242 THE VITAMINS 



materials which give color reactions characterized by absorption at other 

 positions may be either active or inactive, or mixtures of inactive and 

 active chromogens, and the biological technic still remains the only satis- 

 factory method of assay." 



Recently Wokes has observed a similar range of absorption bands 

 in the blue products obtained by treating with antimony trichloride 

 certain cholesterol preparations which are devoid of vitamin A potency, 

 and he considers it doubtful that the blue colors given by arsenic and 

 antimony chloride with cod-Hver oil and other sources of vitamin A 

 are directly due to that vitamin. Even though spectroscopic measure- 

 ments may distinguish between the colors shown by vitamin A and 

 other chromogenic substances, the fact remains that lipochromes disturb 

 the direct method of applying color tests to food products, such as 

 butter, for the estimation of vitamin A. 



Various modifications of early technique were reported by Bertram 

 (1929), Evers (1929, 1929a), and Andersen and Nightingale (1929). 

 The last-named investigators consider it essential to prepare the un- 

 saponifiable fraction of the material to be tested and to dilute the 

 chloroform solution of this fraction to such a point that the blue color 

 with antimony trichloride is just perceptible. Under the conditions which 

 they prescribe they state that "hundreds of tests have confirmed the 

 accuracy of the method from a comparative viewpoint. In addition, many 

 duplicate tests have been made by independent laboratories, and in all 

 cases very reasonable agreement has been obtained. That it is reliable 

 when compared with feeding tests on experimental animals, under the 

 strictly defined conditions now adopted for such tests, has been con- 

 firmed by numerous biological tests. The vitamin potencies determined 

 by it have been in satisfactory agreement with the results obtained in 

 the biological tests conducted by the Pharmacological Laboratories of 

 the Pharmaceutical Society and by other highly qualified observers." 

 This is doubtless to be taken as referring to Hver oils and not to mis- 

 cellaneous food materials. 



Norris and Church (1930), following the technique developed by 

 Norris and Danielson (1929a), tested the unsaponifiable fractions of 

 cod-liver oil and found that the blue color of the extracts was (within 

 limits) a linear function of the percentage concentration, while this 

 had not been true of the original oils. Unsaturated oils and oleic acid 

 added to the extracts were found to accelerate the rate of fading of the 

 blue color, but on dilution of the extract a point was reached at which 

 there was no longer interference. This was thought to explain the 

 findings of Norris and Danielson that at sufficiently low color values 



