GAS

CHROMATOGRAPHY
MAHARSHI ARVIND INSTUTE OF PHARMACY
MANSAROVAR , JAIPUR..

GUIDEDBY
prepared
pr by
Mr kapil sharma
jasmin modi
M
pharma Ist semester
CONTENT:
 Introduction.
HISTORY.
Advantages.
Technique.
PRATICAL REQUIRMENTS.
Application .
Introduction.

Gas chromatography is a widely used technique for the separation

of gaseous and volatile sub which are difficult to separate.

It is similar to column chromatography except that the gas is used

as the mobile phase instead of the liquid.

Here gas as a mobile phase or moving phase is passed through a

column a column containing liquid adsorbent coated on inert solid
support ,thus the adsorption or partition is possible.

Gas solid chromatography(G.S.C) based upon the selective

adsorption on solid, so the component of mixture distributes them
selves between the gas phase and adsorbent and the separation is
due to ADSORPTIVE PROPERTIES.
where as Gas liquid chromatography (G.L.C) is based
upon partition between Gas and immobile liquid coated
on solid,.
•so the component of mixture distributes them selves
between the gas phase and liquid adsorbent coated on
solid and the separation is due to PARTITION PROPERTIES.

HISTORY:
Gas solid chromatography was initially developed by
G.Damkohlar
and H.Thiele in 1943.
•While gas liquid chromatography was originated by Nobel
laureate.A.J.P martin and A.T james in 1952.
 Advantages:
1)This technique has very high resolution power .
Ex: separation of methyl ester of oleaic ,stearic ,and
linoleic acid is possible.
2) the sensitivity of this method is quite high.

3) It involves the relative simple instrument operation of

gas chromatography &related calculation do not required
highly skilled personnel and thus this technique is
suitable for routine analysis.

4) The analysis is complete in short time and give good

accuracy and precision.
Technique:
• Gas chromatography is special form of the
chromatography in which moving or mobile phase is a
gas and the stationary phase is either solid(G.S.C) or
liquid (G.L.C).

• In this method the sample is introduced in moving gas

stream and is carried out through column. The column
contain either adsorbent(G.S.C) or liquid on a solid
support t(this act as e stationary phase)

• The component of mixture sample distribute b/w two

phase .the adsorption or solubility properties may differ
from components to components. and therefore the
components are carried out along the column at different
rate and finally they emerge at the outlet of the column in
distinct zone(peaks) separated by carrier gas.
• On emerging, the vapours of the components are detected by
suitable detector accompanied by automatic recording.

• COLUMN EFFICIENCY:
• Column efficiency is measured by the number of theoretical
plates. The original theory of chromatography i.e .plate theory

• Was able to describe the effect of variable that Influence the

migration rates in quantitative term.

• How ever the plate theory is unable to describe the effect of

factor which are responsible for band broadening.

• Hence plate theory is supplemented by rate theory

a)plate theory:
The plate concept is adapted from the theory of
distillation columns. According to the theory a
chromatographic column is composed of discrete, but
continuous narrow horizontal plate.

It is assumed that during chromatographic process the

equilibration of the solute between mobile and the
stationary phase take place at each theoretical plate. with
step wise transfer of solute and solvent from one plate to
next.

The separation efficiency of chromatographic column

increase with increasing number of theoretical plate .thus,
the number of theoretical plate is can be measured from
the chromatogram.
 N=16(T/W) 2

Where t = distance from injection point to peak

maxima (retention time)and
w= peak width.in unit of time which can be
determine by
drawining the tangents about 2/3 rd of the height
to the peak at the point of inflaction
• The second term affecting column efficiency is H.E.T.P
which is the length of column necessary for attainment
of solute equilibrium b/w mobile and stationary phase

H.E.T.P is related to the number of theoretical plate

”N”
 
H.E.T.P=L/ N
= L/16(W/T)2

Where L=length of column usually

in c.m
b)Rate theory :
The rate theory developed by van Demter success fully describe the
influence of variable that affect the band separation and band
broadening.

•Van demter equation is useful in optimizing the chromatographic

performance and can be expressed as .

• where
A=eddy’s diffusion
B=longitudinal diffusion
C=mass transfer u =linear gas velocity (flow
rate)through
the chromatographic column.it is measured by

µ= length column(c.m)
retention time of air (second)
• Criteria for compound to be analysed by gas chromatography.
• Two important criteria are:

• Volatility :Unless a compound is volatile, it can not be

mixed with mobile phase. Hence volatility is important.

• Thermostability: All the compound are not in the form of

vapour. there will be solid as well as liquid sample. Hence
it is difficult to convert them in to vapour form so they
have to be heated to a higher temperature. At that
temperature the compound have to be thermo stable. If
they are not thermo stable then they can not be analyzed
by gas chromatography.
 PRATICAL REQUIRMENTS:

Filters/Traps Data system

H

RESET

Regulators Syringe/Sampler

Inlets
carrier gas.
Detectors Flow regulator and flow meter.
Gas Carrier

Injection devices.
Hydrogen
Air

Column Columns.
Temperature control device.
Detector.
Recorders and integrators.
• CARRIER GAS:
• The main purpose of carrier gas is to transport the sample
components through the column. For selection of the carrier
gas, following factor must be consider.
• It should be chemically inert and should not interact with the
sample or stationary phase.

• It should be suitable for the detector to be utilized and the

type of the sample or stationary phase.

• It should give best column performance consistent with the

desired speed of analyzed.

• It should be readily available cheap and of high purity.

• It should not cause the risk of fire or explosion hazard.

• Most widely used are Hydrogen, Helium, Nitrogen,
and Argon.

• Hydrogen: It has better thermal conductivity ,low

density.it is useful in case of thermal conductivity
detector and flame ionization detector. The
disadvantages is that it reacts with unsaturated
compounds and it is inflammable.

• Helium: it also has excellent thermal conductivity, but it

is expensive.it is good carrier gas when used with
thermal conductivity detector.

• Nitrogen: it is in expensive but has reduce sensitivity.

• As carrier gas are compressible it must be stored under
high pressure in cylinder and used when required.
 2)Flow regulator and flow meter:
Flow meter are used to measure the flow rate of carrier
gas. They are Rota meter and soap bubble flow meter.

Rota meter: it is placed conveniently before the column

inlet . it has an ordinary glass tube (like burette) with a
float held on to a spring .the level of the float is determine
by the flow rate of carrier gas and is pre calibrated.

Fig: (a) Rotameter

• Soap bubble meter:
• it is similar to rota meter and instead of float , soap bubble
formed indicates the flow rate.it has a glass tube with a inlet
tube at the bottom through which gas comes in .

• A rubber bulb is used to store the soap solution when the bulb
is gently pressed , a drop of soap solution is converted in to a
bubble by the pressure of carrier gas and travel up.
• The distance travelled upwards is
measure of flow rate of carrier gas.

Fig:
soap bubble meter
 3)Sample injection device:

 The sample injection system is very important because

one of the feature of gas chromatography is the use of
very small amount of the sample.

 Liquid samples are generally introduced by

hypodermic syringe through a self sealing rubber
septum in to a small inlet chamber, which may be
heated to cause flash evaporation .

 Solid sample must be dissolved in volatile liquids for

introduction or may be introduced directly if they can be
liquefied.

 Gas samples require special gas sampling valves for

introduction in to the carrier gas stream
• 4)Columns:
For analytical work it has 4.8 meter diameter .
It can be of any length from few centimeter to over a
hundred meter and can be of coiled ,bent or straight.
Three type :
(1)packed column.
(2) open tubular column.
(3) support coated open tubular column.(SCOT)

(5) Temperature control devices 

Preheater: pre heater are used in Gas chromatography
to convert the sample in to its vapour form and mix
them with the mobile phase or carrier gas.
 Thermostatically controlled oven:
For this two type of operation are available
:
(i) Isothermal programming :(iso means same)
In which the same temperature is maintained
throught the process.

(ii)Linear programming: in which the oven is

heated lineary over a period of time Eg 1500C initially to
2000Cat the end of separation .with increase the
temperature rate of 50c/minutes.
This required when the sample contain mixture of low nd
high boiling point temperature.
6)Detectors:
A detector uses some property by which it can detect the
difference between a pure carrier gas and eluted
components.
The requirements of an ideal detector are:
i.) Applicability to wide range of samples.
ii) High sensitivity to even small concentration.
iii) Rapidity of response‘
iv) Linearity i.e less response to low concentration and
proportional response to high concentration.
v) Response should be un affected by the temperature,
flow rate, and character of carrier gas
vi) simple and easy to maintain.
vii). In expensive.
Thermal conductivity detect OR Katharometer:
 The Principle is based upon thermal conductivity difference
between carrier gas and that of the components.
 Kathro meter has two platinum wire of uniform dimension which
form a part of a wheat stone bridge.

 Through one of them pure carrier gas always flows through and
through the other ,the effulents of the column passes.
 The two platinum wires are heated electrically and hence assume
equilibrium condition of temperature and electrical resistance.

 When pure gas passes through both of them , there is no difference

in temperature or resistance of the wire.
 Hence this produce a difference in resistance and so conductivity
between two wires, which is amplified and recorded as a signal.
 Fig thermal conductivity detector

The thermal conductivities of some carrier gases are

givenH as follows.
He N Methane Hexane
2 2

   
32.7 33.9 6.5 5.2 3
• Advantages:
Responds to all compounds
Adequate sensitivity for many compounds
Good linear range of signal
Simple construction
Signal quite stable provided carrier gas glow rate,
temperature, and filament power are controlled
Nondestructive detection

Disadvantages:
Low sensitivity.
Affected by fluctuations in temperature and flow rate.
The response in only relative and absolute.
Biological sample can not be analysed.
 
 
• 2)Flame ionisation Detector.
• A tiny film of hydrogen is maintained at the capillary jet
made of quartz or platinum, air or oxygen is introduced
through a side by inlet for supporting the combustion.
• column effluent are led in to the flame where in
ionization of components may take place.

Fig: flame ionization detector

• An electrode system located close by picks up the
ionization current which is then amplified and fed to
recorder when only carrier gas passing through flame,
there is or very small and constant ionization current
recorded as study base line.

• when the sample components elutes and pass through

the flame, its molecule are ionized and the resulting
ionization current after amplification is fed in to suitable
recorder.

• A FID is sensitive to all organic compound but not

insensitive to the noble
gases,oxygen,nitrogen,co,co2,water,nitrous
oxide,H2s,so2,cs2.
• Advantages:
This detector is extermly sensitive and back ground
noise is low hene µg quantities of sample can be
determine.
Stable and in sensitive to small changes in the flow rate
to carrier gas.
 (3)Electron capture device:
•The electron capture detector has two electrode ,with column
effluent passing between them.
•One of the electrode is treated with radioactive isotope which
emits the electron as it decays. the emitted electron produce
secondary electrons, which are collected by the anode ,
•when the potential of 20V is applied between them .

Fig Electron capture detector

• when carrier gas alone pass through all secondary
electron are collected by the positively polarized electrode.
• Hence a steady base line is recorded. Effluent
molecule which have affinity for electron ,capture these
electrons when they pass through the electrodes. Hence the
amount of steady state current is reduced. This diff is
amplified and recorded as out put signal.
• The carrier gas used in this type of detector depends upon
the electron affinity of the compound analyzed .

• for compound having more electron affinity argon is used as

a carrier gas, compound having less affinity to electron
nitrogen , co2 is used as carrier gas.

• Advantages Highly sensitive. Even Nano gram quantities

can be determine but it has dis advantages that this can be
used only for which has affinity to electron.
• RECORDER:
• Recorders are used to record the response obtained from the
detectors after amplification in gas chromatography generally
potentiometric detector is used.
• In this type of recorder the input response is continuously
balanced by feed back response. a pan connected through this
system moves proportionally along the width of the chart paper,
thus recording the signal .
• At the same time the chart paper moves at a fixed speed along its
length.
Before the operating a recorder ,its zero should be recorded .
Integrators.
• An integrator is employed for simultaneous measurement of areas
under chromatographic peaks by the electric /mechanical means .
• Manually measurement of this techniques is tedious ,time
consuming, and less precise. Electronic integrators print out of the
peak area digital and give highest peak area
• Application :
• 1) Quality analysis : it is nothing but the identification of a
compound .this is done by comparing the retention time of
sample as well as standard. Under identical condition .the
retention time of the standard and sample are same . if there
is deviation than they are not same compound.

• Retention time mean sit the difference between the point of

injection and the peak maxima . it is the time required for 50%
of component to be eluted from the column . it measured in
time in second or minutes.

• 2)checking the purity of the compound: By comparing the

chromatogram of the standard and that of the sample, the
purity of the compound can be reported. If the additional peak
are present and hence the compound is not purified from the
percentage area of the peaks obtained. The percentage purity
can also be reported.
3 presence of impurity:
This can be seen by the presence of additional peaks when
compared to the reference or standard material.
The percentage impurities may also be calculated from peak areas.
4) Quantitative analysis: The quantity of the compound can be
determine by the following method.
(A) Direct comparing method.
By injecting a sample and standard separately and comparing
their peak areas , the quantity of the sample can be determine.
 
A1/A2=W1/W2

Where A1 and A2are peak of the sample and standard

W1 and W2 are the concentration or weight of sample and
standard.
B calibration curve method.
In this method the standard of various concentration are
used to determine their peak areas.
A graph of peak area v/s concentration is plotted .from
the peak area of the unknown sample the concentration
of the unknown is calculated by interpolation method.
C Internal standard method.
In this method , a compound with similar retention
characteristics is used.
A known concentration of the internal standard is added
separately to the standard solution and sample solution
whose concentration is not known.
The chromatogram is recorded and the peak area ratio of
the sample and internal standard is determine.
By using the peak area ratio of standard and internal
standard , the concentration of the unknown solution is
determined.
5 )Multi components analysis or Determination of mixture
of drugs: Similar to the quantification of a single drug ,
multi component, analysis is determined by using any one
of the above methods.
Marketed formulation are available which contain
several drugs and each component can be determined
quantitatively .

6) Isolation and identification of drug or metabolites in

urine , plasma, serum etc can be carried out.

7 Isolation and identification of mixture of components

like amino acids ,plant extracts, volatile oil etc.
REFERENCE:

Instrumental Method of Chemical Analysis

by Gurudeep R. Chatval & ShamK.Anand
Pharmaceutical analysis (Volume II) by
Dr.A.V.Kasture,
Dr, K .R. Mahadik, Dr H.N.More.
Text book of Pharmaceutical Analysis(III rd edition)
By Dr.S.Ravishankar
www.amswer.com
www.wikipedia.com
.