The use of paper as a chromatographic medium is usually regarded as a typically partition system, where the stationary phase is water, held by adsorption on cellulose molecules, which in turn are kept in a fixed position by the fibrous structure of the paper. It is now realized that the paper used for the inert support of the mobile phase.
TYPES OF PAPERS
The major work of paper chromatography has been carried out on standard filter paper material, however there are still commercially available a range of chromatographic papers.
i; Pure cellulose (which on burning does not give ashes)
ii; Silica gel loaded. –
iii; Ion exchange cellulose.
iv; Resin loaded cellulose.
These papers are manufactured to a high specification with controlled porosity, thickness and matting characteristics and are low metal content. Chromatographic paper is available in sheets (18×22 In) or in strips line x several hundred feet. Each manufacturer supplies chromatographic paper in several different grades, the difference being principally in the density and thickness. The primary role of the paper is to act as a support for the stationary phase. In general, Whatman No I or Whatman No 3 filter papers are used for analytical work and Whatman No 3 or Whatman No 3MM for preparative work.
Characteristics of Whatman chromatographic papers are given below:
Table: Rate of flow of liquid on filter paper.
SAMPLE FOR CHROMATOGRAPHY
The mixture to be separated is applied to the paper as a solution. Solids are dissolved in a small quantity of a suitable solvent. The extracts of animal or plant tissues are prepared by grinding them irra suitable solvent. The insoluble material is then removed by filtration. The extract thus obtained can be directly applied or afIer concentrating it. The solution should be 0.5 % and of which 10 pl is applied to the paper. The precise amount and concentration will depend on the complexity of the sample mixture and the sensitivity of the detection system. The solvent is evaporated completely without attacking the paper. Solid samples, such as oils, or biological cell or tissue materials are macerated with the solvent. Many important samples, such as urine or other biological fluids, are already in an aqueous medium. In other cases water is used as the solvent when the substances are soluble in it.
SPOTTING OF THE SAMPLE ON PAPER
After selecting the size and grade of paper to be used, a pencil line is drawn 1.5 cm above the lower edge of the chromatographic japer and at a suitable distance from it a number of small crosses are marked on the line at equal distance corresponding to the numbpr of the samples to be applied. The nature of each sample is written against its cross on the paper with pencil. A drop of each sample is ‘spotted on to the appropriate position with a short length of capillary tubing or platinum loop. Large spots should be avoided as they lead to poorer separations. The spots are dried with the help of a hair dryer. When the spots are dry, the paPer is ready for development. “Development is the name given to the process in which a solvent flows through the paper to produce separation”
CHOICE OF SOLVENT
There are a number of solvents which can be used in paper chromatography. The final selection depends entirely On the nature of the substances to be separated. There are two ways to solve the problem. The first is the knowledge which one could get from literatures and the second is by trial and error method.
In general, chromatographic solvents for paper partition chromatography can he made by simply saturating an organic solvent such as n-butanol with water. Many popular solvents are of this type, but several solvent systems incorporate a small amount of water so that the polar compounds like amino acids, sugars, and phenolic compounds etc. move very slowly or fail to separate in such binary systems. Therefore, a third component may be added which may be an acid, a base or complexing agent. Some of the solvent systems commonly used are given in table below:
SOLVENTS FOR PAPER CHROMATOGRAPHY
Substances to be
Saturated solution 1:1:!
Ethyl acetate-pyridine-water-n-butanoI-1.5M NH3
2:1:2 Saturated solution
Co, Mn, Ni, Cu, Fe (Chlorides)
F. Cl. Br. I (Na salts) 11g. Ph. Cd. Cu, I
As, SI), Sn (Chloride)
Acetyl acetone (saturated solution in water)-acetone-conc. HC1