VI. ESTIMATION 



19 



lytic fuiK'tion for PABA was not excludocl, structural origin of I lie hcn/.cnc 

 rinu; of \-il:niii)i B|j fi'oni PAl^V was ihc faxorod hypothesis. 



VI. Estimation 



LEMUEL I). WRIGHT and PIOTIOH A. TAVORMINA 



A. CHEMICAL METHODS 



PABA, being a primary aromatic amine, diazotizes with nitrous acid, 

 and the resulting diazo compound then couples with various other aromatic 

 compounds to yield colored products that may be determined spectro- 

 photometrically. The Bratton-Marshall method' for the determination of 

 sulfanilamide as applied to the determination of PABA entails diazotization, 

 removal of excess nitrous acid with ammonium sulfamate,^ and coupling 

 of the diazo product with X-(l-naphthyl)ethylenediaminc dih3^drochloride. 

 This affords the basis of the preferred chemical method for PABA deter- 



H,X- 



— COOH 



NaN02_ 

 HCl 



HOOC- 



H 



-N2+CI- 



N— CH2CH,NH2 



HOOC— 



-N2+CI- + 



H 



N— CH2CH2NH2 



N=N- 



-COOH 



mination. The colored compound formed by coupling diazotized sulfanila- 

 mide with N-(l-naphthyl)ethylenediamine has an absorption maximum at 

 o4o m/i.* 



1 A. C. Bratton and E. K. Marshall, Jr., ./. Biol. Chcm. 128, 537 (1939). 



2 E. K. Marshall, Jr., and J. T. Litchfield, Jr., Science 88, S5 (1938). 



