90 VITAMINS A AND CAROTENES 



2. Determination of Vitamins A 



Experiments on animals are costly and allow serial experiments only in 

 special cases. Therefore, analysts have always wanted to go from biological 

 assay to chemical analysis which is quicker and easier to perform. After 

 statistically significant data on pure and crystalline compounds were ob- 

 tained by biological assay of vitamin A, the analytical problem shifted to 

 chemical methods. 



As was proved by careful comparison between the biological and spec- 

 trophotometric vitamin A determinations, especially with whale liver oils, 

 over- and undervaluing was observed when the same conversion factor was 

 used.^ This was because the oils contain substances which also absorb near 

 the ultraviolet maximum of vitamin A and cause a shift of the maximum 

 and also affect the extinction coefficient. This phenomenon is termed 

 "irrelevant absorption." Morton and Stubbs'^ elaborated a method which 

 eliminates the irrelevant absorption. A simplification of this correction 

 method is given by Oser.^^ Whether correction is necessary can be answered 

 only after a careful spectrophotometric survey of the extinction curve. 

 Vitamin A ester concentrates and most good molecular distillates do not 

 need any correction. Cod liver oils nearly always require correction, if the 

 determination is made on the unsaponified oil. Therefore, if possible, in this 

 case the determination is carried out on the unsaponifiable fraction. Whale 

 liver oils or oils stored for a long time nearly always require correction, 

 even if the determination is made with the unsaponifiable material. The 

 technical details of the correction according to Morton and Stubbs may 

 be found in the original papers.- ^"^ 



In cases of strong irrelevancy it is advisable to submit the samples to 

 chromatographic purification and to isolate the vitamin A in as pure form 

 as possible. As the latest methods of determination^^--^ based on this prin- 

 ciple show, a spectrophotometer gives an absorption curve which is com- 

 parable with that of the standard and which allows unhesitating application 

 of the conversion factor of 1900. Considering the great amount of work 

 dealing with the estimation of vitamin A, it is not possible to give more 

 than a few examples. Further details are given in the special literature on 

 this analysis.--'-* The vitamin A determination in fish liver oils is described 



" R. A. Morton and A. L. Stubbs, Analyst 71, .356 (I9i6) ; Biochem. J. 41, 525 (1947); 

 42, 195 (1948). 



18 B. L. Oser, Anal. Chem. 21, 529 (1949). 



19 N. T. Gridgeman, G. P. Gibson, and .J. P. Savage, Analyst 73, 662 (1948). 



20 R. K. liarua and R. A. Morton, Biochem. J. 45, 308 (1948). 



21 W. Hjarde, Acta Chem. Scand. 4, 628 (1950). 



22 F. Gstirner, Chemisch-physikalische Vitamin-Bestimmungs-Methoden, 4th ed. 

 Ferdinand Enke, Stuttgart, 1951. 



23 W. J. Dann, Biol. Symposia 12, 13 (1947). 



