364 ANIMAL BIOCHEMISTRY 



type, containing abnormal calcium-phosphorus ratios, are used for the 

 production of experimental rickets. In some of these biological 

 methods, satisfaction of the first criterion of page 362 is impossible. 



Chemical Methods 



In certain cases satisfactory colorimetric or fluorimetric methods 

 have been standardized against biological assays and are now widely 

 used because they are much quicker and less expensive than the latter. 



Colorimetric methods depend on the formation of stable colored 

 compounds or complexes that can be measured in a colorimeter. 

 Vitamin A forms a blue complex with antimony trichloride in chloro- 

 form. When vitamin A is extracted from a saponified food material, 

 it can be treated with the antimony trichloride reagent, and the color 

 can be compared with that produced by known amounts of pure 

 vitamin A, using the same reagent. The depth of color is proportional 

 to the concentration of vitamin A. 



Vitamin C can be titrated with a purple dye called 2,6-dichloro- 

 benzenoneindophenol. The dye is a mild oxidizing agent which 

 oxidizes L-ascorbic acid to dehydroascorbic acid. In the process the 

 purple dye is reduced to a colorless compound. Thus the dye serves 

 as its own end-point indicator. Pure crystalline ascorbic acid is used 

 as a reference standard, and the vitamin content of the unknown food 

 is determined by comparison with this standard. 



Fluorimetric methods depend upon the presence of a fluorescent 

 substance, one that absorbs light and re -emits light of a different wave 

 length. When the incident light is employed as a beam, the fluores- 

 cent light is emitted in all directions and is readily detected with a 

 photoelectric cell placed at right angles to the incident beam. Oxida- 

 tions of thiamine and riboflavin with a mild agent yield products that 

 fluoresce when irradiated with ultraviolet light. When pure vitamins 

 are used as reference standards, the unknown quantities in foods can 

 be estimated readily. 



Physical Methods 



Vitamins A and D have characteristic absorption spectra and are 

 determined spectrophotometrically. The instrument used for this 

 work is called a spectrophotometer. It measures the amount of light 

 of any chosen wave length absorbed by a solution of the sample. The 

 wave lengths absorbed depend upon the molecular structure, primarily 

 on the number and arrangement of double bonds in the molecule. 

 A range of wave lengths affected by the molecule is called an absorp- 

 tion band. In such an absorption region, the amount of light ab- 



