. RIBOFLAVIN 443 



The bubbles must be small enough so that all spattering is confined 

 in the tube. 



9. Add 0.02 ml. 0.04% bromotliymol blue and titrate with 0.3 N 

 sodium hydroxide from a 0.2 ml. Rehberg burette. Stir by a stream 

 of air bubbles. Neither the air bubbler nor the burette tip should 

 exceed 0.7 mm. diameter. The solubility of the indicator in caprylic 

 alcohol makes it desirable to use very little of the latter. 



10. Plot the calibration curve from the titration values found 

 using the standard riboflavin solns. Obtain the riboflavin content of 

 the unknown from its titration value by reference to the calibration 

 curve. 



11. To minimize the effect of possible interfering substances, 

 and to obtain a more precise assay, prepare the standards as follows 

 if it is possible: Extract some of the tissue to be analyzed as already 

 described. Irradiate the extract, which is in 0.002 N hydrochloric 

 acid, for 30 min. in a Pyrex tube at a distance of 3-4 cm. from a 

 mercury arc lamp, such as the General Electric HB-4, to destroy the 

 riboflavin present. With a minimum of dilution, prepare standards 

 from this extract containing 0, 0.5, 1.0, 1.5, and 2.0 m/xg. riboflavin 

 per 0.1 ml. 



