18 II. METHODS OF INVESTIGATION 



is tlie idea that the use of two pliotocells with one Hght source automatically 

 eliminates the effect of fluctuations of the latter — whereas in fact it only 

 does so if the light falling on the two photocells is equal when readings 

 are made. 



As with spectrophotometers, it is essential for a user of any type of photo- 

 electric equipment to know his instrument — its principles and design, its 

 capabilities and deficiencies — and to check its accuracy periodically. 

 While photoelectric devices are capable of greater accuracy than visual, 

 they are also subject to more sources of error, which are in many cases less 

 evident to the user. 



4.2. Fluorescence 



Measurement of fluorescence is used for the determination of 

 porphyrins and some bile pigments. For many purposes, simple 

 visual comparison of the solution of the substance to be measured 

 with a series of correctly graded standards when viewed under fil- 

 tered ultraviolet illumination is sufficiently accurate. The use of 

 filters between the fluorescing solution and the eye may often be of 

 advantage in removing interfering fluorescence of a different color. 

 The ultraviolet light used to excite the fluorescence is commonly 

 derived from a mercury arc shielded by a Woods glass filter to remove 

 both the greater part of the visual spectrum and the shorter, biologi- 

 cally dangerous, ultraviolet rays. 



For more accurate work, photoelectric comparison is needed, and 

 several of the more elaborate photoelectric absorptiometers are fitted 

 with attachments for exciting and comparing fluorescence. The same 

 precautions are needed in operating these instruments as with photo- 

 electric colorimeters, but also great care must be taken to see that 

 none of the exciting radiation enters the photoelectric cell. 



With fluorescence measurements, proportionality of intensity to 

 concentration is maintained only over a small range, at low con- 

 centration. Further, pH frefjuently influences the fluorescence, as do 

 also some organic compounds and inorganic salts. Precautions must 

 therefore be taken to ensure that the best conditions for the excita- 

 tion of fluorescence are obtained. 



5. THE PHOTOCHEMICAL ABSORPTION SPECTRUM 



An ingenious indirect method of determining the absorption 

 spectrum of an enzyme present in too small concentration to produce 

 any measurable effect with conventional spectrophotometric methods 



