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3.2 PAPER CHROMATOGRAPHY

3.2 PAPER CHROMATOGRAPHY

          In paper chromatography the adsorbant is a sheet of paper of suitable texture and thickness, which may sometimes be impregnated with a liquid phase that is immiscible with the mobile phase.

          The concept of Rf value discussed on thin-layer chromatography is applicable to paper chromatography. Because of the nature of the adsorbant it is possible to carry out paper chromatography in either a descending or an ascending mode.

Descending Paper Chromatography

          APPARATUS The apparatus is as follows:

          A glass chamber of suitable dimensions to accommodate 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 stopper.

          A solvent trough, suspended in the upper part of the chamber, with a device, usually a heavy glass rod, for holding the chromatographic paper. On either 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 chamber.

          The chromatographic 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.

          PROCEDURE Place in the bottom of the chromatographic chamber a layer 2 to 3 cm deep of the stationary phase specified in the monograph. Close the chamber, and allow to stand for 24 hours at constant temperature. Maintain the chamber under these conditions 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 to it. Apply to a spot on the pencil line the volume of the solution specified 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 chamber, close the lid, and allow to stand for 90 minutes. Introduce into the solvent trough, through the hole in the lid, a sufficient quantity of the mobile phase specified in the monograph, close the hole and allow development to proceed for the prescribed distance or time. Remove the paper from the chromatographic chamber and allow to dry in air. The paper should be protected from bright light during the development and drying processes.

Ascending Paper Chromatography

APPARATUS The apparatus is as follows:

A glass chamber of suitable dimensions to accommodate the chromatographic paper used, ground at the top to take a closely fitting glass lid.

A device, in the top of the chamber, to suspend the chromatographic paper and to be capable of being lowered without opening the chamber.

A dish, in the bottom of the chamber, to contain the mobile phase into which the paper may be lowered.

         The chromatographic 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.

         PROCEDURE Place in the dish a layer 2 to 3 cm deep of the mobile phase specified in the monograph. If specified in the monograph, pour the stationary phase between the walls of the chamber and the dish. Close the chromatographic chamber and allow to stand for 24 hours at constant temperature. Maintain the chamber at this temperature throughout the subsequent procedure. Apply the test substance to the paper as described in Descending Paper Chromatography. Insert the paper into the chamber, close the lid, and allow to stand for 90 minutes. Lower the paper into the mobile phase specified in the monograph, and allow development to proceed for the prescribed distance or time. Remove the paper from the chamber and allow to dry in air. The paper should be protected from bright light during the development and drying processes.

APPENDICES • 3.2 PAPER CHROMATOGRAPHY
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หมายเหตุ / Note : TP II 2011 PAGE 391-392