GC introduction and instrumentation part

1. GAS CHROMATOGRAPHY
(INSTRUMENTATION)
Presented by
NIVEDITHA G
1st sem Mpharm
Dept Of pharmaceutics
NARGUND COLLEGE OF PHARMACY

2. CONTENTS:
 Definition
 Classification
 Instrumentation
Carrier gas
Pressure and Flow regulators
Sample injection system
Columns
Detectors
Recorder
 Integrator

3. DEFINITIONS
1) Chromatography:- It refers to a physical method of
separation in which the components to be separated
are distributed between the two phases constituting a
stationary phase and the other being the mobile
phase.
2) Gas Chromatography:- It is a technique in which the
components of vaporized sample are separated on the
basis of their partition/adsorption between the mobile
phase and stationary phase.
Stationary phase may be liquid or solid held in
column.

4. CLASSIFICATION
1. Gas Solid Chromatography:-
In this the stationary
phase is a solid adsorptive material and the separation
is due to differences between adsorptive affinity of
solute particles between stationary and mobile phase.
2. Gas Liquid Chromatography:-
In this the stationary
phase is a non-volatile liquid which is coated on an
inert surface( solid support) and the separation is due
to distribution of solute between stationary and mobile
phase due to difference in partition coefficient.

5. INSTRUMENTATION

6. CARRIER GAS
The purpose of carrier gas is to transmit the
sample from the point of introduction through
the column to the detector.
The carrier gas acts as a mobile phase which
plays the key role in separation process.
They determine the efficiency of column, the
time of analyze and the sensitivity of a given
detector.

7. Factors considered while selecting the carrier gas:-
1. Availability in pure form & stable form before it enters
the chromatography.
2. Inertness.
3. Free from moisture.
4. Should give best column performance & be able to
minimize gaseous diffusion.
5. Inexpensive and safe in handling.

8. MAKE UP GAS
1. Most detector require 30-40 m2/min total gas flow rate
for best sensitivity and peak.
2. To supplement carrier gas flow, make up gas is added
at the column exit to obtain total gas flow of
30-40 m2/min into detector.

9. THE CARRIER GAS USED IN GC
9
Gas Application Comments
Helium General carrier gas or make
up gas
Expensive
Nitrogen General carrier gas or make
up gas
Cheap, not good for
capillary column as it
gives long run time
Oxygen Combustion gas for some
FID
Not normally used
Argon Carrier gas for TCD Determination of
helium
Hydrogen Carrier gas for capillary
column & combution gas
for FID
Cheap, explosive

10. Air Combustion gas
for FID
Cheap, readily
available
Argon/methane Make up &
packed column
carrier gas for
ECD
Better linearity &
selectivity than
N2 but poor
detection limit

11. PRESSURE AND FLOW REGULATORS
 High pressure is preferred as it decreases
the analysis time.
 The carrier gas flow is maintained by:
1. Needle valve
2. Pressure controller
3. Mass flow controllers

12. NEEDLE VALVE:-
It is the simplest method of control and operated manually.
Limitation:-
 It lacks the automotive control.
 Change in temperature which leads to change in
viscosity which gives rise to change in flow rate.

13. PRESSURE CONTROLLER
 This device is used to maintain the constant pressure
at the inlet of the column.
 The pressure is controlled by diaphragm, which is
attached to the orifice for controlling the pressure and
is adjusted through the knob.
 If the outlet pressure rises above the present value
then the orifice closes,
 If less, then it opens completely.
 For optimum pressure, it is kept in balance position.

15. MASS FLOW CONTROLLER
 These are complex and automatically
working devices.

16. MEASUREMENT OF FLOW RATE
 Rotameter:-
These are fixed in the carrier gas line
before the column inlet and outlet.
 Soap-bubble meter:-
The flow meter is connected at the
outlet of the column by the side arm and soap bubbles
are introduced into the gas stream by squeezing the
bulb at the bottom.
The bubbles move up the calibrated
tube and the time taken for a bubble travel between
two marking A and B is measured.

17. SAMPLE INJECTION SYSTEM

18. THREE MAIN METHODS OF INJECTION
I. Pull small volume of air into the syringe ahead of the
sample. This results in all of the liquid in the syringe being
injected with no dead volume remaining in the needle.
II. Trapping the sample between two ‘block’ of air in addition
to eliminating the needle dead volume, it also enables to
determine the exact injection volume by observing it
within the calibrated glass barrel before injection.
III. Aspirate a small volume of solvent into the syringe, the air,
sample and finally air. Thus the sample is entrapped
between two block of air while and residual sample
coating the wall of syringe a needle is flushed into the
instrument by additional plug of solvent.

19. Factors considered while selection of sample inj.
method:
i. Chemical nature & physical state of sample,
ii. The quantity of sample to be used,
iii. Type of analysis to be performed.

20. SYSTEMS USED FOR SAMPLE INJECTION
a) Micro volume syringe
b) Micropipettes
a) Micro volume syringe:-
 It is the most popular method for introduction of liquid
samples, as it does not affects the gas flow rate.
 This method depends on the introduction of sample
with the micro syringe through a self sealing rubber
septum.
 The sample injection point is designed in such a way
that, the sample is swept quickly onto the column
quickly, as it is injected.

21.  The temperature of point is kept higher than column, to
aid volatilization but there is possibility of
condensation of sample as it reaches the cooler
column.
 This is avoided by keeping the temperature of the
column above the ‘decrease point’ of the sample.
 If the sample introduced in the column has high
partition coefficient, then it dissolves more in liquid
stationary phase at the inlet than gas phase, the ideal
technique is by applying sample directly to the column
packing and this is known as on-column injection and
this is preferred when the samples of restricted boiling
range are used.

22. b) Micropipettes:-
 A special system, where it requires very small sample
volumes and the column capacity is very less, i.e. in
the range of 10-3 µl, split stream injectors are used.
 In those, sample is introduced into port and
evaporated and either the sample vapour or it’s
homogeneous mixture with the carrier gas is split into
two highly unequal parts and the sample volumes are
introduced.

23. COLUMNS
 Most important part of GC, in which separation of
sample take place. The column is a tube which holds the
stationary phase in finely dispersed form & providing a
large surface area for mobile phase. The size of column
generally 4mm in diameter & length between 120cm to
150cm.
 Usually columns are made up of steel, glass, copper,
aluminium & may be straight, bent in U shape or coiled.
 Ni & Teflon can also be used.

24. SUPPORT MEDIUM
 The purpose of solid support is to provide large & inert surface
area for holding the liquid. The liquid phase in thin & uniform
film. It must be poor adsorbent & must finely divided porous
substance having large surface area .
 It should be…..
1. Chemically inert
2. Heat stable
3. Sufficient mechanical strength to prevent fractionating with
normal handling & be uniformly wetted by the liquid phase.
 Most common supports available from diatomaceous earth
namely firebrick & kieselgurh.
 Glass beads, porous polymers, unglazed tiles, sand fluorinated
resins are also used as support medium.

25. LIQUID PHASE
There is no well accepted method for selecting the best
liquid phase for a particular separation. The right
selection is based on mainly experience and/or trial &
error method.
Criteria for selecting Liquid phase :-
 Non-volatile
 Compatibility
 low viscosity
 chemically inert.

26. CLASSIFICATION OF LIQUID
 Very polar :- Glycols, glycerols, amino alcohols,
hydroxy acids, polyphenols, dibasic acids etc.
 Polar :- Alcohols, fatty acids, phenols, primary &
secondary amines.
 Intermediate :- ethers, ketones, aldehydes, esters,
tertiary amines.
 Less polar :- Chloroform, dichloro methane,
1,2-dichloro ethane, aromatic hydrocarbons etc.
 Islon-polar :- saturated hydrocarbons,
halohydrocarbons.

27. TYPES OF COLUMN
 Based on their construction columns are divided into
two major groups.
 Packed column
 Open tubular column :-
a) support coated ( SCOT)
b) Wall coated ( WCOT)
 Capillary Columns give much better resolution but their
sample capacity is much lower than the packed columns
(about 1/10 or less).

28. Liquid film
(Sationary Phase)
Support
Solid Staionary Phase
Micro-Packed PLOT SCOT WCOT

29. 1) Packed column :-
These are prepared by packing metal or glass tubing with
granular stationary phase. For GSC the columns are packed
with adsorbants or porous polymers, while in GLC columns
are packed by coating the liquid phase over an inert solid
support.
2) Open tubular column :-
These columns are called as Capillary or Golay columns
& are prepared from long capillary tubing having uniform
& narrow internal diameter ( 0.01 – 0.03 inch). The inside
wall of capillary tubing is coated by liquid phase in the
form of thin & uniform film. The carrier gas faces least
resistance as there is no packing in the column. The sample
capacity of this column is very low.

30. a) Support Coated column :-
SCOT columns are made by depositing a micron size
porous layer of support material on the inside wall of a
capillary column & then coating with a thin film of
liquid phase. These columns are having higher sample
capacity & yield better resolution.
b) Wall coated Column (WCOT) :-
In which the stationary phase is coated directly on to
the inner walls of tubing which is liquid.

31. EQUILIBRATION OF THE COLUMN
 Before introduction of the sample, complete
equilibration or conditioning must be obtained.
 Column packed with stationary phase is attached to the
instrument & desired flow rate of carrier gas is adjusted
by flow regulators.
 The column temperature is set at desired temperature
but below the upper temperature limit of the liquid
phase used.
 Conditioning is achieved by passing carrier gas for at
least 6 hours or generally for 24 hours. A properly
conditioned column will show zero base-line on the
recorder.

32. CONTROL OF COLUMN TEMPERATURE
 Column are usually operated above room temperature
except for gaseous sample.
 A temperature programming is now used where the
column is not kept at constant temperature but it is
subjected to controlled rise which reduces the retention
times of the less volatile samples to be analyzed more
rapidly.
 For this, various methods have been used i.e. vapour
jackets, electrically heated air baths or metal blocks, etc.

33. DETECTORS
 After the resolution of solute, each vaporized component
emerges in turn from column & is carried into detector
mixed with carrier gas.
 The detector receives the impulse from the elute of the
column in the form of solute.
 Vapour is sensed by the detector. It converts this impulse
into an electrical signal proportional to the concentration
of the solute in the carrier gas.
 This signal is amplified & recorded as a peak on the
chromatogram, thus the detectors are considered as brain
centre of the instrument.

34. CRITERIA FOR SELECTING THE DETECTOR
 It should be stable.
 It should give rapid & linear response to change in solute
vapour as the column effluent passes through the
detectors.
 It should have concentration reproducibility & sensitivity
to a wide range of solute vapours.
 High sensitivity to even low concentration.
 Simple, easy to operate and inexpensive.
 Response should not be changed with temp, flow rate &
carrier gas used.

35. THERMAL CONDUCTIVITY DETECTOR

36.  This is also known as Katharometer.
 The TCD is based on the fact that rate of loss of heat
from the body depends upon thermal conductivity of the
surrounding gas is a function of its composition.
 Thus the rate of loss of heat is related to the composition
of the surrounding gas.
 Hydrogen & helium posses higher thermal conductivity,
hence are considered as best carrier gas for katharometer.
 Hydrogen is inflammable.
 Helium is expensive.
 Both gases give good responses.

37. Advantages:
Applicable to most compounds which are not detected by
FID.
Linearity is good.
TCD is conc. dependent detector & hence it does not
destroy the sample, hence it finds use in preparative scale.
Simple, easy to maintain and inexpensive.
Disadvantages:
Sensitivity is low when compared to FID.
Gets affected by fluctuations & flow rate.
The response is only relative & not absolute.
Biological samples cannot be analyzed.

38. FLAME IONIZATION DETECTOR

39. When the sample component elutes & passes through
the flame, its molecules are ionized & the resulting
ionization current after amplification is fed to the suitable
recorder.
A FID is sensitive to almost all the organic compounds
but insensitive to noble gases, oxygen, nitrogen, CO,CO2,
water, nitrogen oxide, H2S, SO2.
Advantages:
The background current is small.
Noise level is low.
Response is greatest for hydrocarbons.
Linearity is excellent.
Disadvantages:
FID is mass dependent detector, hence it destroys the sample.

40. ELECTRON CAPTURE DETECTOR

41. Based on electron affinity of different substances.
examples: chlorinated compounds, unsaturated
compounds.
In this, when a component having affinity for electrons
elutes out of the column & enters the detector, it absorbs
some electrons causing drop in standing current.
This temp is traced by the recorder as a peak.
Parts of instrumentation:
1) Radio active material metal foil.
2) Anode & cathode electrode.
3) Potential difference of 20 v to 100 v.

42. Advantages:
ECD – Halogens, Peroxides, Quinones etc. i.e.,
compounds which have electronegative functional groups.
Nanogram quantities can be detected.
Disadvantages:
Linearity is poor.
ECD – only for molecules which have electron affinity.
ECD – Insensitive towards functional groups like
amines, alcohols & hydrocarbons.

43. THERMIONIC DETECTOR

44. This detector is sensitive towards organic compounds
containing nitrogen & phosphorus.
Response to phosphorus is 10 times more than nitrogen.
As it is designed for nitrogen & phosphorus it is also
called as “NITROGEN PHOSPHORUS DETECTOR”
(NPD).
A solid alkali metal salt is placed just above the flame.
(Rubidium Silicate) (RS)
Hydrogen –Helium gas from the column passes through
flame tip assembly + excess air that gives RS bead.
The heated alkali bead emits electrons by thermionic
emission which is collected at the anode & thus produce
an ion current.

45. When a solute containing nitrogen or phosphorus is
eluted, the partially combusted nitrogen & phosphorus
materials are adsorbed on the surface of the bead.
This adsorbed material reduces the work function of the
surface & as consequence, the emission of electrons is
increased which raises the anode current, which is
recorded.
The sensitivity of the NPD is about 10-12 g/ml for
phosphorus & 10-11 g/ml for nitrogen.
Advantages:
This detector can be widely used in the analysis of
phosphorus containing pesticides.

46. RECORDER:
 The signal from a gas chromatograph is recorded as a
function of time by a potentiometric recorder.
Integrator:
 An integrator is employed for simultaneous
measurement of areas under chromatographic peaks by
mechanical or electronic means.