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1. PAPER
CHROMATOGRAPHY

2. Definition:
 Paper chromatography is defined as the
technique in which the analysis of unknown
substances is carried out mainly by the flow of
solvents on a specially designed filter paper.
 There are two types:
 Paper adsorption chromatography: Paper
impregnated with silica or alumina, it act as adsorbent
(stationary phase) and solvent as mobile phase.
 Paper partition chromatography: in which moisture
water present in the pores of the cellulose paper and
another mobile phase is used as solvent.
In general PC refers to P partition C only since most
separation is based on partition type only.

3. Principle:
 The principle of separation is mainly partition
rather than adsorption.
 The whatmann filter paper is used as
stationary phase. It is made of 99% of
cellulose. Cellulose is very good adsorbent
and it adsorb atmospheric moisture.
 This moisture develop as thin film of liquid on
the surface of the filter paper and this behaves
as stationary liquid.
 Instead of water as stationary phase other
organic solvents can be used by suitable
modification.

4.  Mobile phase – organic solvent or mixture of
organic solvents (mixed in a specific ratio) and
buffers are used.
 It is also called moving liquid or development
solvent.
 The distribution of analyte takes place
between stationary liquid (aqueous phase) and
mobile liquid (organic phase) . Hence called
partition paper chromatography.

5.  In this technique a drop of the test solution is
applied as a small spot on a filter paper and the
spot is dried.
 The paper is kept in a closed chamber and the
edge of the filter paper is dipped into a solvent
called development solvent.
 Now the development solvent moves against the
gravitational force (upwards) by capillary action of
filter paper. The minute pores present in the
stationary phase will provide the capillary action.
 When the solvent reaches the spot (mixture of
2or more substances), the various substances
are moved by solvent system at various speeds
and the solvent is allowed to move upto 3/4th of
the paper, then stopped.

6.  This process of movement of m/p and
separation of analyte mixture is called
development process.
 Now the paper is dried and various spots are
visualized by suitable detection processes.

7. Steps:
 Selection of stationary phase.
 Selection of mobile phase.
 Chamber saturation (preparing the developing
chamber).
 Preparation and application of sample.
 Development.
 Drying.
 Detection.

8. Selection of stationary phase :
 Paper of chromatographic grade consists of α-
cellulose – 98.99%, β-cellulose – 0.3-1%, pentosans –
0.4-0.8%, ether soluble matter – 0.015-0.02%, ash –
0.01-0.07%.
 Whatman filter papers of different grade like no.1,
no.2, no.3, no.3MM, no.4, no.17, no.20 etc are used.
These papers differ in size, shapes, porosities and
thickness.
 Choice of filter paper depends upon thickness, flow
rate, purity, technique, etc.
 Modified papers: acid or base washed, glass fiber
type paper.

9.  Hydrophilic papers: papers modified with
methanol, formamide, glycol, glycerol etc.
 Hydrophobic papers: acetylation of OH group
leads to hydrophobic nature, hence can be used
for reverse phase chromatography.
the filter paper of suitable size that can be kept in
the chamber is taken and a line is drawn leaving
2cm from bottom and also another line at 3/4th
length of the paper is drawn with a pencil.

10. Selection of mobile phase:
 Organic solvents, buffers, or mixture of solvents
are used.
 Generally in PC mixture of organic solvents
prepared in some ratios are used based the
polarity of analyte mixture.
 Hydrophilic mobile phases:
 Methanol : water – 3:1 or 4:1
 n-butanol : glacial acetic acid : water – 4:1:5
 Hydrophobic mobile phases:
 Kerosene : 70% isopropanolol
 Dimethyl ether : cyclohexane

11. Chamber saturation:
 A development chamber is taken and mobile
phase is added to it and closed.
 This setup is kept aside for 20 to 25 minutes.
During which between the components of the
developing solvent and their vapor, an equilibrium
will be established eventually.
 This equilibrium is called chamber saturation.
 It the process of replacing of atmospheric air
present with vapors of mobile phase.
 Use of chamber saturation:
 Edge effect doesn’t occur
 Due to the presence of air the separation favors to
edges of the paper and is not proper. It is prevented
by chamber saturation.

12. Preparation and application of
sample:
 Analyte mixture is dissolved in a suitable solvent
to make about a one percent solution (0.01 g
sample/1g solvent). Less than one milliliter of
solution will be needed for the experiment.
 Spotting:
 Touch the tip of a clean drawn out capillary tube to
the sample and let about 3mm of sample rise into the
tube. To spot the sample, touch the capillary to the
line.
 Make sure the spot does not exceed 1cm in diameter.
Allow the sample to evaporate.
 Spots should be 2 to 2.5cm away from the edges of
the paper and from each other.

14. Development:
 After preparing the chamber and spotting of
the samples, the sample is developed.
 The paper is dipped into the mobile phase and
the mobile phase moves upward due the
capillary action due to pores present in the
paper.
 As the solvent reaches the 3/4th length of the
paper the development is stopped.
 Development technique:
 Ascending:
 conventional type
 Development occurs against the gravity.

15.  Descending:
 Flow of solvent is assisted by gravity and hence
development is faster.
 Solvent holder is on top.
 Ascending – descending:
 Combination of ascending and descending type.
 First ascending takes place then descending.
 Length of separation is increased.
 Circular or radial development:
 Filter paper is circular.
 The spot is kept at the center.
 The solvent flows through a wick at the centre and
spreads in all directions
 Spots appear as concentric circles.

16.  Two dimensional development:
 First the plates are developed in one axis and the plates
after drying are developed in other axis (i.e., the plates are
rotated to 90 degree).
 When large number of compounds or complex mixtures
are need to be separated this method can be followed.
 Either same solvent or different solvent system can be
used.

18. Drying:
 Gently remove the paper out of the chamber
by handling it in the corners
 And dry it in a well ventilated area.

19. Detecting or visualizing agents:
 Colored spots can be visually detected. But for
detecting colorless spots, the following
techniques are used:
 Non specific methods: no. of spots can be
detected but not exact nature or type of
compound.
 Iodine chamber method : where brown or amber
colored spots are observed when the paper are
kept in a tank with few iodine crystals at the
bottom.
 UV chamber for fluorescent compounds: when
viewed under UV chamber at 254 or at 365 nm,
fluorescent compounds can be detected.

20.  Specific methods: specific spray or detecting
or visualizing agents are used to find out the
nature of compound or for identification
purposes.
 Eg.
 Fecl3 for phenolic and tannin compounds
 ninhydrin for amino acids.
 The detecting techniques can also be
categorized as:
 Destructive technique: when specific spray
agents are used the samples are destroyed
before detection.
 Eg. Ninhydrin reagent
 Non destructive technique: methods like UV
chamber, iodine chamber, densitometry method
doesn’t destroy the sample even after detection.

21.  For radioactive materials, detection is by using
autoradiography or Geiger Muller counter.
 For antibiotics, the chromatogram is laid on
nutrient agar inoculated with appropriate strain
and the zone of inhibition is compared.

22. Quantitative analysis:
 Direct technique:
 by densitometer measure density of spots
 This method is also called as in-situ method.
 Indirect technique:
 The papers are cut into portions and eluted with
solvents.
 The solution is analyzed by spectrophotometry,
electrochemical techniques, etc.

23. Qualitative analysis:
 Rf value:
 Rf= dist travelled by solute/dist travelled by solvent front
 value ranges from 0-1
 Ideal values are 0.3-0.8.
 It is characteristic to each compound in a particular
combination of sp and mp.
 The unknown compound can be identified by comparing
its rf values with standard’s.
 Rx value:
 Distance travelled by sample/dist travelled by standard.
 It is always closer to 1.

24.  Rm values:
 To find whether compounds belong to a
homologous series.
 It is a combined value.
 Delta Rm = log (1/Rf – 1).

25. Applications:
 Useful for analysis of amino acids, sugars, natural
products, etc.
 Separation of mixture of drugs of chemical or
biological origin, plant extracts, etc.
 Separation of carbohydrates , vitamins,
antibiotics, proteins, alkaloids, glycosides, amino
acids, etc.
 Study of inorganic metal salts and complex ions.
 Identification of decomposition products.
 Analysis of metabolites of drugs in blood, urine
etc.
 Identification of impurities.