62 . Biological Stains 



of thionin and methylene blue, but occupying an intermediate 

 position in the spectrum. 



This illustration shows how valuable the spectrophotometric 

 analysis may be in determining whether a given product is a simple 

 dye or a mixture of two or more dyes. This fact, together with its 

 use in determining the exact shade of any dye, makes it the most 

 valuable test to apply to a stain, other than to determine by actual 

 use whether the sample will prove satisfactory to the microscopist 

 or not. 



The user of biological stains may well question, however, 

 whether spectrophotometric data have any actual value for him in 

 guiding him in the use of dyes. Such data as are given in this 

 book do in fact have a distinct use, provided he understands the 

 principle underlying them. Let us suppose, for example, that never 

 having heard of pyronin, he did not know its color; in such a case 

 the absorption maximum (552) as given on p. 178 would furnish 

 him this information. By referring to the spectrum and its compli- 

 mentary colors on page 54 he would find that an absorption maxi- 

 mum at 55^ means a primary absorption of green light; and as the 

 complementary color is red, he would thus learn that pyronin is a 

 red dye. In a similar way if he were trying to decide between using 

 one or the other of two closely related dyes, knowledge of their ab- 

 sorption maxima might help him, provided the decision depended 

 on shade. Thus if he were using azure A and obtained a shade 

 somewhat too red, the spectrophotometric data given on pp. 107-110 

 would show him whether to substitute azure B or azure C. It 

 will be seen that the absorption maxima of azures A, B, and C are 

 respectively 628, 652^ and 609. Reference again to the Table 1 on 

 p. 54 shows that the complementary color corresponding to wave- 

 length 65^ would be on the blue side of 628, while 609 would be on 

 the red side; accordingly azure B should be selected if bluer stain- 

 ing than with azure A were desired. 



It should be remarked that the absorption curve of a dye is not 

 necessarily the same in two different solvents. In the following 

 pages aqueous solutions are to be understood unless some other 

 solvent is specified. 



CHROMATOGRAPHY 



Recently more and more stress is coming to be laid on a dif- 

 ferent method of dye analysis known as chromatography. It has 

 no real relation to spectrophotometry, but it is considered in this 

 chapter because it also furnishes a means of separation and iden- 

 tification of dyes on the basis of differences in color that are not 

 shown by simple visual comparison. 



When two different chemicals are in solution together and are 

 brought into contact with porous substance in which capillary rise 

 readily takes place, it is rare that both compounds will pass through 

 the pores of the substance at the same rate. The result is the 



