364 RIBOFLAVIN 



merits by hydrosulfite and stannous chloride. The riboflavin is then reoxi- 

 dized to the fluorescent form by shaking with air. Many other pigments 

 are not reoxidized by this procedure. Slater and MorelP*^ have modified 

 the method of Najjar^^ and applied it to a variety of products. In this pro- 

 cedure (described in detail by Jones^), the riboflavin is extracted into 

 butanol-pyridine mixture after the permanganate oxidation. Anhydrous 

 Na2S04 is used to effect the transfer and separation. This method, which 

 has not had general application, appears to have advantages and to deserve 

 broader trial. 



Pigments and fluorescent materials which are not separated from ribo- 

 flavin by purification treatment must be taken into account by appropriate 

 blanks. The two parts of the method are therefore inseparable and cannot 

 be easily discussed alone. The criticisms of the hydrosulfite reduction, 

 which is usually used later in the analysis to obtain the blank reading, are 

 discussed below. 



(3) Fluorometric Measurement. Although visual comparison of samples 

 with appropriate standards under ultraviolet light has been used,^ it is 

 obviously much less satisfactory than the use of a photofluorometer and 

 may be impossible in the presence of interfering materials. A number of 

 fluorometers are available, equipped with appropriate light source, filters, 

 etc. Optical parts must be of low fluorescent glass. (See Loofbourow'^ for 

 a short discussion of riboflavin fluorometry.) Since riboflavin is sensitive 

 to light, a secondary standard of fluoroscein is used to adjust the sensitivity 

 of the instrument, and readings are performed rapidly. A "standard curve," 

 such as is commonly used in colorimetry, is usually not applicable; there- 

 fore the instrument must yield an essentially straight-line relationship be- 

 tween riboflavin concentration and reading over the range used. This range 

 is limited, since self-quenching occurs at high concentrations, probably 

 above 0.13 7 per milliliter.- Perhaps the instrument developed by Lowry^^ 

 deserves special mention because of its stability and adaptability over wide 

 ranges of riboflavin concentration. 



(4) Standards. ■ Probably the preferred method of relating fluorometer 

 reading to the amount of fluorescence in the unknown is the inclusion of a 

 "recovery sample" with each unknown. The increment in reading caused 

 by the added riboflavin is used to calculate the riboflavin in the sample. 

 This method automatically allows for losses in preparation (assuming that 

 the riboflavin in the sample behaves as that added; see Hoffer ef a/."), 

 adsorption of either incident or fluorescent light by colored samples, and 



20 E. C. Slater and D. B. Morell, Biochem. J. 40, 644 (1046). 



21 V. A. Najjar, /. Biol. Chem. 141, 355 (1941). 



22 J. R. Loofbourow, Vitamins and Hormones 1, 143 (1943). 



23 O. H. Lowry, /. Biol. Chem. 173, 677 (1948). 



