QUANTITATIVF, ANALYSIS 



defined; in other cases neither ihe reactions nor the colored products 

 are defined with certainty; hut emiiirical conditions ha\e heen fixed 

 which relate the color quantitatively to the amount of the substance 

 under analysis. Thus Folin (21) used the color produced by Nessler's 

 reagent as the means for quantitative estimation of ammonia, and 

 hence of nitrogen, through the ammonia obtained by Kjeldahl diges- 

 tion, and of urea through the ammonia obtained by urea hydrolysis. 

 The constitution of the colored compound of ammonia and potassium 

 mercuri-iodide is still under dispute, but the colorimetric results are 

 accurate. Sugars reducing Cu++ to Cu''" were determined by Folin 

 and VVu (21) by letting the cuprous ion thus formed act on a molybdatc 

 solution, with reduction of the colorless hexavalent molybdenum to a 

 lower valence, which shows an intense blue color; the reactions do not 

 appear to be stoichiometric, but quantitative relations can be obtained 

 between colored molybdenum products and the initial sugar. For 

 almost every substance of interest in quantitative biochemical analysis, 

 chromogenic reactions have been devised which can be used for more 

 or less accurate estimation. As a rule these procedures are rapid, and 

 are adapted to minute amounts of material. 



During the first years of the colorimetric epoch, the instrument 

 in general use was the familiar Duboscq colorimeter, in which the 

 depths of colored solution layers in two parallel columns, one of the 

 unknown solution and the other of a standard, are varied until the two 

 fields viewed with the eye appear equal. The simplicity and versatility 

 of the Duboscq colorimeter assisted greatly in the rapid adoption of 

 colorimetric procedures. 



Photometers, in which the percentage transmission of light could 

 be measured without standard solutions for direct comparison, were 

 known long before this period, but were too complicated and ex- 

 pensive for ordinary routine in biochemical laboratories. During the 

 past two decades, however, photometers have been progressively made 

 more adaptable to such routine, and have been gradually displacing 

 colorimeters of the Duboscq type. In the photometer, the concentra- 

 tion of light-absorbing solute is related to the optical density according 

 to the simple linear formula of Beer's law (20,22,23): 



C = kl) (1) 



where C is the concentration and D is the optical density (or extinction). 



