II. CHEMISTRY 307 



moles of formaldehyde, the activity falls off rapidly. As in the case of tri- 

 and tetrasuccinateis, the sulfate is microhiologically active only after pre- 

 \i()iis hydrolysis. Ribofla\iii mono- and disuccinates have vitamin Bo 

 activities for the rat which are 100% and 65%, respectively, of riboflavin's 

 acti\ity. Both the mono- and diaceton(> derivatives of riboflavin are active 

 in the nutrition of rats. Riboflavin-o'-phosphate is fully as active in the rat 

 as riboflavin (oral and parenteral adniinistration), as well as in the micro- 

 biological test. The same is true for flavin adenine dinucleotidc^ 



Neutral solutions of riboflavin ha\'e a greenish-yellow color. The absorp- 

 tion spectrum shows characteristic absorption ma.xima at 475, 445, 359- 

 372, 208, and 223 m/z. •-•'•'' The absorption in the visible part of the spec- 

 trum has been used for (|uantitative determination of riboflavin. 



Neutral aqueous solutions of riboflavin display intense yellowish-green 

 fluorescence, with a maximum at 5G5 m^ which can be used for quantitative 

 determination of the vitamin. The fluorescence vanishes on the addition 

 of acids or alkalis; optimal fluorescence occurs at pH 4 to 8.^^ A recent study 

 of the fluorescence of riboflavin and flavin adenine dinucleotide (FAD) 

 includes the effect of different quenchers. The relatively weak fluorescence 

 of FAD may be caused by internal quenching by interaction of the alloxa- 

 zine and adenine portions of the molecule.^^ 



Riboflavin has an amphoteric character. Its dissociation constants are 

 /v'a = G.3 X 10~^- and /u = 0.5 X 10~^; the isoelectric point corresponds 

 to a pH of 0.0. The pH of the saturated aqueous solution is approximately 

 O.-'-i 



The optical activity of riboflavin in neutral and acid solutions is exceed- 

 ingly small. In an alkaline medium, the optical rotation is strongly de- 

 pendent upon the concentration: [a ]d^° = — 70°(c = 0.06%; 0.1 A'NaOH); 

 [a]l^° = - 117° (c = 0.5 %; 0.1 N NaOH).^^ Borate-containing solutions are 

 strongh^ dextrorotatory: [a]l^° = +340° (pH 12); in this case the rotation 

 depends only slightly upon the riboflavin concentration.^^ 



Neutral aqueous solutions of riboflavin are relatively heat stable if pro- 

 tected from light and can be sterilized by autoclaving for a short time; 

 only slight destruction occurs by heating to 120° for hours. At room tem- 

 perature (27°) decomposition of buffered solutions (pH 5 and 0) takes place 

 at rates of 3 and 1.2 % per month. No appreciable destruction of the vita- 

 min can be observed during the cooking of food,-^ but when milk in bottles 



2^ H. Kuhn and G. Moruzzi, Bcr. 67, 888 (1934); F. Kavanagh, Arch. Biochcm. 20, 



31.5 (1949). 

 "C Wohor, Biorhem. ./. 47, 114 (1950). 

 "-' irKului, H. Rudy, and F. WcyRand, Bcr. 68, 625 (1935). For tho rulo wliicli c,roverns 



the rotation of ditTerent 9-])olyIiydro.\\:iIk\ 1 llavins, sc(> F. Weyjiand. Hi r. 73, 1278 



(1940). 

 " \\. Kuhn and H. Rudy, Ber. 68, 169 (1935). 

 " R. R. Williams and V. H. Cheldelin, Science 96, 22 (1942). 



