PAPER CHROMATOGRAPHY

PAPER CHROMATOGRAPHY

What Is Paper Chromatography?

Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary phase through which a solution is made to pass is called paper chromatography.

It is an inexpensive method of separating dissolved chemical substances by their different migration rates across the sheets of paper.

It is a powerful analytical tool that uses very small quantities of material. Paper chromatography was discovered by Synge and Martin in the year 1943.

Paper Chromatography Principle

It involved partition or adsorption chromatography.

Partition chromatography because the substances are partitioned or distributed between liquid phases. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper. When the mobile phase moves, the separation of mixture takes place.

Adsorption chromatography between solid and liquid phases, wherein the solid surface of the paper is the stationary phase and the liquid phase is the mobile phase.

Stationary phase and paper used

The paper chromatography is very similar to TLC. Difference is, instead of using a thin layer of silica on metal, it uses a special type of chromatography paper as stationery phase. This paper is made of cellulose. Cellulose is a polymer of simple sugar, glucose.

Cellulose contains -OH group similar to the silica or alumina on the TLC plate. The surface of cellulose is thus very polar. So the compounds can form hydrogen bond or can interact by van der waals dispersion forces and dipole dipole forces.

Stationary phase and paper used…

Paper useful :

Wattman paper No. 1, 2, 3, 4, 17, 20 etc

Choice of filter paper depend on Flow rate, thickness, purity & technique

Modified papers: Acid base washed glass fiber type

Hydrophobic paper:

Hydrophilic paper:

Impregnation of silica:

Paper size:

Paper Chromatography Procedure

Selecting a suitable type of development: 
It is decided based on the complexity of the solvent, paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to perform. Also, it is easy to handle, the chromatogram obtained is faster and the process is less time-consuming.

Selecting a suitable filter paper: Selection of filter paper is done based on the size of the pores, and the sample quality.

Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable solvent (inert with the sample under analysis) used in making the mobile phase.

Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a capillary tube.

Chromatogram development: Chromatogram development is spotted by immersing the paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper.

Paper drying and compound detection: 

Once the chromatogram is developed, paper is dried using an air drier. also, detecting  solution can be sprayed on the chromatogram developed paper and dried to identify the sample chromatogram spots.

Paper chromatography works in few steps

Step 1: A horizontal line is drawn near one end (about 1.5 cm from the bottom edge) of the paper.

Step 2: The sample needs to be separated is placed as a small drop or line on to the paper using capillary tube. Labeling the drop by a pencil with an alphabet or number help to identify the compound later. The drops are then soaked on the paper and dried.

Step 3: The paper is then placed into a sealed container with a swallow layer of  suitable solvent. The solvent level must be lower than the pencil line or drop on it. The container need to be covered to stop the solvent to evaporate.

Step 4: The solvent rises up the paper chromatography taking each component of the sample with it. The components travel with the solvent depends on three things:

• The polarity of the sample molecule. The non polar components    travel faster than  the polar component.

• The attraction between the sample molecule and the solvent or solvent mixture.

•  The attraction between the sample and the silica.

Suppose any sample compound mixture contains three colored molecules  blue, Purple and red. According to their polarity, the order of these compounds is blue<purple<red. Thus the most non polar blue will travel first along with the mobile phase. Then purple and at last most polar compound the red one.

Step 5: When the solvent rises near the end of the paper then the paper should be taken out from sealed container and air dried. The paper with separated bands of components are then observed under UV-light or by using detecting agents.

Detecting and visualizing agent

1.Non-specific methods – exact nature of drug is unknown

2.Specific methods – exact nature is known

     1. Non-specific methods

Iodine chamber

UV chamber for fluorescent compound (254nm & 365nm)

2. Specific methods

Ferric chloride – Phenolic comp and tannins

Ninhydrin in acetone – for amino acid

Dragandroff  reagent – for alkaloids

It can further be categorized as

Destructive – Use of reagent

Non destructive – UV, Iodine etc

Rf value

Qualitative Analysis

Rf  Value:   =    It is ratio of distance traveled by solute
               to distance traveled by solvent

Rf value always ranges form 0 to 1.

  Ideal is form 0.3 to 0.8

      Rx value:  =        Distance traveled by sample

                          Distance traveled by standard

Types of paper chromatography (PC)

Ascending PC: Solvent moves in an upward direction.

Descending PC: Solvent moves downwards direction

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Ascending – Descending PC:  Movement of solvent occurs in two directions after a particular point. Initially, the solvent travels upwards on the paper which is folded over a rod and after crossing the rod it continues with its travel in the downward direction.

Radial or Circular PC: The sample is deposited at the center of the circular filter paper. Once the spot is dried, the filter paper is tied horizontally on a Petri dish which contains the solvent.

Two Dimensional PC: Substances which have the same Rf values can be resolved with the help of two-dimensional paper chromatography.

Paper Chromatography Applications

ØTo inspect cosmetics.

ØTo detect the adulterants.

ØTo study the process of fermentation and ripening.

ØTo check the purity of pharmaceuticals.

ØTo detect the contaminants in drinks and foods.

ØTo examine the reaction mixtures in biochemical laboratories.

ØTo determine dopes and drugs in humans and animals.

ØSeparation of mix of drugs of chemical, biologic and plant origin.

ØSeparation of carbohydrate (sugars), vitamins, antibiotics, proteins, alkaloids, glycosides and amino acids .