Introduction, Instrumentation, Injection System, Detector, Application, Advantages, Disadvantages and Programmed temp. gas chromatography.

1. Gas Chromatography
Prepared by- Mr. Ramesh Bhusal
(Asst. Prof. Pharmaceutical Chemistry Dept.)

2. In gas chromatography gas is the mobile phase and solid or liquid is the
stationary phase.
Two major types:
Gas-solid chromatography:
Here, the mobile phase is a gas while the stationary phase is a solid.
GSC principle is ADSORPTION
Gas-liquid chromatography:
The mobile phase is a gas while the stationary phase is a liquid retained
on the surface as an inert solid by adsorption or chemical bonding.
GLC principle is PARTITION

3. Criteria for compounds to be analysed by
GC
 Volatality
 Thermostability

4. How 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.

5. Carrier gas supply
 It should meet the following criteria:
 Should be chemically inert
 Should be cheap and readily available
 Should be of high quality and not cause any fire accidents
 Should give best possible results
 Should be suitable for the sample to be analyzed and for the detector
Example. Helium, Argon, Nitrogen, Hydrogen.
All these gasses is supplied in pressurise metal cylinder along with other
accessories like Pressure regulator, pressure gauge, flow meter and
molecular sieves.

6. Soap Bubble Flow Meter

7. Sample Injection System
In order to get a peak which is sharp enough and provide the accuracy in
the quantification a sample of suitable size should be introduce as a plug
of vapour.
Sampling unit or injection port is attached to the column head.
Since the sample should be in vaporized state, the injection port is
provided with an oven that helps to maintain its temperature at about 20-
50 0 C above the boiling point of the sample.
Gaseous samples may be introduced by use of a gas tight hypodermic
needle of 0.5-10 ml capacity.
For Liquid samples , micro syringes of 0.1-100μL capacity may be used.

8. Following sample injection system is used to place sample onto
column.
 Split injection
 Split less injection
 Direct on column injection
 Pyrolysis

9. Split injection
 0.1 to 1 μL of sample is injected
 1 to 10 % is allowed to pass through column rest is vented to
atmosphere via split vent
 Works if sample concentration is more than 0.1 % v/v

10. Split less injection
 Used for less concentrated material (ppm)
eg. Trace analysis
 Split vent is closed

11. Direct on column injection
 Sample is directly introduce in column
Problems of Direct on column injection
 Total mass of material that a 0.25 mm column can handle without
overloading
 Syringe needle must be smaller than the internal diameter of column
(0.25 μm)
How to overcome problem
 Special syringe are available which can deliver very small volume
 Needle is short length of quartz capillary tube of small diameter
 Can not used normal rubber septum instead of that special pneumatic
injection port have been developed

12. Pyrolysis
 Compound of high molecular mass there is a major problem of
involatility.
 This difficulty is overcome by breaking large molecule into small
volatile fragment and then analyse called PGC.
 Resulting chromatogram is called pyrogram.
 Used for qualitative and quantitative analysis.
PGC is classified into two types
1. Static mode (furnace) reactor
Used heat for pyrolysis
2. Dynamic (filament) reactor
Used direct current for pyrolysis

13. Head Space Analysis

14.  Headspace sampling is essentially a separation technique in which
volatile material may be extracted from a heavier sample matrix and
injected into a gas chromatograph for analysis.
Why headspace analysis?
Sample like blood, plastic and cosmetic contains high molecular weight
non volatile material that can remain in the GC system. Many laboratory
analyst use extensive sample preparation technique to extract and
concentrate the compound of interest from this unwanted volatile
material. This technique are time consuming and costly.
This can be overcome by using head space analysis sampling technique.
Procedure.....
Application.....

15. Column
Heart of GC system where actual separation takes place.
Column
Packed Column Capillary Column
Open Tubular Column
WCOT
SCOT
PLOT

16. Packed Column
• Made up of- Glass, Stainless steel, Copper, Aluminium
• Internal Diameter- 1-9 mm
• Length- 2-20 m
• Offer resistance to the flow of mobile phase
• Pressure drop across two ends of packed column
Ideal solid support characteristic
• Small spherical, uniform particle size
• Inert
• Thermally stable
• Good mechanical strength
• Rapidly wetted by liquid phase
eg. Firebick, Kieselguhr

17. Capillary column/ Open tubular column
• Made up of- Glass, Stainless steel, Copper, Aluminium
• Internal Diameter- 0.3-0.5 mm
• Length- 10-100 m
• Offer negligible resistance to the flow of mobile phase
• Pressure drop across two ends of capillary column is negligible
The loading capacity of capillary column can be increased by coating the
inner wall of the column with a porous material such as graphite, metal
oxide or silicate. It provide increased surface area.

18. In capillary column the stationary phase is coated on the inner wall of the
tube on that basis it is classified as follows.
 Wall Coated Open Tubular (WCOT)
 Support Coated Open Tubular (SCOT)
 Porous Layer Open Tubular (PLOT)

19. Comparison between packed and capillary column

20. Rule of thumb for column selection
 Like separate like
 Long column, long elution but high resolution
N= L/2dp
 Small diameter high efficiency
 Film thickness

21. Column Oven
The column is enclosed in a thermostatically controlled oven so that its
temperature held constant.
 Operating temperature range from ambient to over 400 oC.
 For isothermal operation it kept constant.

22. Detector
 Consider as a brain of instrument.
 Connected to outlet of the column.
 Detector receives the impulse from the elute of the column.
 It convert these impulse into an electrical signal.
 The signal is amplified and recorded as a peak on chromatograph.
Examples...
1) Thermal Conductivity Detector (TCD)
2) Flame Ionisation Detector (FID)
3) Electron Capture Detector (ECD)

23. Properties of an ideal detector
 Sensitive
 Operate at high T (0-400 °C)
 Stable and reproducible
 Linear response
 Fast response
 Simple (reliable)
 Non-destructive
 Uniform response to all analytes

24. Thermal Conductivity Detector (TCD)
Also known as Katharometer or Hot wire detector.
Principle- Rate of loss of heat from the body depends upon thermal
conductivity of surrounding gas and thermal conductivity of surrounding
gas is a function of its composition.

26.  In the detector two pairs of matched filament are arranged in a
Wheatstone bridge circuit.
 One pair of filament(Reference) are surrounded by carrier gas and
another pair (Sample) are surrounded by effluent from column.
 When pure carrier gas passed over both the reference and sample
filament the bridge is balanced.
 When a vapour of sample emerge from the column the rate of cooling
of sample filament changes and bridge become unbalanced.
 The imbalance is a measure of the concentration of sample vapour in
carrier gas.
 Out of balance signal fed to a recorder, producing the chromatogram.

27. Advantages of Katharometer
 Linearity is good
 Applicable to most compounds
 Non destructive
 Simple & inexpensive
Disadvantages
 Low sensitivity
 Affected by fluctuations in temperature and flow rate

28. Flame Ionisation Detector (FID)

29.  This employs hydrogen flame that is maintained in a small cylindrical
jet made up of platinum or quartz.
 Effluent from the column with helium or nitrogen as carrier gas are
fed into the hydrogen flame, gets ignited and undergoes pyrolysis to
produce ions.
 An electrode system picks this ionisation current.
 The current produced in doing so is amplified and fed to an
appropriate recorder.
 When only carrier gas passes through the flame there is no current or
very small current, recorded as a steady base line.

30. Advantages
 Selective
 Sensitive
Disadvantages
 Non linear response
 Flame ionization detectors cannot detect inorganic substances

31. Electron Capture Detector (ECD)

32.  A foil made up of a radioactive metal like Ni 63 (β-emitter) is placed
inside a Teflon coated cell which also contains a cathode and an
anode.
 In the absence of organic species, the produced electrons migrate
towards positive electrode and produce a certain constant standing
current.
 When a sample/eluent is present it captures the electrons, elutes from
column, there is a drop in this constant current.
 This loss of current is traced by the recorder as a peak.

33. Advantages
 Selective
 Sensitive
Disadvantages
 Non linear response

34. Application of GC
 Qualitative Analysis
 Quantitative Analysis
 Elemental Analysis
 Analysis of pharmaceutical product and drug

35. GC Advantages
 This technique has strong power for the separation of even complex
mixture that can be resolved into its constituents.
 The method is highly sensitive
 Small sample is required for analysis.
 It has good precision and accuracy.
 The operation is completed in a very short interval of time.
 Used for qualitative as well as quantitative analysis.
 Automated system

36. GC Disadvantages
 Volatilization is required for the analysis and there may be a chance
for the degradation of the sample.
 It cannot be used for analysis of biological sample because of the high
temperature of the column.

37. Programmed temperature Gas
Chromatography
 In ordinary gas chromatography separation of mixture containing
components of widely different boiling point under constant
temperature column condition is quite unsatisfactory.
 If the column temperature which adequately separates the low boiling
(more Volatile) constituents is selected then the higher boiling
components takes more time to elute out therefore will appear as a
broad peaks.
 These problem can be solved by PTGC.
 In PTGC a lower initial temperature is used and early peaks are well
resolved. As the temperature increases each higher boiling component
is pused out.
 Higher boiling component are eluted earlier and as a sharp peaks
similar to earlier peaks.
 PTGC required less analysis time than isothermal operation.

38. Different method to increase the temperature of
column during chromatographic elution process.

39. Thank You...