Over the years different types of paper chromatography have evolved which have been classified by the direction of flow of the mobile liquid phase
Ascending Paper Chromatography
As the name suggests the developing solvent moves in an upward direction. Sufficient quantity of mobile phase is poured into the development chamber. Sample and reference are spotted on the line drawn a few centimeters from the bottom edge of the paper suspended from a hook or clip at the top. Alternately, the paper can be rolled in the shape of a cylinder and kept inside the development chamber. Solvent moves up through capillary action. The rate of rise of solvent front decreases with time and some chromatograms can be left overnight for the components to effectively separate. It is important to cover the container with a plate so that the atmosphere inside the development chamber is saturated with the solvent vapour. Separation of the atmosphere with the solvent vapour prevents evaporation of the solvent as it rises up the paper.
Descending Paper Chromatography
In this technique the solvent front travels down the length of paper suspended from the top inside the developing chamber. The mobile phase is kept in a trough in the upper chamber. The paper with spotting on the line drawn a few centimeters from the top is clamped to the top. Prior to elution the jar is covered and equilibrated with the mobile phase vapour.
The solvent moves downwards through the combined capillary action and gravitational pull. The advantage of this technique is that development can be continued indefinitely even though the solvent runs off at the bottom end of the paper
Ascending – Descending Chromatography
In the hybrid version the solvent first travels upwards on the paper which is folded over a rod and it continues with its travel downwards after crossing the rod. This arrangement permits longer development period for better resolution of complex mixtures
Two dimensional chromatography helps resolve substances having similar Rf values.
A single spot of the mixture is applied close to one end of the baseline on the square filter paper. The paper is allowed to develop by either ascending or descending mode and on completion allowed to dry. The filter paper is now rotated 90° so that the edge having the series of separated spots is at the bottom and allowed to develop in the same or a different solvent phase
The arrangement helps to resolve the spots in two-dimensions by spreading the spots on the filter paper.
Horizontal or Circular Paper Chromatography
Horizontal or Circular Paper Chromatography permits separation of sample components in the form of concentric circular zones through radial movement of liquid phase. A circular shaped filter paper is employed and different arrangements are used.
Arrangement – 1
A narrow slit about 2mm wide is cut from the circumference to the centre and the wick formed is bent so as to dip in the solvent contained in a petri dish. Sample spotting is done along the circumference of the smaller inner circle. The petri dish is covered after securing the filter paper to the top and allowed to develop. The mobile phase reaches the filter paper through capillary action and spreads radially outwards thereby resolving the sample components radially.
Arrangement – 2
In this arrangement there is no need to cut a wick. A small capillary is inserted through an adjustable collar placed inside the solvent inside the petri dish. After applying the sample spot to the centre of a circular filter paper it is covered with a petri dish cover and allowed to develop. As before the solvent is drawn up by capillary action of the filter paper and spreads radially outwards thereby resolving the sample constituents radially.