370
INDUSTRIAL AND ENGINEERING CHEMISTRY
moles of isoprene units per 100 moles of isobutylene units (mole %), follows from U = 5610/%" (5) ALTERNATIVE METHOD OF ESTIMATING U FROM IODINE CHLORIDE DATA
For a number of years this laboratory has employed a modification (5) of the Kemp-Wijs method for obtaining comparative unssturation values for Butyl polymers. I n this routine procedure a 0.3-gram sample of the rubber is dissolved in 100 cc. of carbon tetrachloride, 5 cc. of 0.2 N Wijs reagent are added, and the mixture is allowed to stand in the dark for 1 hour a t room temperature. Twenty cubic centimeters of 157, alcoholic potassium iodide are then added, the mixture is titrated with 0.1 N thiosulfate to a canary yellow color, 5 cc. of 0.5% starch solution are added, and the titration is completed. A blank determination is made simultaneously, and from the difference in the titers the unsaturation value U(ICl), in mole 7& is calculated from the formula C(IC1) = 0.281 X (cc. of 0.1 N thiosulfate)/grams of sample (6) I t has been found invariably that these values are approximately twice as large as those obtained by the ozonolysis method, and it has been suggested (4) that the difference is due to factors, such as substitution reactions, commonly encountered in iodine chloride determinations. Figure 5 shows the relationship between the two values of U for a number of Butyl rubber samples of varying degrees of unsaturation. The curve for the isoprene copolymer was plotted from a quadratic equation by the method
Vol. 17, No. 6
of least squares. With the aid of this graph it is possible to obtain a t least approximate values of U from corresponding values of U(IC1) determined by the method described above. More recently Kemp and Peters (3) have published an iodine chloride procedure for Butyl rubber in which a different soIvent and different reaction conditions are employed. It is likely that their method would be capable of similar use in estimating U by this indirect method. It x a s considered of interest to include in Figure 5 curves for Butyl polymers containing butadiene and dimethylbutadiene, respectively, as the diolefin units. Over the unsaturation ranges investigated, these materials show linear relationships between U (ozone) and U(ICl), although the slopes differ by about 20%. I n view of the well-known influence of substituent groups on the course of the reaction of an olefin with halogens, these differenres are not surpriqing. ACKNOWLEDGMENT
The writers are indebted t o some of their associates n-ho synthesized the special polymers used in part of this work. LITERATURE CITED
(1) Flory, P. J., J. Am. Chem. SOC.,65, 372 (1943). (2) Flory, P. J., unpublished data. (3) Kemp, A. R., and Peters, H., IZD.E m . CHEM.,AXAL.ED..15 453 (1943).
(4) Rehner, J., J r . , IND. ENG.CHEM.,36, 118 (1944).
(5) Thomas, R. M., unpublished method.
Fluorometric Determination of Riboflavin in Eggs W A L T E R J. PETERSON, R. S. DEARSTYNE, R. E. C O M S T O C K , Department of
A
AND
VIRGINIA WELDON
Animal Industry, North Carolina Agricultural Experiment Station, Raleigh, N. C.
S U M B E R of studies on the riboflavin content of eggs by the biological rat-growth assay (3, 5, 7 , f f )and the microbiological procedure of Snell and Strong (2, 4, 12, 13, 14) have been reported. Application of the fluorometric method has, however, been rather limited (1, 8, 9). The early reports of results by the rat-growth method are difficult to interpret in terms of micrograms of riboflavin. The microbiological method, though generally considered t o provide reliable results, requires specialized techniques which are not familiar to all chemists. Since extracts of some biological materials may exert either inhibitory or stimulating effects (14) in the microbiological procedure, the adaptation of the convenient fluorometric method to many materials would seem desirable for purposes of comparison. Experience with fluorometric techniques has shown that methods which are applicable to certain plant or animal tissues, may need considerable modification before they can be used with success on other materials. The riboflavin content of hard-boiled egg white can be conveniently determined fluorometrically by a method previously proposed for its determination in pork products ( I O ) . When the hard-boiled yolk is used in this determinat'ion, however, the resulting acid extract (after autoclaving and incubating with clarase) is a stable emulaion which does not provide a clear, readable filtrate. It was found that successful clarification of the extract could be accomplished by either of two methods: precipitation of the extract x i t h two volumes of acetone as proposed by Hand (6) for the fluorometric determination of riboflavin in milk, or breaking the emulsion by mixing the extract with a small amount (5% of the total volume) of chloroform in a Waring Blendor for 30 seconds. From the standpoint 'of analytical speed, simplicity of operation, and accuracy, the latter method was found to be preferable. The acetone precipitation method
has the disadvantage of dissolving the yolk carotenols. This necessitates the simultaneous precipitation oi an extra aliquot of extract with acetone containing a known amount of pure riboflavin, in order to determine the increment in the fluorometer reading due to the added riboflavin. I n the following comparison of methods, the eggs used n-ere from Rhode Island Red hens which had been receiving a diet containing 6.2 micrograms of riboflavin per gram. I n addition to duplicate determinations on individual eggs from different birds, analyses were also made of eggs laid on the second successive day for the same birds. All eggs were analyzed within a iew days after being laid. DESCRIPTION OF METHODS
The eggs are hard-boiled for 6 to 10 minutes, peeled, and the whites and yolks separated, since they arc to be determined separately. The first stage in the preparation of the extract is the same for both white and yolk. The entire yolk (weighed) or 10 to 20 grams of white is dropped into 100 cc. of 0.04 N sulfuric acid in a Waring Blendor and macerated for 2 minutes at high speed. The creamy mixture obtained is then transferred quantitatively to a 250- or 300-cc. Erlenmeyer flask, using a minimum amount of water from a wash bottle to effect the transfer. The flask is plugged with cotton and autoclaved 15 minutes a t 6.8-kg. (15-pounds) pressure. -4s soon as the flask has cooled, 20 cc. of a 2.5y0solution of clarase, freshly prepared in a sodium acetateacetic acid buffer, are added. [The buffer solution (pH 4.5) is prepared by adding 54.4 cc. of glacial acetic acid to 66.9380 grams of anhydrous sodium acetate together with sufficient distilled water t o obtain a solution of the reagents, and then transferred to a 1-liter volumetric flask and made up to volume with distilled water.] The contents of the flask are mixed thoroughly and then incubated a t a temperature of 45" C. for 24 hours. The flask is agitated two or three times during the incubation. Following the incubation period, the extract is brought to a volume of 200 cc. At this point the contents of the flask should
ANALYTICAL EDITION
June, 1945
be thoroughly mixed, either by stoppering the flask and shaking thoroughly, or transferring the contents t o a Waring Blendor and mixing for 30 seconds. A t this stage, the extract of the egg white may be filtered or clarified by centrifugation and the riboflavin concentration determined as previously described (IO). The yolk extract, however, is a stable emulsion which cannot be clarified by either filtration or centrifugation. Either of the following treatments n-ill provide clear filtrates. PRECIPITATIOS WITH ACETONE.Into each of tn-o test tubes, -1and B, are pipetted 5 cc. of the well-mixed yolk extract. Ten cubic centimeters of acetone are added t o tube A, and 10 cc. of acetone containing 0.15 microgram of riboflavin per cc. to tube B. Tube B thus contains an increment of 0.1 microgram of riboflavin per cc. over tube -4. The solutions containing the precipitated yolk protein are mixed and filtered through S o . 597 S. and S. filter paper. Fluorescence readings on the filtrates may be made immediately. With the Coleman electronic photometer (Model 12), the instrument was adjusted each day with a sodium fluorescein solution of such strength that, when Its rcading was 100, a n aqueous solution containing 0.2 microgram of riboflavin per cc. read 70. This range was suitable for most yolk extracts prepared in the above manner. Having obtained readings for A and B, 0.5 cc. of sodium hydrosulfite solution is added to each tube and readings are taken again. The average of these two readings constitutes the reading of the sample blank, or C reading. Since acetone extracts become cloudy in a short time when treated with sodium hydrosulfite, it is necessary t o take readings immediately after each addition. Knowing the fluorescence increment due to 0.1 microgram of riboflavin per cc. (B - A), the riboflavin concentration is readily calculated (IO). The hydrosulfite solution was prepared by dissolving 5 grams of sodium hydrosulfite in 100 cc. of an ice-cold sodium bicarbonate solution (2 grams of sodium bicarbonate per 100 cc.). CHLOR~FORX TREATMEXT. The entire volume of yolk extract (200 ec.) is poured into a Waring Blendor, 10 cc. of chloroform are added from a buret and the solution IS mixed for 30 seconds. After being transferred to the original flask, the extract is permitted to stand in the dark for a t least 30 minutes, after which it is filtered and approximately 20 cc. of filtrate are collected. The chloroform treatment breaks the emulsion and provides a clear filtrate. Filtrates may be kept under refrigeration for several days without deterioration or change in riboflavin content. The riboflavin concentration may be determined as previously described for its determination in pork products ( I O ) . It, is important that a “complete blank” containing all the reagents and clarase used in the method be run through the entire procedure, and the resulting concentration subtracted from the v d u e of the sample extract. This applies to both the acetone and chloroform treatments. Itecoverics of riboflavin varied from 97 to 1027, when 10, 20, 30, or 40 microgranis of riboflavin were blended with yolk samples and analyzed by the chloroform t,echnique. Table
I. Typical Results OF Riboflavin Determinations in Egg Yolks
Bird KO.
7 5 13 25 1 10
Acetone Method May 10 M a y 11 Macrogram 9 / g r a m
3 3 3 4 4
48 09 15 65 05 4 14
4 07 2 3 4 4
83 43
74 10
4 26
CHCla Method >fay 10 M a y 11 .\licragrams/gram 3 56 3 43 2 96 2 84 3 24 3 20 4 55 4 36 4 01 3 95 4 03 4 46
DISCUSSION
Two eggs from each of 18 birds were analyzed. The two ezgs were in every case litid on successive days. Table I contains typical results for yolks obtained by the two clarification procedures. If the methods are equally accurate, the variation between eggs by the same bird should be approximately equal, no matter which is used. The standard deviations of eggs from the same bird were 0.36 and 0.26 microgram for the acetone and chloroform t,echniques, respectively. The difference between thece values is not statistically significant but it should be safe to conclude that the chloroform method is a t least as accurate as the other. I t will be apparent to the analyst that from the standpoint of simplicity of operation and analytical speed, the chloroform clarification procedure is much t o be preferred.
371
Duplicate analyses were made on the yolks of each of 12 egg+ by the chloroform technique, using weighed halves of each yolk as duplicates. The standard deviation of duplicates was 0.21 microgram. Comparing this with the standard deviatiqn (0.26 microgram) for eggs from the same bird where each yolk constituted a sample, it is apparent that variation between eggs laid by the same bird on successive days is small. The standard deviation for duplicate analyses on whites was 0.15 microgram, and the standard deviation of eggs laid on successive days was 0.18 microgram. Again the real day-to-day differences must have been small. I n neither case, however, would i t be safe to attempt to deduce the range of values likely t o be encountered in the eggs of a single bird over a period of several days. There might be cyclic changes: in fact, such change> could account for some of the observed variation from bird to bird. There were highly significant differences between birds 111 riboflavin content of yolks, whites, and whole eggs, and in the ratio of riboflavin in yolks to riboflavin in whites. Mean values and standard deviations are listed in Table 11. The riboflavin content of whole eggs was calculated from values obtained on yolks and whites. Table
II.
Typical Results, Riboflavin Content of Eggs Yolk Khite Yolh/White Whole E g g
Mean of 36 egg8 Standard devlation of eggs from different birds
Mzcrograms/gram
Macrograms/gram
4 09
2 67
1.19
0 44
.Vicrogrnms/gram
1 53
3 18
0 48
0 53
~~
SUMMARY
Stable emulsions which interfere in the fluorometric determination of riboflavin in egg yolks can be clarified by mixing the extract in a Waring Blendor rvith a small amount of chloroform. Average riboflavin values for all eggs studied xere (micrograms per gram): yolk, 4.1; white, 2.7; entire egg, 3.2. Variations from day to day in the riboflavin content of eggs from the same bird were extremely small. The difference in the riboflavin content of eggs from different birds on the same ratio11 11-aslarge and highly significant. The average ratio of riboflavin in yolks to riboflavin in whites was 1.53. The standard deviation of the ratio was 0.48. LITERATURE CITED
Bauernfeind, J. C., and Norris, L. C., Poultry Sci., 18, 400 (1939).
Cheldelin, V. H., and Wdliams, R. J., University of Texas, Publ. 4237, 105-24 (1942).
Daniel, E. P., and Munsell, H. E., U. S. Dept. Agr., .UZSC Publ. 275 11937). Engel, R. TG., Phillips, P. H., and Halpin, J. GI, Poultry Sci., 19, 135-42 (1940). Euler, H. V., Adler, E., and Schlotzer, A,, 2. physiol. Chem., 226, 87-94 (1934). Hand, D. B., IND.ENQ.CHEM.,A y . 4 ~ ED., . 11,306-9 (1939) Hund, C. H., Winter, A. R., and Bethke, R. M.,Poultry Sci., 18, 330-6 (1939). Llurthy, G . N., I n d i a n J . N e d . Research, 24, 1083-92 (1937). Norris, L. C., and Bauernfeind, J. C., Food Research, 5, 521-32 (1940). Peterson, IT-. J.,Brady, D. E., and Shaw, A. O., ISD. ESG. CHEM.,ANAL.ED., 15, 634 (1943). Rose, M.S., and Vahlteich, E. &I.> J. Am. Dtetet. Assoc., 14, 593-GI4 (1938). Snell, E. E., and Quarles, E., J . Sutrition, 22, 483-9 (1941). Snell, E. E., and Strong, F. M . , IXD.ESG.CHEM.,ASAL. ED., 11, 346 (1939). University of Texas, Publ. 4137, 4237 (1941 and 1942). PRESEXTED before the Division of Biological Chemistry a t the 108th Meeting of the AMERICASCHEMICAL SOCIETY,New York, N. Y . Published with the approval of the director as Paper 208 of the Journal Series. This investigation was aided by a S a i f t a n d Co. fellowship.
