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S.GOKULAKRISHNAN
M.Pharm (Pharmaceutics) – I Year,
Mother Theresa Post Graduate and Research Institute of Health Sciences,
(A Government of Puducherry Institution)
Puducherry.
CHROMATOGRAPHY
GOKULAKRISHNAN CHROMATOGRAPHY 1
• Chromatography
• Chroma
• Graphein
GOKULAKRISHNAN CHROMATOGRAPHY 2
color
To write
History
 It was first employed in Russia by the
Italian-scientist Mikhail Tsvet in 1900.
 The separation of plant pigments such as
Chlorophyll.
 New types of chromatography developed.
CHROMATOGRAPHY
DEFINITION
 Chromatography is the separation of mixture into individual
compounds using a stationary phase and mobile phase.
GOKULAKRISHNAN CHROMATOGRAPHY 3
PHASE IN CHROMATOGRAPHY ?
GOKULAKRISHNAN CHROMATOGRAPHY 4
STATIONARY PHASE
GOKULAKRISHNAN CHROMATOGRAPHY 5
ADSORBENT
Atoms that accumulate on
the surface of the material
GOKULAKRISHNAN CHROMATOGRAPHY 6
TYPE OF STATIONARY PHASE
MOBILE PHASE
GOKULAKRISHNAN CHROMATOGRAPHY 7
ELUENT
Solvent that carry the
analyte in elution.
MOBILE PHASE
GOKULAKRISHNAN CHROMATOGRAPHY 8
SUPER CRITICAL FLUID:
mobile phase is a fluid above and close to its
critical temperature and pressure.
TYPES OF CHROMATOGRAPHY
1. PAPER CHROMATOGRAPHY
2. THIN LAYER CHROMATOGRAPHY
3. ION EXCHANGE CHROMATOGRAPHY
4. COLUMN CHROMATOGRAPHY
5. GAS CHROMATOGRAPHY
6. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
7. AFFINITY CHROMATOGRAPHYGOKULAKRISHNAN CHROMATOGRAPHY 9
PAPER CHROMATOGRAPHY
 Paper chromatography was first introduced by German scientist
Christian Friedrich Schonbein(1865)
 Paper chromatography to be the simplest and most widely
used of the chromatographic techniques because of its applicability to
isolation, identification and quantitative determination of organic and
inorganic compounds.
GOKULAKRISHNAN CHROMATOGRAPHY 10
PAPER CHROMATOGRAPHY
ANALYSIS OF UNKNOWN SUSTANCES
 It is carried out mainly by the flow of solvents on specially
designed filter paper.
 There are two types of paper chromatography, they are:
GOKULAKRISHNAN CHROMATOGRAPHY 11
PAPER CHROMATOGRAPHY
1.PAPER ADSORPTION CHROMATOGRAPHY
Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as mobile phase.
2.PAPER PARTITION CHROMATOGRAPHY
Moisture / Water present in the pores of cellulose fibers
present in filter paper acts as stationary phase & another
mobile phase is used as solvent
In general P.C – Paper Partition Chromatography
GOKULAKRISHNAN CHROMATOGRAPHY 12
P PAPER CHROMATOGRAPHY
PRINCIPLE OF SEPERATION
The principle of separation is mainly partition rather than adsorption.
Cellulose layers in filter paper contains moisture which acts as stationary phase &
organic solvents/buffers are used as mobile phase
PRACTICAL REQUIREMENTS
1)Stationary phase & papers used
2)Application of sample
3)Mobile phase
4)Development technique
5)Detecting or Visualizing agents
GOKULAKRISHNAN CHROMATOGRAPHY 13
PAPER CHROMATOGRAPHY
STATIONARY PHASE AND PAPERS USED
Whatman filter papers of different grades like No.1,
No.2,No.3,No.4, No.20, No.40, No.42 etc are used. In general this
paper contains 98-99% of α-cellulose, 0.3 – 1% β –cellulose.
Factors that governs the choice of paper:
» Nature of Sample and solvents used.
» Based on Quantitative or Qualitative analysis.
» Based on thickness of the paper.GOKULAKRISHNAN CHROMATOGRAPHY 14
PAPER CHROMATOGRAPHY
 Modified Papers – acid or base washed filter paper, glass fiber type paper.
 Hydrophilic Papers – Papers modified with methanol, formamide, glycol,
glycerol etc.
 Hydrophobic papers – acetylation of OH groups leads to hydrophobic
nature, hence can be used for reverse phase chromatography.
 Impregnation of silica, alumna, or ion exchange resins can also be made.
GOKULAKRISHNAN CHROMATOGRAPHY 15
PREPARATION OF PAPER
 Cut the paper into desired shape and size depending upon work to be carried
out.
 The starting line is marked on the paper with an ordinary pencil 5cm from the
bottom edge.On the staring line marks are made 2cm apart from each other.
GOKULAKRISHNAN CHROMATOGRAPHY 16
PAPER CHROMATOGRAPHY
Preparation of the solution
 Choice of suitable solvent for making solution is very important.
Pure solutions can be applied direct on the paper but solids are
always dissolved in small quantity of a suitable solvent.
 Biological tissues are treated with suitable solvents and their
extracts obtained. Proteins can be precipitated with alcohol and salts
can be removed by treatment with ion exchange resin.
GOKULAKRISHNAN CHROMATOGRAPHY 17
PAPER CHROMATOGRAPHY
APPLICATION OF SAMPLE
 The sample to be applied is dissolved in the mobile phase and applied as
a small spot on the origin line, using capillary tube or micropipette.
 very low concentration is used to avoid larger zone
 The spot is dried on the filter paper and is placed in developing
chamber.
GOKULAKRISHNAN CHROMATOGRAPHY 18
CHOICE OF THE SOLVENT
The commonly employed solvents are the polar solvents, but
the choice depends on the nature of the substance to be
separated.
If pure solvents do not give satisfactory separation, a
mixture of solvents of suitable polarity may be applied.
GOKULAKRISHNAN CHROMATOGRAPHY 19
PAPER CHROMATOGRAPHY
MOBILE PHASE
Pure solvents, buffer solutions or mixture of solvents
Examples-
Hydrophilic mobile phase
Isopropanol: ammonia:water 9:1:2
Methanol :water 4:1
N-butanol : glacial acetic acid : water 4:1:5
Hydrophobic mobile phases
dimethyl ether: cyclohexane
kerosene : 70% isopropanol
GOKULAKRISHNAN CHROMATOGRAPHY 20
CHROMATOGRAPHIC CHAMBER
 The chromatographic chamber are made up of many materials like glass,
plastic or stainless steel.
 Glass tanks are preferred most. They are available in various dimensional size
depending upon paper length and development type.
 The chamber atmosphere should be saturated with solvent vapor.
GOKULAKRISHNAN CHROMATOGRAPHY 21
PAPER CHROMATOGRAPHY
DEVELOPMENT TECHNIQUE
 Paper is flexible when compared to glass plate used in TLC, several types of
development are possible which increases the case of operation.
 The paper is dipped in solvent in such a manner that the spots will not dip
completely into the solvent.
 The solvent will rise up and it is allowed to run 2/3rd of paper height for better
and efficient result.
GOKULAKRISHNAN CHROMATOGRAPHY 22
PAPER CHROMATOGRAPHY
Different types of development techs ,are
1) ASCENDING DEVELOPMENT (go up)
Like conventional type, the solvent flows against gravity. The spots are kept
at the bottom portion of paper and kept in a chamber with mobile phase solvent at
the bottom.
GOKULAKRISHNAN CHROMATOGRAPHY 23
PAPER CHROMATOGRAPHY
2) DESCENDING TYPE (a downward slope)
 This is carried out in a special chamber where the solvent holder is at the top.
The spot is kept at the top and the solvent flows down the paper.
 In this method solvent moves from top to bottom so it is called descending
chromatography.
 ADVANTAGE IS THAT, DEVELOPMENT IS FASTER
GOKULAKRISHNAN CHROMATOGRAPHY 24
PAPER CHROMATOGRAPHY
3)ASCENDING – DESCENDING DEVELOPMENT
 A hybrid of above two technique is called ascending-descending
chromatography.
 Only length of separation increased, first ascending takes place
followed by descending
GOKULAKRISHNAN CHROMATOGRAPHY 25
PAPER CHROMATOGRAPHY
4)CIRCULAR / RADIAL DEVELOPMENT
 Spot is kept at the centre of a circular paper. The solvent flows
through a wick at the centre & spreads in all directions uniformly.
GOKULAKRISHNAN CHROMATOGRAPHY 26
PAPER CHROMATOGRAPHY
5)TWO DIMENSIONAL DEVELOPMENT
In this method the paper is developed in
one direction and after development, the
paper is developed in the second direction
allowing more compounds to be separated
into individual spots.
In the second direction, either same
solvent/different solvent system can be used
for development..
GOKULAKRISHNAN CHROMATOGRAPHY 27
PAPER CHROMATOGRAPHY
DRYING OF CHROMATOGRAM
After the solvent has moved a certain distance for certain
time the chromatogram is taken out from the tank & position
of the solvent front is marked with a pencil.
They are dried by cold or hot air depending on volatility
of solvents. A simple hair dryer is a convenient device to dry
chromatograms.
GOKULAKRISHNAN CHROMATOGRAPHY 28
PAPER CHROMATOGRAPHY
DETECTING / VISUALISING AGENTS
If the substance are colored they are visually detected easily.
But for colorless substance, Physical and chemical methods are used to
detect the spot.
(a)Non specific methods ( Physical methods)
E.g. iodine chamber method,
UV chamber for fluorescent compounds – at 254 or at 365nm.
GOKULAKRISHNAN CHROMATOGRAPHY 29
(b) Specific methods (Chemical methods) or Spraying method
EXAMPLES
 Ferric chloride Phenolic comp. & tannins
 Ninhydrin in acetone
Amino acids
 Dragendroff’s reagent
Alkaloids
 3,5 dinitro benzoic acid
Cardiac glycosides
GOKULAKRISHNAN CHROMATOGRAPHY 30
Following detecting tech. can also be categorized as
1) DESTRUCTIVE TECHNIQUES
Specific spray reagents, samples destroyed before
detection e.g. – ninhydrin reagent
2) NON-DESTRUCTIVE TECHNIQUES
For radio active materials - Geiger Muller counter
uv chamber, iodine chamber
QUANTITATIVE ESTIMATIONS
The method can be divided into two main groups
1.Direct techniques-
2.Indirect techniques-
GOKULAKRISHNAN CHROMATOGRAPHY 31
PAPER CHROMATOGRAPHY
Direct Measurement Method
(i) Comparison of visible spots
A rough quantitative measurements
Component in a mixture can be carried out by comparing the intensity
and size of the spot with a standard substance.
(ii) Photo densitometry
The method is used with the chromatograms of colored
compound, instrument which measures quantitatively the density of the
spots.
GOKULAKRISHNAN CHROMATOGRAPHY 32
PAPER CHROMATOGRAPHY
(iii) Fluorimetry
The compound to be determined by fluorimetry must be fluorescent or
convertible into fluorescent derivatives.
(iv) Radiotracer Method
The compound containing radioactive element is labeled and treated with
locating reagent. Using Geiger Muller counter.
(v) Polarographic & Conductometric methods
Used to measure the amount of material in the spot
INDIRECT MEASUREMENT METHOD
In this technique, the spots are cut into portions and eluted with solvents.
This solution can be analyzed by any techniques of analysis like
spectrophotometry, electrochemical methods, etc.
GOKULAKRISHNAN CHROMATOGRAPHY 33
Rf VALUE (Retardation Factor)
In paper chromatography the results are represented by Rf value
which represent the movement or migration of solute relative to the
solvent front.
Rf value=
the ratio of the distance traveled by the substance
the distance traveled by the solvent
GOKULAKRISHNAN CHROMATOGRAPHY 34
Factors affecting Rf VALUE
 The temperature
 The purity of the solvents used
 The quality of the paper, adsorbents & impurities
present in the adsorbents
 Chamber saturation techniques, method of drying & development
 The distance travelled by the solute & solvent
 Chemical reaction between the substances being partitioned.
 pH of the solution
GOKULAKRISHNAN CHROMATOGRAPHY 35
APPLICATIONS
 Separation of mixtures of drugs
 Separation of carbohydrates, vitamins, antibiotics, proteins, etc.
 Identification of drugs
 Identification of impurities
 Analysis of metabolites of drugs in blood , urine …
ADVANTAGES OF PAPER CHROMATOGRAPHY
 Simple
 Inexpensive
 Excellent resolving power
GOKULAKRISHNAN CHROMATOGRAPHY 36

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Paper Chromatography

  • 1. S.GOKULAKRISHNAN M.Pharm (Pharmaceutics) – I Year, Mother Theresa Post Graduate and Research Institute of Health Sciences, (A Government of Puducherry Institution) Puducherry. CHROMATOGRAPHY GOKULAKRISHNAN CHROMATOGRAPHY 1
  • 2. • Chromatography • Chroma • Graphein GOKULAKRISHNAN CHROMATOGRAPHY 2 color To write History  It was first employed in Russia by the Italian-scientist Mikhail Tsvet in 1900.  The separation of plant pigments such as Chlorophyll.  New types of chromatography developed.
  • 3. CHROMATOGRAPHY DEFINITION  Chromatography is the separation of mixture into individual compounds using a stationary phase and mobile phase. GOKULAKRISHNAN CHROMATOGRAPHY 3
  • 4. PHASE IN CHROMATOGRAPHY ? GOKULAKRISHNAN CHROMATOGRAPHY 4
  • 5. STATIONARY PHASE GOKULAKRISHNAN CHROMATOGRAPHY 5 ADSORBENT Atoms that accumulate on the surface of the material
  • 7. MOBILE PHASE GOKULAKRISHNAN CHROMATOGRAPHY 7 ELUENT Solvent that carry the analyte in elution.
  • 8. MOBILE PHASE GOKULAKRISHNAN CHROMATOGRAPHY 8 SUPER CRITICAL FLUID: mobile phase is a fluid above and close to its critical temperature and pressure.
  • 9. TYPES OF CHROMATOGRAPHY 1. PAPER CHROMATOGRAPHY 2. THIN LAYER CHROMATOGRAPHY 3. ION EXCHANGE CHROMATOGRAPHY 4. COLUMN CHROMATOGRAPHY 5. GAS CHROMATOGRAPHY 6. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 7. AFFINITY CHROMATOGRAPHYGOKULAKRISHNAN CHROMATOGRAPHY 9
  • 10. PAPER CHROMATOGRAPHY  Paper chromatography was first introduced by German scientist Christian Friedrich Schonbein(1865)  Paper chromatography to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds. GOKULAKRISHNAN CHROMATOGRAPHY 10
  • 11. PAPER CHROMATOGRAPHY ANALYSIS OF UNKNOWN SUSTANCES  It is carried out mainly by the flow of solvents on specially designed filter paper.  There are two types of paper chromatography, they are: GOKULAKRISHNAN CHROMATOGRAPHY 11
  • 12. PAPER CHROMATOGRAPHY 1.PAPER ADSORPTION CHROMATOGRAPHY Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase. 2.PAPER PARTITION CHROMATOGRAPHY Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent In general P.C – Paper Partition Chromatography GOKULAKRISHNAN CHROMATOGRAPHY 12
  • 13. P PAPER CHROMATOGRAPHY PRINCIPLE OF SEPERATION The principle of separation is mainly partition rather than adsorption. Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phase PRACTICAL REQUIREMENTS 1)Stationary phase & papers used 2)Application of sample 3)Mobile phase 4)Development technique 5)Detecting or Visualizing agents GOKULAKRISHNAN CHROMATOGRAPHY 13
  • 14. PAPER CHROMATOGRAPHY STATIONARY PHASE AND PAPERS USED Whatman filter papers of different grades like No.1, No.2,No.3,No.4, No.20, No.40, No.42 etc are used. In general this paper contains 98-99% of α-cellulose, 0.3 – 1% β –cellulose. Factors that governs the choice of paper: » Nature of Sample and solvents used. » Based on Quantitative or Qualitative analysis. » Based on thickness of the paper.GOKULAKRISHNAN CHROMATOGRAPHY 14
  • 15. PAPER CHROMATOGRAPHY  Modified Papers – acid or base washed filter paper, glass fiber type paper.  Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.  Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography.  Impregnation of silica, alumna, or ion exchange resins can also be made. GOKULAKRISHNAN CHROMATOGRAPHY 15
  • 16. PREPARATION OF PAPER  Cut the paper into desired shape and size depending upon work to be carried out.  The starting line is marked on the paper with an ordinary pencil 5cm from the bottom edge.On the staring line marks are made 2cm apart from each other. GOKULAKRISHNAN CHROMATOGRAPHY 16
  • 17. PAPER CHROMATOGRAPHY Preparation of the solution  Choice of suitable solvent for making solution is very important. Pure solutions can be applied direct on the paper but solids are always dissolved in small quantity of a suitable solvent.  Biological tissues are treated with suitable solvents and their extracts obtained. Proteins can be precipitated with alcohol and salts can be removed by treatment with ion exchange resin. GOKULAKRISHNAN CHROMATOGRAPHY 17
  • 18. PAPER CHROMATOGRAPHY APPLICATION OF SAMPLE  The sample to be applied is dissolved in the mobile phase and applied as a small spot on the origin line, using capillary tube or micropipette.  very low concentration is used to avoid larger zone  The spot is dried on the filter paper and is placed in developing chamber. GOKULAKRISHNAN CHROMATOGRAPHY 18
  • 19. CHOICE OF THE SOLVENT The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied. GOKULAKRISHNAN CHROMATOGRAPHY 19
  • 20. PAPER CHROMATOGRAPHY MOBILE PHASE Pure solvents, buffer solutions or mixture of solvents Examples- Hydrophilic mobile phase Isopropanol: ammonia:water 9:1:2 Methanol :water 4:1 N-butanol : glacial acetic acid : water 4:1:5 Hydrophobic mobile phases dimethyl ether: cyclohexane kerosene : 70% isopropanol GOKULAKRISHNAN CHROMATOGRAPHY 20
  • 21. CHROMATOGRAPHIC CHAMBER  The chromatographic chamber are made up of many materials like glass, plastic or stainless steel.  Glass tanks are preferred most. They are available in various dimensional size depending upon paper length and development type.  The chamber atmosphere should be saturated with solvent vapor. GOKULAKRISHNAN CHROMATOGRAPHY 21
  • 22. PAPER CHROMATOGRAPHY DEVELOPMENT TECHNIQUE  Paper is flexible when compared to glass plate used in TLC, several types of development are possible which increases the case of operation.  The paper is dipped in solvent in such a manner that the spots will not dip completely into the solvent.  The solvent will rise up and it is allowed to run 2/3rd of paper height for better and efficient result. GOKULAKRISHNAN CHROMATOGRAPHY 22
  • 23. PAPER CHROMATOGRAPHY Different types of development techs ,are 1) ASCENDING DEVELOPMENT (go up) Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. GOKULAKRISHNAN CHROMATOGRAPHY 23
  • 24. PAPER CHROMATOGRAPHY 2) DESCENDING TYPE (a downward slope)  This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at the top and the solvent flows down the paper.  In this method solvent moves from top to bottom so it is called descending chromatography.  ADVANTAGE IS THAT, DEVELOPMENT IS FASTER GOKULAKRISHNAN CHROMATOGRAPHY 24
  • 25. PAPER CHROMATOGRAPHY 3)ASCENDING – DESCENDING DEVELOPMENT  A hybrid of above two technique is called ascending-descending chromatography.  Only length of separation increased, first ascending takes place followed by descending GOKULAKRISHNAN CHROMATOGRAPHY 25
  • 26. PAPER CHROMATOGRAPHY 4)CIRCULAR / RADIAL DEVELOPMENT  Spot is kept at the centre of a circular paper. The solvent flows through a wick at the centre & spreads in all directions uniformly. GOKULAKRISHNAN CHROMATOGRAPHY 26
  • 27. PAPER CHROMATOGRAPHY 5)TWO DIMENSIONAL DEVELOPMENT In this method the paper is developed in one direction and after development, the paper is developed in the second direction allowing more compounds to be separated into individual spots. In the second direction, either same solvent/different solvent system can be used for development.. GOKULAKRISHNAN CHROMATOGRAPHY 27
  • 28. PAPER CHROMATOGRAPHY DRYING OF CHROMATOGRAM After the solvent has moved a certain distance for certain time the chromatogram is taken out from the tank & position of the solvent front is marked with a pencil. They are dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms. GOKULAKRISHNAN CHROMATOGRAPHY 28
  • 29. PAPER CHROMATOGRAPHY DETECTING / VISUALISING AGENTS If the substance are colored they are visually detected easily. But for colorless substance, Physical and chemical methods are used to detect the spot. (a)Non specific methods ( Physical methods) E.g. iodine chamber method, UV chamber for fluorescent compounds – at 254 or at 365nm. GOKULAKRISHNAN CHROMATOGRAPHY 29
  • 30. (b) Specific methods (Chemical methods) or Spraying method EXAMPLES  Ferric chloride Phenolic comp. & tannins  Ninhydrin in acetone Amino acids  Dragendroff’s reagent Alkaloids  3,5 dinitro benzoic acid Cardiac glycosides GOKULAKRISHNAN CHROMATOGRAPHY 30
  • 31. Following detecting tech. can also be categorized as 1) DESTRUCTIVE TECHNIQUES Specific spray reagents, samples destroyed before detection e.g. – ninhydrin reagent 2) NON-DESTRUCTIVE TECHNIQUES For radio active materials - Geiger Muller counter uv chamber, iodine chamber QUANTITATIVE ESTIMATIONS The method can be divided into two main groups 1.Direct techniques- 2.Indirect techniques- GOKULAKRISHNAN CHROMATOGRAPHY 31
  • 32. PAPER CHROMATOGRAPHY Direct Measurement Method (i) Comparison of visible spots A rough quantitative measurements Component in a mixture can be carried out by comparing the intensity and size of the spot with a standard substance. (ii) Photo densitometry The method is used with the chromatograms of colored compound, instrument which measures quantitatively the density of the spots. GOKULAKRISHNAN CHROMATOGRAPHY 32
  • 33. PAPER CHROMATOGRAPHY (iii) Fluorimetry The compound to be determined by fluorimetry must be fluorescent or convertible into fluorescent derivatives. (iv) Radiotracer Method The compound containing radioactive element is labeled and treated with locating reagent. Using Geiger Muller counter. (v) Polarographic & Conductometric methods Used to measure the amount of material in the spot INDIRECT MEASUREMENT METHOD In this technique, the spots are cut into portions and eluted with solvents. This solution can be analyzed by any techniques of analysis like spectrophotometry, electrochemical methods, etc. GOKULAKRISHNAN CHROMATOGRAPHY 33
  • 34. Rf VALUE (Retardation Factor) In paper chromatography the results are represented by Rf value which represent the movement or migration of solute relative to the solvent front. Rf value= the ratio of the distance traveled by the substance the distance traveled by the solvent GOKULAKRISHNAN CHROMATOGRAPHY 34
  • 35. Factors affecting Rf VALUE  The temperature  The purity of the solvents used  The quality of the paper, adsorbents & impurities present in the adsorbents  Chamber saturation techniques, method of drying & development  The distance travelled by the solute & solvent  Chemical reaction between the substances being partitioned.  pH of the solution GOKULAKRISHNAN CHROMATOGRAPHY 35
  • 36. APPLICATIONS  Separation of mixtures of drugs  Separation of carbohydrates, vitamins, antibiotics, proteins, etc.  Identification of drugs  Identification of impurities  Analysis of metabolites of drugs in blood , urine … ADVANTAGES OF PAPER CHROMATOGRAPHY  Simple  Inexpensive  Excellent resolving power GOKULAKRISHNAN CHROMATOGRAPHY 36