Chapter 12

Gas Chromatography

Principles of Gas Chromatography

How does separation take place?

• Partition of molecules between a gas mobile phase

and a liquid or solid stationary phase

Separation technique • Gas is the mobile phase and liquid (GLC) or solid (GSC) is the

stationary phase

• GLC: liquid is coated on an inert solid;

separation is the result of solubility in the liquid phase

• GSC: Particulate solid like molecular sieve is the stationary

phase;

separation is the result of adsorption on the solid surface

Uses of GC

• Separation and analysis of organic

compounds

• Testing purity of compounds

• Determine relative amounts of

components in mixtures

• Compound identification

• Isolation of pure compounds (micro

scale work)

Advantages of the GC

• Speed: minutes or seconds

• Resolution: complex samples

• Sensitivity: 10-9 or even 10-12 g/s

• Versatility: Gases, liquids or solids (Qal & Qant)

• GLC is more common than GSC: flexibility and resolution

• Salts and other ionic compounds + high molecular weight may not be determined by the GC

GC Process

1. Column is selected, packed with liquid phase, and installed

2. Sample injected with microliter syringe into the injection port where it is vaporized and mixed into the carrier gas stream (helium, nitrogen, argon).

3. Sample becomes partitioned between moving gas phase and stationary liquid phase.

4. The time spent by different compounds of the sample in vapor phase is a function of their vapor pressure

5. The more volatile compounds arrive at the end of the column first and pass into the detector

Factors Affecting Separation

Boiling Points of Components in Sample

Low boiling compounds have higher vapor pressures.

Boiling point increases with increasing molecular weight

Flow Rate of Carrier Gas

Choice of Liquid Phase (Solubility in the liquid stationary phase determines the retention time in the stationary 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

GC Instrumentation

• Carrier gas

• Sample Injector

• Column

• Detector

Filters/Traps

Air

Hyd

rog

en

Ga

s C

arrie

r

Column

Gas Chromatography System

• gas system

• inlet

• column

• detector

• data system

Data system

Syringe/Sampler

Inlets

Detectors

Regulators

H

RESET

Carrier Gas System

• Carrier gases, must be chemically inert,

• Include helium, argon, nitrogen, carbon dioxide, and hydrogen.

• The choice of gases is often dictated by the detector used.

• Associated with the gas supply are pressure regulators, gauges, and flow meters.

• The carrier gas system often contains a molecular

sieve to remove water or other impurities. • Detector gases - none or air/H2 (Flame ionization

detector)

Common Carrier Gases

• N2: Low cost, safe, simple purification

higher molecular weight. But low

thermal conductivity

• H2: High thermal conductivity, low viscosity

(low pressure drop in the column), low

cost. But more diffusion of solutes;

danger of explosion on leakage

• He: Combines advantages of N2 and H2. But

high price

• Ar: Important for ionization detector,

relatively low cost, simple purification

Purpose of Carrier gases

1. Carrying the volatile components

through the column

2. Providing a suitable matrix for the

detector to measure the sample

components

Requirements of a Carrier Gas

• Should be inert at the temperature used

• Pure and dry

• Should be compatible (appropriate) with the

detector

• Most common gas is He that is most useful

for TCD but greater floe rates are required to

reduce diffusion and peak broadening

• H2 is hazardous and chemically reactive

toward reducible and unsaturated samples

Purity of Carrier Gases

• Impurities (particularly O2 & H2O) can chemically change the liquid phase and thus the tR and reduce the lifetime of the column. False peaks may appear

• Liquid stationary phases (polyesters, polysiloxanes, polyamides) degrade by O2 & H2O

• Contaminant from column may desorb in

H2O causing a high detector background

(baseline drift and noise)

• Traces of hydrocarbons cause a high background in the FID

• Purity should be >99.995%

Drying of Carrier Gas

• Molecular sieve trap between cylinder

and column is used to remove H2O and

hydrocarbons

• Sieve should be regenerated after each

gas cylinder by heating at 300oC for 3hr

with slow flow of N2 or H2

Two stage pressure regulator

1. It indicates the pressure left in the cylinder (min. 40 psi)

2. It indicates the increasing pressure delivered to the GC

(min. = 20 psi)

Carrier gas: He; N2; H2; Ar

Flow control of the carrier gas

1. Effect on column efficiency

Too slow: peak broadening

Too fast: prevents good partitioning

1/8” (3 mm) O.D packed columns: 25-30 ml/min

Capillary columns: 0.01” (0.25 mm) O.D: 0.74 ml/min

2. Effect on tR

1% change in the flow rate causes a 1% change

in tR

3. Effect on the detector response

It causes a displacement of the baseline making

quantitative analysis difficult

• For 1% accuracy in quant. Analysis fluctuation

in flow rate should not be more than ± 0.2%

• Flow rates are normally controlled by a two-

stage pressure regulator at the gas cylinder

and some sort of pressure regulator or flow

regulator mounted in the chromatograph

• Inlet pressures usually range from 10 to 50

psi, which lead to flow rates of 25 to 150

mL/min with packed columns and 1 to 25

mL/min for open-tubular columns

How a constant flow rate can be assured?

1. Control of carrier gas inlet pressure

2. Control of carrier gas flow rate

• In isothermal operation regulating one of them keeps

the other constant

• In programmed temperature operation, if the inlet

pressure is constant the flow rate will change with

temp.

Thus flow rate must be controlled by using

“Differential Flow Controller”

Variation of flow rate through a column as a function of

temp. at constant inlet pressure (13.9 psig)

Column Temp Measured flow rate

relative to flow at 50oC

(Cm3/min)

50 40.0

100 34.4

150 29.6

200 27.8

250 25.6

300 20.4

Flow rate decreases due to

•The increased viscosity of the carrier gas

•Thermal expansion of liquid phase

Effect of the Differential Flow Controller

• It assures constant mass flow rate independent of

column resistance

• Stable flow rates can be obtained over a wide range

of temperatures

Results of differential flow controller

Column Temp, Inlet Pressure He flow

oC Cm Hg ml/min

20 26.3 60.7

50 27.9 61.5

100 32.4 60.5

150 35.0 60.6

200 41.2 60.7

Injection port

Function

• Introduce sample

• Vaporize sample

• Split sample

Main Components

It is a metal block containing:

•Heaters

•Temperature sensors

•Septum holder on the front

•Connection for the column on the rear

Injection Methods

1. Injection ports

2. Sampling loops/valves

Various Types of Injection ports

• Split - only a portion of injection

goes on column

• Splitless - “all” material injected

goes on column

• On-Column - cold injection (sensitive

materials)

most common method of injection

• It involves the use of a micro-syringe to

inject a liquid or gaseous sample

through a silicone rubber diaphragm or

septum into a flash vaporizer port

located at the head of the column.

• Tinjector >50oC above the column temperature

• Septum must be stable at the Tinjector

(Flash Vaporization Inlet)

• Rubber septum serves for about 30 injections in

ordinary care

• 5-10 injections in case of large syringes

Heated

Metal

Block

Injection

Port

liner

Injection of liquid sample

• 10 µl syringe is the most popular device

• Load the desired volume of liquid, then draw the plunger back to pull the liquid out of the needle

• Insert the needle quickly through the septum as far as it will go

• Depress the plunger and immediately remove the needle from the injection port

• Introducing the sample instantaneously

will avoid “appreciable spreading of the

solute band”

• 10 µl benzene sample 2 ml vapor

• 2ml vapor with 60 ml/min requires 2 sec

i.e., Injection technique would add a

peak width of at least 2 sec.

Larger samples cause broader profiles

• For best peak shape and maximum resolution the smallest

possible sample size should be used

• More components in the sample use larger sample size

• Trace analysis use larger sample size

Gas samples

• Special gas-tight syringes with sealing rings

around the tip of the plunger.

• The syringes have a Teflon plunger which

can form a very high seal around the glass.

• Gas sample valves

Valves may be heated if sample is

absorbed or condensed when the valve is

cold

Solid samples

• Sealed glass or indium tube containing the sample is placed in the heated area of the injection port

• When the plunger pushes the tube into the heated area, the sealed tube melts and the volatile components flash off and are carried out into the column

Purpose of Split Injection

• Rapid vaporization and a short retention time in the liner results in a small injection plug.

• Splitting reduces the size of the sample to an amount compatible with the sample capacity of the capillary column.

• In high resolution capillary chromatography where columns with inner diameters below 100 m are used, split ratios can exceed 1:1000.

• When the split ratio is too low, a broad injection band will be introduced into the column, resulting in broader peaks.

• Column overloading can take place. This usually results in asymmetrical peaks (peaks with leading fronts).

Split mode

Split liner

Disadvantages of Split Injection

• Due to a large loss of sample, split injection is not suitable for trace analysis.

• Depending on the inlet temperature, thermal degradation can also take place, especially when using liners containing a glass frit or packed with glass wool. This means that split injection is not suited for the analysis of components prone to thermal degradation.

• Discrimination is also possible. In the heated liner, additional vaporization of the more volatile sample constituents from the needle cannot be avoided. Hence, the composition of the sample that enters the column is no longer representative of the original sample.

• The reproducibility of split injection is strongly dependent on the geometry of the liner and the injection technique.

• The build up of non-volatile residues on the glass liner may lead to additional decomposition and adsorption problems

Liner overload

This

Purpose of Split Injection

• Rapid vaporization and a short retention time in the liner results in a small injection plug.

• Splitting reduces the size of the sample to an amount compatible with the sample capacity of the capillary column.

• In high resolution capillary chromatography where columns with inner diameters below 100 m are used, split ratios can exceed 1:1000.

• When the split ratio is too low, a broad injection band will be introduced into the column, resulting in broader peaks.

• Column overloading can take place. This usually results in asymmetrical peaks (peaks with leading fronts).

Split mode

Split liner

Disadvantages of Split Injection

• Due to a large loss of sample, split injection is not suitable for trace analysis.

• Depending on the inlet temperature, thermal degradation can also take place, especially when using liners containing a glass frit or packed with glass wool. This means that split injection is not suited for the analysis of components prone to thermal degradation.

• Discrimination is also possible. In the heated liner, additional vaporization of the more volatile sample constituents from the needle cannot be avoided. Hence, the composition of the sample that enters the column is no longer representative of the original sample.

• The reproducibility of split injection is strongly dependent on the geometry of the liner and the injection technique.

• The build up of non-volatile residues on the glass liner may lead to additional decomposition and adsorption problems

Liner overload

This

Pyrolysis Chromatography

• It is used for nonvolatile samples (e.g., Plastics)

• The sample is heated rapidly above its decomposition

temp.

• A group of volatile decomposition products will be

produced

• The products can be chromatographed to yield a

fingerprint which is characteristic of the original

material

• Pyrolyzers are attachments to the standard injection

port with a probe extending into the heated zone

• Separate pyrolyzing chamber with a transfer line to

carry the volatile products into the chromatograph

• Pyrolysis remains a rather empirical technique and

• each unit should have its own fingerprint library

determined by running samples of known composition

On Column Injection

• It is used for samples (biological) that decompose, rearrange, or be adsorbed if they contact the heated metal surface of the port

• The needle extends directly into the column (the end of the needle penetrates the packing; this may cause damage to the needle)

• Packing length is adjusted so that the end of the needle is either or just ahead of the glass wool plug

(Sampling Valves)

•Valves give better reproducibility

•Require less skill

•Can be easily automated

Injection valve

Injection Port Temperature

• The temp. should be 20-30 oC hotter than the boiling point of the least volatile component

But low enough to prevent sample decomposition and septum bleed

• Temp. may be checked by raising it and watching :

Position, or area, or shape of the peaks. Drastic changes mean the temp. setting is high

• It should be 10% above that of the column to ensure rapid volatilization of the sample. The efficiency of the column is almost constant under this condition

• Components may be vaporized at a temp. ~100oC below its atmospheric boiling point

• Very high boiling point or temp. sensitive material can be handled by dilution with volatile solvent that permits lowering the injection temp.

This will lower the sensitivity!

• Try various temperatures until peak broadening becomes apparent

• With temp. programming techniques low injection temps become very practical. No rush to vaporize the high boiling components

Effect of injection port temperature on resolution

a: Methanol; b: Ethanol; c: ispropanol

Boling points: 65 – 82 oC

Problems arising with the injection port

• Cleanliness

• Septums

Cleanliness of the injection port

• Any material collects inside the port may trap

the sample, decompose and release

unwanted components to the column

• A glass wool plug inserted in the vaporizer

section will trap most of this material and

can be removed periodically

Mechanical Problems

• Over tightening creates problems:

It may become impossible to push the needle

through it

It may block the carrier gas flow

• It is best to tighten the retainer by hand

• Repeated puncturing of the septum will destroy its

mechanical strength and cause leakage.

tR becomes longer and sensitivity decreases

• Change the septum regularly, preferably at the end

of the day (any air entered can be swept overnight)

Septums

Chemical Problems

• Silicon rubber septums may adsorb some of the

sample causing: peak broadening and baseline

noise. Good injection technique and proper port

design overcome this problem

• Ports are designed for full insertion of a 2 inch

hypodermic needle; otherwise some material will

deposit on the septum and gradually releases

• The syringe plunger should be drawn after loading;

this leaves the needle filled with air and the liquid

not forced out by expansion when the needle enters

hot septum

Thermal Problems

• Low molecular weight components in the

septum may be driven off when the septum

is first heated causing ghost peaks

especially in the case of the temperature

programming

• Conditioning is necessary

Gas Chromatographic Columns

1. Packed Columns for GLC

2. Packed Columns for GSC

3. Capillary Columns

Types of column • Preparative

>1/4"

>3 m in length

• Conventional

1/8 – 1/4" OD, stainless steel or glass

tube

2-6 m in length

• Capillary

• 0.1-0.5 mm ID

• 10-100 meters in length

Capillary (Open tubular columns)

Packed Columns

Main Components of a Column

1.Column Tubing

• Packed

• OpenTubular

(Capillary)

2. Solid Support

3. Liquid or solid Stationary Phase

Column Material

• Stainless Steel : most common.

adsorbs some Compounds particularly

polar ones & especially water.

• Copper tubing reacts with : amines,

acetylenes, terpens & steroids

Widely used. It is good for trace water

analysis.

• Copper oxide coating is reactive and

can interfere in gas analysis.

• O2 must be excluded from the carrier gas

• Al is used but troublemaker due to reactive

Al-oxide formation

• Plastics: are limited due to permeability &

temperature limit

(used for reactive or highly corrosive chemicals

H2S, HF

• Teflon, polypropylene and nylon tubing are

available.

• Glass:

If glass were not difficult to form into columns &

relatively fragile it would be the very best

choice for tubing ( used for pesticides &

steroid).

Column Size Column Length

It is the shortest length that will give the required

separation

Advantages of Short Columns: • Shorter analysis time

• lower Column temp

• Longer column life time

• Lower noise & drift due to column bleed

• Ability to resolve peaks

i.e. increasing length is not a very effective way to

increase resolution.

• Larger samples may be injected into longer columns.

L

• Increase in Column diameter

cusses:

* increase in capacity

* decrease in column efficiency

* longer tR

Solid Support (Stationary Phase Support)

Characteristics of Good Solid Support

• Large surface area – 1-20 m2/g

• Uniform pore diameter – 10 m or less

• Inertness (no chemical activity, Catalytically inactive)

• Regularly shaped particles

• Mechanical strength (Coating without breaking)

• Thermally stable Diatomaceous earth is the most

common (nearly, all silica).

• When diatomite supports are unsuitable, Glass or

Polymer beads are used

Chromosorb Supports Chromosorb P

• Derived from raw diatomite

• Calcined

• Pink

• Hard

• Separate hydrocarbons; not good for polar compounds

• Most adsorptive surface, strong, efficient

Chromosorb W

• Chromosorb P flux calcined with Na2Co3

• Processed from Celite diatomaceous silica

• White

• Friable

• Separate polar compounds

• Non-adsorptive surface, most inert solid support

But fragile

Adsorption on Column Packings

(or Capillary Walls)

• Polar analyte species such as, Alcohols

or Aromatic hydrocarbons are

adsorbed physically on the silicate

surfaces.

• Adsorption results in distorted peaks

broadened with tail

• This catalytic activity may lead to

sample decomposition

Reasons for adsorption activity

• Silicates + Water Silanol

groups on the silicate surface

SiO

OHOH

Si

O

OH

Si

O

OH

Si

• Si-OH groups have strong affinity for polar

organic molecules

Treatment of Solid Supports

• Non-acid washed (NAW) – an untreated form

• Acid washed (AW) – use HCl – Removes metals, impurities, Reduces surface activity and absorption

• Acid washed – Dimethyldichlorosilane treated (AW-DMCS)

Si

OH

+ (CH3)2SiCl2

HClSi

OSi CH3

CH3OH HCl

Cl

CH3

CH3

Si

OSi

CH3

CH3

Factors upon which the solid support is chosen

1. Nature of the sample

• Acidic Sample acidic support * Non polar compounds Chromosorb-P * Compounds with Treated support polar functional is necessary groups (W&G; HP)

• Injection of water or acids may ruin many good

silinized columns. They hydrolyze the silyle ether groups

2. Nature of Liquid Phase

• Avoid incompatibility between the

support and the liquid phase

3. Intended use for specific need (tailored

closely) or general purpose use

(better grade)

4. Coast

Alternative Solide supports

1. Synthetic silica – based supports

(Volaspher and Quartz), Merck

* For non polar or weakly polar stationary phases. * High mechanical strength, * Uniform pore structure (thin film is possible) * The column will be packed very densely * very pure ( 99 % silicic acid) * > 95% silicic acid in diatomites.) * This material is close to the ideal supports. * It may replace Diatomites for sophisticated analysis.

2. Silica Gel * The fine pores were widened yielding a more uniform

pore size distribution and decreasing the surface area.

* The undesirable adsorption contribution is significant.

3. Micro Glass Beads and Porous Layer

• (Glass beads coated with a thin layer of porous silica) Corning Code 0201 (DMCS) Corning Glass Works USA Glass Beads (DMCS) Applied Science Lab USA Anaport Glass Beads Analabs USA Glass Beads Perkin Elmer USA Howlett – Packard USA Zipax CSP (Porous) Du Pont USA Liqu-Chrom ASL USA Prisorb B Merck Jascosil WG03 Jasco, Japan * Glass beads with liquid phase chemically bonded are also

available

4. Fluoro carbon Supports * extremely inert * For separation of strongly polar or reactive compounds * Excellent for water and corrosive chemicals * Most of these are made of : Teflon-6 powder : Poly(tetrafluoroethylene) resin

• PTFE supports are : soft, liable to electrostatic charge causing them to aggregate and adhere to the walls & accessories

• “This is solved by cooling to 0oC before handling”

• PTFE’s have great thermal resistance & high resistance

to chemical attack. Teflon –6 Du Pont Chromosorb T Johns Manville USA Haloport – F Howlett – Packard USA Shi malite F Shimadzu, Japan

Stationary

Liquid Phase

Function of Stationary Liquid Phase

• The stationary phase should provide separation of the sample with a reasonable column life

• Suitable phase is chosen on the basis of : Experience or Experiment.

• It is desirable to have maximum information

about the sample composition : bp.range,

components expected & their structure

• Stationary phases should have similar chemical structure to the sample components

Criteria for Liquid Phases

1. Maximize differential solubility

2. High absolute solubility for sample (measure as tR)

• Solubility good but not too good.

• (Gas phase is inert & separation occurs only in Liquid Phase).

3. Thermal stability (Temp. Limitations)

(Maximum & Minimum temp.)

4. Chemical inertness towards ample

components at temp. of operation

5. Strong attachment to the Solid Support.

6. Low vapor pressure at the temperature used

(otherwise it will bleed off the column).

7. Reproducibility, availability, cost.

– Same liquid phase produces same results when

bought from any source or from same source

Commonly Used Liquid Phases

PHASE TEM. LIMITS Good for

1. SQUALANE 0/125oC Nonpolar

2. OV-1, SE-30 100/350 oC

3. DEXSIL-300 50/350 oC

(Most thermally stable)

4. OV-17; SP-2250 0-350 Moderately polar

5. QF-1; OV-210; 0-275

SP-2401

6. CARBOWAX-20M 60/225 Strongle polar

7. DEGS 20/200

8. OV-275 20/250

Squalane

• Saturated, highly branches, C-30 hydrocarbon

• Non-polar

• Limited temperature range: 0-125 oC

• Separate hydrocarbons

• Standard reference for Rohrschneider and

• McReynolds constants

• Non-polar

• Temperature range: 100-350oC

• Most widely used liquid phase

• Separate all sample types

OV-1 (SE-30) AND SP-2100

OV-17 (SP-2250)

• 50% methyl, 50% phenylpolysiloxane

• Semi-polar

• Temperature range : 0-350oC

• Widely used to separate drugs,

steroids, carbohydrates

Carbowax 20-M

HO – ( - CH2 – CH2 – O-)n – H

• Polymeric polyethylene glycol

• Polar

• Temperature range : 60-225oC

• Widely used for polar samples

Liquid Phase Selection

1. Intuitive 2. Scientific

Intuition

• Liquid stationary phase separates essentially by boiling point within functional group categories.

e.g. :Silicon or squalene (non polar) separates

n-hydro carbons; and alcohols on b.p. basis.

• How does the separation take place when We have a mixture of polar and non polar Components? On b.p. basis?

Scientific

• When more than 3 or 4 components of various properties are to be separated, the intuition does not work.

• What is the solution?

• Retention Index ( Kovats Index)

• e.g., a sample containing the following components

Compound b.p. Chemical type

Carbon tetrachloride 76oC Chlorinated hydrocarbon

Benzene 80 oC Aromatic hydrocarbon

Cyclohexane 81 oC Saturated hydrocarbon

n-Butanol 118 oC Alcohol

Available Columns • SE-30 non-polar Silicon

• Apiezon L non-polar hydrocarbon

• QF-1 Polar fluorinated silicon

• Carbowax Polar polyether

20M

Retention Index (120 oC )ON

Compound SE-30 Apizon QF-1 Carbowax 20M

CCl4 680 687 733 895

678 683 780 961

677 691 701 756

CH3CH2CH2OH

676 620 821 1111

• How can we deal with a list of > 300 liquid phases?

• How the duplication can be eliminated?

* Liquid phases have been classified in

terms of their separating power

* A set of reference compounds have been

used to determine how much longer the

reference compound is retained by the

liquid phase being tested than by some

standard liquid phase.

• McRynolds constants used to

1. Show the increasing polarity of liquid phases.

2. find identical liquid phases.

* Kovats Retention Index (R.I) has been developed

as a means for qualitative analysis

• Further, the concept has been modified into a means of classifying polarities of liquid phases.

Parameters Affect Separation Efficiency

• Solid support particle diameter

• Column length

• Column diameter

• Flow rate

• Type of carrier gas

• Pressure

• Type of liquid phase

• Amount of liquid phase

• Column tubing

• Temperature programming and isothermal column temperature

Column Temperature

1. As the Column temp. increases a sample component spends more time in the mobile phase.

* This will cause a decrease in the tR (Faster separation)

* tR doubles for every 30oC decrease.

2. Increasing the temp. decreases the band broadening since it

leads to a decrease in the available time for diffusion in the

column.

3. The lower the temperature the better is the separation

* The column temp. should not be less than about 10oC below

the bp of the highest-boiling sample component (other wise

distorted peaks may result).

* Roughly, a temp. equal or slightly above the average b.p of a

sample results in a reasonable elution time (20 to 30 min.).

4. Optimum column temp. depends upon:

b.p. of the sample

Degree of separation required.

Temperature Programming

1. Isothermal separation

The temperature is held constant

during the analysis

2. Programmed temperature separation

The temperature is varied gradually

according to a set program

Column Temperature Effect (Isothermal Analysis)

Isothermal chromatographic analysis is one which is performed

at a constant column temperature.

• Higher temp. enables

rapid analysis but loss

in resolution.

• Lower temp. achieves

better resolution but

longer analysis time

Narrow Boiling Range Samples

• Isothermal column temperature should be used.

• Select temperature 20-50oC lower than boiling range of sample when thin films are handled.

• Use highest temperature that still allows adequate resolution and stability to shorten analysis time.

Temperature Programming

• Within a homologous series, retention time increases exponentially with the number of carbons

• As tR increases, band width increases and the peak height decreases, making detection almost impossible after a few peaks have eluted

• Since solubility of a gas in a liquid decreases as temperature increases, the retention of a sample component can be reduced by increasing column temperature

Conclusion:

1. Better resolution

of earlier peaks

2. Latter peaks elute

more rapidly

3. Peak shapes are

more uniform

Creating a temperature Program

• General steps to create a program assuming that the separation is possible:

1. Determine the initial temperature and time based on best possible separation of first few peaks

2. Repeat step 1 for the last few peaks to find the best final temperature and time

3. Experiment with various ramps to account for the rest of the components

PERCENT LOADING OF STATIONARY PHASE

• How much liquid phase should be coated on the support?

• For analytical columns: 5 – 10%

• Higher loading ( up to ~ 30% ) can be applied for light gases (C1 to C4 hydrocarbons)

• Low Loading (1- 5% ) with very high boiling compounds

• The liquid phase should cover the support almost completely. If the support is inert, this condition is not a must

Conditioning the column Why? Remove: impurities in liquid phase ; residual

solvent from coating

How?

• Install the column but do not connect to the detector

• Set the carrier flow 30 ml/min for 1/8 inch

• Heat for 1 hr at 100oC

• Raise the temperature to slightly below the b.p. of liqid phase (about 30 oC higher than expected operating column operation temp.(. Continue heating over night

• Cap the column when it is removed from the instrument

Gas – Solid Chromatography

(Solid-Stationary Phase)

Gas – Solid Chromatography

Basic Principle:

• Partition within the column is caused

by partial and selective adsorption on a

solid surface rather than solubility in a

liquid phase as in GLC

• “Both packed & open tubular columns

can be used”

Characteristics & Comparison to GLC

• Higher temperatures are possible (500oC).

• No liquid stationary phase is used

• Distribution coefficients, k, are generally much larger than those for GLC. Thus separation of species that are not retained by GLC ( such as : components of air, H2S, CO, CO2 & rare gases) is possible.

• Availability of stable solid surfaces

• High column efficiencies due to no liquid phase contribution to band spreading.

• Elimination of liquid substrate bleed effect

• High flow rates & short analysis time.

• GSC, mainly, used in the analysis of extremely volatile Substances most of which are gases at room temperature

Solid Stationary Phase

(Adsorbents)

Types of Adsorbents

• Molecular Sieves

• Silica Gel

• Alumina

• Carbosieve®

• Spherocarb®

• Carbopacks

Molecular Sieves

• Synthetic Zeolites ( Aluminosilicates)

Al2O3 : SiO2 :H2O

• (Commercially: 3A, 4A, 5A & 13 A).

• Separation is based on the molecular size.

Molecules that have polar or polarizable

properties are adsorbed.

• They are packed easily; very durable in use;

possess high batch to batch uniformity.

• They are used for drying and purification

purposes.

• Synthetic zeolites (alkali metal alumino-silicate)

– A12O3 . 1.92 SiO2 . X H2O

• 5A – 5A pore diameter (better resolution

• 13X – 10A pore diameter (rapid analysis)

• Surface area – 700-800 m2/g (Very large)

• Must be activated at 300oC for two hours

• Separate H2, O2, N2, CH4, CO, Ar

• CO2, H2S, SO2, Cl2, HCl are adsorbed

Separation of light fixed gases by Molecular Sieve 5A

By time H2O will be adsorbed causing poor separation of N2 & O2

Re-activate by heating at 300oC for several hours

CAPILLARY GC COLUMNS

(OPEN TUBULAR COLUMN GC)

( HIGH RESOLUTION GC)

What are capillary columns?

• Long thin tube of glass, fused silica or other

material (stainless steel)

• Diameter: ~ 0.1 to 1 mm internal diameter.

• Internal diameters of ~0.05 mm are

manufactured for super critical fluid

chromatography.

• Length: 10 - 100 m. 15 to 30 m are more

common. Most of the times, we work with

columns much longer than we need.

Capillary (Open tubular) Columns

Capillary (Open tubular columns)

Elution of the sample through a packed column

Eddy diffusion

Why Capillary columns?

• The pathways through the column are practically the same length for all molecules of the sample; that is eddy diffusion is virtually ZERO and sharper peaks are expected

• Resistance to mass transfer is much smaller than the packed columns thus tR is always relatively short

• The very thin and uniform film promotes a rapid approach to equilibrium in the partition process.

• less bleed of stationary phase (thin films and low temperatures)

• Less adsorption of trace compounds due to lower surface area compared to packed columns and deactivation process of the column surface

• Heat transfer to the column is superior to that of the large packed columns.

• Columns are very long

• Very impressive separations are obtained by these columns

• Can be used for trace analysis (subpicogram level)

1.8 m X ¼’’(0.64 cm) packed column

152 m X 0.76 mm stainless steel OTC

50 m X 0.25 mm glass OPT

Separation of peppermint oil

35 m X 0.35 mm glass column coated with SE 30

Isothermal at 200 oC

65 m X 0.30 mm glass column coated

with SE 32 Programmed at 100-300 oC

50 m X0.5mm stainless steel

Column coated with OV-17

Isothermal at 270 oC

22 m X 0.26 mm glass

Column coated with SE 52

Programmed at 100-260 oC

Separation of polycyclic aromatic hydrocarbons

Separation of Styrene Impurities

Packed column, Isothermal at 95 oC,

Analysis time = 34 min, stationary phase

Was 1,2,3 tris (2-cyanoethoxy)propane

68 m X 0.28 mm OTC, isothermal at

80 oC, 7 min anaysis time. Same

stationary phase

Capillary column gas chromatography

• The instrument should be modified to accommodate

the capillary column

• Samples injection and separation optimization

should be modified from those used for

packed columns

• Major difference from packed columns:

• Smaller ID

• Longer

• No packing

• Smaller sample capacity

• These characteristics will allow components to be

retained longer. However, the peak shape is still good

How sensitivity is improved with capillary columns?

TYPE OF CAPILLARY COLUMNS

1. Open Tubular Columns

A. Wall Coated Open Tubular Columns (Wcot)

• Liquid phase is deposited directly on the

glass surface without inclusion of any

additive (sometimes, microcrystalline

deposits are used)

• Columns are made of Glass or fused silica

(FSOT Columns)

• Fused silica columns possess thin walls

and they are made stronger by outside

protective polyimide coating

Glass OT columns

Fused silica Open Tubular Columns FSOTC

B. Supported Coated Open Tubular Columns(SCOT)

• Liquid phase is supported on a surface

covered with some type of solid support material (porous diatomaceous earth)

• Greater sample capacity compared to WCOT but less efficiency

• Coating solution contains the support powder as a suspension (one step coating)

C. POROUS LAYER OPEN TUBULAR COLUMNS (PLOT)

• Inner surface has been extended by

substances such as fused silica, or

heavy crystalline deposits

• Porous layer is deposited followed by

coating (two steps coating)

Typical dimensions of OTC for GC

Al-clad fused silica GC

column

Cross sectional view of wall-coated, support coated

and porous layer columns

Separation of a perfume oil

1.5 m X2mm packed column

30 m X 0.25 mm OTC

Carbowax 20 M stationary phase (Scale is reduced for taller peaks)

BONDED PHASE COLUMNS

• Stationary phase is chemically bonded directly to silicon atoms on the inner surface of the walls of the column (glass or silica columns).

• Glass bonded-phase Columns: metallic sites on the surface can increase the retention of polar compounds. Metals can be removed by leaching but the glass becomes brittle.

• Fused Silica bonded-phase Columns: Fused silica is high purity glass with the composition SiO2 and characterized by high purity. They possess higher resolution and efficiency;

• Less brittle than glass: easier to handle without damage.

How the stationary phase may be bonded to the silicate surface?

Si OH + Cl Si

R

R'

R

OSi

R

R'Si

R

Characteristics of the bonded phase columns

• Considerably higher maximum operating temperature

• Low bleed

• Much longer useful life

• By washing with an appropriate range of solvents, it is possible to recover the performance of a degraded column

• Range of Bonded-Phase Capillary Columns

• Below, several stationary phases used in bonded open tubular columns (According to J & W Scientific Products):

Most Common GC Detectors Most common detectors roughly in order from most common

Flame Ionization Detector (FID),

•Thermal Conductivity Detector (TCD or hot wire detector),

•Electron Capture Detector (ECD),

•Photo Ionization Detector (PID),

•Flame Photometric Detector (FPD),

•Thermionic Detector

•VERY expensive choices: Atomic Emission Detector (AED)

•Ozone- or Fluorine-Induced Chemiluminescence Detectors.

•All of these (except the AED) produce an electrical signal that

varies with the amount of analyte exiting the chromatographic

column.

• Fourier Transform Infared Detector (FTIR)

• Mass Spectrometer (MS)

• Other: UV, FT-NMR

Detector Type Support gases Selectivity Detect

ability

Dynamic

range

Flame

ionization

(FID)

Mass

flow

Hydrogen and

air Most organic cpds.

100

pg 107

Thermal

conductivity

(TCD)

Concen

-tration Reference Universal 1 ng 107

Electron

capture

(ECD)

Concen

-tration Make-up

Halides, nitrates, nitriles, peroxides,

anhydrides, organo-metallics 50 fg 105

Nitrogen-

phosphorus

Mass

flow

Hydrogen and

air Nitrogen, phosphorus 10 pg 106

Flame

photometri

c (FPD)

Mass

flow

Hydrogen and

air possibly

oxygen

Sulphur, phosphorus, tin, boron,

arsenic, germanium, selenium,

chromium

100

pg 103

Photo-

ionization

(PID)

Concen

-tration Make-up

Aliphatics, aromatics, ketones,

esters, aldehydes, amines,

heterocyclics, organosulphurs, some

organometallics

2 pg 107

Hall

electrolytic

conductivity

Mass

flow

Hydrogen,

oxygen

Halide, nitrogen, nitrosamine,

sulphur

Schematic of a thermal

conductivity detector cell

TCD

(TCD)

an arrangement of two sample detector cells and two

reference detector cells.”

• Two pairs of TCDs are used in gas chromatographs.

• One pair is placed in the column effluent to detect

the separated components as they leave the column.

• Another pair is placed before the injector or in a

separate reference column.

• The resistances of the two sets of pairs are then

arranged in a bridge circuit.

• The heated element may be a fine platinum, gold, or

tungsten wire or, alternatively, a semi conducting

thermistor.

• The resistance of the wire or thermistor gives a

measure of the thermal conductivity of the gas.

• Elutionheat lossincreased resistance needed to

balance bridge = recorded

Characteristics of TC detector

• Specificity - very little - will detect

almost anything including H2O - called

the universal detector.

• Sensitivity to 10-7 grams/sec - this is

poor - varies with thermal condition of

the compound.

• Linear dynamic range; 104 - this is poor

- response easily becomes nonlinear.

•sample burned in H2/air

flame

•sample must be

combustible

•must use electrometer

•flame resistance 1012W

•ppm sensitivity

•destructive

(FID)

View of FID

Flame Ionization Detector

Basic Principle • The effluent from the column is mixed with hydrogen

and 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,

and a collector electrode is located above the flame.

• The current resulting from the pyrolysis of any

organic compound is measured which is proportional

to the carbon content of the molecule entering.

• FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response.

• The FID is a useful general detector for the analysis of organic compounds;

– it has high sensitivity,

– a large linear response range,

– low noise.

– robust and easy to use

– unfortunately, it *destroys the sample.

Characteristics of a Flame Ionization Detector

(FID)

• Specificity - most organics.

• Sensitivity - 10-12 g/sec for most organics -- this is

quite good.

• Linear range 106 - 107 -- this is good.

•

GC-MS: The equipment

Gas chromatograph –

• this is an oven that contains a 20-60 meter long capillary column (wound into a coil). The internal diameter is 0.2-0.5 mm.

• The column contains a thin film of a resin inside.

• The column is connected to a pressurized tank of an inert gas (usually helium) that creates a flow of gas through the column.

• The sample (containing a mixture of compounds) is injected at the top of the column when the temperature is low (typically between 40 and 80 C).

• The temperature is then raised and the different compounds are separated based on their volatility and their affinity for the column.

Mass spectrometer –

• The traditional (not TOF) mass spectrometer ionizes the sample (in this case an individual compound) with the use of electrons (electron impact ionization).

• The resulting ions are then accelerated in an electric field and then subjected to a magnetic field that causes the ions to deviate from a straight course.

• The deviation is a function of the mass-to-charge (m/z) ratio.

• Many of the ions fragment upon electron impact.

• Between the m/z ratio of the molecular ion (unfragmented) and the fragmentation pattern that is observed it is possible to determine the identity of the compound that eluted off of the GC column.

Internal Construction of a Gas Chromatograph-

Mass Spectrometer

Inject Fragment and Ionize

Separate Fragment Ions

Detect Fragment Ions

Separate Components Of the Mixture

Display Spectrum

GC-MS: A Separation and Identification

Method

Time since injection. Separation!

Mass spectrum of each Component of the mixture. Identification!

The data

5 10 15 20 25 30 35 40 45 50 55 60 65

2500e3

5000e3

7500e3

10.0e6

12.5e6

15.0e6

0 25 50 75 100 125 1500e3

500e3

1000e3

1500e3

2000e3

2500e3

3000e3

3500e3

4000e3

120

91

65

51

77 105144136

0 25 50 75 100 125 150 1750e3

250e3

500e3

750e3

1000e3

1250e3

1500e3

1750e3

2000e3

2250e3

2500e3 150

135

77

107

51

6389

117 166 175

chromatogram

Retention time

mass spectrum

m/z