ABSORBING POWER AND FLUORESCENCE OF RESORUFIN. 33 



Fig. 28 shows the average coefficient of absorption for the seven wave- 

 lengths as a function of the concentration. This curve is used later for 

 finding the coefficient of absorption for different dilutions, especially care- 

 ful measurements for the concentration ,V, being taken as a basis of cal- 

 culation. The values thus obtained, given in Table 5, were used in all 

 subsequent computations involving the coefficient of absorption. 



The above results agree, in every respect, with those given by Walter. 1 

 Beer's law is true for dilute solutions, but fails for greater concentrations, 

 as is indicated by the deviation of the curve from a straight line. The 

 straight form of the curve (Fig. 28) up to concentration | corresponds to 

 what Walter calls "complete solution," in which he says the molecules are 

 in a "separate state." Concentration may be regarded as his "critical 

 dilution," where a change seems to take place in the condition of the fluo- 

 rescent substance. Solutions more concentrated than correspond to those 

 called by Walter "incomplete," in which "molecular groups" exist. 



Interpreted according to the ionization theory, the curve (Fig. 28) 

 indicates that in dilutions less than a state of complete or nearly com- 

 plete ionization has been reached. At this point a change takes place, 

 more resorufin being contained in the solution than is ionized. As the 

 concentration increases, more and more of the solution remains undis- 

 sociated. It appears that the undissociated resorufin is not only incapable 

 of fluorescence, but is also much less effective in causing absorption than 

 is the dissociated substance. 



FLUORESCENCE AND CONCENTRATION. 



For observation of the fluorescence spectrum the spectrophotometer 

 was adjusted as before. In front of slit D (Fig. 22) was placed a glass cell 

 5.4 cm. long, containing the resorufin solution. This cell was entirely 

 covered with black paper, except for a space about 1.5 cm. high across the 

 bottom of the face next to the exciting lights', and two narrow strips, x and y, 

 The opening x was to allow light to enter the collimator slit, while y, 

 directly opposite, was used only for adjusting. The source of illumination 

 used to produce fluorescence was a bank of four acetylene flames S'. 

 Between these and the fluorescent solution was placed a glass cell filled 

 with water to prevent the heating of the resorufin. The comparison source 

 was another acetylene burner S, light from which was reflected into the 

 slit C by a block of magnesium carbonate n. The cell containing the 

 resorufin was so placed that light from the whole layer of solution next the 

 inside surface of glass came through the slit D of the collimator. 



It is clear that a portion of the fluorescent light is absorbed by the solu- 

 tion before reaching D, and attention has already been called to the fact 

 that this absorption will be different for different parts of the fluorescence 

 spectrum. If the fluorescence is measured in the manner indicated above 

 it is therefore necessary to apply a correction for absorption before the 

 typical fluorescence spectrum can be determined. Two methods of making 

 this correction have been used. In the first method the necessary cor- 

 rection was computed as follows : 



IB. Walter, Wied. Ann., 36, pp. 502 and 518, 1S89. 



