Spedrophotometric Analysis 63 



production of a coluiiiii in which each compound tends to be lo- 

 cated in definite bands. This phenomenon was naturally first 

 observed in the case of colored compounds; hence the name chroma- 

 tography, although it is now used, fully as much for uncolored as 

 for colored compounds. Dyes lend themselves naturally to this 

 method of analysis; although until recently it has not been much 

 used for them. The method is of value not only for showing the 

 various bands of color that develop, but actually to isolate the 

 individual constituents of the mixture, as the column can be cut 

 into its various sections and the chemical there localized can be 

 eluted by the proper reagent and thus obtained in pure form. 



The method as originally developed called for a column of alum- 

 ina in a glass tube, in which the bands are located at points deter- 

 mined by the differing degrees of adsorption taking place between 

 the alumina and the different constituents of the mixture. A more 

 recent development, known as paper partition chromatography, 

 depends on a different principle, the relative solubility of different 

 compounds in two solvents, together with the aflSnity of cellulose 

 fibers for the solvents, which determines the rate of passage through 

 the pores of the filter paper. The paper partition method has 

 several advantages, among them the ease with which the chromato- 

 grams can be preserved and with which sections can be removed for 

 extraction of the individual fractions. Simultaneous papers 

 (Emery and Stotz, 1952; Christman and Trubey, 1952) have des- 

 cribed the application of the technic to dye analysis; and at about 

 the same time, Evans and Walls (1952) have shown the possi- 

 bility of getting very similar chromatographic pictures by the use 

 of filter paper electrophoresis. 



A chromatographic strip obtained in this way from a mixture of 

 dyes can be very striking. Emery and Stotz show that a mixture 

 of three dyes (eosin Y, orange G, and erythrosin B) in a mixture 

 of butanol and water, and allowed to diffuse from a spot at the 

 top of a filter paper strip, develops five zones of color, one orange, 

 one red, two pink and fluorescent, one fluorescent without color in 

 ordinary illumination; of these five, the three fluorescent zones all 

 come from eosin Y, the other two from orange G and erythrosin 

 respectively. These authors, by cutting out the various zones 

 have been able to isolate the individual dyes and to figure the con- 

 centration of each in the original mixture. 



This illustration is given merely to show the probable value of 

 the method. Much more work must be done, before it can be used 

 in routine dye analysis, and it is not likely to replace spectro- 

 photometry; but, as shown by Emery and Stotz it can serve to 

 reveal the identity of dyes in a mixture, even though one of them 

 (in this case eosin Y) is present in such strength as to mask the 

 others when analysis must depend on the spectrophotometer. Ac- 

 cordingly it is felt that chromatography may have an important 

 place in future assay methods for biological stains. 



