210 Instrumental Methods of Analysis

CHAPTER
Gas Chromatography
12

12.1. GAS CHROMATOGRAPHY

12.1.1. Introduction
A.T. James and P. Martin first time used the gas chromatography technique in 1952 for
separating long chain fatty acids. The gases and vaporisable substances can also be
separated by gas chromatography based on differential adsorption.
In gas chromatography, gas is used as the mobile phase and solid or liquid is used as the
stationary phase. When the stationary phase is solid, it is known as Gas Solid
Chromatography (GSC) and when the stat ionary phase is liquid, it is known as Gas
Liquid Chromatography (GLC).
In gas chromatography, a moving gas phase is passed over a stationary sorbent to
separate the mixture components. This technique is similar to that of liquid -liquid
chromatography, wi th the only exception that in the former a moving gas is used as the
mobile phase while in the latter it is a liquid. The stationary phase remains the same, i.e.,
a solid or a liquid.

12.1.2. Principle
In gas chromatography, the substance to be analysed is partitioned between the mobile
and stationary phases. During the separation, the sample is vaporised and carried through
the column by the mobile gas phase (i.e., the carrier gas). The different components get
separated bas ed on their vapour pressure and affinities for the stationary phase. The
affinity of a component towards the stationary phase is termed as distribution constant
(Kc), which is also known as the partition coefficient.
Kc = [A]s / [A]m

Where, [A]s = con centration of component A in the stationary phase and [A] m =
concentration of component A in the mobile phase.
Direction of mobile phase flow Chromatography
Detector
Inject

B
+
A
Concentration of solute in
mobile phase
B A
Time

Concentration of solution
in stationary phase
B A A

B A
B

Figure 12.1: Schematic Representation of the Chromatographic Process

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Gas Chromatography (Chapter 12) 211

Movement of different components through the column is controlled by the distribution

constant (K c), thus the chromatographic se paration occurs based on the differences in
distribution constant. Figure 12.1 shows a schematic representation of gas
chromatography. The distribution constant depends on the temperature and the chemical
nature of stationary phase. Thus, temperature can b e used for enhancing the separation of
different components through the column or a different stationary phase.

12.1.3. Theory
Gas chromatography is carried out in a column containing the mobile phase. Gas-Liquid
Chromatography (GLC) employs liquid as the stationary phase and is a form of
partition chromatography ; while Gas-Solid Chromatography (GSC) employs a solid
surface as the stationary phase and is a form of adsorption chromatography.

The migration rate of solutes through the chromatographic system relies on their affinities
for the stationary phase. Resolution is a function of differential zone migration rates that
separate the zone centres, and of zone broadening processes that merge the zones. The
mobile phase (the car rier gas) in gas chromatography is an inert gas, thus the solute’s
affinity for the stationary phase determines the distribution coefficient. The solute in gas
chromatography moves through the column only if it is a gas. Therefore, the relationship
between the solute’s vapour pressure and its concentration in a liquid solvent (the
stationary phase) is determined. If an ideal solution is used, the relationship is Raoult’s
law [equation (1)].
p = xp0 ….. (1)

Where, p = vapour pressure of solute over the solution, x = mole fraction of solute in the
solution, and p0 = vapour pressure of the pure solute].
This equation is not obeyed by the real solutions, but a similar relationship , termed
Henry’s law can be written as:
p = xp0 ….. (2)
Where,  = Activity coefficient, which is a function of x. The activity coefficient remains
constant at very low concentrations:
x 1
  constant ….. (3)
p γp0
The quantity x/p has the nature of a partition coefficient, and it controls selectivity in gas
chromatography. Rather than work with x/p itself, the partition coefficient is defined by:
gmof soluteper gmof liquidphase
K ….. (4)
gmof soluteper cm3 of gas phase
Equation (3) suggests that the affinity of solute for stationary phase depends on its
vapour pressure and activity coef ficient. This dependence indicates that the solutes
having same vapour pressures can be separated even if their activity coefficients are
different in the stationary phase. A gas or a vapour on coming in contact with an
adsorbent gets adsorbed on the solid surface in a specific amount. This phenomenon takes
place as per the laws of Freundlich (i.e., x/m = Kc 1/n) or Langmuir (i.e., x/m = K 1c +
K2c, where, x = Mass of the gas or vapour adsorbed in mass m of the sorbent; c = Vapour
concentration in the gas pha se; and K, K 1, and K 2 = Constants). A gas or a vapour on
coming in contact with a liquid gets dissolved in the liquid in a fixed amount. This
phenomenon takes place as per the Henry’s law of partition (i.e., x/m = K c). Both the
phenomena are selective and different vapour-sorbent pairs have different K-values.
* *
212 Instrumental Methods of Analysis

12.1.4. Types
The two major types of gas chromatography are:
1) Gas-Solid Chromatography: In this type, the stationary phase is solid (adsorbents
like alumina, silica, active carbon, etc. are used). This method provides a long
column life time; however, catalytic changes are observed in this technique.
2) Gas-Liquid Chromatography: In this type, the stationary phase is an immobilised
liquid coated on the solid support (like polyme rs). In this method, the liquid
gradually bleeds off, and this is the disadvantage of this method.

12.1.5. Temperature Programming

Temperature programming combines the best results of runs at different temperatures. The
sample is introduced into the chromatographic system, and the column is maintained at
temperature below that of the lowest -boiling component of the sample (preferably below
90°C). The column temperature is increased at some pre-selected heating rate. Earlier peaks
that represent the low-boiling components appear as they would form an isothermal column
maintained at a low temperature. With the increase in column temperature, the high-boiling
components are forced through the column at an ever -increasing rate. In this way, the
approximate proper temperature programme can be estimated.

Partition coefficient rapidly increases the solute amount in the vapour phase. Solute
concentration in the liquid phase is always more than in the gas phase, i.e., the fraction
1/(1 + k ) is less than unity; therefore, it is a good approximation to ignore C MVM in the
denominator. Remembering that k = K/, on ignoring the tempera ture dependency of the
first term on the right-hand side from the equation, one gets:
 1  H v
ln     C' ….. (5)
 k'  RT

The average increase in temperature should be determined to halve the value of k  (to
double the total solute in the vapour phase). If k  is halved by increasing the temperature
from T1 to T2, the ratio of k' values can be determined from equation (5) as follows:
– H v RT2 H v  T 
ln 2     ….. (6)
– H v RT1 RT  T 

Which gives:
0.693 R ( T ) 2
T  ….. (7)
H v

Where, T = Geometric mean of the two temperatures

T = T2 – T1 ….. (8)

Trouton’s rule is approximately valid, so  H v /Tb ≃ 23.

The chromatographic process is operated near the solute boiling point, thus T ≃ Tb. The
temperature increase ( T) halves the k  value to be 21°C at an operating temperature of
75°C, 24°C at 125°C, and 30°C at 225°C. The k  value is important because more the
solute in the vapour state, faster the peak migrates. Thus, if the value of k  is 5, one -fifth
of the solute molecules exist as vapour.
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Gas Chromatography (Chapter 12) 213

The final temperature should be near the final solute’s boiling point (if the maximum
temperature limit of the stationary phase is not exceeded). With the increase in
temperature range, individual compounds automatically select their own ideal
temperature to migrate and separate in the column.

On increasing the column temperature linearly, the members of any ho mologous series
are eluted at equally spaced intervals ( figure 12.10), rather than proportional to ln t R as
in isothermal elution.

n-Heptanol
n-Pentanol
n-Hexanol
n-Octanol
n-Heptanol

n-Decanol
n-Pentanol

n-Hexanol

Isothermal
n-Butanol
Detector Signal

Detector Signal
temperature
175°C

n-Butanol

n-Octanol

1 Temperature
1 (°C) 1 1 1 n-Decanol
13 26 9 14 1 62 7 0 3 6 9
0° 0° 20° 5
Time (min) 5° 0 5° Time (min)
(a) (b)
Figure 12.10: Chromatograms of an Alcohol Mixture: (a)
Programmed Temperature from 100 to 175°C and (b)
Isothermal Operation at 175°C.

12.1.6. Advantages
Gas chromatography has the following advantages:
1) It is a reliable technique and provides rapid analysis.
2) It is highly efficient and leads to high resolution.
3) It utilises sensitive detectors.
4) It requires small samples (<1 ml).
5) It is non -destructive as it enables the coupling to mass spectrometers, whic h
measures the masses of individual molecules converted into ions, i.e. molecules that
have been electrically charged.
6) It provides high quantitative accuracy.
7) It is a well-established technique with extensive literature and applications.

12.1.7. Disadvantages
Gas chromatography has the following disadvantages:
1) It is limited to volatile samples.
2) It is not suitable for thermolabile samples (that degrade at elevated temperatures).
3) It is not suited to preparative chromatography.
4) It requires MS detector for structural elucidation of the analyte, since most of the
*
non-MS detectors are destructive. *
214 Instrumental Methods of Analysis

12.2. INSTRUMENTATION
12.2.1. Introduction
The basic instrumentation of gas chromatographic techni que has six components;
however, many commercial variations are available.
Reference Reference detector
flowmeter

Gas regulators Sample

detector Bridge
and gauges

Sample
flowmeter

Sample Amplifier
injection
port

Carrier
Recorder
gas

Fraction collector
Column
Thermal chamber
Figure 12.2: Schematic Representation of a Gas Chromatograph
Source: Adapted from Instrumental Methods of Chemical Analysis (pp 2.677),
by Chatwal G.R. (2006) (Himalaya Publishing House)
The following components make up the instrumentation of gas chromatography (figure 12.2):
1) Carrier gas maintained at a high pressure and delivered at a rapid
and reproducible rate,
2) Sample injector,
3) Separation columns,
4) Detectors,
5) Thermostated chambers for regulating the temperature of column and detectors, and
6) Amplifier and recorder system.
Separation in gas chromatography is carried out in a tubular column (of glass, metal, or
Teflon) filled with an adsorbent (the stationary phase). The adsorbent is packed as fine
size graded powder, while the liquids before being packed in the column are either coated
as a fine film on the column wall or are coated over an ine rt size graded porous support
(such as firebrick powder). A carrier gas (the mobile phase) is continuously made to flow
through the column so that the sample components are distributed in the column.
The sample vapour is introduced in the column through the carrier gas entrance end. The
different sample components adsorb on the stationary phase to different extents, and this
depends on their distribution coefficients. The carrier gas immediately sweeps further the
portion of each component in the gas phas e. Thus, a fraction of the adsorbed amount
desorbs out to maintain the value of distribution coefficient. Simultaneously, out of the
amount swept away, some amount again goes into the adsorbent at the next point in the
column to maintain the distribution c oefficient value. This process continues and the
band for each component moves further in the column attaining the shape of Gaussian
distribution.
* *
Gas Chromatography (Chapter 12) 215

12.2.2. Carrier Gas

Hydrogen, helium, nitrogen, and air are the most widely used carrier gases. Hydrogen
in comparison to other gases is more advantageous and also dangerous to use. Helium is
the next best gas, and is used because of its exceptional thermal conductivity, inertness,
low density, and greater flow rates; but it is expensive. Nitrogen is inexpensive but
reduces sensitivity. Air is used only when the atmospheric oxygen is useful to the
detector or separation. The following considerations should be kept in mind while
selecting a carrier gas:
1) It should be inert, i.e., it should not react with the sample, station ary phase, or
contacted hardware.
2) It should be suitable for the detector used and the type of sample being analysed.
3) It should be available in high purity.
4) It should give best column performance reliable with required speed of analysis.
5) It should not be expensive.
6) It should not cause any fire or explosion hazard.

12.2.3. Sample Injector

The system of sample injector is used for introducing the sample in a reproducible
manner and should vaporise it rapidly so that the sample enters the column as a single
slug.

Figure 12.3: HypodermicSyringe

Liquid samples are introduced into a small inlet chamber using hypodermic syringes
(figure 12.3) through a self -sealing rubber septum. The chamber is heated to cause flash
evaporation, and the temperature should not be very high to avoid sample decomposit ion.
Solid samples are either dissolved in volatile liquids prior to their introduction or are
directly introduced if they are liquefiable. Gas samples are introduced into the carrier gas
stream using a special gas sampling valves.

12.2.4. Separation Columns

The columns used are made of glass or metal tubing, and have a diameter of 4.8mm.
They may be of any length ranging from a few centimetres to a hundred meters. They
may be coiled, bent, or straight. The following six types of analytical columns are used in
gas chromatography:
1) Packed Columns: These columns are prepared by packing metal or glass tubing
with granular stationary phase. In gas liquid chromatography, the packing is prepared
by coating a size graded inert solid support with the liquid phase.
2) Open Tubular or Capillary or Golay Columns: These columns are made of long
capillary tubing (30 -90m) and have uniform and narrow internal diameter (0.025 -
0.075cm). They are of stainless steel (most popular), copper, nylon, glass, etc. The
liquid phase is coated o ver the inner wall of capillary tubing as a thin (0.5 -1µ) and
uniform film. Since there is no packing in these columns, the flow of carrier gas
experiences least resistance.
3) Support Coated Open Tubular Columns: These columns are prepared by coating
the inn er wall of a capillary column with a micron size porous layer of support
material, followed by coating with the liquid phase as a thin film.
4) Wall Coated Open Tubular Columns: These columns are prepared by coating the
*
unmodified smooth inner wall of the tube with the liquid stationary phase. *
216 Instrumental Methods of Analysis

5) Porous-Layer Open -Tubular (PLOT) Columns: These columns are prepared by

coating the inner wall with a porous layer. Porosity can be achieved either by
chemical methods (e.g., etching) or by depositing porous particles on the wall from a
suspension. The porous layer either provides support to the liquid stationary phase or
acts as the stationary phase itself.
6) Support-Coated Open -Tubular (SCOT) Columns: In these columns, the porous
layer consists of support particles and was deposited from a suspension.

12.2.5. Detectors
Gas chromatography employs a wide range of detectors, of which Flame Ionisation
Detector (FID) and the Thermal Conductivity Detector (TCD) are the most common
ones. Both these detectors are sensitive to various com ponents, and both work over a
wide range of concentrations. The TCDs are universal and can be used for detecting
any component ; however, they cannot detect carrier gas till their thermal
conductivities are different from those of the carrier gas at detecto r temperature.

The FIDs are sensitive to hydrocarbons , and their sensitivity is more than that of
TCDs; however, they cannot detect water . Both TCDs and FIDs are quite robust. The
TCDs being non -destructive can be operated in -series before FIDs (destruct ive), thus
providing complementary detection of the same eluents.

Some gas chromatographs utilise a mass spectrometer as the detector; this combination
is known as GC-MS. Some GC-MS utilise an NMR spectrometer as a backup detector;
this combination is kn own as GC-MS-NMR. Some GC -MS-NMR utilise an
IR spectrophotometer as a backup detector; this combination is known as GC-MS-
NMR-IR.
Integral record
ml alkali
Arbitrary scale

Base line

Signal

Base line Differential record

Time or volume of effluent gas

Figure 12.4: Gas Chromatogram of a Mixture of Three Acids using Differential
and Integral Detectors
In Gas-Liquid Chromatography (GLC) , the gas from the column is constantly
monitored and any difference bet ween it and the normal gas is recorded, i.e., detectors in
most apparatus are differential. The chromatogram is a record of detector response
against time, or is a record of the known gas flow against carrier gas volume.

Figure 12.4 shows the trace obtai ned with a detector and also shows the appearance of a
corresponding curve for an integral detector.
* *
Gas Chromatography (Chapter 12) 217

Some desirable properties of a detector are:

1) Its sensitivity should be high and should not show instability at high sensitivities.
2) Its volume should be lo w so that the compound eluted from the column in a small
plug of carrier gas does not undergo further dilution within the detector.
3) Its response should be rapid and linear with the concentration of compound. It should
be calibrated to determine the optimum range.
4) Its response should not be affected by the flow rate of carrier gas and temperature.

Some commonly used detectors in gas chromatography are discussed below:

1) Katharometer: This detector relies on the variation in thermal conductivity of the
carrier gas in the presence of an organic compound. The principle of this detector is
illustrated in figure 12.5. The platinum wires are heated by electric means and
equilibrium conditions of temperature and resistance are attained when the carrier
gas passes ov er them. They are mounted in a Wheatstone bridge arrangement, and
when a compound emerges, the thermal conductivity of the gas surrounding wire
changes; also changing the temperature and resistance of the wire along with the
associated out-of-balance signal, which is amplified and recorded.
Platinum wires connected in
Wheatstone bridge circuit

Carrier
Outlet
gas

To injection point Carrier gas

and column from column

Figure 12.5: Diagrammatic Representation of Katharometer

The sensitivity of katharometer is lower than the sensitivities of other detectors. Its
sensitivity is affected by temperature and flow rate fluctuations.
2) Flame Ionisation Detecto (FID): This det ector is simpler in design ( figure 12.6)
and relies on the change in conductivity of the flame as the compound is burnt . The
change in flame conductivity is not because of simple ionisation of the compounds
emerging from the detector.

The molecule underg oes partial or complete stripping and gives charged hydrogen -
deficient polymers or aggregates of carbon with low ionisation potential.

The carrier gas used is nitrogen or argon mixed with hydrogen before passing to the
burner tip (made of a platinum capil lary). This forms one electrode, and the other
one is silver gauze or brass collector electrode about 1cm above the flame.
* *
218 Instrumental Methods of Analysis

Collector electrode
+

Outlet for gases

Silver gauze

–
Air filter
Hydrogen Air

Carrier gas from column

Figure 12.6: Diagrammatic Representation of Flame Ionisation Detector
3) Thermal Conductivity Detector (TCD): This detector ( figure 12.7) utilises a
heated filament placed in the emer ging gas stream. Thermal conductivity of the gas
phase governs the amount of heat the filament loses by conduction to the detector
walls.

Block

Gas flow out

Gas flow in
Filament

Figure 12.7: Cross-Sectional View of a

Thermal Conductivity Detector

The cavity in the metal block has a tightly coiled filament of tungsten metal,
tungsten-rhenium alloy, or tungsten sheathed with gold. A regulated DC current is
supplied to heat the filament up to a constant temperature till it reaches less than a
dull-red condition. If only carrier gas is flowing through the detector, the heat lost by
the filament to the metal block is constant.
Hydrogen and helium have 6 -10 times greater thermal conductivities than other
organic compounds. Due to this reason, even trace amounts of organic materials
adversely decrease the thermal conductivity of the column effluent . The filament
retains more heat, and its temperature and electrical resistance increases. A standard
detector comprises of four identical filaments (mounted in a brass block), which
form the arms of a Wheatstone bridge.
* *
Gas Chromatography (Chapter 12) 219

A TCD can be constructed with therm istor, which is a metal oxide bead having
attached electrical leads. The bead possesses 8000  resistance at 25°C temperature
and a negative temperature coefficient of resistance. One bead is mounted in the pure
carrier gas stream, and the other in the column effluent.

The Wheatstone bridge circuit is completed by a matched pair of 500 , 3W

resistors. Thermistors are neither mechanically nor electrically exchangeable with
filaments. They can operate at ambient or sub-ambient column temperatures.
4) Thermioni c Emission Detector (TED): This detector utilises fuel -poor
hydrogen plasma and a low temperature flame. This flame suppresses the normal
flame ionisation response of compounds not containing nitrogen or phosphorus
(figure 12.8).

Ceramic
insulators

Signal
Collector
probe

Ceramic
insulators

Ceramic bead with

Bead probe heater coil

Flame tip

Figure 12.8: Diagrammatic Representation of Thermionic Emission Detector

TED has a very small hydrogen flow, and responds well to nitrogen and phosphorus
compounds. It can be made to respond to only phosphorus compounds by increasing
the plasma size and altering the polarity between the plasma tip and collector.
5) Electron Capture Detector (ECD): This detector relies on the electron affinity of
different substances. It responds to compounds whose molecules have electron
affinity, e.g., chlorinated compounds, alkyl lead, etc. It responds less to
hydrocarbons. A diag rammatic representation of an ECD using a metal foil coated
with a tritium-containing compound as a steady source of slow electrons is shown in
the figure 12.9.

ECD shows high sensitivity to some compounds, e.g., chlorinated pesticides can be
determined d own to sub -picogram levels. ECD has a drawback of having a very
narrow linear range; however, this can be solved by proper calibration and
adjustment of the sample size.
* *
220 Instrumental Methods of Analysis

Radioactive Gas exit

foil 65Ni or 3H Insulation

Gas Anode
inlet

Figure 12.9: Diagrammatic Representation of Electron Capture Detector

Source: Adapted from Instrumental Methods of Chemical Analysis (pp 2.684),
by Chatwal G.R. (2006) (Himalaya Publishing House)

ECD is used for detecting trace environmental pollutants. It is highly sensitive to

halogenated compounds and is used for detecting herbicides, pesticides, SF 6 traces in
fuel gases, organometallics ( e.g., lead tetraethyl), polynuclear aromatic carcinogens,
and NO2 and SO2 in chimney stack gases.
6) Alkali Flame Ionisation (The rmionic) Detector (AFID): This is a flame ionisation
detector using a source of alkali metal salt placed between the flame jet and the
collector electrode. The alkali source is generally a glass bead having rubidium
silicate (non -volatile salt) fused on pl atinum wire and maintained at a negative
potential. AFID can be made to respond to compounds containing both nitrogen and
phosphorus or only phosphorus by changing the polarity of jet.
7) Flame Photometric Detector (FPD): This is a special type of flame emiss ion filter
photometer. It is used for determining the volatile sulphur or phosphorus compounds.
The column effluent passes into a dual hydrogen -enriched, low temperature flame
within a shield. The carrier gas is supplied with air and hydrogen as make -up gases.
Only the upper flame is viewed.
Phosphorus forms an HPO species that emit band emissions at 510 and 526nm
around the sides and base of the flame. Sulphur compounds form an S 2 entity that
emits a series of bands centred on 394nm but also overlaps the phosphorus spectrum.
FPD responds linearly to phosphorus, whereas its response to sulphur depends on the
square of concentration. The sensitivity of FPD to phosphorus is about 100 times less
than the TED.
8) Photo-Ionisation Detector (PID): This detector prod uces ionisation of solute
molecules by using UV radiation from lamps with energies ranging between 9.5 -
11.7eV. The ions are collected at a positively charged electrode and the current is
measured. Compounds having ionisation potentials lower than the lamp ionising
energy produce a response.
9) Electrolytic Conductivity Detector (ECD): This detector relies on electrolytic
conductivity. Organic compounds eluting from the gas chromatography column are
burned in a miniature furnace to form simple molecular species . These species ionise
and contribute to the conductivity of deionised water. The changes in electrolytic
conductivity are monitored.
The analyte ions are removed from the liquid by an ion -exchange column, part of a
continuous circulating system that reg enerates conductivity -grade water. On mixing
the combustion products with hydrogen gas and hydrogenating over a nickel catalyst
in a quartz tube furnace at 850°C temperature, ammonia from organic nitrogen, HCl
*
from organic chlorides, and H 2S from sulphur compounds are formed. *
Gas Chromatography (Chapter 12) 221

12.2.6. Substrates
The solid support is coated with a substrate, which is a high boiling liquid that acts as the
immobile phase in gas-liquid chromatography. The general requirements for the liquid
phase are:
1) Good solvent property of the component,
2) Differential partitioning of sample components,
3) Low vapour pressure at the column temperature, and
4) High thermal stability.
Some typical substrates are given in table 12.1:
Table 12.1: Some Typical Substrates
Substrates Solute Types Temperature
(°C)
1) Polyglycols Amines, ethers, alcohols, ketones, 100-200
esters, and aromatics
2) Paraffin oil (Nujol) Paraffins, olefins, and halides 150
3) Silicone oils Paraffins, olefins, esters, and ethers 200
4) Didecyl phthalate Polar compounds 170

12.2.7. Temperature Control

A temperature programming enables controlled increase of temperature during an
analysis. As a result, the latter peaks also become sharp and emerge quickly. Thus in
temperature programming, the components of a wide boiling range mixture can be
efficiently determined. The temperature programming can be carried out in the following
three modes:
1) Natural or ballistic,
2) Linear,
3) Matrix or multi-linear.

The operation with linear temperature programme is more common. The requirements
for good temperature programming are:
1) A dual column system to compensate for bleeding of liquid phase from columns
when temperature is increased,
2) Separate heaters for injector, column, oven, and detector system,
3) Differential flow controllers,
4) Low mass column oven to enable rapid heat transfer,
5) Thin walled columns,
6) Low liquid phase loading,
7) Pure dry carrier gas, and
8) Stable and non-bleeding injection septums.

12.2.8. Data Acquisition System

A data acquisition system for gas chromatography comprises of two basic components.
The analog signal com ing from the detector is amplified and converted into digital data
by the first component . The second component is a computer with a suitable software
program that receives the digitalised signal. Some field portable gas chromatography
systems can be conne cted to a laptop and the chromatography program is run from a
compact disc. The chromatography software set up the run conditions for gas
chromatography, acquires data, integrates peaks, display peaks on the computer screen,
prints chromatograms, and run reports.
* *
222 Instrumental Methods of Analysis

12.2.9. Gas-Solid Chromatography (GSC)

The apparatus and technique for GSC are similar to that for GLC, with the only
difference in the nature of stationary phase and column length. Information on the
columns used in GSC and their applications is illustrated in table 12.2.
Table 12.2: GSC Columns and their Applications
Columns Separation Uses
1) Carbon (Carbosieve B) Light hydrocarbons, H2, and O2
2) Silica (Spherosil, Porasil) Light hydrocarbons, H2, and O2
3) Alumina Hydrocarbons
4) Molecular sieves H2, O2 and N2, and branched chain hydrocarbons
5) Porus polymers (Pora Pak, Aqueous solutions, acids, and other polar
Chromosorb) materials.

12.3. DERIVATIZATION
12.3.1. Introduction
Derivatization reactions transform an analyte for detectability in gas chromatography or
other instrumental analytical methods. Derivatization in gas chromatography analysis is a
technique that modifies the functionality of an analyte to allow chromatographic
separations. The resultant modified analyte is the product, and is known as the
derivative, whose structure may be similar or closely related to but not the same as the
original non-modified chemical compound.
Apart from particular analytes such as pharmaceuticals, biomolecules such as organic acids,
amides, poly-hydroxy compounds, amino acids, pesticides, and other persistent organic
compounds, some new classes of desirable compounds, like fluorinated alkylated substances
and polycyclic ar omatic hydrocarbons, are also being developed. Thus, new chemical
analytical methods need to be developed and the existing ones need to be improvised. For this,
one should be familiar with the derivatization methods applicable to GC analysis.

12.3.2. Derivatization Reagent

Derivatization reagent is a substance used for chemically modifying a compound so that a
new compound, with properties suitable for analysis in gas and liquid chromatography,
can be produced. A suitable derivatization reagent for gas chromatogra phy should be
selected based on the following criteria:
1) It should produce more than 95% complete derivatives.
2) It should not cause any rearrangements or structural alterations in the compounds
during the formation of a derivative.
3) It should not cause any sample loss during the reaction.
4) It should produce a derivative that will not interact with the column used in gas
chromatography.
5) It should produce a derivative that remains stable with time.

12.3.3. Objectives for Derivatization

Derivatization technique has the following objectives:
1) It improves resolution and reduces tailing of polar compounds containing –OH,
–COOH, −NH, –NH2, –SH, and other functional groups.
2) It enables the analysis of relatively non-volatile compounds.
3) It reduces the volatility of compounds prior to gas chromatography analysis.
4) It improves the analytical efficiency and increases detectability.
5) It causes stabilisation of compounds for gas chromatography analysis.
* *
Gas Chromatography (Chapter 12) 223

12.3.4. Types of Derivatization

Derivatization reactions for gas chromatography are of t hree types, i.e., alkylation (the
general process is esterification), acylation, and silylation. These processes make the
highly polar materials ( e.g., organic acids, amides, poly -hydroxy compounds, and amino
acids) sufficiently volatile, thus suitable for gas chromatography analysis. The three
derivatization reactions are discussed below.

[Link]. Alkylation
Alkylation is the first step for further derivatization. It is most widely used as a protection
method for some active hydrogen in a sample molecule. It repre sents esterification in
which the active hydrogen is replaced with an aliphatic or aliphatic -aromatic group ( e.g.,
benzyl). The equation below shows the gene ral reaction of esterification [ X = halogen or
alkyl group (R) and H = another alkyl group (R)]:
RCOOH + PhCH2X → RCOOCH2Ph + HX

The major chromatographic use of alkylation is the transformation of organic acids

into esters (particularly methyl esters) that produce better chromatograms than the
free acids . Alkylation reactions are also used for prepari ng ethers, thioethers and
thioesters, N -alkylamines, amides, and sulphonamides. The polarity of alkylation
products is generally less than that of the starting materials. This is because active
hydrogen has been replaced with an alkyl group. The obtained a lkyl esters provide
exceptional stability, and can be isolated and stored for longer periods. In esterification,
an acid and an alcohol react to form an ester.
Dialkylacetals, diazoalkales, Pentafluorobenzyl Bromide (PFBBr), benzylbromide, Boron
trifluoride (BF 3) in methanol or butanol , and Tetrabutylammonium Hydroxide (TBH) are
the commonly used derivatization reagents in alkylation reactions. These alkylation
reagents can be used either alone to form esters, ethers . and amides or in combination
with acy lation or silylation reagents. The reaction conditions for alkylation range from
strongly acidic to strongly basic; however, stable derivatives are produced under both the
conditions. The alkylation reagents are more limited to amines and acidic hydroxyls. A
drawback is that sometimes the reaction conditions become severe and the reagents
become toxic.

[Link]. Acylation
Acylation reaction involves introduction of an acyl group to an organic compound.
Acylation in carboxylic acid involves addition of an acyl group and loss of a hydroxyl
group. Acylation can convert the compounds with active hydrogen, e.g., −OH, −SH, and
–NH, into esters, thioesters, and amides, respectively. This reaction is popularly used for
the production of volatile derivatives of highly polar and volatile organic materials .
The reaction also improves the stability of thermolabile compou nds by adding protecting
groups into the molecule. Acylation can make the extremely polar materials (such as
sugars) amenable to separation by gas chromatography, and thus, are a valuable
alternative to silylation. An example of acylation reaction between acetic anhydride and
an alcohol to produce acetate ester and acetic acid is shown below.

Acylation reaction has the following advantages in gas chromatography analysis:

1) It improves the stability of analyte by protecting the unstable groups.
2) It provides volatility to compounds having many polar groups (carbohydrates or
*
amino acids), as they are non-volatile and decompose on heating. *
224 Instrumental Methods of Analysis

3) It aids in chromatographic separations which is not possible with compounds

inappropriate for gas chromatography analysis.
4) It can detect compounds at very low levels using an electron capture detector.

Fluoracylimidazoles, fluorinated anhydrides, N -Methyl-bis(Trifluoroacetamide)

(MBTFA), Pentafluorobenzoyl Chloride (PFBCl), and Pentafluoroprop anol (PFPOH) are
the reagents commonly used for acylation. These reagents target the highly polar, multi -
functional compounds, like carbohydrates and amino acids. They also allow the
introduction of electron -capturing groups, and therefore enhance the dete ctability during
analysis. The acylating reagents are available as acid anhydrides, acyl derivatives, or acyl
halides, of which the latter two are highly reactive and can be suitably used where issues
of steric hindrance may be a factor.

Acid anhydrides a re available in a number of fluorinated configurations, which can

improve detection. These fluorinated anhydride derivatives are used for detection using
electron capture and flame ionisation detectors. Fluorinated anhydrides are used in
derivatizing sampl es to confirm drugs of abuse. Still due to their acidic nature, it is
necessary to remove any excess of by -products before gas chromatography analysis. This
is done to prevent column deterioration. Because of the acidic by -products, the
derivatization proc ess has been carried out in pyridine, tetrahydrofuran, or another
solvent that can accept the acidic by-product.

[Link]. Silylation
Silylation is the most predominant derivatization method. It readily turns the sample
volatile, and is very suitable for gas chroma tography analysis of non -volatile samples. In
silylation reaction, a silyl group , such as dimethylsilyl [SiH(CH 3)2], t-butyldimethylsilyl
[Si(CH3)2C(CH3)3], and chloromethyldimethylsilyl [SiCH 2Cl(CH3)2] is added into a
molecule to replace active hydrogen . As a result of this replacement, the polarity of the
compound as well as hydrogen bonding reduces. Silylation reaction has been successfully
used in the gas chromatography analysis of hydroxyl and amino compounds that are
considered non -volatile or unstabl e at 200 -300°C temperature. The silylated derivatives
are more volatile and stable, thus yield narrow and symmetrical peaks.

General Reaction Mechanism: Silylation reaction is driven by a good leaving group

having a low basicity, the ability to stabilise a negative charge in the transitional state,
and little or no π back bonding between the leaving group and silicon atom. In this
reaction, the active hydrogen (in −OH, −COOH, −NH, −NH 2, and −SH groups) is
replaced with a trimethylsilyl group. Silylation o ccurs through nucleophilic attack (SN 2),
where the degree of silylation is better if a good leaving group is used. As a result, a
bimolecular transition state occurs in the intermediate step of reaction mechanism. The
reaction for the formation of trialkyl silyl derivatives is shown below, where X = Cl. The
leaving group in case of trimethylchlorosilane (TMCS) is the Cl atom.

In silylation derivatization, the sample as well as the solvents should be dry. Silyl
reagents should be stored in tightly closed containers as they are moisture -sensitive, and
thus should be used in pure form and as little as possible. This will eliminate excessive
peaks and prevent a large solvent peak.
* *
Gas Chromatography (Chapter 12) 225

The most commonly used solvent in silylation i s pyridine. It may produce peak tailing,
still it is an acid scavenger and drives the reaction forward. In many cases of silylation, a
solvent is not required with silylating reagents. The completion of silylation
derivatization is observed when a sample readily dissolves in the reagent.
According to Regis, the ease of reactivity of the functional group towards silylation
follows the order:
Alcohol > Phenol > Carboxyl > Amine > Amide/Hydroxyl
For alcohols, the order is as follows:
Primary > Secondary > Tertiary

Many reagents need to be heated at temperature not more than 60°C for 10 -15 minutes.
This is necessary to prevent the breakdown of derivative. However, the hindered products
may require to be heated for a long-term.

Hexamethyldisilzane (HMDS), Trim ethylchlorosilane (TMCS),

Trimethylsilylimidazole (TMSI), Bis(trimethylsilyl)acetamide (BSA), Bis
trimethylsilyl Trifluoroacetamide (BSTFA), N -Methyl trimethylsilyl
Trifluoroacetamide (MSTFA), Trimethylsilyl Diethylamine (TMS -DEA), N -Methyl -
N-t-Butyldimeth ylsilyl Trifluoroacetamide (MTBSTFA), and Halo -methylsilyl are
the reagents used for silylation derivatization.

Halo-methylsilyl derivatization reagents can produce silylated and halogenated

derivatives for Electron Capture Detectors (ECDs). Silyl reagent s form trimethylsilyl
ether and trimethylsilyl ester derivatives by reacting with alcohols and acids,
respectively. These derivatives are volatile and can be easily separated. Silyl reagents
show compatibility with most detection systems, but they cause di fficulties with Flame
Ionisation Detectors (FIDs) when used in excess. Silyl reagents can be influenced by the
solvent system and by adding a catalyst ( e.g., trimethylchlorosilane or pyridine), which
increases the reagent reactivity. Reagents that introdu ce a t -butyldimethylsilyl group
instead of trimethylsilyl group impart greater hydrolytic stability to the t -
butyldimethylsilyl derivatives.

These derivatives have better stability against hydrolysis, and also have distinctive
fragmentation patterns, thus are useful in various GC/MS applications. Trimethylsilyl and
t-butyldimethylsilyl derivatives mostly have exceptional thermal stability and are
amenable to a variety of injection and column conditions. Silylation provides the ability
to derivatize various compounds for gas chromatography analysis.

12.4. APPLICATIONS
12.4.1. Introduction
The major applications of gas chromatography are qualitative and quantitative
analysis of liquids, gases, and vapours (mainly of organic com pounds). This
technique can be used for determining a stable compound that can be vaporised
below 300°C temperature.

However, if the compound is not stable to isomerisation and decomposition at these

temperatures, the method will give rise to inaccurate results. The non -volatile
compounds or the ones unstable at this temperature can be analysed by liquid
chromatography.
* *
226 Instrumental Methods of Analysis

12.4.2. Qualitative Analysis

The individual components of a mixture can be qualitatively analysed by either:
1) Comparing the retention times or vol umes of the unknown with that of the series of
standards, or
2) Collecting the individual components emerging from the chromatograph and
identifying them using other methods.

All the experimental parameters of the

Log retention volume, VR

standards and unknowns, mainly the flow
rate and temperature, should be carefully
controlled and duplicated if the analysis is
to be done using retention times or
volumes. There are many compounds
with the same retention time.

Due to this reason, the data from a single

set of conditions are not sufficient for
positive identification; and hence, 2 3 4 5 6 7 8 9
confirmation should be made using Number of carbon atoms
different substrate-solvent combinations. Figure 12.11: Plot of Log of Retention
Volume against the Number of Carbon
Sometimes, the sample mixture should be Atoms for a Homologous Series
Source: Adapted from Instrumental
spiked with a known compound. If the
Methods of Chemical Analysis
area of a previously present band (pp 2.692), by Chatwal G.R. (2006)
increases and no additional elution band is (Himalaya Publishing House)
obtained under several sets of conditions,
positive identification can be practically confirmed.

Under a given set of experimental conditions, the logarithm of retention volume and the
number of carbon atoms in each
homologous series are linear. Each different Alkanes Esters
Log VR on non-polar column

homologous series yields a different line

(figure 12.11). If the series is known, the Ketones
number of carbon atoms can be determined
Alcohols
using these graphs.

The series to which a compound belongs

can be determined by plotting the logarithm
of retention volume on a polar column
against the logarithm of retention volume
on non-polar column (figure 12.12). Log VR on polar column
Figure 12.12: Plot of Log Retention
After collecting the samples, the final
Volume on a Polar Column against
identification can be made through mass the Log of Retention Volume on a
spectroscopy, nuclear magnetic resonance, Non-Polar Column
or infrared absorption techniques. Source: Adapted from Instrumental
Methods of Chemical Analysis (pp
Interfacing devices are mostly required for 2.692), by Chatwal G.R. (2006)
compensating the sample size, sample (Himalaya Publishing House)
composition, and/or the pressure
requirements of different instruments.
* *
Gas Chromatography (Chapter 12) 227

12.4.3. Quantitative Analysis

The quantitative analysis of a chromatogram relies on the basis that the area of a single
component elution peak and the quantity of the detected component are proportional.
Accurate measurements of the peak area can be obtained if the output at the recorder and
the concentration are linear, and also if the time response of the recorder matches the time
response of the detector (if not so an automatic integrator should be coupled directly to
the detector as an alternative). The flow rate of the carrier gas should also be reproducibly
constant to allow a conversion between the flow rate and time.

The automatic integrators are preferably used because of their speed, accuracy, and ease
of result interpretation. If an integrator is unavailable, cut and weigh procedure is used
for area determination. In this pr ocedure, sample of known weight is injected, the paper
of constant thickness and moisture content is cut, and a weight component per weight of
paper factor is determined.

Height (H)

Width
(W)

Log VR on Polar Column

Figure 12.13: Triangulation Method of Determining Peak Area
Source: Adapted from Instrumental Methods of Chemical Analysis (pp 2.693),
by Chatwal G.R. (2006) (Himalaya Publishing House)

The peak area can also be determined by triangulation procedure, in which tangents are
drawn on both sides of the curve ( figure 12.13). The intercept of the two tangents at the
top is the height (H), the distance between the baseline and the intercepts of the two
tangents is the width (W), and the area (A) can be calculated as:
A = ½ WH

12.4.4. Specific Applications

Gas liquid chromatography has a wide range of applications. In the last few decades,
rapid changing complexities of the product ( e.g., drugs, foodstuffs, and consumer
products) and the technology with associated envir onmental problems have been seen.
This has forced the development of systems that can be used for the separation and
identification of a large number of known and unknown species having increased
sensitivity. GLC has an essential role in this task.
Some applications of GLC are:
1) Detection of Steroidal Drugs: GLC is used for detecting steroids in athletes in
international sports competitions and the steroids administered to animals in traces. It
is also used for detecting hazardous pollutants ( e.g., formaldehyde, carbon
monoxide, trichloroethylene, benzene, and acrylonitrile).

It can be used for analysing the volatile fatty acids produced by anaerobic bacteria. It
also enables fingerprinting of particular microorganisms, thus facilitating
identification of the bacteria.
* *
228 Instrumental Methods of Analysis

2) Analysis of Foods: GLC is used for analysing foods. It is also used for
separating and identifying lipids, proteins, carbohydrates, preservatives,
flavours, colourants, texture modifiers, vitamins, steroids, drug and pesticide
residues, and trace elements.

Most of the compounds are non -volatile, thus HPLC is now used for food
analysis; however, GLC is also used for the same purpose by converting the
compounds into a volatile form to make them suitable for separation. This is
termed derivati zation, e.g., lipids are converted to fatty acid methyl esters,
proteins are converted by acid hydrolysis and esterification, and carbohydrates
are converted by silylation.
3) Analysis of Dairy Products: GLC is used for analysing the dairy products,
aldehydes and ketones (for rancidity), fatty acids (by derivatization), and milk
sugars. It is also used for analysing butter (for determining the butter fat content),
and added colours and flavours.
4) Drug Analysis: GLC is used in drug analysis, e.g., analysis of co mmercial drug
preparations, illicit drug samples, blood, urine samples, and stomach contents.
5) Separation of Metal Chelates: GLC is used for separating various metal chelates,
e.g., separation of -diketonates of chromium (III) and other thermolabile di -, t ri-,
and tetravalent metals that are soluble in organic solvents and volatile.

Acetyl acetone, trifluoroacetyl acetone, and hexafluoroacetone are the most

commonly used ligand for derivatization of metal ions. Increasing fluorination of the
ligand yields a more volatile metal chelate.

12.5. SUMMARY
The details given in the chapter can be summarised as follows:
1) A.T. James and P. Martin first time used the gas chromatography technique in 1952
for separating long chain fatty acids.
2) When the stationary phase is solid, it is known as Gas Solid Chromatography
(GSC) and when the stationary phase is liquid, it is known as Gas Liquid
Chromatography (GLC).
3) The affinity of a component towards the stationary phase is termed as distribution
constant (Kc).
4) Gas Liquid Chromatography (GLC) employs liquid as the stationary phase and is
a form of partition chromatography.
5) Gas-Solid Chromatography (GSC) employs a solid surface as the stationary phase
and is a form of adsorption chromatography.
6) Resolution is a function of differ ential zone migration rates that separate the zone
centres, and of zone broadening processes that merge the zones.
7) Hydrogen, helium, nitrogen, and air are the most widely used carrier gases.
8) Packed columns are prepared by packing metal or glass tubing wi th granular
stationary phase.
9) Open tubular or capillary or golay columns are made of long capillary tubing (30 -
90m) and have uniform and narrow internal diameter (0.025 -0.075cm).
* *