GAS

CHROMATOGRAPHY

NAUREEN SHEHZADI
M.PHIL. (PHARMACEUTICAL CHEMISTRY)
UNIVERSITY COLLEGE OF PHARMACY,
UNIVERSITY OF THE PUNJAB, LAHORE, PAKISTAN
 OUTLINE
• History • How GC works?
• Definition • Factors affecting sample
• Principle of separation separation
• Requirements for GC • Chromatogram analysis
• Types • Derivatization of samples
• Instrumentation • Compound identification
• The carrier gas • Applications
• The flow regulators or flow
meters
• The injection port
• The column
• Stationary phases
• Supports
• Temperature control devices
• Detectors
• Recorders and integrators
 HISTORY
• German graduate student “Fritz Prior” developed solid state
gas chromatography in 1947.

• Father of modern gas chromatography is considered to be

“Archer John Porter Martin” who laid the foundation for the
development of gas chromatography. He produced liquid-
gas chromatograph in 1950.
 DEFINITION
Gas chromatography is a technique whereby the components
of a mixture in the gaseous state are separated as the sample
passes over a stationary liquid or solid phase. Differences in the
interaction between the sample components and the stationary
phase account for the separation.

Stationary phase:
1. Solid
2. Liquid

Mobile phase:
Gas
PRINCIPLE OF SEPARATION

Principle of separation
in gas
chromatography

Partition Adsorption
 REQUIREMENTS FOR GC
• Low molecular weight
• Thermally stable compound
• High vapor pressure
• Low boiling point
 TYPES

Types of gas
chromatography

Gas-liquid Gas-solid
chromatography chromatography
1- Gas-liquid chromatography

Gas liquid chromatography consists of mobile gas phase and

stationary liquid that is coated on to either;

• A solid matrix (diatomaceous earth)

• Wall of a capillary tube

Typically the stationary phase has sufficiently low vapor pressure

(mm) at column temperature so that it can be considered as
non-volatile.
Sample mixture in gaseous form is run through the column with a
carrier gas.

Separation can be achieved by the differences in distribution

ratios of the components of the sample between the mobile
and stationary phases causing them to move through column at
different rates and with different retention times.
2- Gas-solid chromatography

Gas solid chromatography consists of mobile gas phase and

stationary solid phase. It is not used commonly because of less
wider applications.
 INSTRUMENTATION
A typical gas chromatograph consists of following components;

§ The carrier gas

§ The flow regulators or flow meters
§ The injection port
§ The column
§ Stationary phases
§ Supports
§ Temperature control devices
§ Detectors
§ Recorders and integrators
1- The carrier gas
It is the mobile phase of system.

A high pressure gas cylinder is used as a carrier gas reservoir.

REQUIREMENTS OF A CARRIER:

• Suitable for the detector

• Highly pure
• Easily available
• Cheap
• Should not cause the risk of fire
• Should give best column performance
 CARRIERS ADVANTAGES DISADVANTAGES
Hydrogen 1. Better thermal 1. Explosive hazards
conductivity 2. Reacts with unsaturated
2. Low density compounds

Helium 1) Excellent thermal Expensive

conductivity
2) Low density
3) Greater flow rates

Nitrogen Inexpensive Reduced sensitivity

Air Used only when oxygen in
the air is useful to the
detector or separation
2- The flow regulator/flow meter
It is employed to deliver the gas with uniform pressure/flow rate.

Flow meters

Rotameter Soap-bubble meter

Rotameter

• It is placed before column inlet.

• It has a glass tube with a float held on to a spring.
• The level of the float is determined by the flow rate of carrier
gas.
Soap bubble meter

It is similar to Rota meter but instead of a float, a soap bubble

formed indicates the flow rate.
3- The injection port
It is designed for instantaneous injection and vaporization of a
sample so that the sample is introduced immediately into the
column.

Sample is usually injected using a calibrated syringe.

How sample is introduced?

Gases can be introduced into the column by using gas-tight

syringe or sample loop.

Liquids can be injected through loop or septum devices.

Solids can be injected in two ways;

• Material can be dissolved in suitable solvent and then solution

is injected.
• Solid is packed into the end of syringe and then introduced
into column through a septum using a plunger.
4- The column
Column container may be made of;

§ Glass
§ Stainless steel
§ Copper tube

Glass columns are inert and highly fragile. These are used for
biological samples or for compounds which undergo reaction
with copper or stainless steel.
Types of columns (based on content)

Porous packing
Packed column
Non-porous
packing Liquid coating
Column

Wall-coated open
tubular column

Capillary Porous-layer open

column tubular column

Support-coated open
tubular column
(i) Packed column

• Columns are available in a packed manner.

• Stationary phase may be an adsorbent solid or a liquid

coated onto a solid support material.
(ii) Capillary column

These are also called open tubular, capillary column or Golay

column.

These columns are not packed but inner walls are coated with a
liquid layer. These are used for partition chromatography.

Advantage is that it provides a high degree of efficiency

Disadvantage is that more sample cannot loaded

Types of capillary columns:

There are three common types of capillary columns, which differ

in how the stationary phase and/or solid support are coated
into the column.

§ WCOT
§ PLOT
§ SCOT
5- Stationary phases
The phase chosen must meet following requirements;

§ Low vapor pressure

§ Thermally stable
§ Chemically inert
 STATIONARY APPLICATIONS
PHASES

Adiponitrile Hydrocarbons
Apiezon L Alcohols, Aldehydes, Ketones, Fatty acids,
Asphalt Pesticides
Aromatics

Beeswax Essential oils

Carbowax Aldehydes and ketones
Silicon gum Alcohol, Aromatics, Bile and urinary compounds,
rubber SE30 Drugs and alkaloids, Fatty acids, Pesticides,
Gases, Sugars, Vitamins
6- Supports
Main purpose of solid support is to provide support to the thin,
uniform film of liquid phase.

It shall meet following requirements;

§ Provide large surface area

§ Inert
§ Provide mechanical strength
7- Temperature control devices
PRE-HEATERS:
These devices convert sample into its vapor form. These are
available along with injecting devices.

THERMOSTATICALLY CONTROLLED OVEN:

Temperature maintenance in a column is highly essential for
efficient separation. So a thermostatically controlled oven can
be used to maintain temperature.
8- Detectors
A detector is the heart of the apparatus.

AN IDEAL DETECTOR:

The requirements of an ideal detector are;

• Applicability to wide range of samples
• High sensitivity
• Linearity
• Response should be unaffected by temperature, flow rate etc.
• Non destructive
• Simple
• Inexpensive
Types of detectors
(based on selectivity)

Detector

Selective detectors Non-selective detectors

• A non-selective detector responds to all compounds except

the carrier gas.

• A selective detector responds to a range of compounds with

a common physical or chemical property.
Types of detectors
(based on dependency)

Detector

Concentration dependent
Flow dependent detectors detectors

• The signal from a concentration dependent detector is related

to the concentration of solute in the detector and does not
usually destroy sample.

• Mass flow dependent detectors usually destroy the sample,

and the signal is related to the rate at which solute molecules
enter the detector.
 GC detectors

DETECTORS TYPE SELECTIVITY

Thermal conductivity (TCD) CDD Universal

Halides, nitrates, nitriles,
Electron capture (ECD) CDD peroxides, anhydrides,
organometallics

Aliphatics, aromatics, ketones,

esters, aldehydes, amines,
Photo-ionization (PID) CDD
heterocyclics, organosulphurs,
organometallics
 DETECTORS TYPE SELECTIVITY

Nitrogen-phosphorus FDD Nitrogen, phosphorus

Sulphur, phosphorus, tin, boron,

Flame photometric (FPD) FDD arsenic, germanium, selenium,
chromium
Hall electrolytic Halide, nitrogen, nitrosamine,
FDD
conductivity Sulphur

Flame ionization (FID) FDD Most organic compounds

Mass spectrometer
9- Recorders and integrators
• RECORDERS record the baseline and all the peaks obtained

• INTEGRATORS record the individual peaks with retention time,

height etc.
 HOW GC MACHINE WORKS?
• First, a vaporized sample is injected onto the chromatographic
column.

• Second, the sample moves through the column through the

flow of inert gas.

• Third, the components are recorded as a sequence of peaks

as they leave the column.
 FACTORS AFFECTING
SEPERATION
FACTORS EFFECT
Particle size Small particle size cause ↑ in surface area ↑ in
number of theoretical plates
Carrier gas flow Too slow flow, peak tend to be broad
rate
Too fast flow, peak will not be resolved
Type and amount Excess liquid support , tailing in column
of stationary
phase
Column length ↑ in column length ↑ in number of theoretical
plates
Column diameter ↓ in column diameter ↑ in number of theoretical
plates
 FACTORS EFFECT
Column ↑ in temperature will speed up all the compounds
temperature in a mixture.

Film thickness Thicker film increase retention time.

Thicker films increase stationary phase “bleed”
from the column especially at high temperatures.

Thick film is undesirable particularly with mass

spectrophotometric detection where a specific
type of capillary column is used.

Volatility of Low boiling point compound will travel faster

compound through column than high boiling point
compound.
 FACTORS EFFECT

Polarity of Polar compound will move more slowly especially

compound when column is polar

Polarity of All compounds will move slower on polar columns

column packing but polar compounds will show larger effect.
CHROMATOGRAM ANALYSIS
• The number of components in a sample is determined by the
number of peaks. The amount of a given component in a
sample is determined by the area under the peaks.

• The identity of components can be determined by the given

retention times.
DERIVATIZATION OF SAMPLE
Derivatization refers to conversion of non-volatile form of
compound to volatile form that is suitable for separation.

Or

Treatment of sample to improve the process of separation by

column or detection by detector is called derivatization.

EXAMPLES:
• Low vapor pressure organic acids can be converted to low
boiling acid chlorides
COMPOUND IDENTIFICATION
There are two techniques that are used to identify compounds
eluting from the column.

RETENTION TIME COMPARISON:

• A known pure standard is injected and the retention time is

recorded
• Sample is then analyzed and retention time is matched to the
standards
• Gas flow rate must be constant
• The temperature used must be the same for both the standard
and the sample
COMPOUND IDENTIFICATION
SAMPLE SPIKING:

This approach is more reliable than retention time matching,

especially for complex mixtures.

• The sample is run first to get a suitable separation

• A small volume of standard is added to the sample prior to
injection
• The sample is then analyzed and one peak on the sample will
increase and the rest will decrease (because of dilution)
APPLICATIONS

Qualitative analysis
Gc applications

Quantitative
analysis

Miscellaneous
Applications

Headspace GC
analysis
1- Qualitative analysis
Qualitative identification by GC is achieved by two ways;

§ Comparison of retention time of the unknown to the retention

times for a series of standards

§ Characterization of peaks through use of chemical and

instrumental tests. For example effluent can be directed to IR
spectrometer or mass spectrometer
2- Quantitative analysis
It is based on principle that the detector signal is proportional to
the concentration/ mass of a component received by detector
at that instant.

A peak is traced by continuous integration of this signal while

the total amount of component passes through the detector.

Amount of compound is related to total peak area.

3- Miscellaneous applications
Food industry

Pharmaceutical analysis

Drug detections

Petrochemical industry

Identification of volatile materials

MICELLANEOUS Metal chelates and inorganic

APPLICATIONS materials

Forensic analysis

Environmental sciences

Agrochemical industry

Paint industry

Biomedical field
Food industry

GC is being used successfully for separation and identification of

various agents in food.

For example;
• Lipids, Proteins, Carbohydrates & Vitamins etc.
• Steroids
• Preservatives
• Colorants & Flavors
• Texture modifiers
• Drugs
• Pesticide residues
• Trace elements
Food industry

Dairy products:

GC is used for checking;

• Presence of fatty acids in dairy products e.g. butter

• Milk sugars
• Presence of aldehyde and ketones in dairy products (For
rancidity)
• Added colors and flavors
Pharmaceutical analysis

It involves analysis of;

• Illicit drug samples

• Commercial drug preparations
Drug detections

GLC is used for;

• Detection of steroid and other banned drugs used by athletes

in international sports competitions
• Detection of steroids administered to animals in races
Petrochemical industry

GC finds application in petrochemical industry for separation

and identification of various compounds as well as analysis of air
for presence of various hazardous pollutants.
Identification of volatile materials

Pyrolysis GC is used for separation and identification of volatile

materials like plastics, synthetic and natural polymers, paints etc.
Metal chelates and inorganic materials

Inorganic compounds are usually non-volatile. Only some metal

hydrides and chlorides have sufficient volatility for GC.

Organometallics other than chelates that can be detected

using GC are;

• Boranes
• Silanes
• Germanes
• Organotin
• Lead compounds
Forensic analysis

Gas chromatography (GC) is one of the primary analytical

techniques used in every forensic laboratory.

Examples are;

• Analysis of body fluids for the presence of illegal substances

• Testing of fiber and blood from a crime scene
• Detection of the residue from explosives
• Detection method for ignitable liquids
Environmental sciences

Hazardous pollutants can be monitored using GLC.

For example separation and identification of

• Formaldehyde
• Carbon monoxide
• Trichloroethylene
• Benzene
• Acetonitrile
• Polycyclic aromatic hydrocarbons
• Biocides
• Organilead
• Organomercury compounds
• Organophosphorous compounds
Agrochemical industry

GC is used for identification of pesticides e.g. chlorinated

pesticides DDT, BHC.
Biomedical field

It helps in solving various clinical problems by analyzing

biological fluids e.g. blood levels of various drugs can be
determined for dose adjustments.
 ADVANTAGES
• Low viscosity of gas allows for use of long columns (up to 60M)
• Great efficiency and separation power
• High gas flow rate allows fast analysis
• Can be automated
• Many different detection methods
• Allows analysis of molecules containing specific functional
groups e.g. halogens
• Easily interfaced to mass spectrophotometry that provides
structural information about analyte
• Can detect very small quantities (10-12g)
• Widely applicable to many different compounds
 LIMITATIONS
• Not applicable to important non-volatile compounds e.g.
proteins
• Expensive equipment
• Skilled operator is required