1. Gas chromatography is a technique used to separate components of a mixture using their volatility. It involves two phases - a stationary phase and a mobile gas phase. 2. The basic components of a gas chromatograph are an injection port, column, detector, and recorder. The sample is injected and carried by the mobile gas phase through the column where separation occurs. 3. Separation is based on the difference in partitioning behavior of analytes between the stationary and mobile phases. Components with higher partition coefficients have longer retention times.

1.  Rayat Shikshan Sansthas Veer Wajekar ASC College ,Phunde

Department of Chemistry
T.Y.B.Sc. Analytical Chemistry
Paper-IV Sem-VI
CHROMATOGRAPHY
Dr.Gurumeet C Wadhawa
29 March 2023 1

2. Chromatography
Chromatography - from Greek word chromos= colour and graphy= Writing
Russian botanist- Mikhail Tswett (1903)
Separation of plant pigment (Leaf extract) –Chlorophylls, xanthophylls
Glass column packed with finely divided calcium carbonate stationary phase
David Day- Geologist ,separation of crude oil on ( Fractional distribution)
A.J.P. Martin and R.L.M. synge - 1952 Nobel Prize
Separation,isolation,Identification of closely related components of Complex-
mixture. All these methods involve the relative movement of two phases-
stationary phase, Mobile phase

3. Mikhail Tswett
Born
14 May 1872
Asti, Italy
Died 26 June 1919 (age 47)
Nationality Russia
Fields Botany
Known for
Adsorption
chromatography

4. Classification based on phases involved
Solid Stationary Phase
(Adsorption)
Liquid Stationary Phase
(Partition)
Mobile phase liquid
TLC
Column
Gel
Mobile phase gas
GSC
Mobile
Phase liquid
Paper
HPLC
Mobile
phase gas
GLC

5. Classification of Chromatographic methods
According to mechanism of separation:
The mechanism of separation depends mainly on the nature of the stationary
phase. Based on separation mechanisms chromatography can be classified into:
1- Adsorption Chromatography:
It is the oldest and most common type of chromatography. The stationary phase is
a solid with adsorption power. Mixture components will be adsorbed on the
surface of the stationary phase with different powers and that account for
separation. Silica gel is the most common stationary phase in adsorption
chromatography.

6. Classification of Chromatographic methods
According to mechanism of separation:
The mechanism of separation depends mainly on the nature of the stationary
phase. Based on separation mechanisms chromatography can be classified into:
1- Adsorption Chromatography:
It is the oldest and most common type of chromatography. The stationary phase is
a solid with adsorption power. Mixture components will be adsorbed on the
surface of the stationary phase with different powers and that account for
separation. Silica gel is the most common stationary phase in adsorption
chromatography.

7. 2- Partition Chromatography:
The stationary phase is a liquid forming a thin film on an inert solid acts as
support. The stationary liquid is usually more polar than the mobile liquid. The
two liquids must be immiscible with each other. Cellulose powder and wet silica
gel are examples of supports in partition chromatography that carry film of
water act as stationary phase.

8. 3- Ion Exchange Chromatography:
Ion–exchange can be described as the process of the reversible stoichiometric
exchange of ions of same charge between a mobile liquid phase and an
insoluble solid stationary phase
An ion exchanger is an insoluble material liberating the counter ions (mobile
ions) by electrolytic dissociation.

9. 4- Size –Exclusion Chromatography
Gel filtration is used in fractionation mode, uses porous particles to
separate multiple components in a sample on the basis of differences in
their size.
Molecules that are smaller than the pore size can enter the
particles and therefore have a longer path and longer transit
time than larger molecules that cannot enter the particles
Schematic of a size-exclusion
chromatography column

11. GAS
CHROMATOGRAPHY

12. Contents
 3.1.1 Introduction, Basic Principle, Terms involved in GC
 (Numerical Problems Expected)
 3.1.2 Instrumentation of Gas Chromatography: Block
Diagram and
 components.
 3.1.3 Columns and their packing in GSC and GLC
 3.1.4 Different types of detectors :TCD,FID,ECD
 3.1.5 Quantitative and Qualitative analysis
 3.1.6 Comparison between GSC and GLC
 3.1.7 Applications of GC
29 March 2023 12

13. Contents
 3.1.1 Introduction, Basic Principle, Terms involved in GC
 (Numerical Problems Expected)
 3.1.2 Instrumentation of Gas Chromatography: Block
Diagram and
 components.
 3.1.3 Columns and their packing in GSC and GLC
 3.1.4 Different types of detectors :TCD,FID,ECD
 3.1.5 Quantitative and Qualitative analysis
 3.1.6 Comparison between GSC and GLC
 3.1.7 Applications of GC
29 March 2023 13

14. GAS SOLID
CHROMATOGRAPHY
SOLID + GAS
GAS LIQUID
CHROMATOGRAPHY
LIQUID + GAS
GAS
CHROMATOGRAPHY
ADSORPTION PARTITION
29 March 2023 14

15. GLC at
Glance
Petroleum and refineries
Food and Cosmetics
Pharmaceuticals
Environmental pollution
control
Forensic science
Laboratories
R&D
Rubber Industries
Polymer Industries
And Many More
29 March 2023 15

16. GAS
CHROMATOGRAPHY
THEORY
INSTRUMENTATIONS
APPLICATIONS
29 March 2023 16

17. Partition Chromatography
L+L
Paper
Chromatography
L+L
Gas Liquid
Chromatography
L+Gas
HPLC
L+ L
29 March 2023 17

18. 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.
29 March 2023 18

19. 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
29 March 2023 19

20. Base line
Retention Time------------
Detector
Response
Retention Time------------
B
C
A
Chromatogram
29 March 2023 20

21. 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 mini volts as a function of
time. Usually, time is the abscissa and mini volts 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.
The retention time for solute A is depicted in the figure as the
distance from
29 March 2023 21

22. Important
Terms Involved In
GLC
29 March 2023 22

23. 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
29 March 2023 23

24. b) Retention Volume:
(VR):
It is used for qualitative analysis. Volume of mobile phase
requires to the solute component to elute 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
29 March 2023 24

25. 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.
29 March 2023 25

26. 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.
29 March 2023 26

27. 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+W2 of two peaks.
29 March 2023 27

28. *
* *
* *
*
*
carrier gas,
Pressure regulator
and flow control
Sample Injection
port
column
Column oven
Recorder
Detector
GAS CHROMATOGRAPHIC INSTRUMENT
*
Most Volatile
Least Volatile
29 March 2023 28

29. Components of GLC
Instruments
Carrier Gas
Flow control and
Pressure
Regualator
Sample Injection
Port
Column
Column Oven
Detector
Readout Device
29 March 2023 29

30. • Characteristics Of a Carrier Gas
1
• It should be inert
2
• ii) It should be pure and dry.
3
• iii) It should not be inflammable
4
• iv) It should be chemically inert towards solutes of
interest at the column temperature.
29 March 2023 30

31. • The basic requirements of a liquid phase are as under
1
• It should exhibit different solubility for the components
present in the mixture.
2
• ii) It should have low vapour pressure (0.01–0.1mm) at
operating temperatures for a reasonable column life.
3
• iii) It should be thermally stable.
4
• iv) It should be chemically inert towards solutes of interest
at the column temperature.
29 March 2023 31

32. (+)
(-)
CATHODE
ANODE
Air
COLUMN
EFFLUENT
H2
Collector
Flame
Electrical
Igniter
FLAME IONISATION DETECTOR
FID
29 March 2023 32

33. 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.
29 March 2023 33

34. Limitations of FID
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.
29 March 2023 34

35. Heated
Metal
Box
THETHERMALCONDUCTIVITYDETECTOR
Column
Effluent In
Column
Effluent out
Carrier Gas In
Carrier Gas Out
R S
Leeds to Wheatstone bridge
29 March 2023 35

36. 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.
29 March 2023 36

37. 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.
29 March 2023 37

38. 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
29 March 2023 38

39. ELECTRON CAPTURE DETECTOR
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.
29 March 2023 39

40. Column Effluent
Collector Electrode
Beta Source (+)
(-)
29 March 2023 40

41. 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.
29 March 2023 41

42. 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.
29 March 2023 42

43. Applications of
Gas Liquid
Chromatography
Qualitative
Analysis
Quantitative
analysis
Applications
in various
fields
29 March 2023 43

44. Qualitative
Analysis
Retention
Time
Retention
Volume
29 March 2023 44

45. 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 March 2023 45

46. i) Area normalization method:
Area normalization method:
Individual peak area
Percentage Composition of Component= ------------------------------ X 100
• Total area of all the peaks
29 March 2023 46

47. 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.
29 March 2023 47

48. 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.
29 March 2023 48

49. Other
Applications
29 March 2023 49

50. A) 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.
29 March 2023 50

51. 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.
29 March 2023 51

52. 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.
29 March 2023 52

53. 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.
29 March 2023 53

54. Other Applications
d) Forensic toxicology:
It is highly specialized 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 utilizing 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.
29 March 2023 54

55. •Thank you
29 March 2023 55

56. Numerical based on
Gas Chromatography
UNIT 3

57. Numerical 1
• Following data are obtained on a
chromatographic column having a length of
30 cm
– Retention time of unretained species = 1.40 min
– Retention time of component X = 12.22 min
– Bandwidth at the base = 1.5 min
• Calculate
– Average rate of movement of mobile phase
– Average rate of movement of solute
– Number of theoretical plate
– HETP

58. Solution 1
• Average rate of mobile phase( )
• Average rate of solute ( )
• Number of plates =
• HETP =

59. Numerical 2
• A chromatogram of a mixture A & B provided
following data:
• Calculate number of plates in each peak and
HETP if column length is 25.0 cm
Species
Retention time
(min)
Peak width (min)
Unretained 4.2 -
A 6.4 1.75
B 9.0 2.07