J. 5. Knypl
and R. Antoszewski University of L6di Poland
II
Method for Determining the Area of spots in Paper Chromatography
Present methods used to measure the areas of spots in quantitative chromatographic and chemichromatographic analysis are laborious, inaccurate, and dependent on subjective factors. Among these is the planimetric method,' the method in which the spots are drawn on coordinate paper,2 and the method in which the spots, or copies of them, are cut out and weighed.8 These methods each have their own errors. Some destroy the chromatogram, and none yields permanent records. The present method uses photography to provide a simple, objective way of measuring the area of a spot in a paper chromatogram. Between two pieces of glass which have been cleaned with soap and alcohol, there is formed a multiple "sandwich" consisting, from the top down, of the paper with the spot to he tested, under this a piece of transparent drawing paper with a millimeter grid drawn on it, under this the photosensitive material (paper or film), and then the lower glass plate. The whole sandwich is held tightly compressed by two rubber bands. It is illuminated from the top with light, preferably complementary to the color of the spot to be measured. White light may also be used, and in this case, results are improved if a suitable filter is introduced between the paper hearing the spot and the paper hearing the millimeter grid. The time of exposure, depending as it does on the intensity of the light, the type of paper or film used, and the transparency of the sandwich, is chosen by trial. The accompanying photographs were made by the method described. The lamp used was the ultraviolet lamp, "Minerallight" with filter SL 2537, and a type of photographic paper "Fotopan" C 26' 111 C, made in Poland was employed. It must he stressed that the conditions of copying must be kept uniform through a series of copies. The length of exposure, as well as the conditions of development of the paper or film, influence the magnitude of the systematic error. EDITOR'S NOTE: The assistanoe of Professor Harold G . Cmsidy, Yale University, in the preparation of this manuscript is gratefully acknowledged. 'SEILER,H., SORKIN,E.,AND ERLENMEYER, H., Helu. Chim. Acta, 35, 120 (1952). l L ~ w ~ ~ ~ o w sA,, x r "Chromatografia," , red. J. OpiehskaBlauth, Warszawa, P. W. N., 1957, p. 723. BEERSTEHER, E., Anal. Chem., 22, 1200 (1950).
I n working with the semicircular spots (Fig. 1) it is suggested that the paper be lengthened a t the bottom, using a clean piece of impregnated paper so as to make the millimeter grid more visible. Then the lower edge
Figure 1. (left1 Chemichromatogrophic spot from o 3eries made in the impregnating technique for determining phorphorur. Figure 2. (right) Chernichromatogrophic spot from the o n ~ l y r i rof 0 pro. tein hydrolyrote on paper impregnoted with copper orthophorphote.
of the paper should he aligned exactly with one of the heavier lines of the millimeter grid, as shown. Good results with fluorescent spots are obtained by using a filter impervious to ultraviolet between the chromatographic paper and the grid. Thus, an inverse image is obtained with the millimeter grid plainly visible on the spot and the rest of the paper remaining white. The photographic paper or film is then preferably chosen to have reduced sensitivity for ultraviolet light. The same results are obtained in recording transparent spots. Of course, in the case of spots which absorb in the ultraviolet or which fluoresce under ultraviolet, the upper plate must pass ultraviolet light. (Fig. 2) This method gives a permanent record for measurement and calculation of surface area. The subjective element in measuring the spot is reduced to a minimum. The method may he used also to gauge retentiograms. The method has the added advantage that it is independent of any changes in size that the paper or film may undergo during chemical processing, since the scale is subject to the same changes. The method may be used to measure the areas of other irregular surfaces, such as the surfaces of leaves or of microscopic preparations photographed by the lanametric method, etc.
Volume 37, Number I , Jonuary 7960
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