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GAS LIQUID CHROMATOGRAPHY (GLC)

Introduction and principle-

Gas – Liquid chromatography (GLC) is one of the most useful techniques in

analytical chemistry. Claesson published one of the first important accounts of gas
liquid chromatography in 1946. Gas – liquid chromatography is a form of partition
chromatography in which the stationary phase is a film coated on a solid support and
the mobile phase is an inert gas like Nitrogen (N2) called as carrier gas flowing over the
surface of a liquid film in a controlled fashion. The sample under analysis is vaporized
under conditions of high temperature programming. The components of the vaporized
sample are fractionated as a result of partitioning between a mobile gaseous phase and a
liquid stationary phase held in a column.
Principle: When the vapours of sample mixture move between the stationary phase
(liquid) and mobile phase (gas) the different components of a sample mixture will
separate according to their partition coefficient between the gas and liquid stationary
phase.
Partition coeff.(Kg) = Concn. of solute in liquid (w/cc) Concn of solute in gas (w/cc)

It is general assumption that if partition coefficient is low the emergence of the

component is fast and vice versa. The substances having low boiling point (B.P) i.e.
more volatility and higher vapour pressure will have more concentration in the mobile
phase and thus will elute or emerge first and so on. For example, lower carbon number
compounds have low B.P and higher volatility and vapour pressure will elute first than
the higher carbon number compounds e.g. lower chain fatty acids emerge first than long

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chain ones. Therefore, less polar substances elute fast than polar substances. More polar
substances are more retained in the column and therefore move slowly as compared to
less polar substances which move at faster rate.
In chromatographic analysis there are two terms commonly used (i) Retention Time and
(ii) Retention volume.

Retention Time ( tR): It is the time required for the maximum for a solute peak (the
peak of that particular component) to reach the detector in a gas chromatographic
column. The retention time (tR) is characteristic of that component and the area under
the peak is proportional to its quantity. These parameters yield qualitative and
quantitative data, respectively.

The characterization of mixture in as unknown sample is done through retention

time by comparing with those of reference compounds. The relative proportion of
varouis components in a mixture is determined by calculating their peak areas and then
calculating the percentage of peaks are out of the total area of various peaks obtained.

Retention volume (VR) is defined as the volume of the gas required to carry a
component maximum through the column
VR = tR Fc
Where Fc is the volume flow rate of the gas at outlet.

4. Apparatus:

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The basic components of a typical gas chromatograph (GC) are as:

Carrier Gas Supply: The gaseous mobile phase must be inert. Helium is the most
common mobile phase, although argon, nitrogen, hydrogen are also used. Most of these
gasses in highly pure form – including mixtures such as nitrogen with hydrogen are
available in cylinders. Generally the gasses used in GC must be thoroughly dried
because moisture entrapped in the gasses leads to background noise. Now, a day’s GC
suppliers are providing desiccant cartridges and other filters along with the machines
which take care of these problems as well as other impurities. Otherwise, the best
desiccant is a molecular sieve (Linde 5A) activated at 200 – 300 o C. the flow rate of
these gases is controlled by the pressure gauges and flow meters.

Detector Carrier Gas

Thermal conductivity Helium

Flame ionization Helium or nitrogen

Electron capture Very dry nitrogen

Detector Carrier Gas

Thermal conductivity Helium

Flame ionization Helium or nitrogen

Electron capture Very dry nitrogen

Sample injection Systems:

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Method of sample injection depends on the type of sample

i.e. gaseous, liquid or solid. In GLC the requirement is that the suitable amount of
sample should be injected as a “plug” of vapors. It has been noticed that slow injection
or oversized samples cause band spreading and poor resolution. Sample size depends
upon the sensitivity of the detector; when an
ionization detector is used a liquid sample should not be greater than 0.5 l.
Liquid Samples: Liquids are injected by means of micro syringes through a silicon
septum into a heated sample port located at the head of the
column. The sample port is ordinarily above 50 o C above the boiling point of the least
volatile components of the sample. For ordinary packed analytical columns, sample
sizes range from a few tenth of micro liter to 20 l .
Capillary columns require samples that are smaller by a factor of 100 or more. Here a
sample splitter is often required to deliver only a small known fraction (1:100 to 1:500)
of the injected sample, with the remainder going to waste.
Solid samples: Solid samples are generally weighed into thin glass ampules which are
placed in the gas stream and then crushed.
Columns: Efficiency of any gas chromatograph is very much dependent on the

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columns used in GLC, which may be of glass or metal. These columns are mainly of
two types – packed columns and open – tubular (capillary) columns.
Packed columns: These columns can accommodate larger samples and are generally
more convenient to use. They are normally 2 –3 m long and have inside diameters of 2
–4 mm. The tubes are ordinarily formed as coils with diameters of roughly 15cm to
permit convenient thermo stating in an oven. The packing or support, for a column hold
the liquid stationary phase in place, so that the surface area exposed to the mobile phase
is as large as possible. The ideal particle size of packing material for gas
chromatography is in the range of 60 – 80 mesh (250 – 170 m) or 80 – 100 mesh (170
– 149 m

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Capillary columns: Capillary columns are generally made up of glass or fused silica.
These columns have inside diameters of 0.25 – 0.50 mm and lengths of 25 – 100 m.
Silica capillaries which have much thinner walls than their glass or metal counter parts,
have outside diameters of about 0.3 mm.

Column Oven: The oven used in GLC is usually having a high precision thermostat to
control the temperature of the column fitted inside the oven to get the reproducible
retention time. Range of temperature may vary from 0 - 400ᵒC

MOBILE PHASE FOR GLC

The most common mobile phases for gas chromatography are He, Ar, and N 2,
which have the advantage of being chemically inert toward both the sample and the
stationary phase. The nature of the carrier gas has no significant influence on K, the
partition coefficient, but it does have an effect on the solutes dispersion (has an effect
on Neff and LOD). The choice of carrier gas often is determined by the needs of
instrument’s detector. For a packed column the mobile phase velocity usually is 25–150
mL/min. The typical flow rate for a capillary column is 1–25 mL/min.

STATIONARY PHASES FOR GLC:

Hundreds of liquids have been proposed as stationary phases in the development
of GLC. The important factors governing the selection of a liquid phase are its polarity
and operational temperature range. The later can be determined from tables or supplier.
From practical point of view, the greater the polarity of the liquid stationary phase, the
greater the retention of a polar solute relative to that of a non-polar solute with a similar
boiling point. Like is dissolved by like is the general rule. Thus if sample components

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are similar in chemical structure but have different volatilities, a non polar liquid phase
is generally better .
Conversely the components have different functional groups, but are of similar boiling
points then polar phase is generally more suitable.
These stationary phases are mainly of two types – polar and non-polar.
Polar (Selective) stationary phases: ZThey contain functional groups such as - CN, -
CO, and –OH. These phases include Polyethylene glycol and polyester which retain
polar solutes. Theses phases separate solute molecules on the basis of functional groups.
Selective stationary phases show selective retention for carbon – carbon double bonds.
Such phases are used to separate carbonyls, lactones and fatty acid esters.

Non-polar (non- selective) phases: They are generally hydrocarbon type (dialkyl
siloxane) such as methyl silicons, Apiezen greases and squalene. They tend to
fractionate solutes by B.P. Generally used for triglyceride.
All type of stationary phases has the following properties:
(1) Low volatility (ideally boiling point of the liquid should be at 100ᵒ C higher than the
maximum operating temperature of the column (2) Thermal stability (3) Chemical

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inertness (4) Solvent characteristics.

Some Common Liquid Stationary phases: Polydimethyl siloxane, Phenyl –
polydimethyl siloxane, Polyethylene glycol, Cynopropyl – polydimethyl siloxane.
Selection of stationary phases depend upon the compounds, researches want to
study.

Stationary phase Type Analyte type

100% Dimethyl polysiloxane Non- polar Solvents, Petroleum
products, flavors, saturated
hydrocarbons
5% : 95% Diphenyl : dimethyl Non- Polar flavors, pesticides,
polysiloxane aromatic hydrocarbons
35% : 65%; Diphenyl :Medium polarity Nitrogen containing
dimethyl pesticides
polysiloxane
Polyethylene glycol Polar Fatty acid methyl esters,
fatty acids, flavours,
alcohols.
Conditioning of column: To remove low molecular weight residues from stationary
phase so as to maintain the minimum bleeds of liquid phase throughout the working
range of temp. This is normally accomplished by heating the column to a temperature
about 20 – 30°C above its proposed operating temperature with a stream of carrier gas
passing through it. This is essential to ensure that the complete removal of any solvent,
water and volatile contaminants that may have been retained during the preparation of
the column material.

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Derivatives for GLC: Most common derivatization is done prior to G.C. which makes
the sample more volatile; e.g.
1. Methyl esters: Both acidic and basic catalysts are used for esterification reactions of

fatty acids and fats.

2. Silyl ethers: The silyl ethers are preferred for partial glycerides, sterol, carbohydrates

etc.
3. 2,4 DNPH: Volatile carbonyl compounds are important flavour compounds in food

stuffs, accounting for flavour & off flavour. These are converted to their 2, 4
dinitrophenyl hydrzones.

PROCEDURE/WORKING OF GLC
SAMPLE INTRODUCTION
Three factors determine how we introduce a sample to the gas chromatograph.
First, all of the sample’s constituents must be volatile. Second, the analytes must be
present at an appropriate concentration. Finally, the physical process of injecting the
sample must not degrade the separation. Each of these needs is considered in this
section.
Preparing a Volatile Sample
Not every sample can be injected directly into a gas chromatograph. To move
through the column, the sample’s constituents must be sufficiently volatile. A solute of
low volatility, for example, may be retained by the column and continue to elute during
the analysis of subsequent samples. A nonvolatile solute will condense at the top of the
column, degrading the column’s performance.
Sample can be separated a as a volatile analytes from its nonvolatile components
using any of the extraction techniques. A liquid–liquid extraction of analytes from an

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aqueous matrix into methylene chloride or another organic solvent is a common choice.
Solid-phase extractions also are used to remove a sample’s nonvolatile components.
An attractive approach to isolating analytes is a solid-phase microextraction (SPME).
In one approach, which is illustrated in Figure below, a fused-silica fiber is placed
inside a syringe needle. The fiber, which is coated with a thin film of an adsorbent
material, such as polydimethyl siloxane, is lowered into the sample by depressing a
plunger and is exposed to the sample for a predetermined time. After withdrawing the
fiber into the needle, it is transferred to the gas chromatograph for analysis.

Figure

Two additional methods for isolating volatile analytes are a purge-and-trap and
headspace sampling.

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In a purge-and-trap, we bubble an inert gas, such as He or N2, through the sample,

releasing—or purging—the volatile compounds. These compounds are carried by the
purge gas through a trap that contains an absorbent material, such as Tenax, where they
are retained. Heating the trap and back-flushing with carrier gas transfers the volatile
compounds to the gas chromatograph.
In headspace sampling we place the sample in a closed vial with an overlying air space.
After allowing time for the volatile analytes to equilibrate between the sample and the
overlying air, we use a syringe to extract a portion of the vapor phase and inject it into
the gas chromatograph. Alternatively, we can sample the headspace with an SPME.

STEPS INVOLVED IN GLC

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DETECTORS IN GLC

Detectors: Detectors are very sensitive and respond quickly to minute concentrations of
solutes exiting the columns. Detectors have the linear response stabile and uniform
response for a wide variety of chemical species. There are many
types of detectors are available.
(a) Thermal conductivity (b) Gas density (c) Flame ionization (d) ß – ray ionization
( Cross section, Argon, Helium, Electron capture, Electron mobility) (e) Photo
ionization. (f) Glow discharge (g) Flame temperature (h) Dielectric constant. Out of the
above- mentioned detectors the two are most commonly used.

Thermal Conductivity Detector (TCD): this is also called as Katharometer. This is

madeup of four filaments arranged in a electrical bridge network. The carrier gas
flowing around these filaments through cavities. The temperature of filament is
determined by the rate of heat loss by conduction through the carrier gas. As the
components elute out from the column, the composition of gas changes with the
consequent changes in the thermal conductivity. This in turn, produces change in
temperature of filaments which generate electrical output from the bridge circuit.
Merits: i) Simple ii) can be used in all applications iii) non destructive and thus suitable
for preparative fraction collection work.
Limitation: Low resistance ii) Low sensitivity ( 10 –9 g/ mL carrier ).

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Flame Ionization Detector (FID): Most popular detector due to its high sensitivity,
wide

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range and greater reliability. It responds only to organic compounds. . It works on the
principle that most organic compounds, when pyrolize in a hot flame, produce ionic
intermediates that conduct electricity through the flame. It consists of a small hydrogen
flame burning in an excess of air and surrounded by an electrostatic field. Column
effluent is mixed with hydrogen entering the burner. Organic components eluted from
the column are burnt producing CO2. During this oxidation process, some ionizing
particles and electron are formed as intermediate products of oxidation. These ionizing
particles are quantitatively proportional to the amount of carbon in original compounds.
These ionizing particles are collected and neutralized by the polarizing electrodes
generating an electric current which is picked up by electrometer and forms a peak on
the recording chart. The ionization detector exhibits a high sensitivity (10 – 13 g / mL ),
a large linear response and low noise. It is also rugged and easy to use. It responds only
to organic compounds.

Detection limit: 5 ppb for light hydrocarbon gases and 10 picograms for higher organic
liquids and gases. When column temperature of 200°C or higher is used, the practical
detection limit is about 1 nanogram (10-7 - 10-8g.).

Disadvantage: It destroys the sample.

Modified Flame ionization detector (FID): It is also called as thermo ionic alkali
flame detector (TID). In this case an alkali metal salt is added to the flame to enhance
the ionization of compounds containing P. Cl and N. The salts mainly PO4 and halides
of Na, K, Rb or CS are fed to the flame by (i) fixed wire (ii) heated capillary (iii) pallet
fastened on jet. Salts should be replaced when consumed.

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Electron Capture Detector (ECD): It is based on the principal of electron capturing

by various substances being sensed which causes a reduction in the ion current.
Chemicals containing an electronegative element or group have a strong affinity for
electrons. In the gaseous state, they tend to capture electrons to form negative ions.
Exposing these compounds to a source of low energy electrons forms the basis of an
extremely sensitive selective detector for such compounds. The radioactive source
employed for ECD include: Tritium (H3)and Ni63 . Methane or argon are also used as a
quench gas (mixed with carrier ga) to reduce the energy.

Non – radio active sources: Glow discharge in helium high sensitivity and high
operating temperature. (400°C). In all types of ECD, more important is specificity or
selectivity than sensitivity. Non capturing compounds (compounds which do not

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contain electronegative group) should go undetected.

Advantages : Very high sensitivity and specificity for molecules such as oxygen,
halogens oxygen – containing and halogen containing compounds, which have high
electron affinities.

NITROGEN/PHOSPHOROUS THERMIONIC DETECTOR

The Nitrogen/Phosphorous (NPD) detector is based on ceramic bead containing
RbCl or CsCl placed inside a heater coil. As shown in Figure 12.1.1512.1.15, the bead
is situated above a hydrogen flame. The heated alkali impregnated bead emits electrons
by thermionic emission which are collected at the anode and provides background
current through the electrode system. When a solute that contains N or P is eluted, the
partially combusted N and P materials are adsorbed on the surface of the bead. The
adsorbed material reduces the workfunction of the surface and, thus, e- emission is
increased and the current collected at the anode rises.
The NPD has a very high sensitivity, i.e., about an order of magnitude less than that of
the electron capture detector (ca.10-12 g/ml for phosphorus and 10-11 g/ml for nitrogen).
Relative to the FID, the NPD is 500x more sensitive for P-bearing species and 50x more
sensitive for N-bearing species
The Nitrogen Phosphorus Detector (NPD): This detector is very-very sensitive and a
specific detector. It is highly responsive to organic compounds containing nitrogen
and/or phosphorus. Sensor in this detector is made up of a rubidium or cesium bead
contained inside a small heater coil. One side of the detector has the anode. The heated
alkali Rubidium Sulphate emits electrons by thermionic emission which are collected at

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the anode and thus produce ions. When a solute containing nitrogen or phosphorus is
eluted, the partially combusted nitrogen and phosphorus materials are adsorbed on the
surface of the bead. This adsorbed material reduces the work function of the surface
and, as consequence, the emission of electrons is increased which raises the anode
current. The sensitivity of the NPD is about 10-12 g/ml for phosphorus and 10-11 g/ml
for nitrogen).

MASS SPECTROMETER DETECTOR

A mass spectrometer is an instrument that ionizes a gaseous molecule using sufficient
energy that the resulting ion breaks apart into smaller ions. Because these ions have
different mass-to-charge ratios, it is possible to separate them using a magnetic field or
an electrical field. The resulting mass spectrum contains both quantitative and
qualitative information about the analyte.

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There are several options for monitoring a chromatogram when using a mass
spectrometer as the detector. The most common method is to continuously scan the
entire mass spectrum and report the total signal for all ions that reach the detector
during each scan. This total ion scan provides universal detection for all analytes. We
can achieve some degree of selectivity by monitoring one or more specific mass-to-
charge ratios, a process called selective-ion monitoring. A mass spectrometer provides
excellent detection limits, typically 25 fg to 100 pg, with a linear range of 10 5 orders of
magnitude. Because we continuously record the mass spectrum of the column’s eluent,
we can go back and examine the mass spectrum for any time increment. This is a
distinct advantage for GC–MS because we can use the mass spectrum to help identify a
mixture’s components.

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RAPID SCANNING FOURIER TRANSFORM INFRARED DETECTOR

A Fourier transform infrared spectrophotometer (FT–IR) also can serve as a detector. In
GC–FT–IR, effluent from the column flows through an optical cell constructed from a
10–40 cm Pyrex tube with an internal diameter of 1–3 mm. The cell’s interior surface is
coated with a reflecting layer of gold. Multiple reflections of the source radiation as it is
transmit- ted through the cell increase the optical path length through the sample. As is
the case with GC–MS, an FT–IR detector continuously records the column eluent’s
spectrum, which allows us to examine the IR spectrum for any time increment.

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APPLICATIONS OF GAS-LIQUID CHROMATOGRAPHY

Gas liquid chromatography is generally used for both qualitative and quantitative
analysis of organic compounds. This technique is much sought technique in
Agricultural Science, Agriculture Industry, Food industry, Environmental field,
Forensic field,
Biotechnology field, Perfume and fragrance
industry i.e. cosmetic industry and chemical industry. This technique is very useful for
the estimation of (i) pesticide and insecticide residues in food and other consumables

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(ii) estimation of pollutants in water and other food stuff (iii) Banned and controlled
drugs in urine, blood, tablets, energy drinks etc.

Gas liquid chromatography has a very wide field of application in the separation and
analysis of multi component mixtures such as essential oils, hydrocarbons and solvents.
It is one of the primary analytical techniques, which was used in forensic laboratory.
 GLC is combined with mass spectroscopy (GC/MS) for drug detection, fire
investigation, environmental analysis, explosives investigation, and identification of
unknown samples.
 GC/MS is also used in airport security to detect unwanted substances. Non
derivatized sugars and sugar alcohols are successfully analyzed by GC/MS using
atmospheric pressure chemical ionization (APCI) in negative ion mode.
 GC is widely used by forensic scientists for an analysis of body fluids for the
presence of illegal substances, testing of fiber and blood from a crime scene, and to
detect residue from explosives.

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