Chromatogrpahy is a technique widely used to separate compound mixtures in to its components .Paper chromatography involves water present in paper as stationary phase and mobile phase is solvent used

1. PAPER CHROMATOGRAPHY
PRESENTED BY:
PRASANTH
PROFESSOR
AL SHIFA COLLEGE OF PHARMACY

2. DEFINITION
Paper chromatography is the technique in which the
separation of an unknown substance is mainly carried out
by the flow of solvents on the specially designed
chromatographic paper.

3. PRINCIPLE
 Principle involved is –PARTITION COEFFICIENT
PHENOMENON.
 Substances are distributed between two liquids
 STATIONARY PHASE ( moisture held in the fibers of the
paper)
 MOBILE PHASE (moving liquid or developing solvent).
 Components of the mixture to be separated migrate at
different rates & appear as spots at different points on the
paper.

4. TYPES OF PAPER CHROMATOGRAPHY
 PAPER PARTITION CHROMATOGRAPHY
 PAPER ADSORPTION CHROMATOGRAPHY

5. OPERATIONAL TECHNIQUE
1. CHOICE OF PROPER TECHNIQUE.
2. CHOICE OF FILTER PAPER.
The choice depends on:
-whether used for quantitative or qualitative analysis.
-whether used for analytical or preparative chromatography.
-whether substances used are hydrophilic or
lipophilic,neutral or charged species.

6. -Generally WHATMAN filter paper is used.
-Choice of particular WHATMAN paper depends upon the
type of separation.
-Coarser and faster paper. eg. WHATMAN 31 –used when Rf
value is wide apart.
-Slow papers are used ,eg. WHATMAN 20 – when Rf value is
closer.

7.  WHATMAN filter paper contain:
 98-99% -cellulose
 0.07-0.01% mineral content
 -cellulose
 ether soluble matter
 ammonia
 lipophilic substances (waxes,fats)
 Specially treated or modified filter papers are used like
Whatman-phosphate,Whatman-citrate etc.

8. 3. SOLVENT
General criteria for a good solvent system:
 Rf value should lie between 0.05-0.85
 Difference between Rf values –must be 0.05
 Solvents should not undergo chemical reaction.
 Solvents should not interfere with detection of spots.
 Composition of solvent system should not alter with time.

9. Eg. Of solvents :
n-hexane
cyclohexane
benzene
n-butanol ,etc..
Eg of solvent system
stationary phase mobile phase
water isopropanol-ammonia-water
(9:1:2)
water n-butanol-acetic acid-water
(4:1:5)

10. 4. PREPARATION OF SAMPLES
-The sample volume of 10-20  having as many g of
substance is the ideal quantity to be spotted.
5. SPOTTING
- With the help of a graduated micro pipette,the test solutions
are applied.
6. DRYING THE CHROMATOGRAM
- dried in special drying cabinets being heated electrically
with temperature controls.

11. 7. VISUALISATION
-by chemical methods
-chromatogenic or visualising reagents,eg.ninhydrin
in acetone for amino acids
-by physical methods
-iodine chamber,uv chamber for flourescent
compounds.

12. DEVELOPMENT TECHNIQUES
1.ASCENDING DEVELOPMENT
-solvent flows against gravity.
-spots kept at bottom portion of paper.
-the development of paper is done by allowing the solvent to
travel up the paper.
-in this mobile phase is placed in a suitable container at the
bottom of the chamber and the paper is suspended from a
hook.

14. 2.DESCENDING DEVELOPMENT
- solvent holder at top & also spot at top of paper
- Solvent flow assissted by gravity.
- The development of paper is by allowing the solvent to
travel down the paper.
- The apparatus consist of a well sealed glass tank
provided with mobile phase at upper portion.
- Development is faster.

16. 3.ASCENDING -DESCENDING
-combination of both
-upper part of ascending chromatography –folded over a
glass rod allowing to change into descending .
4. RADIAL PAPER CHROMATOGRAPHY
-also known as circular paper chromatography.
-radial development takes place.

18. 5. TWO DIMENSIONAL DEVELOPMENT
-square or rectangular paper is used.
-sample applied to one of the corners.
-2nd development is done at right angle to the direction of the
first run using another solvent system.

19. MIGRATION PARAMETERS
RF VALUE
RF = distance travelled by solute
distance travelled by solvent front
value ranges from 0-1
RX VALUE
RX = distance travelled by sample
distance travelled by standard
value is closer to 1

20. RM VALUE
RM = log (1/R F-1)
RF values of chemical related compounds are very close.
RF values depend on:
 Solvent employed
 Medium for separation
 Nature of mixture
 Temperature
 Size of chamber

21. APPLICATIONS
1.Analysis of polar compounds like amino
acids,sugars,natural products.
2.Separation of mixtures of drugs of chemical or biological
origin,plant extracts etc..
3.Separation of
carbohydrates,vitamins,antibiotics,proteins,alkaloids,glycosi
des.
4.Identification of drugs
5.Identification of impurities

22. 6.Identification of foreign substances in drugs.
7.Identification of decomposition products
8.Analysis of metabolites of drugs in blood,urine etc