COLORIMETRY AND FLUORESCENCE 17 



4. COLORIMETRY AND FLUORESCENCE 

 MEASUREMENT 



4.1. Colorimetry 



Although strictly speaking the laws of light absorption hold only 

 for monochromatic light, it is possible with many substances to 

 utilize comparatively wide wave bands, even white light, for color- 

 imetric comparisons. The technique by which the depth of a column 

 of solution of unknown concentration is varied until its color matches 

 that of a standard solution is too well known to need elaboration 

 here. Readers are referred to works such as those of Gibb (991) and 

 Weissberger {3026) for detailed discussion. 



Methods of comparison whereby the two optical fields are not 

 contiguous — that is, the simple comparator type of instrument — 

 have been used extensively for such purposes as hemoglobin estima- 

 tion, but are suitable only for the roughest work. Where accuracy is 

 needed, a good colorimeter, or a comparator incorporating a dividing 

 prism, is an essential. 



For colorimetry against glass or other artificial standards to be 

 reasonably accurate with white light, it is necessary that the absorp- 

 tion spectrum of the substance possess no very sharp bands. On this 

 account oxyhemoglobin and carboxyhemoglobin are not suitable for 

 this technique but acid and alkaline hematin are. Even when stand- 

 ards of the same substance are used, simple colorimetry is not always 

 suitable for sharp-banded substances. The difficulty is often resolved 

 by the use of color filters, thus reducing the width of the spectral 

 band transmitted by the instrument. In some cases spectrocolorime- 

 try offers a simple alternative. By ob.servation of the colorimeter 

 field with a spectroscope, the strength of corresponding absorption 

 bands can be matched. This technique is particularly useful for 

 porphyrins and hemochromes. 



The tendency in modern work is for absolute photometry, usually with 

 photoelectric methods, to replace optical comparison methods. Further, 

 accuracy is improved by using filters to narrow the spectral region employed, 

 the colorimeter thus approaching the spectrophotometer. A very large 

 number of instruments of this type has been described, many being com- 

 mercially available. While there can be no question of the advantages of 

 photoelectric methods, it must be said that most instruments have their own 

 disadvantages, sometimes serious. Some of these are undoubtedly due to bad 

 design, or to various misconceptions. One of the most serious of the latter 



