Paper chromatography is a type of planar chromatography that uses paper as the stationary phase. It involves separating mixtures based on how compounds partition between the stationary phase (cellulose fibers in the paper) and a mobile liquid phase. When a sample mixture is applied to the paper and an appropriate solvent is run up the paper, the different compounds will travel different distances depending on how they partition between the paper and solvent. Their positions can then be detected visually or through chemical detection methods to separate and analyze the compounds in the original mixture. Paper chromatography is useful for separating and analyzing colored pigments, performing qualitative analyses, and examining complex mixtures in fields like pathology and forensics.

1. Paper chromatography
By: Asst. Prof. Sanjaykumar Uchibagle

2. INTRODUCTION:
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary (stationary phase) while the other (the mobile phase)
moves in a definite direction.
In 1906, Mikhail Tswett, the Russian botanist discovered
chromatography.
Chromatography, a group of methods for separating very small quantities
of complex mixtures, with very high resolution, is one of the most important
techniques in environmental analysis

3. Chromatographic terms
The analyte is the substance to be separated during chromatography.
A chromatogram is the visual output of the chromatograph.
The eluate is the mobile phase leaving the column.
The eluent is the solvent that carries the analyte
The detector refers to the instrument used for qualitative and
quantitative detection of analytes after separation.

4. FLOW CHART DIAGRAM OF TYPES OF CHROMATOGRAPHY

5. Paper chromatography is a type of Planar chromatography in which
paper is the support for stationary phase.
 It is a partition chromatography.
-Stationary Phase : Liquid, deposited in network of cellulose
fibers.
-Mobile Phase : Liquid, solvent(single or mixture)
 It is a liquid – liquid chromatography

6. Principle
 It is based on the principle of partition of a compound between two liquid
phases.
 The water hold in cellulose fibers of the paper acts as the stationary phase
and acts as one of the solvents (liquids).
 When a non–aqueous mobile phase moves along the paper, the components
of the loaded mixture distribute themselves between two phases in the ratio
characteristic of their distribution coefficients.
 The component which is more soluble in stationary phase moves slowly
while the component which is more soluble in mobile phase moves fast.
 As a result of this differential movement, components get separated.

7. Important Aspects of Chromatography
1. Filter Paper
2. Solvent System
3. Spraying reagents

8. Filter Paper
 Usually Whatman paper No. 1 is used.
 Characters : Uniform flow rate Grain size and texture
 Some times the papers are acid washed to remove traces of
impurities which may affect the analysis.
 Strip of whatman No. 1 paper

9. Solvent system
 Solvent is prepared by saturating organic solvent with water.
 But several organic liquids incorporate only small amount of water.
In such binary solvent system polar components of the mixture fail
to separate.
 In such cases a third liquid like acid(HCl, AcOH, HCOOH) or
base(NH3 or pyridine) or a complexing agent is often added to
retain more water in organic solvents.
 Hence, generally three component solvent systems are used

10. Some important solvent

11. Spraying reagents
 Coloured components do not require spraying reagent, they are
visible.
 Colourless components are located by spraying with specific
reagents. These reagents chemically react with the components
and develop colour in the region where they are present.

12. Methodology-1
 Sample loading Preparation of sample solution : 1-2% solution is
prepared in suitable solvent.
 A reference line at 2cm .
 Distance from the edge of the paper is drawn and points of application are
marked. The solution of mixture under test is spotted either as a fine line
or fine spot. Spotting is repeated twice or thrice.
Reference Line .

13. Methodology 2
 Developing Saturation of the paper strip: The sample loaded strip is
hanged for some time in the chromatographic jar containing the solvent
but without dipping in the solvent.
 The strip is developed by running the solvent through the paper.
 Depending on the direction of flow, there are two types paper
chromatography.
A. Ascending paper chromatography.
B. Descending paper chromatography

14. Ascending P. C.
 Solvent moves upward(ascends) from the bottom to top by capillary
action.
 The paper strip is placed vertically in the solvent keeping the reference
line above the solvent level.

15. Descending P. C.
 Solvent moves downward(descends) from the top to bottom.
 The paper strip is hanged vertically downward from the trough containing
solvent, keeping the spotted end at the top.
 The flow of solvent is faster than that in ascending P.C. But A.P.C. is
preferred than D.P.C. due to simplicity of set up.

16. Ascending-Descending Development.
 A hybrid of above two techniques is called Ascending-Descending
chromatography.
 Only length of separation increased first ascending takes place followed by
descending.
 Ascending-Descending Development

17. Radial modeCircular Development.
 Here the solvent travels from center(mid point) towards periphery of
Circular chromatography paper. The entire system is kept in a covered
petri dish for development of chromatogram.
 The wick at the center of paper dips into mobile phase in a Petri dish by
which the solvent drains on to the paper and moves the sample radially to
form the sample spots of different compounds as concentric rings. Radial
modeCircular Development.

19. Two Dimensional Development.
 Here the chromatogram development occurs in two directions at right
angles.
 In this mode the samples are spotted to one corner of rectangular paper
and allowed for first development. Then the paper is again immersed in
mobile phase at right angle to previous development for second
chromatogram.

20. Spiral/Ascending Descending.
 It is a hybrid of two techniques.
 The upper part of ascending chromatography can be folded over a glass rod
allowing the descending development to change over into the descending
after crossing the glass rod.
 The chromatogram is repeatedly developed in the same direction and thus
the complete resolution of two or more substances which have Rf values
close together can be obtained.
 As the mobile phase one can use either the same solvent system or different
solvent systems.

21. Analysis of Spot Detection.
 Colorless Spots.
 Non destructive
 Destructive
 Nondestructive spots can be viewed using physical methods
which is by UV detection
 UV light makes them visible and glow.

23. Destructive types of spots can be viewed by chemical methods through locating
agents
Locating agents are of 2 types (a) Specific locating agent (b) Non specific locating
agents
Basically chemical methods involve application of locating agents . Reagents give
coloured compounds after reacting.
Specific locating agents are basically of 2 types oNinhydrin : forms coloured
complexes with amino acids.
2,4 Di nitro phenol hydrazine : used o locate aldehydes and ketones oReagents are
those which react with specific components having specific functional groups
Non specific types form colored compounds with various organic compounds.
Example of non specific L.A are iodine , ammonia , H2SO4 and H2S oLocating
agents are applied by (a) Dipping method (b) spraying method.

24. Application.
 Separating Colored Pigments
 Qualitative Analysis
 Pathology and Forensic Science
 Analyzing Complex Mixtures Applications

25. Thank You!