ESTIMATION 



3. ESTIMATION OF p-AMINOBENZOIC ACID 



Chemical Methods 



H. Tauber and S. Laufer ^ observed that when j!)-dimethylamino- 

 benzaldehyde was reacted with ^-aminobenzoic acid in glacial acetic 

 acid, a yellow colour was produced, the intensity of which was propor- 

 tional to the concentration of j^-aminobenzoic acid. They suggested 

 that the reaction might be used for the estimation of ^-aminobenzoic 

 acid. E. R. Kirch and O. Bergeim^ proposed the use of a colour 

 reaction with diazotised aneurine for the estimation of j5)-aminobenzoic 

 acid in urine, whilst H. W. Eckert ^ used the colour formed with di- 

 methyl-a-naphthyl amine for its estimation in blood. Conjugated 

 _/)-aminobenzoic acid was estimated after hydrolysis with acid. Another 

 method of assay, which appears to be more in the nature of an identi- 

 fication and purity test, comprises the dissolution of the ^-amino- 

 benzoic acid in dilute hydrochloric acid and titration with bromine, as 

 in the estimation of phenol.^ 



Microbiological Methods 



Chemical methods of assay do not appear to have found favour 

 in the estimation of _^-aminobenzoic acid, and microbiological methods 

 have been extensively used, just as they have in the case of other 

 members of the vitamin B complex. The first method was due to 

 M. Landy and D. M. Dicken,^ who proposed the use of Acetohacter 

 suboxydans as the test organism. The basal medium consisted of 

 casein hydrolysate, glycerol, pantothenic acid, nicotinic acid, trypto- 

 phan, cystine and salts, and the growth of the organism was measured 

 turbidimetrically. The method was used for the estimation of 

 ^^-aminobenzoic acid in animal tissues, blood, body-fluids, cereals and 

 yeast, and appeared to be highly specific, J. C. Lewis ^ suggested 

 the use of Lactobacillus arabinosus 17-5 with a basal medium similar 

 to that of Landy and Dicken,* but supplemented with additional 

 members of the vitamin B complex. The growth response in this 

 instance, however, was measured by titrating the lactic acid produced. 

 Digestion with dilute alkali was used to liberate bound _/)-aminobenzoic 

 acid, strong alkali or acids resulting in partial inactivation. The 

 method was used to estimate ^-aminobenzoic acid in foodstuffs and 

 was said to be very specific. 



According to Mitchell et al.,'^ the method of Landy and Dicken 

 gave a response equivalent to only a fraction of the total p-diUiino- 

 benzoic acid present after acid or alkaline hydrolysis ; even after 

 enzymic hydrolysis or autolysis, low results were obtained. They 

 advocated hydrolysis with 6N-sulphuric acid at 115° C. for one hour. 



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