gas chromatography
Introduction :-
Gas Chromatography is an analytical technique use for separation
of thermally stable and volatile substance.
In Gas Chromatography the mobile phase is gas and the stationary
phase is solid or liquid.
Principle :-
The principle involved in gas Chromatography is adsorption or
partition which is dependent on the stationary phase used.
If the stationary phase used is liquid it is called as gas
liquid Chromatography (GLC).
In GLC mostly kiesulghur or diatomaceous earth.
If the stationary phase is solid it is called as gas solid
Chromatography ( GSC).
In GSC mostly granular silica, alumina are used as stationary
phase.
The components which is having lesser affinity to ward s.p
travel faster and of greater affinity is travel slowly.
Theory :-
In this technique the sample is to be vapourised and is carried
a long a prepared column of suitable temperature by means of a carrier gas
which act as mobile phase.
When the vapour of the sample is passed through the column the
components are separated by adsorption or partition basing on the stationary
phase used.
The sample to be analysed by G.C. should be stable when
vapourised and when passed through prepored or packed column in order to avoid
decomposition of components and complex chromatogram if unstable or non
volatile components are to be separated by G.C by preparing their derivatives.
Example :- Trimethyl silyl derivatives of carbohydrates.
Due to limited availability of solid adsorbent gas solid
Chromatography is not widely used.
In gas liquid Chromatography the stationary liquid phase is
located on a solid support or the wall of capillary tube.
The stationary phase used in GLC should have low vapour pressure
at the column temperature and almost non volatile.
INSTRUMANTION
OF GAS CHROMATOGRAPHY :-
- carrier
gas :- Transmits
the sample form the point of introduction through the column to the
detector. examples of carrier gas is hydrogen, helium, or nitrogen flow
rate is depends upon column i.d.→2mm/min, i.d.→15-20mm/min
- purification
gas :- two stage of
purification is recommended i.e. 1. a sintered metal filter to remove
particulate matter and 2. a drying cartridge containing a desiccant to
remove water vapor from the CG cartidge also contains a molecular sieve
which remove small and trace molecules such as water and low MW
hydrocarbons.
- sample injection device :- it will be receive sample vaporize, it instantly
and deliver the sample to the head of the column in a sharp concentrated
band with minimum fronting or tailing. usually sample in liquid state is
introduced by microsyring/hypodermic syring(sample size 1 to 50 μl). the
gaseous sample can be introduced in the same way(0.5 to 5 ml) with the
help of microsyringe or loop injector.
- columns
:- important of GC it is
made up of glass or stainless steel.
columns can be
classfied
- depending
on its use :-
- analytical column :-
1-1.5 meters length and 3-6 mm d.m.
- preparative column :- 3-6
meters length and 6-9 mm d.m.
- depending
on its nature :-
- packed column :- column are
available in a packed manner .
s.p
for GLC :- polyethylene glycol,
esters, anides
2. open tubular or
capillary column or golay column :-
- long capillary tubing 30-90 m
in length
- uniform & narrow d.m of
0.025-0.075 c.m
- made up of stainless steel
& from of a coil
3. SCOT column
(support coated open tubular column)
- improved version of golay / capillary
columns have small sample capacity
- made by depositing a micron
size porous layer of supporting material on the inner wall of the
capillary column
- Then coated with a thin film of
liquid phase.
5. DETECTOR
:- decides
sensitivity of GC
- Working
principal :- difference
in property of CG alone and CG with solute is measured.
- TCD(thermal
conductivity detector /hot – wire detector):-
- based on a principal that
thermal conductivity will vary with the composition of GC.
- the actual detecting elements
may be either an electrically heated wire filament or thermister. they
both function by detecting change in electrical resistance which can be
ultimately measured by WB circuit.
- temp. change→ change in
resistance→ measured by wheatstone bridge
- CG should be of high thermal
conductivity: H/He
- TC is expressed as caloria/ cm
- used for measurement of trace
amount of water
- FID
(Flame ionization detector):-
• Burning of compounds
in a flame will lead to ion poduction which are collected at opposite
electrodes → current produced is directly proportional to concentration of
solute.
• Burning H2 is
provided as fuel which is supported by air in the form of pure oxygen.
• C.G + OXYGEN →
few electron
• C.G +
sample + oxygen → more ions → more electron
• About 90% of drug GC
analysis are analysis are analyzed by FID.
6. FLOW METER
:- Measure flow of
CG limit is 20-60 ml\min. Soap bubble flow meter is most commonly used.
7. MODIFICATION IN
GC :-
DERIVATIZATION
:- To convert polar to
non polar , to increase the thermal stability , to increase the ability of
detection, Aid in identification.
Temperature
programming :- when the mixture
contain compounds of widely differing volatility use of chromatograph in iso
thermal condition gives unsatisfactory results.
- High volatility compound are
eluted first , where as the rest need more time for elution leading to
band broadning.
- If separation is carried out at
high temperature , the lower B.P compound will be co-eluted &
over lapping peaks would be obtained.
- to avoid these problem
temperature programming is done where increase in temp is regulated
systematically.
APPLICATION
OF GC :-
- Quality control and analysis of
drug products like anti biotics (penicilin), anti viral (amantidine),
general anesthetics, etc.
- ASSAY of drugs – purity of a
compound can be determined for drugs like :-
- ATROPINE SULPHATE
- CLOVE OIL
- STEARIC ACID
- in determining the levels of
metabolites in body fluids like plasma, urine, serum etc.
3. miscellaneous :-
- analysis of food likes
carbohydrates, proteins,lipdis , vitamins, steroids ,and trace elements
- polluent like formaldehyde,
carbon monoxide , benzene , DDT etc.