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Chromatography is a separation of mixture into individual
components by using a stationary phase and a mobile phase.
There are various advanced chromatographic techniques, widely
used for the estimation of multicomponent drugs in their formulations
High Performance Liquid Chromatography
High Performance Thin Layer Chromatography
In Gas chromatography, the components of a
vapouraised sample are fractionated as a consequence of a
partition between a mobile gaseous phase and a stationary
phase held in a column.
According to the nature of stationary phase, Gas
chromatography may be
(a). Gas solid chromatography [GSC]
(b). Gas liquid chromatography [GLC]
Basic chromatographic arrangement
GSC not used because of limited no. of S.P
GSC principle is ADSORPTION
GLC principle is PARTITION
Mobile Phase – inert gas used as carrier.
Stationary Phase– liquid coated on a solid or a solid within a
The mixture of compounds to be separated is converted into vapor
And mixed with gaseous M.P
Component more soluble in the S.P → travels slower
Component less soluble in the S.P → travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by G.C
How a Gas Chromatography Machine Works
First, a vaporized sample is injected onto the chromatographic
Second, the sample moves through the column through the flow of
Third, the components are recorded as a sequence of peaks as they
leave the column.
In GLC, the main principle is partition. Gas is used as
mobile phase, liquid which is coated on to a solid support is used as
The mixture of components to be separated is converted to vopour and
mixed with gaseous mobile phase.
The component which is more soluble in stationary phase travels
slower and eluted later.
The components which is less soluble in stationary phase travels faster
and eluted out first.
No two components has the same partition co-efficient for a fixed
combination of stationary phase , mobile phase.
Hence the components are separated according their
THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid)
(solid or heavy liquid coated onto a solid or support system)
Flow regulators & Flow meters
Temperature control devices
Recorders & Integrators
o Carrier Gas: He, Ar, N2, H2
o Flow regulators & flow meters:
o Injection Port: Rubber septum barrier (usually maintained at a higher
temperature than the boiling point of the least volatile component in the
o Column: (fused silica with a thin coating of stationary phase on the
o Oven: Thermostat controlled forced air oven
o Data System: recorders & integrators
Gas Supply Unit
Carrier gas should be:
Inert, high purity, easily available.
Low cost, due to large quantities are used
Allow the detector or respond in an adequate
less risk of explosion or hazards.
Should not cause the risk of fire, Should give best
CHOICE OF CARRIER GAS:
Name of the
Gives the most time efficient
Still very efficient at high gas
velocities i.e.. 60 cm/ sec
Very inert, will not react with
Gives a very time efficient separation
Very inert, will not react with
Can form an explosive mixture with
is a reductive gas
A non-replenishable resource
Very slow velocity to achieve good
Narrow range for maximum
FLOW REFULATORS & METERS
Flow regulators are used to deliver the gas with uniform pressure or
Flow rates of carrier gas:
– Linear flow rate (cm/s): u = L/tr
– Volumetric flow rate (mL/min): u (π r2)
L is length of column, tr is retention time, r is the internal radius of
Flow rate depends on type of column
– Packed column: 29-150 mL/min
– Capillary column: 1 to 25 mL/min
To regulate pressure& control the gas flow through the separation
They are two types :
2. soap bubble flow rate
It is like a burette with a float held on to a
placed before column inlet it has a glass
tube with a float held on a spring.
the level of the float is determined by
the flow rate of carrier gas
Soap bubble meter
soap bubbles formed indicates the
Glass tube with a inlet tube at the
Rubber bulb-----store soap solution
When the bulb is gently pressed of
soap solution is converted into a
bubble by the pressure of a carrier gas
Soap bubble flow meter
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
Micro syringes injection port
Gas tight syringe
Important part of GC
Made up of glass or stainless steel
Glass column- inert , highly fragile
COLUMNS can be classified
Depending on its use
1. Analytical column
1-1.5 meters length & 3-6 mm diameter
2. Preparative column
3-6 meters length, 6-9mm diameter
Columns in GC are two types
based on its nature:
1) packed column
2) capillary column
(open tubular column)
Types of open tubular column:
Solid support coated
with liquid phase
open tubular column)
2.Open tubular column
MADE UP OF Glass or
columns are available in a
Better resolution – efficient mass
transfer between gas and SP
Tubing – fused silica, glass,
copper, stainless steel
Long capillary tubing 30-90 M in
Uniform & narrow diameter of
0.025 - 0.075 cm
Disadvantage: more sample
3.SCOT columns (Support
coated open tubular column
Improved version of Capillary
columns, have small sample
Made by depositing a micron
size porous layer of supporting
material on the inner wall of the
Then coated with a thin film of
Type of Column
Sample size (ng)
Equilibration of the column
Column is attached to instrument & desired flow rate by flow
Set desired temperature.
Conditioning is achieved by passing carrier gas for 24 hours
Temperature Control Devices
Pre heaters: convert sample into its vapor form, present along with
Thermostatically controlled oven:
temperature maintenance in a column is highly essential for efficient
Two types of operations:
Isothermal program:Linear programming:- this method is efficient for separation of
Instrumentation - Oven
Temp (deg C)
Heart of the apparatus
The requirements of an ideal detector are Applicability to wide range of samples.
High sensitivity even small concentrations.
Response should be unaffected by temperature, flow rate…
Simple & inexpensive.
Instrumentation - Detectors
• Thermal Conductivity (TCD)
• Electron Capture (ECD)
• Argon ionization detector ( AID)
• Flame Ionization (FID)
Thermal Conductivity Detector
(T.C.D) or Katharometer:
• TCD is based upon changes in thermal conductivity of gas stream.
• It consists of two cells i.e. reference cell and sample cell.
• It is in the form of a wheat- stone bridge.
• When carrier gas is passing there is no deflection in the galvanometer.
• But when the column effluent is allowed to pass there is deflection in
• This deflection is recorded which is due to change in the thermal
Linearity is good
Applicable to most compounds
Simple & inexpensive
Affected by fluctuations in temperature and flow rate
Biological samples cannot be analyzed
Flame Ionization Detector
Organic compounds are readily pyrolysed when introduced into a
hydrogen-oxygen flame and produce ions in the process.
The ions can be collected at a charged electrode and the resulting
current measured by electrometer amplifier.
In FID the effluent gases are mixed with hydrogen and burned in
presence of oxygen
•µg quantities of the solute can be detected
•Responds to most of the organic compounds
•Linearity is excellent
Disadvantages: destroy the sample
Argon ionization detector
Depends on the excitation of argon atoms to a metastable state, by
using radioactive energy.
Argon→ irradiation Argon + e- →collision
collision of sub. → Ionization →↑Current
1.Responds to organic compounds
1.Response is not absolute
2.Linearity is poor
3. Sensitivity is affected by water
Electron Capture Detector:
ECD is based upon electron affinity of the molecule.
It is composed of a radio active source which emits electrons.
When the effluent from the column is allowed to flow through the
chamber the electron are absorbed and current is observed.
1) Radio active material
2) Anode and cathode
3)Potential difference of
If a compound is present that contains electronegative atoms,
those electrons are captured and negative ions are formed, and
rate of electron collection decreases
The detector selective for compounds with atoms of high
This detector is frequently used in the analysis of chlorinated
– pesticides, polychlorinated biphenyls
Used only for compounds with electron affinity
RECORDERS & INTEGRATORS
Record the baseline and all the peaks obtained.
Record the individual peaks with Retention time, height.
Derivatisation of sample
Treat sample to improve the process of separation by column or
detection by the detector
They are 2 types
Pre column derivatisation : this is done to improve some properties
of the sample for separation by column. By this method Components
are converted to volatile & thermo stable derivative.
in following Conditions - Pre column derivatisation is done
if Component is less volatile
if Compounds are thermo labile( heat sensitive)
to decrease tailing & to improve separation
Ex: sugars, COOH, alcohols , phenols are converted to less polar by
using( bis trymethyl silyl acetamide reagent )
They can also be converted to acetyl derivative or triflouro acetyl
Post column derivatisation
This is done to Improve response shown by detector
This is online technique where flow rate is neither stopped nor
The components may not be detected by detector unless
derivatisation is done.)
The components may be converted in such away that their ionization
or affinity towards electrons is increased.
Pretreatment of solid support
To overcome tailing .
Generally doing separation of non polar components like esters,
Techniques: 1. use more polar liquid S.P.
2. Increasing amount of liquid phase.
3.Pretreatment of solid support to remove active sites.
Derivatisation prior to GC is often desirable to
1.improve the thermal stability of compounds, particularly
compounds that contain polar functional groups.
2.Change the separation properties of compounds by the purposeful
adjustment of their volatility.
TYPES OF DERIVATISATION METHODS:
(A cancer suspect agent).
(a) This is the most common type of derivation techniques used in GC.
(b) It involves replacing an active hydrogen on the solute (i.e. R-OH,
RCOOH, R-NH2, etc.) with an alkylsilyl group (usually –SiMe3). The
result of this reaction is that the solute is converted into a less
polar, more volatile and more thermally stable form.
(c) The most common reagent used in silylation is trimethylchlorosilane
(TMS). Examples of its use are shown below:
(d) Besides trimethylchlorosilane, a number of other silylation reagents
can also be used. These reagents have slightly different reactivity from
The byproduct of
BSTFA is highly
BSA and BSTFA are highly stable TMS derivatives, with most organic
functional groups, under mild reaction conditions.
I. Alkylation involves the addition of alkyl group to some active
function group on the solute. A common example is esterification of a
carboxylic acid, forming a volatile methyl ester. This is commonly done
using borontrifluoride in methanol as the reagent.
RCOOH + BF3/MeOH
I. Acylation involves the conversion of a solute into an acylate
derivates. This is often used to improve the volatility of alcohols,
phenols, thiols and amine (e.g., -OH, -SH and -NH) containing
compounds. As is true for other GC derivations, acylation can also
be used to increase the response of a solute to a given detector (e.g.,
allowing the use of electron capture in solute’s detection by including
fluorine atoms in the derivitizing agent.
ii. Trifluoroacetic anhydride (TFAA) is one common reagent used for
N-CO-CF3 + HOCOCF
Drug-of-abuse confirmation testing by GC
iii. Another set of reagents used for solute with primary and secondary
amines, as well as hydroxyl and thiol groups are N-Methylbis[trifluoroacetamide] (MBTFA). The reaction is under mild nonacidic
Byproduct is volatile
Very high resolution power, complex mixtures can be resolved into its
components by this method.
Very high sensitivity with TCD, detect down to 100 ppm
It is a micro method, small sample size is required
Fast analysis is possible, gas as moving phase- rapid equilibrium
Relatively good precision & accuracy
Qualitative & quantitative analysis is possible
G.C is capable of separating, detecting & partially characterizing the
organic compounds , particularly when present in small quantities.
1, Qualitative analysis
Rt & RV are used for the identification & separation
2, Checking the purity of a compound
Compare the chromatogram of the std. & that of the sample
3, Quantitative analysis
It is necessary to measure the peak area or peak height of each
4, used for analysis of drugs & their metabolites.
Instrumental Methods Of Chemical Analysis By Gurdeep R
Chatwal &Sham K Anand pg no
Principles Of Instrumental Analysis By Skoog Holler Niemen
Willard merit, Dean settle, instrumental methods of analysis,