16 II. METHODS OF INVESTIGATION 



another on addition of increasing quantities of a reagent is frequently 

 followed spectrophotometrically. Full data are provided by taking 

 a series of complete absorption curves, but frequently sufficient data 

 may be obtained from readings at a single wavelength where the 

 most pronounced change in absorption occurs. If the molar extinc- 

 tion coefficients of both the initial and final products are known, the 

 percentage transformation at any stage of the titration can be com- 

 puted directly; however, these constants can also be found from the 

 data of the titration itself {Jt-53). The study of Hogness and collab- 

 orators (1307) on cyanide ferriprotoporphyrin is an excellent example 

 of the application of spectrophotometric titration methods. 



3.2. Methods of Measurements 



For the purposes of spectrophotometry the optical spectrum is 

 usually considered as comprising three regions, the ultraviolet, the 

 visible, and the infrared, although both the first and last of these may 

 be subdivided (c/. 3026, p. 745). The technique of measurement of 

 an absorption spectrum is determined by the wavelength range 

 under consideration. In the case of hematin compounds and their 

 derivatives, the most significant absorption bands occur principally 

 in the visible portion of the spectrum, with the near ultraviolet as the 

 next significant region. A few compounds have pronounced infrared 

 bands, but comparatively little attention has so far been paid to this 

 region. 



Information on the design and construction of various instru- 

 ments and on the technique of spectrophotometric measurement, 

 together with bibliographies, will be found in the works of Heilmeyer 

 {1'213), Weissberger (3026), Twyman and Allsopp (28J^0), and in 

 numerous papers and reviews. 



Users of spectrophotometers, particularly elaborate commercial photo- 

 electric types, should clearly realize that such instruments require regular 

 calibration of the density and wavelength scales. This is a simple procedure 

 in most cases, but is one which should under no circumstances be neglected. 

 With automatic recording instruments a daily check can be made without 

 any trouble, by taking the absorption curve of a suitable standardized glass 

 filter. With visual instruments the wavelength scale can be checked occa- 

 sionally by means of a neon lamp, and the density scale may be checked by 

 a reading on a standardized glass filter, at one or two selected wavelengths. 

 Elementary precautions of this nature will preclude the possibility of such 

 confusion as recently overtook one well-known German school. If an instru- 

 ment is originally properly designed and constructed, a little regular care 

 will suffice to maintain its working at the highest possible accuracy. 



