1. Dnyanasadhana College, Thane.
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Department of Chemistry T.Y.B.Sc. Analytical Chemistry Paper-IV Sem-VI
GAS CHROMATOGRAPHY
Dr.Bhagure G.R.

2. GAS CHROMATOGRAPHY ADSORPTION PARTITION GAS SOLID CHROMATOGRAPHY
SOLID + GAS
GAS LIQUID
LIQUID + GAS
ADSORPTION
PARTITION

3. THEORY
GAS
CHROMATOGRAPHY
INSTRUMENTATIONS
APPLICATIONS

4. Partition Chromatography Gas Liquid Chromatography
L+L
Paper Chromatography
Gas Liquid Chromatography
L+ Gas
HPLC
L+ L

5. Principles of GC Sample A,B,C GSC Adsorption GLC Partition A
Most Volatile C
Least
Volatile B
Less Volatile
Separate
First
Separate
at Last
Separate after A

6. PRINCIPLE;-
Gas chromatography consists of gas as mobile phase and stationary phase may be solid or liquid. The time require for the separation of component is decided by large no. of factor, but it primarily dependant on extent of adsorption of solute In GSC or its partition in liquid phase and gas mobile phase in GLC. If the solute shows more affinity for solid surface or liquid stationary phase it will take more time to move over the entire length of column i.e. it will take more time for separation and vice –versa.
Consider a small length of column, sample contains three components A, B, and C is injected from sample injection port. It will be carried by mobile phase in the column. In the column the sample is get vaporized. The most volatile component will separate out first where as least volatile component will separate out later on.

7. The distribution coefficient shows the distribution of the molecules of the solute in two phases.
Concentration of solute in the stationary phase Cs
K= = CM
Concentration of solute in the stationary phase
A smaller value of K means that concentration of solute in mobile phase is more; it will require less time to come out from the column. Other hand a larger value of K means that concentration of solute in stationary phase is more; it will require more time to come out from the column. The time require to the elute the solute component from the column is called as Retention time, it’s a characteristic of every species .Qualitative analysis can be performed using retention time. Quantitative analysis can be performed from the area of the peak is calculated from the peak

8. Retention Time------------ Retention Time------------
A: Most volatile
B: Less Volatile
C: Very less Volatile
Detector Response B A C
Base line
Retention Time 
Retention Time 

9. In the operation of a gas chromatograph the, solutes in a mixture are completely vaporized in the injection port and they are moved through the column by a carrier gas under pressure. It is in the column where separation takes place. From the column, the separated solutes pass through a detector where they are sensed generating an electronic signal. The signal is then amplified and normally displayed on a strip chart recorder. The trace plotted on the recorder is called a "Chromatogram". It is a plot of the detector response in minivolts as a function of time. Usually, time is the abscissa and minivolts the ordinate. From the chromatogram, several general observations can be made.
Under a given set of experimental conditions, each peak has a characteristic retention time (tR) and the retention volume (VR) that are useful in qualitative analysis of solutes.

10. Retention Time: (tR) =------------------------------- = -----------
a) Retention Time: (tR)
It is used for qualitative analysis. It is defined as the time between the point of injection of sample and appearance of solute peak at the detector. OR
The time require to the elute the solute component from the column
Length of column Packing L
Retention Time: (tR) = =
Velocity of the solute Rs

11. Retention Volume: (VR):
It is used for qualitative analysis. Volume of mobile phase requires to elutes the solute component from the column is called as Retention volume.
Retention time and retention volume are related by equation,
VR = tRF
Where F is the flow rate of mobile phase
Length of column Packing L
Retention Volume: (VR) = = Velocity of the solute RS

12. tR = Retention time of the substance .
C) Relative Retention: It is defined as the ratio of the retention time or retention volume for the substance, after correction for tM and VM to the corrected retention time or retention volume of a reference compound.
tR – tM VR – VM
O< = =
tRef– tM VRef – VM
tR = Retention time of the substance .
tM = Retention time of the mobile phase
tRef = Retention time of the reference compound.
VR = Retention volume of the substance.
VM = Retention volume of the mobile phase
VRef = Retention volume of the reference compound.

13. d) Height Equivalent to Theoretical Plate (HETP):
Efficiency of column depends upon no. of theoretical plates that column is supposed to made of. HETP is length of column corresponding to a single theoretical plate. L
HETP = Where L= length of column. n
n= number of theoretical plates
** An efficient column is one for which ‘n’ is large or ‘H; is small.

14. e) Peak resolution :
Resolution is a measure of the separation between adjacent peaks in chromatogram
*** As the difference between the retention time of the peaks increases, the separation increases i.e. Resolution is directly proportional to the difference in retention time of the peaks.
2[ (tR)2--(tR)1] Where (tR)1, (tR)2 are retention times
R = of two peaks & W1+W2 are width
W1+W of two peaks.

15. GAS CHROMATOGRAPHIC INSTRUMENT
Pressure regulator
and flow control
Sample Injection
port
Detector * * *
* *
Recorder
carrier gas,
column
Column oven
Most Volatile
Less Volatile

16. Working Of Gas Chromatography:
In the operation of a gas chromatograph the, solutes in a mixture are completely vaporized in the injection port and they are moved through the column by a carrier gas under pressure. It is in the column where separation takes place. From the column, the separated solutes pass through a detector where they are sensed generating an electronic signal. The signal is then amplified and normally displayed on a strip chart recorder. The trace plotted on the recorder is called a "Chromatogram". It is a plot of the detector response in minivolts as a function of time. Usually, time is the abscissa and minivolts the ordinate. From the chromatogram, several general observations can be made.
Under a given set of experimental conditions, each peak has a characteristic retention time (tR) and the retention volume (VR) that are useful in qualitative analysis of solutes.

17. Approximate Limit of Detection (gs-1) Approximate Linear Range
Properties of Selected Gas Chromatography Detectors
Type
Approximate Limit of Detection (gs-1)
Approximate Linear Range
Comments
Thermal conductivity (TCD)
Universal detector -measures changes in heat conduction
Flame ionization (FID)
10-12
Universal detector -measures ion currents from pyrolysis
Electron capture (EC or ECD)
10-14
Selective detector for compounds containing atoms with high electron affinities
Flame photometric (FPD)
10-13
102
Selective detector for compounds containing S,P
Nitrogen-phosphorus
Selective for N,P containing compounds
Photoionisation (PID)
105
Universal (some selectivity due to identity of gas in lamp)
Hall Detector
10-11
Specific detector for compounds which contain halogen, S, or N
Mass spectrometer (MS)
variable, depends on MS
Universal
Fourier-transform infrared (FTIR)
10-10
Polar molecules

18. - + FID Collector Flame (-) CATHODE (+) Electrical Igniter ANODE Air
COLUMN
EFFLUENT H2
FLAME IONISATION DETECTOR

19. The principle of FID detector is, organic compound when reaches to flame produces ionic species that conduct electricity through flame. Hydrogen is as carrier gas in this detector.
In FID the eluate coming from the column is combined with hydrogen (Fuel) and air to form combustible mixture. This mixture forms a flame which provides sufficient energy for ionization.
The gaseous cations form in flame are attracted to negative electrode and repelled by positive electrode. Upon striking the collector electrode, the positive ion causes a current to flow in an external circuit. The current flow is proportional to the concentration of ionisable sample component.

20. Limitations :
1) FID responds to only ionisable substances.
2) It does not respond to inorganic compounds containing Nitrogen gas, oxygen gas and carbon dioxide gas.
3) It destroys the sample entered in to the flame.

21. THERMAL CONDUCTIVITY DETECTOR
Leeds to Wheatstone bridge
Carrier Gas In
Heated
Metal
Box
Column
Effluent In R S
Carrier Gas Out
Column
Effluent out
THERMAL CONDUCTIVITY DETECTOR

22. The thermal conductivity detector is based on the difference between thermal conductivities of the pure gas and carrier gas containing sample.
TCD consist of two identical brass cells fitted with platinum or tungsten wires. These resistance wires consist of reference and sensing elements which forms two arms of Wheatstone bridge. Both these wires are heated by an electric current. When pure carrier gas flows through both the cells, the temperature and hence resistance of both the filaments are in Wheatstone bridge is same which shows balanced circuit.

23. When column effluent allowed to flow through one cell and pure gas through the other cell, the resistance of both wire changes due to unequal cooling which results in increase in current.
This current is directly proportional to the quantity of solute present in the sample.
Advantages of TCD:
It response to organic and inorganic substances.
Its non destructive detector, the solute after separation can be collected.
It’s simple and gives large linear dynamic range

24. The basic principle of electron capture detector is based on electron absorption by compounds having an affinity for free electrons. It responds to compounds having an electronegative element or functional group. Methane gas is used in this detector because it easily undergoes ionization.
In ECD Ni63 foil is used as source of beta rays. In presence of beta rays carrier gas undergoes ionization. This forms positive carrier gas ions and electrons.
The electrons emitted during ionization are captured by positive collector electrode.
When a sample component enters the detector, electrons emitted by carrier gas are captured by the component. The NET result is removal of electron from the system and decrease in standing current. The decrease current is recorded as negative peak on the recorder.

25. Electron Capture Detector
(-)
Column Effluent
Collector Electrode
Beta Source
Ni63 foil is used as source of beta rays
(+)

26. Advantages Of ECD
It responds to compounds that capture the electrons.
Organic compound containing electronegative group’s ex. Nitro groups, phosphorous, oxygen and halogen.
ECD is very good detector for insecticides, pesticides, polychlorinated biphenyls.
It is non destructive in nature.

27. Applications of Gas Liquid Chromatography
Qualitative Analysis
Quantitative analysis
Applications in various fields

28. Quantitative analysis
The chromatogram obtained on a recorder chart can be used to measure quantitatively the concentration of components in a mixture. In general, three methods are used for quantitative evaluation:
i) Area normalization method:
In this method, it is assumed that the entire sample is eluted from the column. The area of each peak is measured and percent composition is obtained by dividing the individual peak area by the total area of all the peaks and multiplying by 100. The value so obtained will be acceptable only if the detector response is the same (particularly for FED) for all the components of the mixture. If not, the detector response factor for each component needs to be established and appropriate corrections made in the measured areas.

29. ii) Internal standardization method:
In this method, known amounts of sample and standard are mixed and chromatographed. The peak areas for sample component and for standard are measured and ratios of both peak areas are determined. Either area ratios are plotted against weight ratios to obtain a graph. Chromatographor area ratios for unknown are compared directly with those for the known amounts.
Thus, accurately known quantity of the internal standard is added the unknown sample and this mixture is chromatographed and area ratios are measured.
The requirements for a suitable internal standard are summarized as follows:
• The standard must be a compound, which is well separated from all components of the mixture being analyzed.
• The standard must not react with any component of the sample, nor it
should influence the physical properties of the other components, e.g.volatility.
• It should yield a symmetrical chromatographic peak.
• It must be chemically similar to the unknown.
• It should have concentration comparable to the components of interest.

30. iii) Comparison method:
In this method, a synthetic mixture containing known quantities of the components of interest in the range of concentration expected in the unknown sample is prepared and analyzed.
The values for the peak areas for different known volumes of synthetic blends are estimated and a calibration curve is plotted.
An exact quantity of the unknown sample is then injected and the peak areas so calculated are used to read from the calibration curve the
concentration of the component in the unknown mixture is then determine.

31. Some Examples of Applications
Bacterial identifications
Long chain fatty acids found in the bacterial cell can be used to distinguish between various microorganisms. Fatty acids with chain length from Cl0 to C20 can be separated and estimated on a 3 m glass column of 2 mm internal diameter packed with 3% SP–2100 DOH at oven temperature of 150°C to 225°C with nitrogen gas at a flow rate of 20 rnL/min.
2. Environmental analysis
a) Water analysis: The organic pollutants in water are concentrated from
water samples by solvent extraction or by purge and trap technique. The
volatile pollutants are analysed on 80/100-mesh carbopack C/0.2 %
carbowax 1500 column.
b) Air analysis: The analysis of organic vapours in the industrial air
environment for the assessment of exposure to workers is done as follows:
i) Organic vapours are collected on a charcoal adsorbent with a portable pump.
ii) Desorption from charcoal is done in a closed vial with carbon disulphide.
iii) Analysis of the desorbed sample is done on a GC using a 6 m, 3 mm internal diameter S.S column packed with 10 % SP–1000 by temperature programming from 100°C to 200°C. By this method, pollutants such as vinyl chloride, xylenes and aromatic hydrocarbons can be estimated

32. c) Clinical and toxicological analysis: Toxicologists have recognised the
usefulness of GC for the analysis of toxic substances. The analysis of lidocaine and diphenhydramine has been done using flame ionisation detection. A 15m x 0.25mm i.d. 5% methylsilicone (DB-5) column has been used, temperature programmed from 180°C to 230°C at 5°C min–1 and helium used as a carrier gas.
d) Forensic toxicology: It is highly specialised branch of analytical chemistry concerned primarily with the analysis of specimen from different organs of the human body for toxic substances. A simple GC system utilising four columns and three liquid phases ( SE–30 , Hallcomid M-18, and Carbowax 6000), complemented by a direct solvent extraction scheme designed to detect common poisons, drugs, and human metabolites to a sensitivity limit of 2 μg/ml in blood, urine and tissue is developed.

33. Thank you