GC and its application

1. Prepared by
Sneha A.Chavan
Department of Pharmaceutics
M pharmacy 1st year IInd semester
GAS CHROMATOGRAPHY
AND ITS APPLICATION
1

2. What is gas chromatography
 It is a technique where by the components of a
mixture in the gaseous state are separated as the
sample passes over a stationary liquid or solid
phase and a gaseous mobile phase.
2

3. Gas
chromatography
Gas solid
chromatography
(GSC )
Gas liquid
chromatography (
GLC )
Based on stationary
phase
3

4. Basic principle
4

5. Gas solid chromatography
(GSC)
 Mobile phase – gas
 Stationary phase – solid
 Limited application as compared to GLC
1. Interaction of solute with stationary phase is in the
form of their adsorption on it. And adsorption is non
- linear. K = Cs / Cm this
ratio does not remain constant during separation.
K = distribution coefficient of the solute
Cs = concentration of drug in stationary phase
Cm = concentration of drug in mobile phase
2. K (GSC) >>> K (GLC) hence, solute in the gaseous
form are not eluted out completely from solid
stationary phase.5

6. Cont….
 This leads to semi permanent retention of solute
on the stationary phase.
 longer time require for elution of solute.
 It result in problem of tailing or broadening of
elution peak.
 Tailing brings about remixing of separated
compound.
 Stationary phase is molecular sieves or porous
polymer
6

7. Cont…
 Molecular sieve
 Molecular sieves are resin like zeolite which are
manufactured carefully so as to control the pore
size in them.
 Pore or holes are of molecular dimension.
 The molecule that are smaller than pore,
penetrate into sieve of molecule, where they can
be adsorbed.
 Bigger molecule cannot enter the interstices of
the sieves.
 For smaller molecule, larger surface area is
available for the adsorption as compared to that
for larger molecule.7

8. Cont….
 The molecules having smaller size, e.g., O2, CO,
H2, N2 and methanol, are able to enter interstices
of molecular sieve but can be separated from
each other because of the difference in their
adsorption coefficient.
 Problem
the molecular sieve as the stationary phase is
that the polar compound like CO2 are permanently
adsorbed in the pores of the sieves and thus block
the surface to other species.
8

9. Cont….
 Porous polymer
 Another stationary phase is in the form of porous
polymeric beads of uniform size.
 A polymer can be in the form of styrene cross
linked with divinyl benzene.
 Marketed trade name Porapak
 Pore size of polymer is controlled by controlling
degree of polymerization.
 Being hydrophobic, this polymer find its main use
in the separation of gases, which are polar in
nature ( oxides of nitrogen, methanol, vinyl
chloride, CO2, H2O )
9

10. Application
 Technique is useful for separation of species such
as nitrogen oxide, H2S, CS2 CO, CO2 and rare
gas that are not retained on liquid column.
10

11. Gas liquid chromatography
 Mobile phase – Gas
 Stationary Phase – liquid which is immobilized by
supporting on some solid matrix
 Mobile phase (gas) does not interact much with the
solute to be separated.
 Role of mobile phase is to push the solute, which are
desorbed from stationary phase, out of the column.
 Hence, mobile phase is referred to as carrier gas
 Sample should be in the form of vapor. This vapor is
introduced at head of column.
 The solute having greater solubility in the stationary
liquid phase remain in smaller concentration in it.
11

12. Cont…
 Thus solute distribute themselves between the
phases according to their distribution coefficient.
 Solute having lesser interaction with stationary
phase are easily desorbed from it and eluted out
faster.
 The elution of desorbed compound is achieved by
forcing an inert carrier gas such as nitrogen or
helium through the column.
 Chromatogram obtained in GLC, also record
elution curves for the eluted compounds, which
are bell shaped.
12

13. Application
 Peak height or peak area of an elute from GC
column ha been widely used for quantitative and
semi quantitative analysis
 GLC is commonly used for analysis of volatile
compound or compound which can be vaporized
at temperature of the sample port of the gas
chromatograph.
 Applied for separation of complex mixture,
organometallic compound and biochemical
system.
 Powerful fractionating tool in conjunction with
superior analytical instruments such as mass, IR,
UV and NMR spectrometer.
13

14. Quantitative Analysis
14
Quantitative analysis involves the following steps
:-
 The generation of signal for each compound in
the mixture by the detector.
 The measurement of some parameter for the
peak obtained in a chromatogram.
 Calculation of separated compound from
measured parameter.

15. Measurement of peak parameters
in chromatography
15
A. Analysis based on Peak Height

16. Cont…
16
 Advantage
 Measurement of the peak height gives accurate
result, provided the width during the period
required to obtain chromatogram for a sample
and a standard, the column temperature, the flow
rate of the mobile phase and rate of sample
injection are controlled properly.
 Suitable for narrow peak (height measured
properly and accurately)

17. Cont…
17
 Disadvantage
 Variation in peak width observed which result in
change in peak height leading to inaccuracy in
measurement.

18. 18
B. Analysis Based On Peak Area
 Advantage
 Preferred parameter for asymmetric or skewed
peak.
 Measurement of peak area is independent of
band broadening and offer advantage of
reproducibility. This is because band broadening
is inversely proportional to height.
 Disadvantage
 Measurement of peak area is not easy as
measurement of peak height

19. Measurement of peak area
19
a) Peak measurement by triangulation technique
A = ½ × w × h
w - width of peak
h – height of peak
Advantage
 Simple formula for calculation of area under the curve
Disadvantage
 Useful only for symmetrical peak
 Tedious job for putting tangent on side of triangle.
 Require operator skill
 Slight error in drawing tangent can have profound
effect on height measurement.

20. 20

21. 21
b) Peak measurement by width at half the height
method
A = h × (½ w)
Advantage
 Method reduces error due to tailing, in the
measurement of area.
 More accurate, rapid, simpler compared to
triangulation technique.
Disadvantage
 Useful only for symmetrical peak

22. 22

23. 23
c) Area measurement by planimetry
Advantage
 Does not provide precise measurement of peak
area.
 More accurate than triangulation technique.
 Useful for unsymmetrical and skewed peak.
Disadvantage
 Precision and accuracy depend on skill of
operator
 Time consuming and tedious method.
 Same peak is traced several times and average
of all value is calculated.

24. 24

25. 25
d) Area measurement by cut and weight method
Advantage
 Superior than triangulation method
 Useful for asymmetric or skewed peak
Disadvantage
 Accuracy depend on accuracy of cutting chart
paper.
 Time consuming
 Very high quality chart paper is essential in order
to get reproducibility in the measurement.

26. 26
e) Area measurement using ball and disc
integrator
Advantage
 Used for skewed peak also.
 Provide high degree of automation and higher
accuracy as compared to other technique.

27. Method of quantitative analysis
27
 Area normalization technique
 Internal standard method
 External standard method
 Preparation of calibration curve

28. Area Normalization Method
28

29. Cont…
29
 For this method to be accurate, the following
criteria must met
1. All analyte must be eluted
2. All analyte must be detected
3. All analyte must have same sensitivity
(response/mass)
 Advantage
i. Simple technique to calculate % of compound
when limited number of compound present in
mixture
ii. Does not require reference standard for finding

30. Cont…
30
 Disadvantage
a. Elution of all compound in the sample is
required even when % of only one compound is
to be determined.
b. Method is not used when number of compound
is more.

31. Internal standard method
31
 Amount of each compound in mixture is calculated
by using a reference standard.
 The reference compound is mixed along with
mixture to be separated and this mixture is injected
in column. Hence reference compound is called is
called as internal standard.
 Choice of internal standard is made based on the
following criteria
a. It must have completely different retention time
than for the compound to be separated.
b. Must be elute out near the peak of interest.
c. Must be in similar in concentration to compound
of interest.

32. 32

33. Cont…
33
 Internal standard and compounds in mixture separate
depending on their retention time value.
 From height or area of peak for reference standard and eluted
compound, the concentration of each compound in mixture
can be found out as follows :
h (std) × c (std) = h (sample) × c (sample)
Advantage
 Higher accuracy.
 Error associated with apparatus and procedure can also be
eliminated by use of an internal standard.
Disadvantage
 Sometime difficult to get suitable internal standard. And it may
not elute out near compound of interest.
 It may not soluble in the solvent in the concentration range
corresponding to compound to be separated.

34. External standard method
34
 Standard is not mixed along with the sample to
be separated.
 Separate solution of standard and sample are
prepared.
 External standard should be 100% pure (AR
grade) or it should be of known purity.
 Both solution are injected separated in the
column which result in produces two separate
chromatogram.
 The height of sample is compared with height of
standard and their concentration are determined.
Advantage

35. Cont…
35
Disadvantage
 Error may occur during weighing and dilution,
which make the process less accurate as
compared to internal standard.
 Require precise control on analytical technique,
particularly while injecting the solution in the
column.
 Two chromatogram are required to be recorded.
 More time consuming.

36. Preparation of calibration
curve
36
 Standard solution of compound of interest are
prepared.
 Equal volume of solution injected in
chromatograph and recorded.
 Area under the curve is measured and
concentration plotted against the area.
 Unknown concentration of sample can be
obtained by measuring area under the peak and
then dropping a perpendicular on concentration
axis.

37. 37

38. Qualitative analysis by GLC
 Useful tool for identifying separated compounds.
 Retention time
o Retention time (RT) is a measure of the time
taken for a solute to pass through a
chromatography column.
o Characteristics parameter of solute which help in
identification.
o Change with change in temperature of column
and flow rate and pressure of carrier gas.
o Precise control needed.
o Chromatogram recorded number of time hence,
process become tedious and expensive.38

39. Cont…
39
 Relative retention time (α)
 It describes the retention time of solute in relation
with the retention time of standard.

40. Cont…
40
 Retention volume (Vr)
o It is defined as the total volume of carrier gas
required to elute out the compound completely
from the column.
o
o If column temperature is increased, the volume of
gas is increased because of which flow rate of
carrier gas is also increased, but retention time is
reduced.

41. 41
 Specific retention volume (Vg)
o Volume of gas per unit weight of the stationary
phase, required to elute the compound
completely is called specific retention volume.

42. Gas Chromatography
Environmental Analysis
42
 used in quantification of pollutants in drinking and
waste water.
 identification of unknown organic compound in
hazardous waste.
 used in analysis of industrial products as well
identification of reaction products.
 Environmental toxins can be identified with gas
chromatography.
 Air samples can be analyzed by gas
chromatography for quality control.
 Pesticides , fertilizers can by analyzed by GC.

43. Gas Chromatography Clinical
Analysis
43
 Blood , Saliva and other secretions which
contains large amount of organic volatiles can be
easily analyzed by gas chromatography.
 Isolation of volatile proteins , lipids,
carbohydrates by gas chromatography.
 used to analyze the chemicals and drugs present
in the body.
 Various biological volatile organic compounds can
be analyze by gas chromatography.
 Urine sample is also analyzed by gas
chromatography.

44. Gas Chromatography Food
Analysis
44
 Food products can be analyzed by gas
chromatography.
 used in analysis of alcohol beverages.
 Analysis of various solvents in food preparation.
 analysis of dairy products such as milk, butter,
cheese etc.
 Quantification of volatile organic food products

45. Gas Chromatography
Forensics
45
 determination of blood alcohol content in the
body.
 Certain drugs are prohibited in certain states, so
in order to check whether person has taken drugs
in the body
 used to test samples found at crime scene.

46. Reference
46
1. Instrumental method of analysis, Supriya
Mahajan, page no. 283 - 293
2. Principles of Instrumental Analysis, Douglas A.
Skoog, F. James Holler, Stanley B. Crouch, page
no.865 – 892
3. Basic Gas Chromatography, Harold Mcnair,
James Miller, 2nd edition, page no. 138- 143
3. http://frndzzz.com/Applications-of-Gas-
Chromatography/GC-Pharmaceutical-Analysis

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