Appendix III E. Paper Chromatography

Apparatus The apparatus consists of a glass tank of suitable size for the chromatographic paper used, ground at the top to take a closely fitting lid. In the top of the tank is a device which suspends the chromatographic paper and is capable of being lowered without opening the chamber. In the bottom of the tank is a dish to contain the mobile phase into which the paper may be lowered. The chromatographic paper consists of suitable filter paper, cut into strips of sufficient length and not less than 2.5  cm wide; the paper is cut so that the mobile phase runs in the direction of the grain of the paper.

Method Place in the dish a layer 2.5  cm deep of the mobile phase prescribed in the monograph. If prescribed in the monograph, pour the stationary phase between the walls of the tank and the dish. Close the tank and allow to stand for 24  h at 20  °C to 25  °C. Maintain the tank at this temperature throughout the subsequent procedure. Draw a fine pencil line horizontally across the paper 3  cm from one end. Using a micro pipette, apply to a spot on the pencil line the volume of the solution prescribed in the monograph. If the total volume to be applied would produce a spot more than 10  mm in diameter, apply the solution in portions allowing each to dry before the next application. When more than one chromatogram is to be run on the same strip of paper, space the solutions along the pencil line at points not less than 3  cm apart. Insert the paper into the tank, close the lid and allow to stand for 1  h  30  min. Lower the paper into the mobile phase and allow elution to proceed for the prescribed distance or time. Remove the paper from the tank and allow to dry in air. Protect the paper from bright light during the elution process.

Apparatus The apparatus consists of a glass tank of suitable size for the chromatographic paper used, ground at the top to take a closely fitting glass lid. The lid has a central hole about 1.5  cm in diameter closed by a heavy glass plate or a stopper. In the upper part of the tank is suspended a solvent trough with a device for holding the chromatographic paper. On each side of the trough, parallel to and slightly above its upper edges, are two glass guide rods to support the paper in such a manner that no part of it is in contact with the walls of the tank. The chromatographic paper consists of suitable filter paper, cut into strips of sufficient length, and of any convenient width between 2.5  cm and the length of the trough; the paper is cut so that the mobile phase runs in the direction of the grain of the paper.

Method Place in the bottom of the tank a layer 2.5  cm deep of the solvent prescribed in the monograph, close the tank and allow to stand for 24  h at 20  °C to 25  °C. Maintain the tank at this temperature throughout the subsequent procedure. Draw a fine pencil line horizontally across the paper at such a distance from one end that when this end is secured in the solvent trough and the remainder of the paper is hanging freely over the guide rod, the line is a few centimetres below the guide rod and parallel with it. Using a micro-pipette, apply on the pencil line the volume of the solution prescribed in the monograph. If the total volume to be applied would produce a spot more than 10  mm in diameter, apply the solution in portions, allowing each to dry before the next application. When more than one chromatogram is to be run on the same strip of paper, space the solutions along the pencil line at points not less than 3  cm apart. Insert the paper in the tank, close the lid, and allow to stand for 1  h  30  min. Introduce into the solvent trough, through the hole in the lid, a sufficient quantity of the mobile phase, close the tank and allow elution to proceed for the prescribed distance or time. Remove the paper from the tank and allow to dry in air. The paper should be protected from bright light during the elution process.