilibration definition
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Definition of resolution
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Chromatographic methods
Chromatography
Gas
Solid (GSC)
Liquid (GLC)
Supercritical Fluid
Solid (SFC)
Liquid
Solid
Partitioning Size Exclusion
(SEC) Ion Exchange
(IEC)
Cation &Anion
Affinity (AC) Adsorption
(LSC)
Liquid (LLC) Micelles (MEKC)
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Which separation technique for which compound?
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Method selection
based on the Sample
The type of the sample including its
– Phase type; gas / liquid / solid
– Solubility in; aqueous / organic
– Stability features in; time / temperature
– Interaction and decomposition potency
– Contaminants percentage
– pH or EC of the final solution
based on the Facilities
Separation techniques can be replaced by each other according to the sample features and user facilities.
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Examples for liquid sample
Soluble in MeOH:H2O
14 7 1
HPLC No compatible with contaminants (400nm)
HPTLC compatible with contaminants
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Liquid Chromatography
Ion Exchange (IEC)
Size Exclusion (SEC)
Partitioning
Bonded Phase
Liquid-Liquid
LSC SEC Reversed
Phase (RPC)
Ion-pair Micellar-liquid
Metal-complexation
Hydrophilic interaction
Ion suppression
IEC AC
Affinity (AC) Adsorption
(LSC)
> 85% of all analytical separations were performed in RPLC
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Normal Phase
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Ion Exchange
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Size Exclusion
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HPLC Is ….
• High Performance Liquid Chromatography
• High Pressure Liquid Chromatography (usually true)
• High Priced Liquid Chromatography
It also named as:
• High Speed Liquid Chromatography
• High Resolution Liquid Chromatography (HRLC)
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HPLC Instrumentation 1. Pumps
2. Columns and stationary phases
3. injection
4. Detection
5. Additional parts
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Liquid chromatography is a separation technique that involves: • the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity. • The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. • The separated components are collected at the exit of this column and identified by an external easurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount.
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Pumps
• The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
• Normal flow rates in HPLC are in the 1- to 2-mL/min range.
• Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar).
• During the chromatographic experiment, a pump can deliver a constant mobile phase composition
• (isocratic) or an increasing mobile phase composition (gradient).
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Pump instrumentation; Check valves Do not break the parts unless is necessary wash it using MeOH with the help of sonication
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1- Constant pressure pump
It is free from pulsation resulting in smooth baseline
2- Constant flow pump
It is able to give constant flow rate of mobile phase
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Pump Module – types
Pump Module – types
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Isocratic pump - delivers constant mobile phase composition; solvent must be pre-mixed; lowest cost pump
Gradient pump - delivers variable mobile phase composition; can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
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Injection
… how is a sample actually put into an LC system
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Manual Injectors
Front View Inject
Load - Inject
Sample Loop
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Loading Vs. Injection mode
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Injector; LOOP
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Columns and stationary phases
Columns and stationary phases
Made from: Stainless
Shape: Straight
Length: Variable
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Columns features
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Packing materials
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Packing material
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Column selection
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Note that;
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1. If your column do not have guard you have to use pre-column compatible with your separation system.
2. Adjust the column in the right direction
Industrial columns
Pay attention to the column certification and care condition
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Detection
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Spectroscopic Detection
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Wavelength
Time
Absorbance Spectra
Refractive Index Detection
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• Not used in case of gradient elution
• It measures the difference in RI between pure mobile phase and the column eluate (mobile phase + solute).
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Ideal Detector
Should have; 1- High sensitivity
2- Low noise (straight base line)
3-Wide range of response to different compounds
4- Unaffected by temperature or mobile phase
5- Non destructive to the compounds
6- Provides qualitative and quantitative information about the detected sample
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Additional parts
De gasser Mixing chamber Fraction collector
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Why temperature control needed?
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Mobile Phase
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Importance of right choose
The best mobile phase is:
1. Pure
2. Low viscosity
3. Chemically inert
4. Low price
5. Compatible with detector
6. Solubility of the sample
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Solvent compatibility chart
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Isocratic Vs. Gradient Elution
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Gradient Methods
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Example
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Solvent preparation
Both samples and solvents should filtered through 450 nm.
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Solvent Inlet Filter
• Filtration before entering the column.
• Degassing using degasser. - Heating with stirring
- Applying vacuum,
- Passing nitrogen or helium
- Ultrasound
• Pre-saturation with the stationary phase in case of liquid liquid chromatography.
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How Starting the Analysis?
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The person best prepared to choose, will know the likely outcome.
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Particle size importance
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Suggested optimum condition
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Remember that
• The pH of the solvent (water) may be adjusted using phosphate or perchlorate or trifloroacetate acid or sulphate buffer.
The selectivity of HPLC is affected by :
1- Type of mobile phase, organic or aqueous.
2- The composition of the mobile phase, whether one solvent or more.
3- The pH of the mobile phase.
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Buffer effect example
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TFA
Incomplete separation
HClO4
No separation for 2 and 3
H2SO4
Best separation
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Column purity and TFA effect
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Gas Chromatography
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Introduction: Why we need GC?
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– Separation and analysis of organic compounds
– Testing purity of compounds
– Determine relative amounts of components in mixture
– Compound identification
– Isolation of pure compounds (microscale work)
• Similar to column chromatography, but differs in 3 ways:
– Partitioning process carried out between Moving Gas Phase and
Stationary Liquid Phase
– Temperature of gas can be controlled
– Concentration of compound in gas phase is a function of the vapor
pressure only.
• GC also known as Vapor-Phase Chromatography (VPC) and Gas-
Liquid Partition Chromatography (GLPC)
Introduction: What information can be found?
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• Chromatograph of known/unknown mixture
• Determine Retention Times
• Calculate Peak Areas
• Adjust Peak Areas for Thermal Response
• Calculate Total Area from Adjusted Areas
• Calculate Mole Fraction
• Calculate Mole Percentage
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Introduction: What is GC and how its works?
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Typically, components with similar polarity elute in order of volatility. Thus
alkanes elute in order of increasing boiling points; lower boiling alkanes will
have shorter retention times than higher boiling alkanes.
• Low resolution (case sensitive)
• Separation ability of volatile compound in the range of 350 – 400 C SO it is limited for Inorganic and non volatiles
• Separation of 10 – 20 % of molecules
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Introduction: GC limitations
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Introduction: GC Terms …
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• Chromatogram
• Retention Time
• Dead volume
• Adjusted retention time Relative
• Retention factor
• Relative retention/velocity
• Capacity factor
• Scale Up
• Theoretical Plate
• Number of Theoretical Plates
• Separation
• Resolution
• Peaks types
• Thermal Response Factor
• Chromatogram - The instrumental output. A signal as a function of time (or volume)
• Retention Time - How long a compound stays in the column. (tr) or could be
expressed in terms of volume (Vr)
• Dead volume Vm or could be expressed as a time (tm)
• Volume to get through the system even without any interaction. A constant for a given column.
• Adjusted retention time tr’ = tr - tm
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Terms of …
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Preparative vs Resolution vs Speed vs Expense
Terms of …
Silica support Stationary phase
Gas (mobile phase)
Terms of …
N = L
H
Length of the column
Height equivalent
of a theoretical
plate
Theoretical Plates and its number
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Terms of …
• Resolution:
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Terms of …
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The influence of column
characteristics on separation
parameters:
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Terms of …
Retention Factor Phase Relation Film Thickness Diameter
-
-
-
-
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Column Capacity of injection:
The maximum injection possibility which is not affected on
the peck shape and separation resolution.
Column Capacity of separation:
The relation between the retention time of each peck and
its width, which shows the maximum peck separation of
the column.
Terms of …
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Oven • Programmable
• Isothermal- run at one constant temperature
• Temperature programming - Start at low temperature and gradually ramp to higher temperature
• Better sensitivity for components that are retained longer
• Much better chromatographic resolution
• Peak refocusing at head of column
• More constant peak width
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Terms of …
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KOVATS Index (RI): the retention of
samples in the comparison with Normal
Hydrocarbons (NH) at the specific
stationary phase and temperature:
If: modified retention
X: Sample
Y: NH with lower number of carbon than X
Z: NH with higher number of carbon than X
Terms of …
𝐼 = 100 𝑌 + 100 𝑍 − 𝑌log 𝑡𝑟
′ 𝑋 log 𝑡𝑟′ 𝑌
log 𝑡𝑟′ 𝑍 log 𝑡𝑟
′ 𝑌
𝑡𝑟′
Factors Affecting Separation – Boiling Points of Components in Sample
• Low boiling point compounds have higher vapor pressures.
• High boiling point compounds have lower vapor pressures requiring more energy to reach equilibrium vapor pressure, i.e., atmospheric pressure.
• Boiling point increases as molecular weight increases.
– Flow Rate of Carrier Gas
– Choice of Liquid Phase
• Molecular weights, functional groups, and polarities of component molecules are factors in selecting liquid phase.
– Length of Column
• Similar compounds require longer columns than dissimilar compounds. Isomeric mixtures often require quite long columns
Terms of …
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Thermal Response Factor • The areas of gas chromatogram peaks are proportional to
the molar content of the mixture • Compounds with different functional groups or widely
varying molecular weights do not all have the same thermal conductivity. This can cause the instrument to produce response variations, which cause deviations (non-linearity) in the relationship between peak area and molar content
• A correction factor called “The Thermal Response Factor” for a given compound can be established from the relative peak areas of an equimolar solution
• Equimolar mixtures contain compounds with the same molar content, i.e., the same number of moles
• Thus, equimolar mixtures should produce peaks of equal area, if the instrument response is linear
Terms of …
Instrumentation
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• Hardware to introduce the sample
• Technique to separate the sample into components
• Hardware to detect the individual components.
• Data Processing to process this information.
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Sample requirements
1. Volatile and acceptable vapor pressure in the range of 350 – 400 C
2. Become volatile with the fast increasing of temperature
3. Stable
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What is the sample? • Usually a mixture of several components
• Sample usually introduced as a liquid
• Components of interest (analytes) usually in low concentrations (<1% to ppb levels)
• Samples dissolved in volatile solvent
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GC is best for separation of volatile compounds which are
thermally stable.
Chemical derivatization prior to analysis is generally done to:
• increase the volatility and decrease the polarity of
compounds;
• reduce thermal degradation of samples by increasing their
thermal stability;
• increase detector response
• improve separation and reduce tailing
Derivatizing Reagents Common derivatization methods can be
classified into 4 groups depending on the type of reaction
applied:
• Silylation
• Acylation
• Alkylation
• Esterification
Why is chemical derivatization needed?
Sample Inlet
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Injection methods
• Manually
• Automated injection
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Injection apparatus
Syringe
Auto sampler
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Injection apparatus: Manual
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Syringe selection
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Syringe selection
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Syringe selection
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Syringe Cleaning
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Short
Concentrated
Long
Diffuse
Solute Bands
Same column, same chromatographic conditions
Influence of Injection Efficiency
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Injection apparatus: Automated
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Injection apparatus: Automated
GC Septa
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GC Septa types:
GC Septa types:
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Injection apparatus
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Split & Splitless Injection
• Most common method of Injection into Capillary Columns
• Most commonly misunderstood also!
• Same injector hardware is used for both techniques
• Electronically controlled Solenoid changes Gas Flow to determine Injector function.
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Split Injection
Mechanism by which a portion of the injected solution is discarded.
Only a small portion (1/1000 - 1/20) of sample goes through the
column.
Used for concentrated samples (>0.1%).
Can be performed isothermally.
Fast injection speed. Injector and septa contamination not usually noticed
Splitless Injection
Most of the sample goes through the column (85-100%)
Used for dilute samples (<0.1%)
Injection speed slow
Should not be performed isothermally
Solvent focusing is important
Controlled by solenoid valve
Requires careful optimisation
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Split vs Splitless
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b) split injection (350℃) (only 0.1-10% sample)
- concentrated sample
- high resolution
- dirty samples
- could cause thermal decomposition
c) splitless injection (220℃) (80%)
- dilute sample
- high resolution
- solvent trapping (Tsolvent < 40℃)
- cold trapping (Tsolute < 150℃)
On Column Injection
• All of the sample is transferred to the column
• Needle is inserted directly into column or into insert directly above column
o Trace analysis
o Thermally labile compounds e.g Pesticides, Drugs
o Wide boiling point range
o High molecular weight
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Large Volume Injection
• To enhance sensitivity in Environmental applications.
• Uses 100 µL syringe: Inject up to 70 µl
• Very slow injection with injector temperature a few degrees below solvent boiling point, split open, flow at about 150 ml/ min
• Solvent vents out of split vent, thus concentrating the analyte
• Close split
• Fast temperature ramp to top column temperature +20°C
• Column programming as per sample requirements
Cross Section of PTV Injector
Modern Temperature Programmable Injector (Varian 1079)
Programmable Temperature Vapourising Injector
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Dial 1-816-650-0741 for e-Seminar Audio
*see Knaus, Fulleman & Turner, HRC 4(1981)643
At Injection (To)
Needle At Injection (To)
Carrier Gas
Carrier Gas
Solvent Vapors
Column
Column
Temperature Profile High volatility solute
Low volatility solute
n-Paraffin standard showing distillation range from C6 to C110 on DB-HT Sim Dist
Column: DB-HT Sim Dist
5 m x 0.53 mm I.D., 0.15 µm
Carrier: Helium at 18 mL/min, measured at 35°C
Oven: - 30 - 430°C at 10°/min
Injector: OPTICTM PTV
55 - 450°C at 2°/sec
0.5 µL of about 2% n-Parraffins in CS2
Detector: FID, 450°C
Nitrogen makeup gas at 15 mL/min
Time(minutes) 0 5 10 15 20 25 30 35 40 45 50
6
7
8
9
10
16
11 12
70
50
40
90 80
60
14
18
20 24 28 30
32
110
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What are the influential parameters:
• Syringe blocking or non homogeneity injection.
• Injection leaking or non homogeneity injection.
• Sample widening in column
• Vaporization speed
• Vapor transfer speed
• Split ratio
• The art of your hand
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Liner Selection
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Liner Selection
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Split Injection
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Liner Maintenance
Ferrules
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Ferrules and cutter
Ferrules
130
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Gold seals
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Column
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Columns: Columns type
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Gas-liquid Chromatography: Classical packed column,
and WCOT
Gas-Solid Chromatography: Classical packed column,
and PLOT
Partition mechanism
Adsorption mechanism
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Columns: Columns type
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1. Packed-bed column (d > 2 mm, packing particle from 100 to 250 micron)
2. Micro-packed column (d < 1 mm, dp/dc less than 0.3)
3. Packed capillary column (d < 0.6 mm, packing particle 5-20 micron)
5. Support coated open tubular (SCOT) Liquid phase + glass powder or particle support
6. Porous layer open tubular column (PLOT) Particle support
4. Wall coated open tubular columns (WCOT)
Thin layer of stationary phase coated directly on the wall of the tube.
Columns: Columns type
Packed GC Columns
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Columns: Columns type
Columns: Stationary Phases
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PDMS: PolyDiMethylSiloxane Organic stationary phases must have sufficient molecular weight to achieve low volatility
Or by de-shielding the oxygen and forming a wax. (Carbowax)
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Columns: Stationary Phases
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Columns: Selection
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Companies….
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Columns: Stationary Phases
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Columns: Stationary Phases
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Columns: Stationary Phases
Non-Bonded: Liquid phase is used to coat the inside
walls of the column to a thickness of about 0.1 – 1.0 μm
Bonded: Silanol groups on the column wall form a
chemical bond with the stationary phase
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Advantage of Bonded Phases:
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1. High Reproducibility
2. Low Bleed
3. Fast Equilibration
4. Temperature Stability
5. Easy to Back Flush
6. Long Life
Column Bleed
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Bleed increases with film thickness
Polar columns have higher bleed
Bleed is excessive when column is damaged or degraded Avoid strong acids or bases
Adhere to manufacturer’s recommended temperature
limits Avoid leaks
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Column Bleed
Cutting & setting the column
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Choosing a Column
• Internal Diameter
• Film Thickness
Small ID:
Good resolution of early eluting compounds
Longer analysis times
Limited dynamic range
Large ID:
Have less resolution of early eluting compounds
Shorter analysis times
Sufficient resolution for complex mixtures
Greater dynamic range
Amount of stationary phase coating
Affects retention and capacity
Thicker films increase retention and capacity
Thin films are useful for high boilers
Standard capillary columns typically 0.25µm
0.53mm ID (Megabore) typically 1.0 - 1.5µm
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Choosing a Column
• Length
• Phase
isothermal analysis Retention more dependant on length Doubling column length doubles analysis times Resolution a function of Square Root of Length Gain 41% in resolution Is it worth the extra time and expense?-
programmed analysis Retention more dependant on temperature
Marginally increases analysis times
Run conditions should be optimised
The maximum amount that can be injected without
significant peak distortion
Column capacity increases with
film thickness
temperature
internal diameter
stationary phase selectivity
If exceeded, results in :-
peak broadening
asymmetry
leading
Column Capacity
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Summary - Effect of ID, Film Thickness, and Length
ID • Choice based on
capacity and resolution • Use 0.25mm for MSDs • Use 0.32mm for
split/splitless & DI • Use 0.53mm for DI & • purge & trap
Film Thickness • Thick film for low
boilers • Thin film for high
boilers
• Thicker films for larger
ID's
Length
Gain in resolution is not double
Isothermal: tR L
Programmed: tR is more dependent on temperature
Carrier Gas
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Column possible damages:
• Physical damages:
high temperature, acidic/basic compounds, … borosilicate coated silicate
• Oxidation:
O2 impurity of carrier gas, leaking spaces, time & temperature of oxidation process
1. Peck shape variation
2. Tailing for active compounds
3. Upper baseline
4. No tailing for hydrocarbons
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Column Bleeding:
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Column possible damages: • Chemical damages: H2O and organic solvents on the non covalent columns.
= cut the 10-20 cm of the columns occasionally OR
= use the pre-column
Alkaline (NH4OH, NaOH, KOH, …)
Acids (H3PO4, H2SO4, HCl, HNO3, … )
BUT (No water, analysis at 100 C for HCl
Column Bleeding
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Detectors
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Characteristics of the Ideal Detector
A short response time that is independent of flow rate.
High reliability and ease of use.
Similarity in response toward all solutes or a highly selective
response toward one or more classes of solutes.
Nondestructive of sample.
Qualitative analysis :
Mass spectrometer, IR Quantitative analysis :
Area of a chromatographic peak.
Detectors
Detector noise is any perturbation on
the detector output that is not
related to an eluted solute. It is a
fundamental property of the
detecting system and determines the
ultimate sensitivity or minimum
detectable concentration. Detector
noise has been divided into three
types, 'short term noise, 'long term noise' and ‘drift'
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Detectors
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Types of the detectors:
FID: Flame Ionization Detector
TCD: Thermal Conductivity Detector
ECD: Electron Capture Detector
FPD: Flame Photometric GC Detector
NPD: Nitrogen Phosphorus Detector
PAD: Photoionization Detector
Thermal conductivity detector:
-most general way
-responds to everything
-not sensitive enough for high resolution.
Flame ionization detector :
-most popular
-mainly responds hydrocarbons (C-H)
Comparison
Electron capture detector :
- for compounds containing atoms with high electron
affinities.
- sensitive for halogen, C=O, NOx, & orgaometallic
compounds.
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The flame ionization detector is the most widely used and generally applicable detector for gas chromatography.
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Flame Ionization Detectors (FID)
• The effluent from the column is mixed with hydrogen and air and
then ignited electrically.
• Most organic compounds, when pyrolyzed at the temperature of
a hydrogen/air flame, produce ions and electrons that can
conduct electricity through the flame.
• A potential of a few hundred volts is applied.
• The resulting current (~10-12 A) is then measured.
• The flame ionization detector exhibits a high sensitivity (~10-13 g/s), large linear response range (~107), and low noise.
• A disadvantage of the flame ionization detector is that it is destructive of sample.
Flame Ionization Detectors (FID)
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Thermal Conductivity Detectors(TCD)
A very early detector for gas chromatography, and one that still finds wide application, is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules.
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• The sensing element of TCD is an electrically heated element whose temperature at constant electrical power depends upon the thermal
conductivity of the surrounding gas.
• The heated element may be a fine platinum, gold, or tungsten wire or a
semiconducting thermistor.
• The advantage of the thermal conductivity detector is its simplicity, its
large linear dynamic range(~105), its general response to both organic
and inorganic species, and its nondestructive character, which permits
collection of solutes after detection.
• A limitation of the katharometer is its relatively low sensitivity (~10-8 g
solute/mL carrier gas).
• Other detectors exceed this sensitivity by factors as large as 104 to 107.
Thermal Conductivity Detectors(TCD)
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Relative Thermal Conductivity
Compound Relative Thermal
Conductivity
Carbon Tetrachloride 0.05
Benzene 0.11
Hexane 0.12
Argon 0.12
Methanol 0.13
Nitrogen 0.17
Helium 1.00
Hydrogen 1.28
Electron-Capture Detectors(ECD)
The electron-capture detector has become one of the most widely used detectors for environmental samples because this detector selectivity detects halogen containing compounds, such as pesticides and polychlorinated biphenyls.
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• The effluent from the column is passed over a emitter, usually nickel-63. An
electron from the emitter causes ionization of the carrier gas and the
production of a burst of electrons. In the absence of organic species, a constant
standing current between a pair of electrodes results from this ionization
process. The current decreases markedly, however, in the presence of those
organic molecules that tend to capture electrons.
• The electron-capture detector is selective in its response being highly sensitive
to molecules containing electronegative functional groups such as halogens,
peroxides, quinones, and nitro groups.
• It is insensitive to functional groups such as amines, alcohols, and
hydrocarbons.
• An important application of the electron-capture detector has been for the
detection and determination of chlorinated insecticides.
•
Electron-Capture Detectors (ECD)
Atomic Emission Detectors (AED)
The atomic emission detector is available commercially. In this device the eluent is introduced into a microwave-energized helium plasma that is coupled to a diode array optical emission spectrometer. The plasma is sufficiently energetic to atomize all of the elements in a sample and to excite their characteristic atomic emission spectra.
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Thermionic Detectors (TID)
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The thermionic detector is selective toward organic compounds containing phosphorus and nitrogen. Its response to a phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 to 106 larger than a carbon atom. Compared with the flame ionization detector, the thermionic detector is approximately 500 times more sensitive to phosphorus-containing compounds and 50 times more sensitive to nitrogen bearing species. These properties make thermionic detection particularly useful for detecting and determining the many phosphorus-containing pesticides.
FPD: Flame Photometric GC Detector
The overall design of a nitrogen-phosphorus detector (NPD) is similar to a flame-ionization detector (FID). The major difference is that the hydrogen/air flame of the FID is replaced by a heated rubidium silicate bead in the NPD. The effluent from the GC column passes through the hot bead. The hot rubidium salt emits ions when nitrogen and phosphorus-containing compounds pass over it. The ions are collected on a collector above the heated bead to produce a current, similar to the FID.
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NPD: Nitrogen Phosphorus Detector
The overall design of a nitrogen-phosphorus detector (NPD) is similar to a flame-ionization detector (FID). The major difference is that the hydrogen/air flame of the FID is replaced by a heated rubidium silicate bead in the NPD. The effluent from the GC column passes through the hot bead. The hot rubidium salt emits ions when nitrogen and phosphorus-containing compounds pass over it. The ions are collected on a collector above the heated bead to produce a current, similar to the FID.
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Photoionization GC Detector (PID)
The reason to use more than one kind of detector for gas chromatography is to achieve selective and/or highly sensitive detection of specific compounds encountered in particular chromatographic analyses. The selective determination of aromatic hydrocarbons or organo-heteroatom species is the job of the photoionization detector (PID). This device uses ultraviolet light as a means of ionizing an analyte exiting from a GC column. The ions produced by this process are collected by electrodes. The current generated is therefore a measure of the analyte concentration.
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If the energy of an incoming photon is high enough (and the molecule
is quantum mechanically "allowed" to absorb the photon) photo-
excitation can occur to such an extent that an electron is completely
removed from its molecular orbital, i.e. ionization.
A Photoionization Reaction
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Photoionization GC Detector (PID)
Since only a small (but
basically unknown) fraction
of the analyte molecules are
actually ionized in the PID
chamber, this is considered
to be a nondestructive GC
detector. Therefore, the
exhaust port of the
PID can be connected to
another detector in series
with the PID.
In this way data from two different detectors can be taken
simultaneously, and selective detection of PID responsive compounds
augmented by response from, say, an FID or ECD. The major challenge
here is to make the design of the ionization chamber and the
downstream connections to the second detector as low volume as
possible (read small diameter) so that peaks that have been separated
by the GC column do not broaden out before detection.