58 Biological Stains 



the other hand, simultaneous inspection of two beams of light is 

 not necessary, nor is the analyzer for reducing the light intensity 

 needed; hence a single beam of light passing through the instru- 

 ment is all that is needed, with resulting greater simplicity of the 

 optical system. The chief bulk of a non-recording photoelectric 

 spectrophotometer is occupied by a potentiometer to record the 

 strength of the current generated by the photoelectric cell. 



In measuring the complete visible absorption of a dye, a series 

 of measurements is made over the portion of the spectrum in 

 which any appreciable absorption may be noted. In doing this 

 with such an instrument as the Beckman, one of the absorption 

 cells is filled with the solvent alone, as a control, another with the 

 dye dissolved in the same solvent, in quite dilute solution. Then, 

 after checking the instrument as to its zero point the control cell 

 is placed in the beam of light coming from the exit slit, the mono- 

 chromator set to a point somewhat to one side of that part of the 

 spectrum where absorption is expected, and a reading taken as to 

 the intensity of the light reaching the phototube. Then a similar 

 reading is taken with the unknown sample. The setting is then 

 changed to a slightly different wavelength, and new readings taken. 

 In practice such readings are taken about every 5 m/x in the re- 

 gions of slight absorption but at least every 1 m/x near the peak. 

 The difference between the readings of the control and the un- 

 known at any point indicates the absorption of light at that 

 point. With the Beckman, four absorption tubes are provided, 

 which allows for readings to be obtained one after the other with 

 three different unknowns. 



If measurements are carried out under suitable standardized 

 conditions, the spectral position and the general form of the ab- 

 sorption curve are characteristic of the individual dye, while the 

 magnitude of absorptive indices (the height of the curve) varies 

 directly with the amount of dye present. The absorption curves 

 of dyes which are very closely related in structure are sometimes so 

 similar as to be practically identical. In such instances the in- 

 dividual dyes may be recognized by means of quantitative deter- 

 minations of the degree in which their absorption is modified under 

 the influence of suitable variations in conditions. 



The absorption curves of typical yellow, orange, red, violet, and 

 blue dyes are recorded in Figure 4. It will be noted that their maxi- 

 mum absorption in each case falls within the range of the comple- 

 mentary color (cf. Table 1). The great majority of dyes of these 

 colors, in the usual solvents and under the usual conditions, show 

 but one absorption band in the visible spectrum. The curves are 

 seldom perfectly symmetrical, however, and usually give indica- 

 tions of localized secondary absorption in some portion of the band. 

 It has been shown that this secondary absorption is due, in numer- 

 ous instances, to a tautomeric form of the dye. It should never be 



