Paper chromatography apparatus

Figure 11.22 represents a paper electrophoresis apparatus. The soaked cellulose sheet is sandwiched between two horizontal glass plates with the ends dipped into vessels containing more electrolyte solution. The electrodes are also dipped into these vessels, as shown. The sample is spotted in the center of the sheet, and the oppositely charged ions then have room to migrate in opposite directions on the sheet. Qualitative analysis is performed much as with paper chromatography, by comparing the distances the... 

A very efficient thin layer form of circular paper chromatography makes use of a circular glass disc coated with an adsorbent (silica, alumina or cellulose). The apparatus is called a Chromatotron (available from Harrison Research, USA). The disc is rotated by a motor, and the sample followed by the eluting solvent are allowed to drip onto a central position on the plate. As the plate rotates the solvent elutes the mixture, centrifugally, while separating the components in the form of circles radiating from the central point. When elution is complete the revolving circular plate is stopped and the circular bands are scraped off and extracted with a suitable solvent. 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

Classic paper chromatography like distribution chromatography is also carried out today in most cases in layers on a support sheet. To obtain low standard deviations extensive automatization from sample application to evaluation is necessary. The essential advantage in comparison to liquid column chromatography is the possibility of separating several samples side by side, because therefor usually no apparatus is required. 

Paper chromatography has now, to a great extent, replaced alumina or similar absorbent columns. An advantage is that much smaller quantities can be used. The apparatus is illustrated diagrammatically in Fig. 25.13. The sheet of filter paper is suspended so that its bottom dips into a vessel containing the eluent and the upper end is attached by clips to a frame (Fig. 25.14). 

Paper chromatography is usually used for highly polar compounds such as sugars, amino acids and natmal pigments. Some advantages of paper chromatography are a small amount of sample is required, a high level of resolution, ease of detection and simphcity of the apparatus. 

Figure 21-1. Apparatus required for ascending paper chromatography.

Figure 21-2. Apparatus required for descending paper chromatography.

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. 

Study of the literature of paper chromatography reveals many views on the establishment of equilibrium in the tank and some authors have given it special attention [70]. It is not possible to generalise because the degree of equilibration needed depends on the size of the apparatus, the solvent system and the nature and purpose of the separation. The object is to prevent evaporation of the solvent from the paper. For most applications it seems that it is not necessary to go to extreme lengths, and some of the meticulous and elaborate equilibration carried out by earlier workers was probably not really necessary. 

A paper strip, suitably supported, dips at its ends into electrode vessels containing a buffer solution, acting as the electrolyte. The paper is soaked in the buffer, and the sample is applied at some point on the strip as a thin transverse streak (by the same method as used in paper chromatography). The electrodes are connected to a d.c. source, and field applied for a predetermined time see Section 3.3.4 for further details. The strip is removed from the apparatus, dried, and bands located—again as in paper and thin layer chromatography. 

The use of apparatus which has been developed for centrifugal paper chromatography (Firms 11, 58) can be extended to TLC also. Kokzun and Bbady [375] have thus separated in 10 min a mixture of dyes on circular glass or aluminium plates (20 cm diameter) at a speed of 500—700 revs/min. In the ordinary way, this separation requires 35 min. 

Bij and Lederer (39) used thin TLC (0.1 mm thick) and had no elongated spots as when he used paper chromatography. The separations were fast (10 min), with good theoretical plates (5000) at Rjs under 5.5 cm. No HPLC or complicated apparatus was required, and no buildup of background radiation occurred due to TLC disposal. Chromatography required ammonium sulphate (0.2-5.0 M). Salts [many less ionized than (NH4)2S04) contributed little. pH (borax, acetate, HCI, ammonium) contributed little to separations achieved with ammonium sulphate. 

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