Paper chromatography descending 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... 

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. 

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

When using a mobile phase that formed two layers when it was prepared, it is considered good technique to use the nonmobile phase layer to establish equilibrium with the paper in descending chromatography. Why is this ... 

The apparatus used for continuous-flow paper electrophoresis can be used for separation with an elution rather than a continuous-flow method. The sample is spotted at the top of the paper and only the buffer solution is continuously added. If the electrophoresis is stopped before the sample components drip from the bottom of the paper, the technique combines the separating ability of descending paper chromatography (separation occurring vertically) with that of electrophoresis (separation occurring horizontally) to yield spots simultaneously developed in two dimensions. 

Descending. The descending technique, though more common in paper chromatography, can be used in TLC. The top of the plate, where the spots are located, has solvent from a trough fed onto it via a wick some solvent of the same composition is placed in the bottom of the tank but the plate is supported above the solvent level. In order to improve the resolution for particularly difficult separations a number of modifications to the above technique have been developed. 

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. 

General. Pyridine is distilled from p-toluenesulfonyl chloride and then from calcium hydride and stored over potassium hydroxide. N,N-Di-methylformamide is distilled from calcium hydride and stored over molecular sieves. Dioxane is distilled from phosphorus pentoxide. Tri-n-butylamine, triethylamine, diphenyl phosphorochloridate, and o- and p-fluorobenzoyl chlorides are distilled before use. Tri-n-butylammonium pyrophosphate is prepared at room temperature according to the method of Moffatt and Khorana and stored at 5°. Paper chromatography is carried out by the descending technique on Whatman No. 1 paper in (A) isobutyric acid-1 M NH4OH (60 40) and (B) 1-propanol-water (7 3). Electrophoresis is carried out on Whatman No. 1 paper at pH 3.5 in 0.035 M citric acid-0.0148 M sodium citrate (1 1). Evaporations are carried out under reduced pressure at bath temperatures below 30°. Phosphate analyses of nucleoside triphosphates are performed by the method of Lowry and Lopez after treatment of approximately 1 / mole of these compounds for 60 min at 22° in 1 ml of Tris chloride at pH 10.4 containing 0.02 mg of alkaline phosphatase of calf intestinal mucosa (type VII, Sigma Chemical Co.). 

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. 

---