Gas Chromatography

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• Gas Chromatography (GC) is a column
chromatographic technique in which the mobile
phase is a gas and stationary phase is a liquid or
solid and the sample components are separated as
vapours.

• AJP Martin is credited for development of GC technique.

He did it in 1952.

• GC is one of the most widely used powerful analytical

tool available for separation of mixture of components.

• This is due to its speed, high resolving power and

extreme sensitivity.

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• Gas chromatography has a potential role both in
the qualitative and quantitative analysis.

• GC can be used for compounds which are

thermally stable and volatile (readily goes into a
gas phase).

• It can also be used for compounds which can be

converted into a volatile derivative.

• In other words, for separating and analyzing

compounds that can
be vaporized without decomposition.

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• It is thus used to separate and detect small
molecular weight compounds in the gas phase.

• The sample is either a gas or a liquid that is

vaporized in the injection port. The mobile phase
for gas chromatography is a carrier gas, like
helium, nitrogen, hydrogen.

• The pressure is applied and the mobile phase

moves the sample through the column. The
separation is accomplished using a column coated
with a stationary phase.

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 Types of GC
• Gas-liquid chromatography (Stationary phase
is liquid)

• Gas-solid chromatography (Stationary phase is

solid)

• GLC is most widely used

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Gas-liquid chromatography
• In this type, the stationary phase is a thin layer
of non-volatile liquid bound to an inert solid
support

• Separation is on the basis of partitioning.

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 Gas-solid chromatography

• In this type, a solid adsorbent is used as a

stationary phase

• Separation is on the basis of adsorption

• It is not so popular as gas-liquid

chromatography.

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 Principle
• During a GC separation, the sample is vaporized and
carried by the mobile phase (the carrier gas) through the
column. Separation of the different components is
achieved based on their relative vapor pressure and
affinities for the stationary phase. The affinity of a
substance towards the stationary phase depends upon
distribution constant Kc, or partition coefficient of sample
between stationary phase and mobile phase

Kc = Cs/Cm,

• Cs = concentration of sample in SP, Cm = Concentration

of sample in MP
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 Principle
• The distribution constant (Kc) controls the movement of
the different compounds through the column, therefore
differences in the distribution constant allow for the
chromatographic separation.

• Distribution constant is dependent upon temperature

and chemical nature of the stationary phase. Thus, apart
from changing stationary phase, temperature can be
used as a way to improve the separation of different
compounds through the column.

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 Instrumentation
• Carrier gas (maintained at high pressure)

• Flow regulator

• Sample injection system

• Column

• Detectors

• Thermo stated chamber or oven

• Amplification and recording system

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Schematic Diagram of Gas
Chromatograph

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 Carrier gas (Mobile Phase)
• It should be chemically inert

• Suitable for detector and sample analysed

• Readily available in high purity

• Should not cause the risk of fire or explosion

• Eg.: helium, nitrogen, hydrogen, argon

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 Carrier gas (Mobile Phase)
• The role of the carrier gas or mobile phase in GC is to
carry the sample molecules along the column while
they are not dissolved in the stationary phase.

• The carrier gas is inert and does not interact with the
sample.

• Thus, separation selectivity in GC can be attributed to

the stationary phase alone.

• However, the choice of carrier gas is important to

maintain high efficiency.
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 Flow/Pressure regulator
• Flow control and/or a pressure regulator is used in GC to
assure a constant flow through the column and detector.

• The flow of the carrier gas is dependent on density and

viscosity of the gas and the resistance of the column.

• In order to maintain a constant flow within the GC system,

the pressure varies. In a pressure regulated system, the
pressure is kept constant and the flow will vary.

• Both systems can be used in isothermal analyses. Since

temperature does not change, the flow will remain
constant.
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 Flow/Pressure regulator
• However in temperature-programmed analyses a flow-
regulated system is preferred, because the decrease in flow
at higher temperatures can be dramatic.

• Flow-regulated system are used with packed columns (gas

flows 20 - 120 ml/min).

• Pressure-regulated systems are used with capillary

columns.

• The gas flow through capillary columns is so small (0.1 -

10 ml/min) that flow controllers are not necessary.

• Rotameter, soap bubble flow meter

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 Sample injector
• The accuracy and reproducibility of GC analysis depend to
a great extent on the technique adopted for sample
injection.

• Very small amount of sample should be injected

• Introduced in a reproducible manner

• Sample must be vapourised instantaneously

• Solid sample is dissolved in volatile liquid prior to injection

• The most common injection method is where a

microsyringe is used to inject sample through a rubber
septum into a flash vapouriser port at the head of the
column. 17
 Sample injector
• The temperature of the sample port is usually about 50°C
higher than the boiling point of the least volatile
component of the sample.

• For packed columns, sample size ranges from 1/10th of a

μl up to 20 μl.

• Capillary columns, on the other hand, need much less

sample, typically around 0.001 mL.

• The injector contains a heated chamber containing a glass

liner into which the sample is injected through the septum.

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 Sample injector
• The carrier gas also enters the chamber.

• The sample vapourises to form a mixture of carrier gas,

vapourised solvent and vapourised solutes.

• A proportion of this mixture passes onto the column

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Injecting syringe Insertion point

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Sample injection system

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 Column
• There are two general types of column used in GC -
packed column and capillary column (open tubular).

• Packed columns contain a finely divided, inert, solid

support material (commonly based on diatomaceous
earth) coated with liquid stationary phase.

• Packed column - Most packed columns are 1.5-10m in

length and have an internal diameter of 2-4mm.GC
columns can be of glass or metal.

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• Capillary columns have an internal diameter of 0.1-0.5
mm. They can be one of two types; wall-coated open
tubular (WCOT) or support-coated open tubular (SCOT)
column. Wall-coated columns consist of a capillary tube
whose walls are coated with liquid stationary phase.

• In support-coated columns, the inner wall of the

capillary is lined with a thin layer of support material
such as diatomaceous earth, onto which the stationary
phase has been adsorbed.

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• SCOT columns are generally less efficient than WCOT
columns. Both types of capillary column are more
efficient than packed columns.

• Packed columns produce broad peaks and capillary

columns produce sharp peaks.

• In addition, capillary columns produce taller peaks,

which allows the detection of lower concentrations (high
detection sensitivity).

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 GC columns
glass stainless steel copper

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Thermostatic Chamber or Oven
• For precise work, column temperature must be
controlled to 1/10th of a degree.

• The optimum column temperature is dependent upon

the boiling point of the sample.

• As a rule of thumb, a temperature slightly above the

average boiling point of the sample results in an elution
time of 2 - 30 minutes.

• Minimal temperatures give good resolution, but

increase elution times.
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Thermostatic Chamber or Oven
• If a sample has a wide boiling range, then temperature
programming can be useful. (Gradient temperature
programming)

• The column temperature is increased (either

continuously or in steps) as separation proceeds.

• Thus, oven are needed in GC.

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Inside GC oven

HIMALAYAN PHARMACY 30
INSTITUTE
 Detectors
• Flame ionization detector

• Electron capture detector

• Thermal conductivity detector

• Mass detectors

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 Thermal Conductivity Detector
• Katharometer or hot wire detector

• TCD is based upon change in thermal conductivity of

the gas stream.

• Thermal conductivity of most of the organic samples are

less than the commonly used carrier gases like
hydrogen, helium or nitrogen.

• TCD consists of a temperature controlled metal block in

which two cylindrical chambers are present, which
consist of two filaments made up of platinum or
tungsten.
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Thermal Conductivity Detector

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 Thermal Conductivity Detector
• Both the filaments are connected to the arms of
wheatstone bridge arrangement.

• When only carrier gas is passing through them, the

resistance of the filaments are constant. But when the
effluent from the column passes through them their
thermal conductivity will change and it will be recorded.

• Difference in THERMAL CONDUCTIVITY between

carrier gas and carrier gas+sample

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 Flame Ionisation Detector
• FID is the most widely used and generally applicable
detector

• The effluent from the column is directed into small air-

hydrogen flame

• The sample produces ions and electrons when

pyrolyzed at the temperature of air-hydrogen flame.

• Detection involves monitoring the current produced by

collecting this charge carriers

• The potential applied between the burner tip and a

collector electrode located above the flame causes the
ions and electrons to move towards the collector.
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FID

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 Flame Ionisation Detector
• The resulting current is then measured which is
directly related with the concentration of eluent eluted
from the column.

• FID exhibits a high sensitivity, large linear response

and low noise.

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 Electron Capture Detector
• Is used for detecting electron-absorbing components
having high electronegativity such as halogenated
compounds and nitro compounds

• The ECD uses a radioactive beta particle (electron)

emitter, radionuclide 63Ni

• The electrons emitted from the electron emitter collide

with the molecules of the carrier gas resulting in many
more free electrons.

• The electrons are accelerated towards a positively

charged anode, generating a current.
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 Electron Capture Detector
• Thus, a constant standing current exist between the
pair of electrodes.

• As the sample is carried into the detector by the carrier

gas, electron-absorbing analyte molecules (sample)
capture electrons and thereby reduce the current
between the collector anode and cathode.

• Rate of electron capture or decrease in current is

proportional to the analyte concentration.

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Electron Capture Detector

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 Derivatization
• It has been estimated that 10-20% of known
compounds can be directly analysed by GC

• Remaining 80-90% are not suitable for analysis directly

by GC, because they are thermally unstable or non-
volatile

• Various techniques have been developed so that low

volatile and polar compounds can be analyzed.

• Derivatization converts the molecules with polar

functional groups into less polar derivatives which
become volatile and increases the thermal stability of
the compound and its detectability by detector.
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 Various Derivatization
Techniques used in GC
• Silylation

• Alkylation

• Acylation

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 Alkylation
• A reactive hydrogen such as –OH or -NH is replaced by
alkyl group to reduce the molecular polarity.

• Derivatize compounds with acidic hydrogens such as

carboxylic acids, phenols, amines etc

• It is generally used to convert organic acids into esters.

• Diazomethane is generally preferred as alkylating

reagent because it completes the reaction in few
seconds itself.
RCOOH +CH2N2 → RCOOCH3 + N2

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 Silylation
• It is the most versatile and widely used derivatisation
technique in GC

• In silylation, active hydrogen is replaced with a

trimethylsilyl group

• Silylation reduces the polarity of the molecules and

decreases the possibility of hydrogen bonding

• Nearly all functional groups can be converted to

alkylsilyl ethers or esters

R-OH + (CH3)3 SiCl → R-OSi(CH3)3 + HCl

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 Acylation
• Acylation with acid anhydrides or acyl imidazoles
converts primary and secondary amines to stable
derivatives.

• Alcohols, primary amines and secondary amines can be

derivatised by acylation

• Acylation converts compounds with active hydrogens

into esters, thioesters, and amides

• Acetylation is the most common acylation reaction

R-OH + (R1CO)2O → R1COOR + R1COOH

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 Advantages
• Due to its high efficiency, GC allows the separation of
the components of complex mixtures in a reasonable
time.

• Accurate quantitation of sample can be done

• Multiple detectors with high sensitivity (ppb) are

available

• Very small quantity of sample is needed for analysis

• Mature technique with many applications notes

available for users.

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 Disadvantages
• Limited to thermally stable and volatile compounds.

• Most GC detectors are destructive, except for mass

detector.

• During injection of the gaseous sample proper attention

is needed for reproducible result

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 Applications
• Measurment of drugs and other metabolites in
biological fluid

• By using thermal conductivity detector estimation of

water and other solvent residue

• Determination of chloroquine, tolbutamide, codeine in

plasma by GLC-FID

• Analysis of nitrogenous drugs and their metabolites in

biological specimen

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 Applications
•Determine the sub-micro drug level in biological
specimen like blood, urine.

•Hydrochlorothiazide has been determined in plasma.

•Aminoglycoside antibiotics and carbohydrate can be

analyzed.

•Analysis of fatty acids produced by bacteria, particularly

anaerobic bacteria.

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