Concentration of Solutes for Paper Chromatography OSCAR F. WIEGAND and A. R. SCHRANK Department of Zoology, The University o f Texas, Austin 72, Tex.
shapes or subdivisions may be used, but each portion of paper should have one solute-free area attached. This arrangement is illustrated in Figure 1, D. These paper sections, with the indicated cuts through the dikes, are used as wicks; the solutes contained in them are transferred to the paper to be chromatographed as illustrated in Figure 1, E and F . h small fold (0.5 mm.) is made in the solutecontaining end of the wick and pressed into a slit in the desired location on the chromatographic paper. The solute is transferred to the paper by elution. By immersing only the solutefree area of the wick in the solvent, loss of the solute is prevented and a quantitative transfer is obtained. Short aide x-icks are eluted quickly; long narrow ones more slowly. This means that the spot size around the slit can be controlled. To obtain the transfer conveniently, to prevent the saturation of the paper with vapor from the eluting solvent, and to provide adequate support for the paper and wick over the solvent, the dish containing the solvent should be covered with aluminum foil with a p p r o p r i a t e 1 y spaced slots for the wicks (Figure 1, F ) . A current of dry air passed over the paper will e v a p o r a t e t h e rising s o l u t i o n w i t h negligible evaporation of the solvent in the I 2 3 4 dish. This technique has Figure 2. Diagram of been most useful in dechromatograms positing solutes from 1. 2. Solutes deposited i n dikes verv dilute solutions. 3. Solutes deposited from micronine*. The solut,es are de= -=-"~ 4. Solutes deposited from paper posited in a narrow
Extreme concentration of small volumes of solution (2 ml.) leads to inaccuracy in analysis because of losses by evaporation, wetting, transfer, chemical reactions, increasing ionic strength, and other factors. Paraffin dikes were devised to hold and concentrate the solutions of extracts from small amounts of plant tissue directly on the chromatographic paper. Aliquots of 50 to 100 pl. were deposited at once in the area normally given to 5 pl. Handling of solutions is simplified and more precision is obtained. The method is versatile and simple; reactions may be carried out directly on the paper.
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REQUENTLY, in the chromatographic assay of crude e\tracts, a special technique is required to get enough solute from dilute solutions onto the paper. The custoniar? use of micropipets of small capacity ( 5 pl. ) becomes time-consuming and tedious. Extensive evaporation of solvent may sometimes be impractical when the extract yields are limited, or nhen such treatment causes changes in the amounts of constituents because of chemical reactions. Three simple techniques have been devised which have proved useful in concentrating ektracts Two of the techniques involve the use of paraffin dikes. For the first method the edge of a heated glass microscope slide is touched to a block of paraffin and then to the chromatographic paper to surround a square or rectangle vith an area of 1 to 2 sq. em. I t is important to deposit just enough paraffin to permeate the paper completely, but this is easily accomplished and can be checked by looking a t the underside of the paper. The solution to be analyzed is placed on the square area of paper surrounded by the dikes in successive aliquots of 50 to 200 pl. and each aliquot is evaporated by a stream of warm, dry air This effect is illustrated in Figure 1, -4. Sheets containing the solute material m a r be stored in a dry atmosphere or developed immediately. Prior to developing, a number of razor blade cuts are made through the paraffin dikes on two sides of the square to permit the solvent to move into and out of the solute-containing area (Figure 1, B ) . Ri values remain unaltered and no additional spots are contributed in the usual solvents by the paraffin. In case of doubt, controls may be run simultaneously. In the second method the extract is placed in a rectangular area surrounded by paraffin dikes. The area is cut out after drying, as indicated by the dashed lines in Figure 1, C. Other 50-200,d.
wicks
band around the slit so that there is a preliminary separation. Chromatograms made in this way shon- compact spots with little "tailing," and the method gives unusually good resolution. The proper choice of solvents will help to eliminate undesirable constituents of the extracts originally deposited. These dike and wick techniques have been used in analysis of amino acids. I t is also possible to carry out enzymatic reactions a t low temperatures directly on the paper area surrounded by the dikes by keeping that portion of the paper in a moist atmosphere between two Petri dish tops. The third method, which is represented in Figure 1, G, involves the transfer of the solution from a bent micropipet onto the paper bv capillarity. The size of the spot is controlled by the rate of evaporation and by the rate of delivery, which is determined b r the size of the orifice in the pipet tip. This is a simplification of a method previously reported ( 1 ) . Figure 2 shovis the pattern of movement of the spots in each of the described methods. T h e solvent front may be depressed for a short distance above the spots because of the slow-er movement through the dikes.
CUT OUT ON DOTTED L I N E
Paraffin dikes
A Wick of Soln S0ln.Y Y olaced in slii
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ACKNOWLEDGMENT cowar
This work was supported in part by the Atomic Energy Commission.
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LITERATURE CITED
Figure 1. Schematic diagrams illustrating several methods for concentrating extracts for paper chromatography
(1) Urbach, K. F., Science 109, 259 (1949). RECEIVED for review September 26, 1965.
259
Accepted November 14, 1955.
