General Nature of Dyes 41 



ASSAY METHODS 



When the project of stain standardization was undertaken, in 

 the early '20's, it was realized that reliable methods of assay were 

 needed. They were so definitely lacking at that time that in the 

 early certification work, primary stress was laid on performance of 

 the batches tested; chemical and optical assay methods were at the 

 start more or less incidental. This was regarded as an unsatis- 

 factory and temporary expedient; but it was adopted because 

 tests for performance were then regarded as more reliable than the 

 assay methods then available. 



These methods were crude. Dye content had to be determined 

 bv means of the rather trickv method of reduction with titanous 

 chloride. This procedure depends on the fact that reducable rad- 

 icals are present in almost all dj^es, so that the dye strength of a 

 sample can be determined by TiCls titration, provided that: (1) 

 the end point is sharp; (2) the molecular weight of the dye is 

 known. These provisos make the method sometimes inapplicable. 

 It often happens that the correlation is very poor between titra- 

 tion and colorimetric comparison of various samples of the same 

 dye. As a result considerable research is often needed to establish 

 a reliable assay method; nitrogen determinations or other more 

 absolute procedures must be resorted to in order to establish a 

 satisfactory norm for actual dye content determinations. 



In addition to assay for dye content, the spectrophotometer 

 was adopted quite early in the work for determining whether or 

 not any sample was true to type. The original spectrophotom- 

 eters (see Chapter III) were visual instruments, and it was quite 

 laborious to make accurate readings. The later introduction of 

 photoelectric spectrophotometers has made for greater accuracy, 

 but it has also necessitated some research before standards based 

 on the new type of instrument were well established. 



One advantage of the photoelectric instruments is the greater 

 ease in obtaining with them a reading of the density at the peak of 

 adsorption (Dp). This has made it possible to use the spectro- 

 photometer not only for determining the nature of the dye but 

 for comparing the strength of various samples in cases where the 

 titration method has proved inadequate. 



The methods at present adopted in the Commission assay labo- 

 ratory are given in the appendix of this book (pp. 292-328). Con- 

 stant efforts are being made to improve the methods, and it is 

 realized that they are much better than those adopted in the '20's. 

 Nevertheless they are still unsatisfactory as methods for evalu- 

 ating the performance of stains; and it is still necessary to con- 

 tinue the practice of testing them in actual use. 



HISTOCHEMICAL REAGENTS 



The possibility that dj^es might some day serve as histochemical 

 reagents was realized quite early. The well known fact that basic 



