GAS CHROMATOGRAPHY

INTRODUCTION : Gas chromatography is a process by which a mixture is separated into its

constituents by a moving gas phase passing over a stationary sorbent. Gas chromatography is
divided into two major categories: gas-liquid chromatography (GLC), where separation occurs
by partitioning a sample between a mobile gas phase and a thin layer of non-volatile liquid
coated on an inert support, and gas-solid chromatography (GSC), which employs a solid of large
surface area as the stationary phase.

Apparatus/Instrumentation: A gas chromatograph consists essentially of the following parts :

Block diagram of GC Typical Chart Recorder

1) A supply of carrier gas from a high-pressure cylinder. The carrier gas used is either
helium, nitrogen, hydrogen or argon, the choice of gas depending on factors such as availability,
purity required, consumption and the type of detector employed. Thus helium is preferred when
thermal conductivity detectors are employed because of its high thermal conductivity relative to
that of the vapours of most organic compounds. Associated with this high pressure supply of
carrier gas are the attendant pressure regulators and flow meters to control and monitor the
carrier gas flow; the operating efficiency of the apparatus is very dependent on the maintenance
of a constant flow of carrier gas.

It is appropriate to emphasise here two important safety considerations:

(a) free-standing gas cylinders must always be supported by means of clamps or chains;
(b) waste gases, especially hydrogen, must be vented through an extraction hood.

2) Sample injection system: Numerous devices have been developed for introducing the
sample, but the major applications involve liquid samples that are introduced using a
microsyringe with hypodermic needle. The latter is inserted through a self-sealing silicone
rubber septum and the sample injected smoothly into a heated metal block at the head of the
column. Manipulation of the syringe may be regarded as an art developed with practice and the
aim must be to introduce the sample in a reproducible manner. The temperature of the sample
port should be such that the liquid is rapidly vaporized but without either decomposing or
fractionating the sample; a useful rule of thumb is to set the sample port temperature
approximately to the boiling point of the least volatile component. For greatest efficiency, the
smallest possible sample size (1-10 µL) consistent with detector sensitivity should be used.

3) The column. The actual separation of sample components is effected in the column where
the nature of the solid support, type and amount of liquid phase, method of packing, length and
temperature are important factors in obtaining the desired resolution.

The column is enclosed in a thermostatically controlled oven so that its temperature is held
constant to within 0.5°C, thus ensuring reproducible conditions. The operating temperature may
range from ambient to over 400°C and for isothermal operation is kept constant during the
separation process.
Although many types of column have been developed for gas chromatography, they may be
divided into two major groups:

(A) Packed columns. Conventional analytical columns are usually prepared with 2-6 mm
internal diameter (I.d.) glass tubing or 3-10 mm outer diameter metal tubing, which is normally
coiled for compactness. Glass columns must be used if any of the sample components are
decomposed by contact with metal.
The material chosen as the inert support should be of uniform granular size and have good
handling characteristics (i.e. be strong enough not to break down in handling) and be capable of
being packed into a uniform bed in a column. The surface area of the material should be large so
as to promote distribution of the liquid phase as a film and ensure the rapid attainment of
equilibrium between the stationary and mobile phases. The most commonly used supports (e.g.
Celite) are made from diatomaceous materials which can hold liquid phases in amounts
exceeding 20 per cent without becoming too sticky to flow freely and can be easily packed.

Commercial preparations of these supports are available in narrow mesh range fractions; to
obtain particles of uniform size the material should be sieved to the desired particle size range
and repeatedly water floated to remove fine particles which contribute to excessive pressure drop
in the final column. To a good approximation the height equivalent of a theoretical plate is
proportional to the average particle diameter so that theoretically the smallest possible particles
should be preferred in terms of column efficiency. Decreasing particle size will, however, rapidly
increase the gas pressure necessary to achieve flow through the column and in practice the best
choice is 80/100 mesh for a 3 mm i.d. column. It may be noted here that for effective packing of
any column the internal diameter of the tubing should be at least eight times the diameter of the
solid support particles.

The selection of the most suitable liquid phase for a particular separation is
crucial. Liquid phases can be broadly classified as follows:
a) Non-polar hydrocarbon-type liquid phases, e.g. paraffin oil (Nujol), squalane, and silicone-
gum rubber; the latter is used for high temperature work (upper limit being around 400 °C).
b) Compounds of intermediate polarity which possess a polar or polarisable group attached to a
large non-polar skeleton, e.g. esters of high-molecular weight alcohols such as dinonyl phthalate.
c) Polar compounds containing a relatively large proportion of polar groups, e.g. the carbowaxes
(polyglycols).
d) Hydrogen-bonding class, i.e. polar liquid phases such as glycol, glycerol, hydroxyacids, etc.,
which possess an appreciable number of hydrogen atoms available for hydrogen bonding.

The column packing is prepared by adding the correct amount of liquid phase
dissolved in a suitable solvent (e.g. acetone or dichloromethane) to a weighed quantity of the
solid support in a suitable dish. The volatile solvent is removed either by spontaneous
evaporation or by careful heating, the mixture being gently agitated to ensure a uniform
distribution of the liquid phase in the support. Final traces of the solvent may be removed under
vacuum and the column packing re-sieved to remove any fines produced during the preparation.
The relative amount of stationary liquid phase in the column packing is usually expressed on the
basis of the percentage by weight of liquid phase present, e.g. 15 per cent loading indicates that
100g column packing contains 15 g of liquid phase on 85 g of inert support. The solid should
remain free flowing after being coated with the liquid phase.

Micropacked columns, sometimes referred to as packed capillary columns, have been used in gas
chromatography, e.g. for the determination of trace components in complex mixtures. These
columns are characterised by small interna1 diameters (id. < 1.0 mm) and packing densities
comparable with conventional packed columns. In general, the column packing technique
requires higher pressures and constant vibration (e.g. ultrasonic) to achieve the necessary
packing density. Micropacked columns give high efficiency but practical problems, especially
sample injection at high back-pressures, have limited their use.

B) Open tubular columns. These capillary columns (id. < 1 mm) are increasingly used in GLC
because of their superior resolving power for complex mixtures. This results from the high
theoretical plate numbers which can be attained with long columns of this type for a relatively
small pressure drop. In these capillary columns the stationary phase is coated on the inner wall of
the tube, two basic types of capillary column being available:
1. wall-coated open tubular (WCOT), in which the stationary phase is coated
directly on to the inner wall of the tubing;

2. support-coated open tubular (SCOT), which have a finely-divided layer of solid support
material deposited on the inner wall on to which the stationary phase is then coated These SCOT
columns are not as efficient as WCOT columns but have a higher sample capacity, which enables
them to be used without a stream splitter.

Capillary columns are fabricated from thin-walled stainless steel, glass, or high-purity
fused silica tubing (the last is preferred for its inertness). Typical dimensions of the columns,
which are coiled, are 25-200m long and 0.2-0.5 mm i.d. Excellent open tubular columns may
now be purchased, providing a number of stationary phases of differing polarity on WCOT and
SCOT columns, and whose efficiency, greatly improved sample detectability, and thermal
stability surpass those exhibited by packed columns; their chief disadvantage is that they have a
lower sample capacity than packed columns
4. The detector. The function of the detector, which is situated at the exit of the separation
column, is to sense and measure the small amounts of the separated components present in the
carrier gas stream leaving the column. The output from the detector is fed to a recorder which
produces a pen-trace called a chromatogram. The choice of detector will depend on factors such
as the concentration level to be measured and the nature of the separated components. The
detectors most widely used in gas chromatography are the thermal conductivity, flame-ionisation
and electron-capture detectors.

Some of the important properties of a detector in gas chromatography are briefly discussed
below.
(a) Sensitivity. This is usually defined as the detector response (mV) per unit concentration of
analyte (mg/mL). It is closely related to the limit of detection (MDL) since high sensitivity often
gives a low limit of detection. The sensitivity also determines the slope of the calibration graph
(slope increases with increasing sensitivity) and therefore influences the precision of the
analysis.
(b) Linearity. The linear range of a detector refers to the concentration range over which the
signal is directly proportional to the amount (or concentration) of analyte. Linearity in detector
response will give linearity of the calibration graph and allows the latter to be drawn with more
certainty.
(c) Stability. An important characteristic of a detector is the extent to which the signal output
remains constant with time, assuming there is a constant input.
(d) Universal or selective response. A universal detector will respond to al1 the components
present in a mixture. In contrast, a selective detector senses only certain components in a sample
which can be advantageous if it responds only to those which are of interest, thus giving a
considerably simplified chromatogram and avoiding interference.

Thermal conductivity detector. The most important of the bulk physical property detectors is
the thermal conductivity detector (TCD) which is a universal, non-destructive, concentration-
sensitive detector. These detectors employ a heated metal filament or a thermistor (a
semiconductor of fused metal oxides) to sense changes in the thermal conductivity of the carrier
gas Stream. Helium and hydrogen are the best carrier gases to use in conjunction with this type
of detector since their thermal conductivities are much higher than any other gases; on safety
grounds helium is preferred because of its inertness.

In the detector two pairs of matched filaments are

arranged in a Wheatstone bridge circuit; two
filaments in opposite arms of the bridge are
surrounded by the carrier gas only, while the other
two filaments are surrounded by the effluent from
the chromatographic column.

Thermal conductivity detector

When pure carrier gas passes over both the reference and sample filaments the bridge is
balanced, but when a vapour emerges from the column, the rate of cooling of the sample
filaments changes and the bridge becomes unbalanced. The extent of this imbalance is a measure
of the concentration of vapour in the carrier gas at that instant, and the out-of-balance signal is
fed to a recorder thus producing the chromatogram. The differential technique used is thus based
on the measurement of the difference in thermal conductivity between the carrier gas and the
carrier gas lsample mixture.

Ionisation detectors. An important characteristic of the common carrier gases is that they
behave as perfect insulators at normal temperatures and pressures. The increased conductivity
due to the presence of a few charged molecules in the effluent from the column thus provides the
high sensitivity which is a feature of the ionisation based detectors. Ionisation detectors in
current use include the flame ionisation detector (FID), thermionic ionisation detector (TID),
photoionisation detector (PID) and electron capture detector (ECD) each, of course, employing a
different method to generate an ion current. The two most widely used ionisation detectors are,
however, the FID and ECD.

Flame ionisation detector. The basis of this detector is that the effluent from
the column is mixed with hydrogen and burned in air to produce a flame which
has sufficient energy to ionise solute molecules having low ionisation potentials.
The ions produced are collected at electrodes and
the resulting ion current measured; the burner jet is
the negative electrode whilst the anode is usually a
wire or grid extending into the tip of the flame.

The combustion of mixtures of hydrogen and air

produces very few ions so that with only the carrier
gas and hydrogen burning an essentially constant
signal is obtained. When, however, carbon
containing compounds are present ionisation occurs
and there is a large increase in the electrical
conductivity of the flame. Because the sample is
destroyed in the flame a stream-splitting device is
employed when further examination of the eluate is
necessary; this device is inserted between the
Flame ionisation detecto column and detector and allows the bulk of the
sample to by-pass the detector.

The FID has wide applicability, being a very nearly universal detector for gas chromatography of
organic compounds, and this, coupled with its high sensitivity, stability, fast response and wide
linear response range, has made it the most popular detector in current use.
Electron capture detector. Most ionisation detectors are based on measurement of the increase
in current (above that due to the background ionisation of the carrier gas) which occurs when a
more readily ionised molecule appears in the gas Stream. The electron capture detector differs
from other ionisation detectors in that it exploits the recombination phenomenon, being based on
electron capture by compounds having an affinity for free electrons; the detector thus measures a
decrease rather than an increase in current.
A β-ray source (commonly a foil containing 3H or 63Ni) is used to generate 'slow'
electrons by ionisation of the carrier gas (nitrogen preferred) flowing through the detector. These
slow electrons migrate to the anode under a fixed potential and give rise to a steady baseline
current. When an electron-capturing gas (i.e. eluate molecules) emerges from the column and
reacts with an electron, the net result is the replacement of an electron by a negative ion of much
greater mass with a corresponding reduction in current flow.

The response of the detector is clearly related to the electron affinity of the eluate
molecules being particularly sensitive to compounds containing halogens and sulphur,
anhydrides, conjugated carbonyls, nitrites, nitrates and organometallic compounds. The ECD is
the second most widely used ionisation detector due to its high sensitivity to a wide range of
compounds. It is, for example, used in trace analysis of pesticides, herbicides, drugs, and other
biologically active compounds, and is of value in detecting ultratrace amounts of metals as their
chelate complexes.

Element-selective detectors. Many samples, e.g. those originating from environmental studies,
contain so many constituent compounds that the gas chromatogram obtained is a complex array
of peaks. For the analytical chemist, who may be interested in only a few of the compounds
present, the replacement of the essentially non-selective type of detector (i.e. thermal
conductivity, flame ionisation, etc.) by a system which responds selectively to some property of
certain of the eluted species may overcome this problem.

The most common selective detectors in use generally respond to the presence of a characteristic
element or group in the eluted compound. This is well illustrated by the thermionic ionisation
detector (TID) which is essentially a flame ionisation detector giving a selective response to
phosphorus- and/or nitrogen-containing compounds. Typically the TID contains an electrically
heated rubidium silicate bead situated a few millimetres above the detector jet tip and below the
collector electrode. The temperature of the bead is maintained at 600-800 OC while a plasma is
sustained in the region of the bead by hydrogen and air support gases. A reaction cycle is so
produced in which the rubidium is vaporised, ionised and finally recaptured by the bead. During
this process an electron flow to the positive collector electrode occurs and this background
current is enhanced when nitrogen or phosphorus compounds are eluted, due it is thought to the
production of radicals in the flame or plasma which accelerate the rate of rubidium recycling.
The selectivity of this detector can facilitate otherwise difficult analyses, e.g. the determination
of pesticides such as Malathion and Parathion.

Finally, a high degree of specific molecular identification can be achieved by the

interfacing of the gas chromatograph with various spectroscopic instruments. Although the
quantitative information obtained from a chromatogram is usually good, the certainty of
identification based only on the retention parameter may be suspect. In the case of spectroscopic
techniques, however, the reverse situation applies since these provide excellent qualitative
information, enabling a pure substance to be identified, but less satisfactory quantitative
information is often obtained from their signals. The combination of chromatographic and
spectroscopic techniques thus provides more information about a sample than may be obtained
from either-instrument independently. The chief combined techniques are gas chromatography
interfaced with mass spectrometry (GC-MS), Fourier transform infrared spectrometry (GC-
FTIR).

Derivatisation: Many samples are, however, unsuitable for direct injection into a gas
chromatograph because, for example, of their high polarity, low volatility or thermal instability.
In this respect the versatility and application of gas chromatography has been greatly extended
by the formation of volatile derivatives, especially by the use of silylation reagents. The term
'silylation' is normally taken to mean the introduction of the trimethylsilyl, -Si(CH3)3 or similar
group in place of active hydrogen atoms in the substance under investigation. A considerable
number of such reagents is now available, including some special silylating agents which give
improved detector response, usually by incorporating a functional group suitable for a selective
detector system. Reagents containing chlorine and bromine atoms, for example, in the silyl group
are used particularly for preparing derivatives injected on to gas chromatographs fitted with
electron-capture detectors. Derivatisation can also give enhanced resolution from other
components in a mixture and improved peak shape for quantitative analysis.

For compounds of high molecular mass, however, the formation of derivatives does
not help to solve the problem of involatility. This difficulty may be overcome by breaking the
large molecules up into smaller and more volatile fragments which may then be analysed by gas-
liquid chromatography, i.e. by using the technique known as pyrolysis gas chromatography
(PGC).

Pyrolysis Gas Chromatography (PGC): Pyrolysis GC is a technique in which a non-volatile

sample is pyrolysed under rigidly controlled conditions, usually in the absence of oxygen, and
the decomposition products separated in the gas chromatographic column. The resulting
chromatogram (pyrogram) is used for both qualitative and quantitative analysis of the sample. If
the latter is very complex, complete identification of the pyrolysis fragments may not be
possible, but in such cases the program may be used to 'fingerprint' the sample. PGC has been
applied to a wide variety of samples, but its major use has been in polymer analysis for the
investigation of both synthetic and naturally occurring polymers. The various PGC systems
can generally be classified into two distinct types:

(a) Static-mode (furnace) reactors which typically consist of a quartz reactor tube and a Pregl
type of combustion furnace. Solid samples are placed in the reactor tube and the system is
closed. The furnace is then placed over the combustion tube and the sample heated to the
pyrolysis temperature. In this type of pyrolysis system, the time required to reach the necessary
temperature is much longer (up to 30 seconds) than in dynamic pyrolysis, resulting in a greater
number of secondary reactions. An important advantage of the static-mode system, however, is
the usually larger sample capacity.
(b) Dynamic (filament) reactors in which the sample is placed on the tip of a filament or wire
igniter (platinum and nichrome wires have been used) which is then sealed in a reactor chamber;
in PGC the latter is typically the injection port of the gas chromatograph. As the carrier gas
passes over the sample, a direct current charge is applied, and the sample is heated rapidly to the
pyrolysis temperature. As the sample decomposes, the pyrolysis products are carried away into a
cooler area (reducing the possibility of secondary reactions) before entering the gas
chromatographic column.

PROGRAMMED TEMPERATURE GAS CHROMATOGRAPHY (PTGC): Gas

chromatograms are usually obtained with the column kept at a constant temperature. Two
important disadvantages result from this isothermal mode of operation.
1. Early peaks are sharp and closely spaced (i.e. resolution is relatively poor in this region of the
chromatogram), whereas late peaks tend to be low, broad and widely spaced (i.e. resolution is
excessive).
2. Compounds of high boiling point are often undetected, particularly in the study of mixtures of
unknown composition and wide boiling point range; the solubilities of the higher-boiling
substances in the stationary phase are so large that they are almost completely immobilised at the
inlet to the column, especially where the latter is operated at a relatively low temperature.

The above consequences of isothermal operation may be largely avoided by using the technique
of programmed-temperature gas chromatography (PTGC) in which the temperature of the whole
column is raised during the sample analysis.
A temperature programme consists of a series of changes in column temperatures which
rnay be conveniently selected by a microprocessor controller. The programme commonly
consists of an initial isothermal period, a linear temperature rise segment, and a final isothermal
period at the temperature which has been reached, but may vary according to the separation to be
effected. The rate of temperature rise, which may vary over a wide range, is a compromise
between the need for a slow rate of change to obtain maximum resolution and a rapid change to
minimise analysis time.
Programmed-temperature gas chromatography permits the separation of compounds of
a very wide boiling range more rapidly than by isothermal operation of the column. The peaks on
the chromatogram are also sharper and more uniform in shape so that, using PTGC, peak heights
may be used to obtain accurate quantitative analysis.

Applications:
1) QUANTITATIVE ANALYSIS. The quantitative determination of a component in gas
chromatography using differential-type detectors of the type previously described is
based upon measurement of the recorded peak area or peak height; the latter is more
suitable in the case of small peaks, or peaks with narrow band width. In order that these
quantities rnay be related to the amount of solute in the sample two conditions must
prevail:
(a) the response of the detector-recorder system must be linear with respect to the concentration
of the solute;
(b) factors such as the rate of carrier gas flow, column temperature, etc., must be kept constant or
the effect of variation must be eliminated, e.g. by use of the interna1 standard method.

Peak area is commonly used as a quantitative measure of a particular component in

the sample and can be measured by one of the following techniques.

a) Planimetry. The planimeter is a mechanical device which enables the peak area to be
measured by tracing the perimeter of the peak. The method is slow but can give accurate results
with experience in manipulation of the planimeter. Accuracy and precision, however, decrease as
peak area diminishes.

b) Geometrical methods. In the so-called triangulation methods, tangents are drawn to the
inflection points of the elution peak and these two lines together with the baseline form a
triangle, the area of the latter is calculated as one-half the product of the base length times the
peak height, the value obtained being about 97 per cent of the actual area under the
chromatographic peak when this is Gaussian in shape.

Measurement of peak area by triangulation

c) Integration by weighing. The chromatographic peak is carefully cut out of the chart and the
paper weighed on an analytical balance. The accuracy of the method is clearly dependent upon
the constancy of the thickness and moisture content of the chart paper, and it is usually
preferable (unless an automatic integrator is available) to use geometrical methods.

d) Automatic integration. The older methods for computing peak area are time-consuming and
often unsatisfactory in terms of accuracy and reproducibility of results. The greater use of
capillary column chromatography, with its resulting sharp, closely spaced peaks has accentuated
the need for a rapid, automatic instrumental method for data processing. The instrument
currently most widely used in quantitative gas chromatography is the digital integrator; real-time
digital automatic integrators process the analytical signal as the analyses are being run. These
systems automatically identify peaks, compute peak areas and/or peak heights, and provide the
results either in printed form or in one of the various computer-compatible formats.
2) ELEMENTAL ANALYSIS: An important application of gas chromatography is its use for
determination of the elements carbon, hydrogen, nitrogen, oxygen and sulphur in organic and
organometallic samples.

3) DETERMINATION OF SUCROSE AS ITS TRIMETHYLSILYL DERIVATIVE

4) Assay of Drugs such as Ethyloestrenol, Lincomycin Hydrochloride

5) Determination of specific organic compounds as impurities in official pharmaceutical
substance, such as Determination of N, N-dimethylaniline in Cephalexin

6) Determination of related substances in official drugs, such as Bromopheneramine maleate ;

Bronopol ; Cepahloridine ; Chlorocresol ; Chloroform ; Chloroxylenol.,
Cindamycin hydrochloride ; Griseofulvin ; Isometheptene mucate ; Levomenthol