Paper chromatography ascending technique

The substance having same Revalue can be separated by two dimensional paper chromatography. Descending technique is better because compounds with low Ry-values which cannot be completely separated by ascending method can be separated by descending technique. 

The different methods of arranging the filter paper in the chromatographic chamber have been applied to sugar separations descending, ascending,39 40 and circular or horizontal development.41 These methods and other techniques will be discussed below. Detailed descriptions of the equipment needed for paper chromatography are readily available.8... 

With paper chromatography a descending technique may be more convenient (though an ascending method can of course also be used). 

The range of development techniques available in paper chromatography is similar to those employed in TLC, namely, ascending, descending, radial. 

The simplest and cheapest technique is paper chromatography, where the chromatographic bed consists of a sheet of paper, i.e., cellulose. The stationary phase consists of water adsorbed to the cellulose as well as of the polymer itself, although ion exchange and complexation processes may play an important role. The sample solution is applied as a spot near one end of the paper. A few centimeters of the sheet are dipped into the mobile phase which then ascends (or descends, as descending mode is also possible) into the stationary phase. When the mobile phase has almost reached the other end of the sheet the paper is removed from the developing tank and dried. If the analytes are not visible because they are not colored, the sheet is treated with a reagent to visualize the spots. 

The choice of solvents (Table 11.2) and the methods of elution are much the same as for paper chromatography. The procedure must of course be conducted in a closed ehamber to prevent evaporation of the solvent and the technique used is ascending out of necessity. Two dimensional chromatography may also be carried out much in the same way as described for paper chromatography. One of the greatest advantages of TLC is the speed at which the separation is achieved. Generally 10-30 minutes are sufficient. However, with certain compounds about 90 minutes may be required. 

Paper chromatography is one of the earlier techniques for the separation of cobal-amins and corrinoids. Whatman nr.2 paper with xcc-butanol/glacial acetic acid/ water (100 3 50) (10) or n-butanol/isopropanol/water (10 7 10) (11) is most frequently used. Whatman 3MM paper has been found useful for the separation of acid hydrolysis products of cyanocobalamin using 2-butanol/glacial acetic acid/water (440 4.1 150) with 0.07% KCN as the ascending solvent (79). A similar system was applied by Kolhouse and Allen (80) for the preparation of various cobalamin analogs and by Kondo et al (81) for the analytical separation of analogs from animal tissues. 

In addition to cellulose papers, modified-cellulose papers and glass-papers are also used for specific purposes. It is difficult to decide whether some self-supporting layers such as micropore membrane filter sheets or powders held in glass-paper sheets should be included among papers or thin layers. Paper chromatography is carried out in a descendant, ascendent or radial (circular) fashion. The diversity of the technique and the innumerable purposes to which it can be applied are discussed fully by textbooks and bibliographies, to which the reader is referred. 

VI.19 APPARATUS AND TECHNIQUE FOR CHROMATOGRAPHIC SEPARATIONS In paper and thin layer chromatography we may use ascending or descending technique. In the former the solvent moves upwards, while in the latter downwards. 

---