1. GOKARAJU RANGARAJU COLLEGE OF
PHARMACY
Presented by :
Ch.Nikhila
170213884003
Dept. of Pharmaceutical
chemistry.
Under the guidance of
M/s. CEEMA MATHEW,
Asst.Prof.
1

2. CONTENTS :
 INTRODUCTION
 PRINCIPLE
 INSRUMENTATION
 ADVANTAGES&APPLICATIONS
 DERIVATISATIO METHODS
2

3. INTRODUCTION:
 CHROMATOGRAPHY:
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
like:
 High Performance Liquid Chromatography
 High Performance Thin Layer Chromatography
 Gas Chromatography
3

4. Gas 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]
Carrier gas
column
detector
Basic chromatographic arrangement
4

5. GSC not used because of limited no. of S.P
GSC principle is ADSORPTION
GLC principle is PARTITION
Chromatographic Separation
Mobile Phase – inert gas used as carrier.
Stationary Phase– liquid coated on a solid or a solid within a
column.
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
1.VOLATILITY:
2.THERMOSTABILITY:
5

6. HO-CH2-CH2-(O-CH2-CH2)n-OH
polyethylene glycol
6

7. How a Gas Chromatography Machine Works
 First, a vaporized sample is injected onto the chromatographic
column.
 Second, the sample moves through the column through the flow of
inert gas.
 Third, the components are recorded as a sequence of peaks as they
leave the column.
7

8. PRINCIPLE:
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
stationary phase.
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
partition coefficients.
8

9. THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid)
MOBILE PHASE
Sample in
Sample out
STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)
9

10. PRACTICAL REQUIREMENTS
Carrier gas
 Flow regulators & Flow meters
 Injection devices
 Columns
 Temperature control devices
 Detectors
 Recorders & Integrators

10

11. SCHEMATIC DIAGRAM
11

12. Hardware and Columns
12

13. INSTRUMENTATION:
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
sample mixture)
o Column: (fused silica with a thin coating of stationary phase on the
inner surface)
o Oven: Thermostat controlled forced air oven
o Detector:
o Data System: recorders & integrators
13

14. CARRIER GAS:
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
manner
 less risk of explosion or hazards.
 Cheap
Should not cause the risk of fire, Should give best
column performance
14

15. CHOICE OF CARRIER GAS:
Name of the
carrier gas
Hydrogen
Advantages



Helium



Nitrogen



Disadvantages
Cheap
Gives the most time efficient
separation
Still very efficient at high gas
velocities i.e.. 60 cm/ sec

Very inert, will not react with
analytes
Gives a very time efficient separation
Non flammable

Cheap
Very inert, will not react with
analytes
Non flammable




Can form an explosive mixture with
air
is a reductive gas
Expensive
A non-replenishable resource
Very slow velocity to achieve good
efficiency
Narrow range for maximum
efficiency
15

16. FLOW REFULATORS & METERS

Flow regulators are used to deliver the gas with uniform pressure or
flow rate

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
column
Flow rate depends on type of column
– Packed column: 29-150 mL/min
– Capillary column: 1 to 25 mL/min

16

17. FLOW REGULATORS:
To regulate pressure& control the gas flow through the separation
column.
They are two types :
1. Rotometer
2. soap bubble flow rate
ROTOMETER:
It is like a burette with a float held on to a
spring
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
17

18. Soap bubble meter




soap bubbles formed indicates the
flow rate.
Glass tube with a inlet tube at the
bottom.
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
&travel up.
Aqueous
solution of
soap or
detergent
18
Soap bubble flow meter

19. inlet tube
19

20. Injection Devices


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
microsyringe
20

21. 21

22. Micro syringe
22

23. COLUMNS
Packed column
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)
Capillary column
23

24. Types of open tubular column:
Solid support coated
with liquid phase
Liquid phase
Wall-coated Open
Tubular
(WCOT)
Support-coated Open
Tubular(porous layer
open tubular column)
(SCOT)
24

25. 1.Packed column:
2.Open tubular column
MADE UP OF Glass or
metals
columns are available in a
packed manner
Better resolution – efficient mass
transfer between gas and SP
Tubing – fused silica, glass,
copper, stainless steel
Long capillary tubing 30-90 M in
length
Uniform & narrow diameter of
0.025 - 0.075 cm
Disadvantage: more sample
cannot loaded
3.SCOT columns (Support
coated open tubular column
Improved version of 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
25

26. CHARACTERISTICS
Type of Column
FSOT
WCOT
SCOT
Packed
10-100
10-100
10-100
1-6
0.1-0.3, 0.53*
0.25-0.75
0.5
2-4
2000-4000
1000-4000
600-1200
500-1000
Sample size (ng)
10-75
10-1000
10-1000
10-106
Relative pressure
Low
Low
Low
High
Relative speed
Fast
Fast
Fast
Slow
Flexible
Yes
No
No
No
Chemical inertness
Best
Length (m)
ID (mm)
Efficiency (Plate/m)
Poor
26

27. Equilibration of the column

Column is attached to instrument & desired flow rate by flow
regulators
 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
injecting devices
Thermostatically controlled oven:
temperature maintenance in a column is highly essential for efficient
separation.
Two types of operations:
Isothermal program:Linear programming:- this method is efficient for separation of
complex mixtures.
27

28. The oven
Inside here
Column
28

29. Instrumentation - Oven
Temperature Control
• Isothermal
• Gradient
240
Temp (deg C)
200
160
120
80
40
0
0
10
20
30
40
50
60
Time (min)
29

30. DETECTORS

Heart of the apparatus
The requirements of an ideal detector are Applicability to wide range of samples.
 Rapidity.
 High sensitivity even small concentrations.

Linearity.



Response should be unaffected by temperature, flow rate…
Non destructive.
Simple & inexpensive.
Instrumentation - Detectors
• Thermal Conductivity (TCD)
• Electron Capture (ECD)
• Argon ionization detector ( AID)
• Flame Ionization (FID)
30

31. 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
the galvanometer.
• This deflection is recorded which is due to change in the thermal
conductivity.
31

32. Advantages
Linearity is good
Applicable to most compounds
Non destructive
Simple & inexpensive
Disadvantages
Low sensitivity
Affected by fluctuations in temperature and flow rate
Biological samples cannot be analyzed
32

33. 33

34. 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
Advantages:
•µg quantities of the solute can be detected
•Stable
•Responds to most of the organic compounds
•Linearity is excellent
Disadvantages: destroy the sample
34

35. Argon ionization detector
Depends on the excitation of argon atoms to a metastable state, by
using radioactive energy.
Argon→ irradiation Argon + e- →collision
MetastableArgon→
collision of sub. → Ionization →↑Current
ADVANTAGES
1.Responds to organic compounds
2.High sensitivity
DISADVANTAGES
1.Response is not absolute
2.Linearity is poor
3. Sensitivity is affected by water

35

36. 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.
Construction :
1) Radio active material
metal foil.
2) Anode and cathode
electrode.
3)Potential difference of
20V to100V.
36

37. 
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
electron affinity.
This detector is frequently used in the analysis of chlorinated
compounds

 e.g.
– pesticides, polychlorinated biphenyls
ADVANTAGE:
Highly sensitive
DISADVANTAGE:
Used only for compounds with electron affinity
37

38. RECORDERS & INTEGRATORS
Record the baseline and all the peaks obtained.
INTEGRATORS
Record the individual peaks with Retention time, height.
38

39. 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
derivative.
39

40. Post column derivatisation

This is done to Improve response shown by detector
 This is online technique where flow rate is neither stopped nor
changed.
 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,
ethers….
Techniques: 1. use more polar liquid S.P.
2. Increasing amount of liquid phase.
3.Pretreatment of solid support to remove active sites.
40

41. DERIVATISATION METHODS
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:
1.Silylation
2.Alkylation
3.Acylation
41

42. SILYLATION
OH
O
Cl
O
2,4-dichlorophenoxyacetic acid
(A cancer suspect agent).
Cl
(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:
R OH
+
Cl
Si
Si
R O
Me3
Me3
+
HCl
SiMe3
OH
O
Cl
Cl
O
+
Cl
Si
Me3
O
Cl
Cl
O
42

43. (d) Besides trimethylchlorosilane, a number of other silylation reagents
can also be used. These reagents have slightly different reactivity from
trimethylchlorosilane.
The byproduct of
BSTFA is highly
Volatile.
N, O-Bis(trimethylsilyl)acetamide
N,O-bis(Trimethylsilyl)trifluoroacetamide
BSA and BSTFA are highly stable TMS derivatives, with most organic
functional groups, under mild reaction conditions.
Me3Si
R OH
+
F3C
Si
O
N
SiMe3
R O
O
Me3
+
F3C
N
SiMe3
43

44. (e) Alkylation
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
RCOOMe
(f) Acylation
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.
44

45. ii. Trifluoroacetic anhydride (TFAA) is one common reagent used for
acylation.
NH
+
O
COCF3
N-CO-CF3 + HOCOCF
3
COCF3
methamphetamine
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
conditions.
H
Me
CF3
N
Byproduct is volatile
O
45

46. ADVANTAGES






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
46

47. Applications :
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
component
4, used for analysis of drugs & their metabolites.
47

48. REFERENCES :





Instrumental Methods Of Chemical Analysis By Gurdeep R
Chatwal &Sham K Anand pg no
Principles Of Instrumental Analysis By Skoog Holler Niemen
Page No.686
Willard merit, Dean settle, instrumental methods of analysis,
7th ed.
www.Chromatography-Online.com
www.Registech.com
48

49. 49