28 CONTROL BULLETIN NO. 125 



Methods Used for Determination of Carotene and Riboflavin in Mixed Feeds 



Carotene 



A 5 gram sample was refluxed for one -half hour with 50 ml of freshly prepared 

 alcoholic KOH (50 g in 200 ml alcohol) and 5 grams anhydrous Na2S04. The 

 solution was cooled to room temperature and decanted into a 500 ml separatory 

 funnel. The residue was extracted by shaking and decanting with at least 3 

 portions of 35 ml of Skelly-solve B until extracts were colorless. The extracts 

 were combined with the alcoholic KOH extract and washed with 90% methanol, 

 shaking thoroughly after each addition, until washings were colorless. The 

 Skelly-solve was then washed with water until free of KOH and filtered through 

 anhydrous Na2S04. The filtrate was passed through an adsorbent column of 

 activated magnesium oxide and Hyflo Super-Cel according to the method of 

 Wall and Kelley, Ind. & Eng. Chem., Anal. Ed., 15, 18 (1943). The eluate was 

 diluted to a convenient volume and the carotene content determined by measuring 

 its per cent transmittance in a spectrophotometer. 



Riboflavin 



The method used is a modification of the Hodson and Norris method for the 

 fluorometric determination of riboflavin. 



A 5 gram sample was weighed into a 250 ml amber extraction flask, 50 ml of 

 0.25 N H2SO4 were added and the mixture was refluxed for one hour. After 

 cooling, the pH of the mixture was adjusted to 6.8 with Na3P04.12 H2O (6% 

 solution) using a glass electrode, diluted to a volume of 200 ml and mixed thor- 

 oughly. The precipitate formed was allowed to settle for at least one-half hour. 

 A 50 ml aliquot was taken from the more or less clear supernatant layer and 2 ml 

 of 4% KMn04 were added while swirling the solution. After about 4 minutes 

 0.2 gram of solid Na2S204 and 2 ml of dilute SnCU (1 ml of 4% SnCl2 in HCl 

 diluted to 250 ml) were added and the solution was mixed thoroughly. 5 ml of 

 acetone were then added if necessary to reduce foaming and solution made to 

 volume of 200 ml with water, mixed and allowed to stand at least 10 minutes. 

 The solution was filtered through No. 41 Whatman filter paper into a 1000 ml 

 amber Erlenmeyer flask until filtrate measured about 100 ml. The unstoppered 

 flask and contents were placed in a shaking machine and shaken for 10 minutes. 

 A 25 ml aliquot was pipetted into a small Erlenmeyer flask (solution A) and the 

 remaining solution was divided into two approximately equal portions (solutions 

 B and C). To solution A, 1 ml of a standard riboflavin solution containing .002 

 mg riboflavin per ml was added and the solution was well mixed. To solution C, 

 0.1 gram of solid Na2S204 was added. 10 ml portions of each solution were 

 pipetted into 3 matched cuvettes and the fluorescence of each solution was meas- 

 ured. The calculation involved, using the weight of sample and dilutions given 

 here, is: 



64 (B - C) . 



• • =p.p.m. ribonavm 



5.2 (A -B) 



