Gas Chromatography

Gas chromatography - Introduction, theory, instrumentation,

derivatization, temperature programming, advantages,
disadvantages and applications
 Introduction to Gas Chromatography
• Gas chromatography (GC) is a powerful analytical technique that can be used to separate,
identify, and quantify individual chemical components in complex mixtures.

• The word “gas” in GC does not refer to the type of samples the technique applies to, but rather
the fact that a gas carries the sample through the instrument. This is called the carrier gas or
mobile phase and is commonly high-purity helium, hydrogen, or nitrogen. Indeed, most GC
methods target liquid samples and there are even applications for solids.

• The fundamental requirement for samples to be analyzed by gas chromatography is the

compounds of interest in the sample must volatilize without thermally decomposing.

• Used in environmental analysis, food and flavor testing, forensics, and pharmaceuticals to detect
and quantify mixtures of volatile compounds.

• High sensitivity, fast analysis, and precise quantification are the strengths of GC.
 Principle of Gas Chromatography
• The principle of separation in GLC is “partition”.
• Gas is used as mobile phase.
• Liquid which is coated on to a solid support is used as stationary phase.
• The mixture of compounds to be separated is converted to vapour and mixed with gaseous
mobile phase.
• The component which is more soluble in the stationary phase travels slower and eluted later
and the component which is less soluble in the stationary phase, travels faster and eluted out
first.
• No two components has the same partition co-efficient for a fixed combination of stationary
and mobile phase and other conditions.
• Hence the components are separated according to their “partition co-efficient”.
 Theory of Gas Chromatography
There are two theories that explain the separation of compounds in gas chromatography-Plate
Theory and Rate Theory.
• The plate theory, introduced by Martin and Synge in 1941, provides a simplified, hypothetical
view of column efficiency in chromatography.
• It conceptualizes the column as a series of "theoretical plates" or layers where equilibrium is
established between the mobile and stationary phases. As the mobile phase moves through the
stationary phase, analytes in the mobile phase equilibrate repeatedly, allowing separation based
on their distribution. Higher numbers of theoretical plates (N) improve resolution, narrowing
peak width and enhancing separation of sample components. Column efficiency is indicated by
the Height Equivalent to a Theoretical Plate (HETP), with a lower HETP value indicating
better efficiency.
 Measuring Efficiency with Plate Theory

• The efficiency of a column is measured by the number of theoretical plates (N).

• Higher N means more "steps" or plates, leading to better separation and sharper

peaks on the chromatogram.

N=16 (Retention Time/Peak Width)2

Retention Time: How long it takes a compound to travel through the column.

Peak Width: The width of the compound's peak in the chromatogram

• The rate theory of chromatography is crucial for understanding analyte movement within
the column.
• It explains how solute elution impacts band shape, affected by the elution rate, providing a
realistic view of peak dispersion and factors contributing to band broadening.
• Rate theory focuses on the time required for analytes to equilibrate between the
stationary and mobile phases, directly influencing chromatographic peak shape.

u = Mobile phase velocity

 INSTRUMENTATION
Flow Regulators
Top view
Flame
Injection Port Ionizatio
n
Detector

Column

Oven
Front view
 CARRIER GAS
• The purpose of carrier gas is to transmit the sample from the point of introduction through the
column to the detector.”

The requirements of a carrier gas are:

It should be inert and not interact chemically with the sample.
It should be easily available.
Cheap.
Less risk of explosion (or) fire hazards.
It should give best column performance consistent with the required speed of analysis.

It should be of high purity because impurities such as oxygen and water can chemically
attack the liquid phase and destroy it.

 The carrier gases used in GC are Hydrogen , Helium & Nitrogen.

Carrier gas Advantages Disadvantages

Hydrogen  Cheap  Can form an explosive

 It has better thermal conductivity, low mixture with air
density. (inflammable)
 Gives the most time efficient separation
(TCD & FID)
 Very efficient even at high gas velocities
i.e.. 60 cm/ sec
Helium  Very inert, will not react with analytes  Expensive
 Gives a very time efficient separation  A non-replenishable resource
 Non flammable

Nitrogen  Itis not expensive  Very slow velocity to

 Very inert, will not react with analytes achieve good efficiency
 FLOW REGULATORS

 As carrier gases are stored under high pressure, flow regulators are used to deliver the gas
with uniform pressure and flow rate

These are of three types

 Rotameter

 Soap bubble meter

 Digital electronic flow measuring device.

 Injection ports
• Handle gas or liquid samples. Often heated to vaporize liquid samples. Liquid or gas syringes
are used to insert the sample through a septum into the carrier gas stream.

• The injection port introduces the sample into the carrier gas

stream, allowing it to enter the chromatographic column for

separation and analysis.

• It vaporizes the sample (if needed) and ensures consistent and

reproducible injection volumes.

Design: The injection port is usually heated to facilitate the rapid vaporization of liquid samples,
ensuring that they transition into the gas phase quickly and efficiently.
Type of Port: There are several designs, including:
• Split Injection Port: Allows a portion of the sample to enter the column while venting the
excess, ideal for concentrated samples.
• Spitless Injection Port: Introduces the entire sample into the column without venting,
suitable for trace analysis.
• Pneumatic Injection Port: Utilizes gas pressure for injection, enhancing precision and
reducing contamination.
• Programmable Temperature Vaporization (PTV) Port: Heats samples gradually to
optimize vaporization, particularly for high-boiling-point compounds.
 COLUMN
 Column is one of the important part of GC which
decides the separation efficiency.

 GC columns are generally available in lengths

ranging from 5 to 60 meters and are usually made of
stainless steel or glass or fused silica or Teflon.

 Stainless steel columns have the advantage of long

life and can be easily handled.

 But some samples like steroids react with stainless

steel. In such cases glass columns are used.
Columns in GC can be classified according to the nature as well as its use.
1. Open Tubular or Capillary Columns
• Material: Typically made of fused silica (a high-purity glass).
• Coating: Covered with a thin layer of polyimide on the outside to provide flexibility and protect
against breakage.
• Stationary Phase: The inner wall is coated with a thin film of stationary phase (e.g., polysiloxanes,
polyethylene glycol) suited for the specific application.
• Use: These columns are popular due to their high efficiency and are used for detailed separation in
applications requiring high resolution.
Open Tubular or Capillary Column:
 Basically it is of 2 types
 Wall-coated open tubular columns(WCOT)
 Support coated open tubular columns (SCOT)
• In wall coated open tubular columns , the stationary
phase is coated directly on the inner walls of the tubing.
• The newest WCOT are made up of specially purified
silica (fused silica open tubular columns).
• Most widely used fused silica open tubular columns
have inside diameter of 0.1 to 0.53 mm and length of
10 -100 meters.
Packed column
2. Packed Columns
• Material: Often made of stainless steel, glass, or Teflon.
• Packing Material: Filled with a solid support material, commonly
diatomaceous earth coated with a liquid stationary phase.
• Use: Packed columns are generally more robust and are used in
applications requiring larger sample sizes or when high efficiency is not
critical.
 DEPENDING ON ITS USE
ANALYTICAL COLUMN

 Analytical columns have a length of 1-1.5 metres

 Outer diameter: 3-6 mm

 These are packed columns and made up of glass or stainless steel.

 Only small amount of sample can be loaded on to the column.

PREPARATIVE COLUMN

 Larger compared to analytical column

 Length: 3-6 metres

 Out side diameter: 6-9 mm

 Large amount of sample can be loaded on to the column.

 STATIONARY PHASES

Organic high boiling liquids such as polydimethylsiloxanes coated on surface of diatomaceous

earth is used as stationary phase in gas chromatography.
Liquid phases can be broadly classified into:
Non polar hydrocarbon-type Liquid phases :
Eg: Poly methyl siloxane, Squalane, paraffin oil, silicone gum rubber,
Poly(di phenyl)di methyl siloxane.
Compounds of intermediate polarity:
Eg: Poly cyno propyl phenyl dimethyl siloxane.
Polar compounds:
Eg: Carbo waxes (Poly Ethylene Glycol , poly Alkaline Glycol)
Very polar compounds:
Eg: Glycols, Glycerol, polyphenols.
The column oven

Inside here Column

 COLUMN OVEN

Column temperature is an important variable that must be controlled for precise work.

The optimum column temperature depends upon the boiling point of the sample and degree of
separation.

Roughly a temperature equal to or slightly above the average boiling point of a sample results in a
reasonable elution time (2-30 minutes).

Types of operations:

(a) Isothermal programming: same (constant) temp.is maintained.

(b) Linear programming: heated linear over a period of time (a sample has a mixture of low boiling
and high boiling point compounds.

GC oven is maintained from ambient to 360ºC.

 DETECTORS
Detector is situated at the exit of the separation column.

It is used to sense and measure the small amounts of the separated
components present in the carrier gas stream leaving the column.

Some requirements of GC detectors are:

It should have high sensitivity to even small concentration.

It should have large linear range.

It should have good stability.

It should show universal or selective response

Inexpensive

Simple and easy to maintain


 DETECTORS

 Flame ionization detector (FID)

 Thermal Conductivity detectors (TCD) /Katharometer

 Electron Capture detector (ECD)

 Nitrogen-Phosphorus (NPD)

 Photo Ionization Detectors (PID)

 Mass Spectral (CI/EI)

 Flame Photometric (FPD)

Electrolytic Conductivity (Hall/ELCD

FLAME IONISATION DETECTOR
FID is the most widely used GC detector.
PRINCIPLE:
• The principle of ionization detector is based on the conduction of
electricity by gases.
MECHANISM:
Compounds are burned in a hydrogen- air flame carbon containing
compounds produce ions that are attracted to the charged electrodes.
The number of ions will be measured and a signal will be generated.
• Carrier gases : Nitrogen or Helium.
• Selectivity : compounds with C-H bonds, a poor
response for some nonhydrogen
containing organics( eg: hex chlor benzene)
• Sensitivity : 0.1 -10 ng.
• Linear range : 105 to 107.
Temperature : 250- 300ºc.
ADVANTAGES:

Extremely sensitive and background noise is low.

Stable and in sensitive to small changes in the flow rate of carrier gas.

Responds to most of the organic compounds

Linearity is excellent.

The following are some compounds that are not detected

by

the FID.
Ex: H2, O2, N2, Sicl4, H2O, CO, SO2, CS2, Ar, Kr, Ne, XE.
 THERMAL CONDUCTIVITY DETECTOR:
Principle:The principle of TCD is based on the measurement of the
difference in thermal conductivity between the carrier gas and carrier gas
(sample vapor) mixture.
Mechanism:
It works on the principle of whetstone bridge, which has four
resistances in the four arms of the bridge.
The carrier gas passes over these two resistances.
o Carrier gases : Hydrogen or helium.
Selectivity : It responds to all compounds (Universal)
o Sensitivity : 5-20 ng.
Linear range : 105 to 106.
Temperature : 150-250ºc.
 Suitable for preparative work as the eluted compound is
not destroyed.
-Non destructive to the sample in case of preparative
work.
- Inexpensive.
 ELECTRON CAPTURE DETECTOR:
The electron affinity of different substances can be used as the basis for an ionization detector
known as the ECD.
It responds to only those compounds whose molecules have an affinity for electrons. Eg:
chlorinated compounds, alkyl lead etc.
Mechanism:
Generally, 63 Ni, tritium foils are used as radioactive source. The foil is placed inside the cell.
The radioactive 63 Ni emits β – particles. 63Ni → β -. These negatively charged particles
collide with the nitrogen carrier gas and produce more electrons.
β- + N2 → ee- + N2+
The e- released is attracted towards anode as a result high potential is seen. When an analyte is
eluted from the column it enters the detectors, it captures some of the free e - and the standing
current decreases giving a negative peak. A + e- → A-
The process obeys Beer's law thus the extent of capture is proportional to concentration of
analyte.
The ECD is most frequently used for detection and measurement of trace environmental
pollutant.
Carrier gases : nitrogen or argon/methane.
Selectivity: halogens, nitrates and conjugated carbonyls.
Sensitivity: 0.1-10 pg. (halogenated compounds),
1-100 pg (nitrates), and 0.1- 1 ng (carbonyls).
Linear range: 103 to 104.
Temperature: 200ºc.
NIROGEN PHOSPHOROUS DETECTOR:
MECHANISM:
Compounds are burned in a rubidium bead supplied with hydrogen and air.
Nitrogen and phosphorous containing compounds produce ions that are
attracted to the collector. The number of ions getting the electrode is measured and
a signal will be generated.
Gases : hydrogen or helium.
Selectivity : nitrogen and phosphorous containing
compounds.
Sensitivity : 1-10 pg
Linear range : 10 4 to 106
Temperature : 250- 300ºc
PHOTO IONIZATION DETECTOR:
PRINCIPLE:
The principle involved in this detector is “ionization’’.
It induces ionization via photons emitted by ultra violet lamp.
MECHANISM:
Compounds eluting in a cell are bombarded with high energy photons emitted from a
lamp. compounds with a ionization potential below the photon energy are ionized. The
resulting ions are attracted to an electrode , measured and a signal will be generated.
Carrier gases: helium or nitrogen
Selectivity: depends on lamp energy , usually used for aromatic and olefins.(10 ev
lamp).
Sensitivity:25-50 pg ( aromatic) , 50-200 pg (olefins)
Linear range: 105 to 106
Temperature: 200ºC.
RECORDERS AND INTEGRATORS:
RECORDER:
• The signal from a gas chromatograph is
continuously recorded as a function of time
generally by a Potentio metric detector.
• In potentiometric detector the input response.
continuously balanced by a feed back response thus
recording the signal.
INTEGRATOR:
• An integrator is employed for simultaneous
measurement of area under chromatographic peaks
by a mechanical /electronic means.
 DERIVATIZATION
“Derivatization is a process of chemically modifying a compound to produce a
new compound which has properties that are suitable for analysis using GC”.

• Purpose of derivatization:

• To permit analysis of compounds not directly amendable to analysis, due to

inadequate volatility or stability.

• Improve chromatographic behavior or detectability.

• Many compounds do not produce a useable chromatography or the sample of

interest goes undetected. As a result it may be necessary to derivatize the
compound before GC analysis is done.

Used to impart volatility to non-volatile compounds.

Used to improve thermal stability and detectability.

Derivatization reactions can be classified into 4
types:
• 1. Silylation
• 2. Alkylaton
• 3. Acylation
• 4. Co-ordination complexation
Silylation: most prevalent methods, readily volatized the sample
 This process produces silyl derivatives which are more volatile, less stable& more
thermally stable.

 Replaces active hydrogens with TMS (trimethyl silyl groups)

 Silylation occurs the nucleophile attack (SN 2). The betters the leaving group, the better
the sialylation.

 Silylation reagents will react with H2O & alcohols first care must be taken to ensures
that both sample & solvent are dry.

 Solvent should be as pure as possible. This will eliminate excessive peaks. Toy using as
little solvent as possible as this will prevent a large solvent peak.

 Pyridine is the most commonly used solvent. Although pyridine may produce peak
tailing it is an acid scavenger & will drive the reaction forward.
• Advantages:

- Ability to sovlate a wide variety of compounds.

- Large number of silylating reagents available.

- Easing prepared.

• Disadvantages:

- Silylation reagents are moisture sensitive

- Must use aprotic (no proton available) organic solvents.

- Examples : TMSI (Tri Methyl Silyl Imidazole)

BSA (Bistrimethyl Silyl Acetamide)

BSTFA (Bistrimethyl Silyl Tri Fluoro Acetamide)

MSTFA (N-Methyl trimethyl Silyl Tri Fluoro Acetamide)

TMCS (Tri Methyl Chloro Silane)

TMS-DEA (Tri Methyl Silyl – Di Ethyl Amine)

• Alkylation’s: (used as a first step to further derivatization or as a method of protection
of certain active hydrogen’s)

• Alkylation’s reduces molecular polarity by replacing active hydrogens with an alkyl

group.

• These reagents are used to modify compounds with acidic hydrogen, such as carboxylic
acids & phenols.

• These reagents make esters, ethers, alkyl amines & alkyl amides that they can be used in
conjunction with acylation or silylation.

• The principle reaction employed for perception of these derivatives is nucleophilic

displacement.

• It is generally used to convert organic acid into esters. As the acidity of the active
hydrogen, the strength of the alkylating reagent must be

• Alkyl esters have excellent stability & can be isolated & stored for long
periods of time.
• Alkylating reagents:

- Dialkyl acetals (DMF)

- Tetra Butyl ammonium Hydroxide (TBH)

- Penta Fluoro Benzoyl Bromide (PFBB)

- BF3 in methanol or butanol

• Advantage:

- Wide range of alkylation reagents available.

- Reaction conditions can vary from strongly acidic to strongly basic.

- Some reaction can be done in selections.

- Alkylation derivative are generally stable.

• Disadvantage:

- Limited to amines & acidic hydroxyls

- Reagents are of ten toxic

- Reaction conditions are frequently severe

• Acylation:

• Acylation reduces the polarity of amines hydroxyl & thiol groups & ads halogenated
functionalities for ECD. In comparison to silylatiion reagents, the acylation reagents target
highly polar, multi functional components such as carbohydrates &

- Acyl derivatives are formed with acyl anhydrides, acyl halides & activated acyl amide
reagents.

- The anhydrides & acyl halides form acid by products which must be removed before GC
analysis.

- Fluorinated acyl groups, going from tri flrore acetyl to hepta fluoro butyryl can be used
to retention times.

- Acylation converts those compounds with active hydrogen’s into esters, thioesters &
amides.
- Acylation are normally carried out in pyridine, tetra hydro furan con another solvent capable of
accepting the acid-by-products.

- The presence of a carbonyl group next to the halogenated carbons enhances the ECD

• Examples of acylating agents: 1. Fluorinated anhydrides:

- Tri Fluoro Acetoic Anhydride (TFAA)

- Penta Fluoro Propionic Anhydride (PFPA)

- Hepta Fluoro Butyric Anhydride (HFBA)

• 2. Fluoro acyl imidazoles:

- Tri Fluoro Acetyl Imidazoles (TFAI)

- Penta Fluoro Propanoyl Imidazoles (PFPI)

- Hepta Fluoro Butyryl Imidazoles (HFBI)

• 3. N-Methyl Bis (Trifluoro Acetamide) – MBTFA

• 4. Penta Fluoro Benzoyl Chloride – PFBCI

• 5. Penta Fluoro Propanol - PFPOH

Advantage:
- Addition of halogenated carbons detectability by ECD
- Derivatives are hydrolytically stable
- sensitivity by adding molecule weight
- Acylation can be used as a 1st step to activate COOH prior to esterification
(ackylation)
Disadvantage:
- Acylation derivatives can be difficult to prepare
- Acylation reagents are moisture sensitive, hazardous & odorous
- Reaction products (acid-by-products) often need to be removed before
analysis.
4. Co-ordination complexation:
Used with metals which can be converted to volatile
compounds after derivatization.
Derivatization reactions can be classified into 4 types:

ALKYLATION:
Used to protect certain active hydrogen's

ACYLATION
Commonly used to add fluorinated groups (for ECD detector)

CO-ORDINATION COMPLEXATION:
Used with metals which can be converted to volatile compounds after derivatization.
SILYLATION:
Readily volatizes the sample and most prevalent method.
Alcohols ,Phenols ,Carboxylic acids ,Amines , etc can be silylated.
Some silylating reagents are: Bis-trimethyl-silyl-acetamide (BSA), (Tri Methyl Silyl
Imidazole) (TMSI).
 Applications of Gas chromatography
1. Qualitative analysis:
Retention time and retention volume of an eluate is characteristic of a particular
component. These can be used for qualitative analysis.
2.Purity of components:
Purity of solvent and purity of drug(thermally stable).
3.Quantitative analysis:
Measuring the peak area or peak height of compound.
Pharmaceutical applications:
i. Steroids and alkaloids
ii. Bile acids and cholesterol
iii. Excipients like magnesium stearate.
iv. Vegetable oils like castor oil, soyabean oil etc.
iv. Volatile oils.
v. Drugs and their metabolites. Ex: Tetracyclines,
penicillin's,
cephalosporins and
sulpha drugs.
vi. Residual solvents or organic volatile impurities:
Dichloromethane and dimethyl alanine in ampicillin sodium.
Ethyl acetate and chloroform in colchicine.
Methanol in gentamycin sulphate.
Prponol-2-ol in warfarin.
vii. medicinal gases
viii. Solvents like ethanol, acetone, isopropyl alcholol.etc.
It can also used for the volatile samples such as human breathe , blood and saliva etc.
which contain organic volatiles.
A quality control units also use GC in order to determine the compounds of a given air
sample.
GC is used in cosmetics to analyze the how much quantity is present in each product.
 GC is used in analysis of methyl testosterone in tablets.
 GC is used in analysis of atropine in eye drops.
 In determination of manufacturing and degradation residues GC is applied.
Eg 1. Determination of pivalic acid in olipivefrin eye drops.
2. Determination of dimethylaniline in bupivacaine.
GC is used to analyze the dairy products for aldehydes and ketenes. (for rancidity)
Applications in pharmaceutical analysis:
 Antibiotics: penicillin's and derivatives, gentamycin ,

kanamycin, neomycin, tetracyclines .

 Anti T.B drugs: Isoniazid, ethambutol.
 Anti virals: Amantadine, idoxuridine, cytarabine.
 Anti neo plastics: Fluorouracil, 6-mercaptopurine.
 General anesthetics: Ether, ethanol, chloroform.
 Sedative hypnotics: Barbiturates, glutethimide.
 Tranquillizers: Diazepam, flurazepam, chlordiazepam.
 CNS stimulants: Nikethamide, Caffeine, theophylline.
TEMPERATURE PROGRAMMING IN GC
Temperature Zones in GC
Three temperature zones should be adjusted before a GC separation can be
done.

 The injector temperature should be such that fast evaporation of all sample
components is achieved.

 The temperature of the injector is always more than that of the column, which
depends on the operational mode of the separation.

 The detector temperature should be kept at same level so as to prevent any

solute condensation in the vicinity of the detector body.
11/19/2024 55
 Temperature Programming
Gas chromatographs are usually capable of performing
what is known as temperature programming gas
chromatography (TPGC).

 The temperature of the column is changed according to

a preset temperature isotherm.

TPGC is a very important procedure, which is used for

the attainment of excellent looking chromatograms in the
least time possible.
11/19/2024 56
 Isothermal at 45°

Isothermal at 145°

Programmed 30 to 180°

57
58
59
60
 The General Elution Problem
Look at the chromatogram below in which six components are to be separated by an elution
process using isothermal conditions at for example 120 oC

Figure 1 Figure 2

• It is clear from the figure 1 that the separation is optimized for the elution of the first two
components. However, the last two components have very long retention and appear as broad
peaks. Using isothermal conditions at high temperature (say for example 200 oC) can optimize
the elution of the last two compounds but, unfortunately, results in bad resolution of the earlier
eluting compounds as shown in the figure 2 where the first two components are coeluted while
the resolution of the second two components becomes too bad. 61
One can also optimize the separation of the middle too components by adjusting the
isothermal conditions (for example at say 160 oC). In this case, a chromatogram like the one
below can be obtained:

62
• However, in chromatographic separations we are interested in fully separating all components
in an acceptable resolution. Therefore, it is not acceptable to optimize the separation for a single
component while disregarding the others. The solution of this problem can be achieved by
consecutive optimization of individual components as the separation proceeds. In this case,
temperature should be changed during the separation process. This is called temperature
programming gas chromatography (TPGC).
• First, a temperature suitable for the separation of the first eluting component is selected, and
then the temperature is increased so that the second component is separated and so on. The
change in temperature can be linear, parabolic, step, or any other formula. The chromatographic
separation where the temperature is changed during the elution process is called TPGC. A
separation like the one below can be obtained:

63