Chromatography is a technique used to separate mixtures by differential partitioning between two phases. There are several types including liquid-solid chromatography, gas-liquid chromatography, and thin layer chromatography. The document discusses the principles, procedures, and applications of various chromatography techniques like column chromatography, paper chromatography, thin layer chromatography, and gas chromatography. These techniques are used for separation, purification, and identification of mixtures in various fields like chemistry, biochemistry, medicine, food analysis, and more.

1. Dr. T. Geetha
St. Mary’s College Thrissur

2. Chromatography
Chromatography – colored writing
Separation
Purification
Identification
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

3. Principle
• Differential distribution of sample components between
two mutually immiscible phases –
stationary phase - fixed
mobile phase - moving
• Movement of mobile phase causes differential migration of sample
components
• Components possess different affinity for a adsorbent
• Migrates at different rates through the mobile phase
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

4. Principle
•Different components emerge at different time
from the stationary phase
•Component having the weakest interaction with the
stationary phase comes out first
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

5. Types of chromatography
Based on types of phases used
 Gas Liquid chromatography (GLC)
 Gas solid chromatography (GSC)
 Liquid liquid chromatography (LLC)
Liquid solid chromatography (LSC)
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

6. Classification based on mechanism
•Adsorption chromatography
•LSC
•GSC
•Partition chromatography
•LLC
•GLC
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

7. Classification based on technique
1. Column chromatography
- adsorbent column
2. Paper chromatography
- stationary liquid film on paper strip
3. Thin layer chromatography
- stationary film of liquid held on adsorbent
coating on a glass plate
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

8. Liquid solid adsorption chromatography
•Column chromatography
•Selective adsorption of sample
components on a adsorbent column
like silica gel, alumina or cellulose
which constitutes stationary phase
•Petroleum ether, acetone, benzene –
mobile phase
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

9. Column chromatography
•Adsorbent column – long glass tube with tap at
lower end packed uniformly with adsorbent
•Solution of mixture of components in solvent
introduced at top of column
•Different components adsorbed to different extends
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

10. Column chromatography
•More readily adsorbed constituent held at the top
•Others with decreasing adsorbabilities held in
different bands along column
•Partial separation improved by passing additional
solvent – Development Of Chromatogram
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

11. Column chromatography
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

12. Column chromatography
•Individual component extracted from adsorbent by
passing suitable solvent down the column
•Components washed down one by one in the
increasing order of absorbability & collected in
separate fractions
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

13. Column chromatography
•Most weakly absorbed component emerges first
•Strongly adsorbed component emerges last
Elution – process of dissolving out components from
adsorbent using a suitable solvent; solvent - eluent
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

14. Column chromatography
o-nitroaniline & p-nitroaniline
Column – Alumina
Solvent – Benzene
Ortho component eluted first
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

15. Application
• Quantitative separation of two or more similar organic
components of a mixture
• Purification of organic substance from contaminants
• Concentration of solutes from dilute solution
• Identification, separation & purification of natural
products
• Identification of industrial products
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

16. Liquid Liquid Partition Chromatography
•Martin & Synge - 1941
•Nobel prize -1952
Archer J. P. Martin
R.L.M. Synge
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

17. Liquid Liquid Partition Chromatography
Stationary phase
 Thin layer of liquid held on
the surface of porous inert
solid support
Cellulose, silica gel, starch,
calcite, kieselguhr
Mobile phase
Liquid immiscible with
stationary liquid phase
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

18. Liquid Liquid Partition Chromatography
Principle
 different components of sample mixture have
different partition coefficients for a given pair of
immiscible liquids – distribute themselves to different
extends between two immiscible liquid phase – move
at different rates - separation
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

19. Partition Column chromatography
•Stationary phase prepared
•Solution containing components made into a slurry
with solid adsorbent transferred to top of column
• Mobile phase introduced on top of column
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

20. Partition Column chromatography
•As mobile phase moves down, partitioning of components
between two immiscible liquids take place
•Components which moves readily to stationary liquid phase –
retarded movement
•Components that moves readily to mobile phase – faster
movement
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

21. Application
•Identification of organic & inorganic mixture
•Laboratory & industrial purification
•Quantitative separation of components – organic or
inorganic substance
Separation of Co & Ni
stationery phase - water supported on cellulose
Mobile phase - acetone containing HCl
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

22. Paper chromatography
STATIONARY PHASE
• Film of liquid adsorbed on
paper mat made of highly
purified cellulose
MOBILE PHASE
• Another liquid percolating
over it
Dr. T. Geetha, Asst. Professor, St. Marys College, Thrissur
Liquid liquid Partition chromatography –Differential migration of the component

23. Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur
Apparatus
1. Support for paper
2. Solvent trough
3. Air tight glass cylinder
• Sample solution spotted near the lower edge of the paper strip &
dried
• Paper suspended in glass cylinder with lower end just dipping in
mobile phase
• Spotted position above solvent level

24. • Solvent ascends through paper by capillary action
• Components move at different rates on account of differential
distribution between the pair of miscible liquids
• Paper strip removed, solvent front marked & dried
• Coloured components appears as coloured spots at different
distance
• Position of colourless compounds determined by spraying with
sensitive reagent capable of forming coloured compounds
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

25. Paper chromatography
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

26. Applications
• Separation & identification of sample compounds present in trace quantity
• Separation of closely related compounds
• Quick check of purity of organic chemicals
• Detection of impurities in pharmaceuticals & food
• Detection of drugs & dopes
• Analysis of cosmetics
• Resolution of isomeric metal complexes
• Determination of metal in ore samples
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

27. Thin Layer Chromatography
• Principle- Differential distribution or partition of components
between two immiscible phases brings about a difference in
their rate of migration – separation
• Stationary phase – film of liquid adsorbed on a uniform thin
layer of an adsorbent –silica gel, alumina or cellulose layer –
coated on glass or plastic plate
• Mobile phase – suitable solvent, immiscible with stationary
phase percolating over it
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

28. Procedure
• Slurry of adsorbent in liquid selected as a stationary phase
spread on flat & uniform glass plate –chromoplate
• Spotted near edge with solution containing the
component
• Placed in a slanting manner in a glass tank containing a
layer of solvent
• Sample spots above solvent
• Tank closed with lid
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

29. • Mobile phase ascends by capillary action
• Sample components carried at different rate
• Components separated – compact spots
• Plate is taken out, solvent front marked, dried
• If components not coloured – sprayed with
suitable agents
• Spots may also be scraped, eluted, investigated
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

30. Rf values
•Retention or retardation factor
•Ratio of distance traveled by a particular component to the
distance travelled by the solvent front during the same time
Rf =
Rf value depends on
• Solvent employed
• Temperature
• Nature of substance
• Quality of medium
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur
distance travelled by the components from origin line
distance travelled by the solvent front from the origin line in the same time
If other conditions are fixed, Rf is a
characteristic of a substance

31. Advantages
•Simple & efficient
•Versatile
•More reproducible
•Rapid separation, purification & identification even
if component is present in minute amounts
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

32. Applications
1. Analysis of complex organic mixtures
2. Checking purity of samples & detection of contaminants
3. Identification & separation of drugs & plant extracts like
alkaloids
4. Separation & isolation of biochemical preparations
5. Mixtures of amino acids separated using a mixture of
chloroform, methanol, ammonium hydrate as solvent -
Detected by using reagent - ninhydrin
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

33. Gas Chromatography
•Gas liquid chromatography
•Gas solid chromatography
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

34. Gas Liquid Chromatography
• Mobile phase – gas –pure chemically inert gas – He, Ar, N2, CO2 –
carrier gas
• Stationary phase – liquid immobilized on surface of inert solid
support – nonvolatile high boiling liquid – methyl silicone,
polyethylene glycol- support is often a coiled capillary column 15 to
100m long & 0.1 to 0.53 mm internal diameter
• Based on differential partitioning of the components of a sample in
the vapour phase between the mobile phase and stationary liquid
phase – differential migration of components - separation
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

35. Procedure
Injection of sample
– microsyringe if sample is liquid
- gas tight syringe if sample gas
Sample in vapour state is carried onto column by carrier gas
Column maintained at constant T higher than B.Pt. of least boiling component in
sample mixture
Components differentially retained in the stationary phase reach the outlet at
different time
Components that comes out of the column are automatically detected by
detector
Intensity of detector signals recorded as a function of time – gas chromatogram
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

36. Gas Chromatography
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

37. Chromatogram
No. of peaks
Area under each peak
Retention time
No. of components in the
sample
Relative concentration of that
component in sample
Identify components
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

38. Applications
• Separation of complex organic & biochemical systems
• Qualitative analysis of mixture of organic compounds like
natural products by retention time
• Confirms presence or absence of suspected compounds
• Separation of benzene and cyclohexane (nearly impossible
by conventional distillation) – easily done by GLC
• Separate detection of hundreds of different hydrocarbon in
gasoline
• Detection of traces of pesticides in food
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

39. Applications
•Check the criteria of purity of organic compounds
•Superior fractionation qualities of GC combined
with Mass spectrometer, IR spectrometer, NMR etc
for qualitative & quantitative analysis
• Commonly used detectors – TCD(Thermal
conductivity Detector), FID (Flame Ionization
Detector)
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

40. Relative merits
LSC
• Adsorbents commonly used
are commercially available at
low cost
• Choice of adsorbent limited
LLC
• Wide variety of liquid
combination available
• Grater selectivity ideal for
separation of compounds
ranging from small organic
molecules to large polymers,
at trace or macro levels
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

41. Advantages of paper & thin layer
chromatography
• Simplicity
• Ease of performance
• High sensitivity
• Low cost
• Hassel free operation
• Minimum operation attention
• Can be combined with other methods of identification
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

42. Relative merits of TLC over Paper
Chromatography
• Faster separation
• Higher sensitivity & resolving power
• Smaller sample size
• High reproducibility
• Withstands strong & corrosive solvents, components & spraying
reagents
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

43. TLC & Paper chromatography
Planar Chromatography –
low quantitative accuracy
Precision
Qualitative & semi qualitative work
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

44. GLC
Advantages
• Faster, efficient
• High sensitivity
• Low sample size
• Automation allows
unattended operation
• Predictability, accuracy
Disadvantage
• Suitable only for
compounds which are
itself thermally stable
& volatile or can be
converted to
derivatives with this
property
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur

45. Thank You
Dr. T. Geetha, Asst. Professor, St. Marys College,
Thrissur