APRIL 15. 1940
lNALYTICAL E D I T I O S
nounced than in the latter and i h improved by the presence of a higher concentration of perchloric acid to stabilize the complex formation. The use of perchloric acid together TTith organic reagents such as the alcohols introduces no hazards so long as the solutions are not heated. T o heat' such solutions would rapidly destroy the color complex through oxidation, so that, this error is not likely to be permitted in the application of the test.
Summary A new and improved classification test for alcoholic hydroxyl groups is based upon the production of a red coloration in the yellon- solutions of the nit,rato or perchlorato cerate
203
anions in the presence of the corresponding nitric and perchloric acids. Alcohols, hydroxy acids, hydroxy esters, halogenated alcohols, glycols, and hydroxy aldehydes containing less than ten carbon atoms h a r e been tested with positive results. Aromatic amines, amine hydrochlorides, and compounds containing chromophoric groups interfere by giving colors or precipitates with the reagent. Cornpounds vhich are readily oxidized may decolorize the test reagent before the red color of the test can be noted. Phenols interfere by formation of conflicting colors.
Literature Cited (1) Smith and Getz, IVD. ENG.CHEM.,Anal. Ed., 10, 191 (1938)
Riboflavin Content of Yeasts Determined Photometrically and Biologically i. E. SCHUJLiCHER AND G. F. HEUSER, Department of Poultry Husbandry, Cornel1 University, Ithaca, N.
Y
EASTS and yeast products have been used for a long time as a source of bhe vitamin B complex, since yeast is believed to contain all the vitamins of the B group. It is the purpose of this paper t'o present a chemical method for the estimation of riboflavin in yeasts and yeast products, to show the relation between chemical and biological determinations, and to point out the variations found in the riboflavin content of different yeasts.
Photometric Method for Determination of Riboflavin i n Yeasts The apparatus used is a photoelectric photometer designed principally for the determination of riboflavin. A discussion of the principles of the apparatus and l a w governing its application to riboflavin are given by Sullivan (2) and Sullivan and Sorris (5). For the extraction of riboflavin a 10-gram sample of the material to be assayed is weighed into a 250-ml. Erlenmeyer flask and 100 ml. of a solution of 5 per cent hydrochloric acid are added. (Repeated extractions of the residue or lengthening the time of extraction did not alter the phot,ometric measurement of riboflavin in yeast.) The mixture is refluxed gently for 40 minutes and allowed to cool. The flask is then tightly stoppered and placed in a refrigerator for 1 hour. Finally, the supernatant liquid is filtered through a fluted filter and the filtrate is ready for aliquot' sampling. For the determination of the riboflavin a 25-ml. aliquot of the filtrate is placed in a 50-ml. volumetric flask and the pH adjusted to 3.5 to 4.0 by the addition of sodium hydroxide. This is necessary in order to get clear solutions and may be conveniently done in most instances by adding 2.25 ml. of a 5 N solution of sodium hydroxide. The volume is then made up to 49 ml. by the addition of distilled water. Since reducible interfering pigments may be present in the filtrate, it has been found necessary to reduce these pigments in order to obtain an accurate determination of the riboflavin content of the sample. This is accomplished by adding 1 ml. of sodium hydrosulfite solution (1 gram of sodium bicarbonate plus 1 gram of sodium hydrosulfite dissolved in 20 cc. of cold distilled water), making the total volume 50 ml. The riboflavin is also reduced to the colorless form by this treatment. [This modification was obtained from the modified method of Hodson and Sorris ( 1 ) for the determination of riboflavin in milk products. ] The solution is transferred to a 250-ml. Erlenmeyer flask, stoppered, and allowed to stand for 10 minutes. The stopper is then removed and the solution shaken vigorously for 5 minutes. In the authors' experience this treatment will reoxidize the riboflavin to the colored form, while the interfering pigments are not reoxidized. Finally, the solution is filtered through a Gooch crucible with an asbestos mat, moderate suction being used. The clear filtrate is t,hen ready for reading by means of the photometer. 2 , detailed description of the photometer and its
Y.
operation is given by Sullivan arid Sorris ( 3 ) . The same procedure was followed. TTwnty milliliters of the clear filtrate are placed in the optical cell and an initial reading, 11,is made to determine the quantity of light absorbed by the riboflavin present and by any colored impurities. Then the riboflavin is reduced to the colorless form by adding 0.6 nil. of sodium hydrosulfite solution, after which a second reading, I z , is made to determine t,he quantity of light absorbed by the color remaining after reduction. The value obtained after reduction is subtracted from the initial value. Since only 90 per cent of the riboflavin is reduced by the sodium hydrosulfite, the reading must be corrected. This corrected value is then converted into total riboflavin by multiplying by the factor 1.036, to correct for the reduced readings obtained at pH 3.5 to 4.0 (5). The micrograms of riboflavin per gram of yeast are finally obtained by multiplying by 20. Since the photometer gives a linear response to changes in light intensity and it has been shown that solul ions of riboflavin follow Beer's law, it is possible to calculate the concentration of extracts directly from the readings. The light transmitted by one of the absorption cells when filled viith water is taken as lo. The light transmitted by a solution of riboflavin is measured and represented by II.The light transmitted by the riboflavin solution after the reduction of the riboflavin is represented by 1 2 . Thus applying Beer's and Lambert's laws and correcting for the per cent reduction and dilution, the folloxing formula was arrived at and employed in the calculations:
1
( 0.90
Io = blank reading I , = initial reading I2 = reduced reading 1.0333 = dilution due to addition of 0 . 6 ml. of sodium hydrosulfite 1.036 = correction for pH reading of 3.5 t o 4.0 20 = sample dilution 0 . 9 0 = correction for 90 per cent reduction 36.7 = absorption coefficient obtained in Etandardizing the photoelectric photometer The above formula may be condensed to the following form for simplification of calculations : Micrograms of riboflavin per gram
==
36.7 X 1.036 X 20 0.90
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INDUSTRIAL AND ENGINEERIKG CHEMISTRY
204
Micrograms of riboflavin per gram = 844.9 [log ( q
2 - 2)
(1.0333)]
Chick and Hen Bioassays Chick and hen bioassays were also conducted on several of the yeasts. I n assaying the yeasts with chicks the following procedure was followed :
A basal diet low in riboflavin but complete in other known factors was supplemented with the yeast to be assayed. As a positive control a standard dried skim milk was used, which previously had been assayed biologically as well as chemically. It was fed a t different levels in order t o increase the accuracy of comparison with the yeast groups. The quantity of riboflavin present in the different yeasts was determined by comparison of the growth responses of the chicks fed the yeast with those fed the dried skim milk. Knowing the quantity of riboflavin added by the standard dried skim milk, the quantity present in the yeast could be calculated. The method of determining values in the hen bioassays was the same as that used in the chick bioassays, except that hatchability instead of growth was used as the criterion.
Table I11 shows the wide variations in riboflavin content of different yeasts. Primary yeasts in general have a higher riboflavin content than residual yeasts. The riboflavin content of the yeast concentrates is determined by the degree of concentration. TABLE111. VARIATIONSIS RIBOFLAVIN CONTEXT OF YEASTS Sample No.
1 2 3 4 5
6
7 8 1 2 3 4 5
1 2
Rat Assays The rat assay values are those reported from the laboratory supplying samples. They were determined by the regular Bourquin and Sherman procedure for vitamin G.
R a t Assay Bourquin and Sherman units per gram Primary Yeasts 35 35 35 42 35 40-50
-
60 50-60 Residual Yeasts 15-18 20 20 20 30 Yeast Concentrates 60 91
++ +
Chemical Analysis Micrograms per gram 34.2 49.5 52.2 57.2 60.9 68.4 74.4 78.2 34 2 38.8 44.3 50.1 60.2 105.4 146.8
Discussion
The bioassays with chicks and hens are in general agreement with those obtained photochemically. The different Results samples of yeast have wide variations in their riboflavin conUsing the chemical procedure described, the riboflavin content-for example, the primary yeasts show values ranging tent of numerous samples of yeast and yeast products was from 34.2 to 78.2 micrograms of riboflavin per gram. This determined. Table I shows the close agreement obtained indicates the importance of knowing the potency of the yeasts, by this method, and indicates that i t is possible to obtain especially when they are used in quantitative experiments. duplicate results and that the range of variation is very narrow. Further analysis of the data reveals that no single conversion I n Table I1 are given the riboflavin values of yeast and yeast factor for yeasts can be employed to convert Bourquin and products as determined chemically as well as biologically. Sherman units into micrograms of riboflavin as determined by T h e chemical analysis is expressed in micrograms of riboflavin the photometric method here employed. This is probably per gram of sample as measured by the procedure herein debecause of the variation in different yeasts of some factor or scribed. The rat assay is expressed in Bourquin and Sherman factors which affect growth and hence the results obtained by units, while the chick and hen bioassays are expressed in means of the biological assay. In general, residual yeasts micrograms of riboflavin per gram of sample. give a conversion factor of approximately 2.0 micrograms of riboflavin per Bourquin and Sherman unit, while primary grown yeasts have a conversion factor of approximately 1.3 O F RIBOFLAVIN DETERMINA- micrograms. TABLEI. AGREEMENTO F RESULTS The yeast feeds studied appear to act like TIONS BY PHOTOMETRIC METHOD primary grown yeasts Tvith respect to the conversion factor. Determination WO. Sample 4 Sample 1 The practical importance of the photochemical method Micrograms per gram of yeast for determining the quantity of riboflavin present in yeast 10.9 49.7 and yeast products is based upon the fact that it is relatively 12.1 51.5 11.5 49.6 inexpensive, is not difficult nor complex, and determinations 10.9 49.2 can be made in a relatively short period of time. 11.5 50.2 7 8 9
Av.
11.25
-
11.5 ii.5 10.9 11.5 0 . 3 5 -I-0 . 8 5
50.19
-
50.2 50.2 49.6 51.5 0.99
+ 1.31
TABLE 11. RIBOFL.4VIN CONTENT O F YEAST AND YEAST PRODUCTS Sample No.
Rat Assay Bourquin and Sherman units
Chemical Analysis -Micrograms Yeast Feeds
Chick Bioassay
Hen Bioassay
Summary A photochemical method for the determination of riboflavin in yeast and yeast products is presented. The photochemical and biological evaluations of riboflavin in yeast’s and yeast products were in general agreement. Wide variations were found in the riboflavin content of different samples of yeast.
Acknowledgment
per gram--
Acknowledgment is made of the cooperation of the AnheuserBusch Company, St. Louis, hlo., which made this investigation possible by establishing a fellomhip a t Cornell University.
Literature Cited Primary Yeasts
1 2
20 20
++
Residual Yeasts 50 1 50.3
43.0 55.0
45.0
..
(1) Hodson, A. Z., and Norris, L. C., “Modification of Method of Sullivan and Norris for Determining Riboflavin Content of Milk and Its By-Products”, unpublished data, Cornell University, 1937. ( 2 ) Sullivan, R . A., “Studies of Flavin”, thesis, Cornell University, 1937. (3) Sullivan, R. A , , and Norris, L. C., ISD. EHQ.CHEM.,Anal. Ed., 11, 535 (1939).
