Thiamine in Beef A
Muscles
Comparison of Values by the Thiochrome Reaction A p p l i e d with and without Adsorption
WINIFRED F. HINMAN, EVELYN G. HALLIDAY,
AND
MARGARET H. BROOKES,
The Harris and Wang nonadsorption thiochrome technique and the Hennesry and Cerecedo base-exchange technique have been compared, as applied to beef muscle. Although 40 to 100% discrepancies were frequently found, only up to 20% differences between the two values could b e explained b y incomplete adsorption, resulting in low Hennessy values when 2 to 5 micrograms of beef thiamine were placed on a column. Comments are made on enzyme digestions and on the Harris thiochrome reaction. A few comparisons with yeast fermentation values are also included.
Older reports based on bioassay methods include more figures on beef in the lower range-bradycardia method (6),Sherman Chase growth method (8), Cow ill calculation from pigeonprotective and rat-growth data ?I$), rat-growth method calculated as Sherman units by Daniel and lllunsell (13, 31)-than in the higher range-rat-curative method, one value, calculated as Sherman units by Daniel and Munsell (IS,1 7 ) ; bradycardia method, one figure, highest, stated to have probable error of 50%
(4, 6). Thus, with bioassay values in the literature falling in ranges which are 100% apart, it was not possible to 6nd support for either of the thiochrome techniques exclusively.
lN
T H E first comparisons of the applications of the Harris and Wang (14, S 4 ) and the Hennessy and Cerecedo (15, 50) thiochrome techniques to identical digests of beef muscle, the authors found the values obtained from the former exceeding those from the latter by 40 to 100%. These discrepancies raised the questions as to whether the direct oxidation technique applied to beef results in the inclusion of nonthiochrome fluorescent compounds which would be eliminated in the Hennessy base-exchange technique and whether the latter procedure can be depended upon to adsorb all the thiamine from beef digests. The data here presented indicate that in some samples of beef, nonthiochrome substances do interfere in direct oxidation, but that, on the other hand, the adsorption procedure is likely to give results which ctre around 10 t o 20% too low if an amount of beef digest containing from 2 to 5 micrograms of thiamine is placed on a 7- t o 8-cm. Decalso column. Although no data were obtained on other foods, this experience with beef would indicate a need for caution in the indiscriminate application of the Harris and Wang technique to low thiamine foods as well as in the application of the Hennessy and Cerecedo technique to such foods when this is done in the same quantitative relation of thiamine to Decalso that has been described as optimal for cereals (30). Nonadsorption methods of thiochrome determination in both animal and vegetable tissues, which vary in details of digestion, of the final oxidation reaction, and of the reading of the fluorescence, have been described in a few American reports (2, 16,22) and in an earlier German one (%), as well as in many British reports (?, 14, 19, $6-19, 34). On the other hand, applications of the Hennessy and Cerecedo base-exhange technique have been widely reported in this country (1,R,9,10,18,81,25) and a similar adsorption procedure was described in one earlier Dutch study (55). Figures on beef muscle are included in only one paper from each of the two groups listed above (18, Z9), and all their fi ures are in the range covered by the authors’ Hennessy vaues. Lane, Johnson, and Williams (18) reported verification of their thiochrome results on beef by the curative bioassay. Further comparison of the authors’ results with other recent bioassay reports on beef showed that the Hennessy value on their first sample of round-namely, 1.0 to 1.1 micrograms per gram-was in good agreement with one figure on lean top round obtained by Booher (6) by a rat-growth method, 1.14 micrograms per gram whereas their Harris value on this round, though 40 to 50% higher than the Hennessy, was just a t the lower end of the range of values obtained on raw round by Waisman and Elvehjem (55)with the rat-growth method of Arnold and Elvehjem (3). The authors’ first Hennessy values on rib ranged between 0.6 and 0.8 microgram per gram and Harris values between 1.2 and 1.3 micrograms per gram. Yet they learned that McLaren and Cover (ZS), using papain-takadiastase digests, were getting Harris values on beef rib which varied between 1.9 and 2.8 micrograms per gram, most of them in the upper end of this range and thus close to the highest value reported for raw round by Waisman and Elvchjem, 2.8 micrograms per gram.
University of Chicago, Chicago, 111.
DISCUSSION OF TECHNIQUES
No important modifications were made in most of the details of the assay methods and for this reason the exact procedure is not given here. In the Hennessy method the authors combined details from three sources (10, 24, SO) and in the Harris method they used essentially the revised Wang and Harris procedure (S4). However, it seems pertinent to discuss a few points in the techniques. SAMPLINGAND DIGESTION. There has been some general opinion among authorities that the complete extraction of thiamine from tissues is difficult ($6). For this reason, the authors felt it desirable with ground beef to keep the sam lesolvent ratio down t o the middle range recommended for cereaithat is, about 1 to 25 or 1to 20 during acid extraction. They now feel, from some later experience with more concentrated digests of beef samples blended in the Waring Blendor, that a concentration of 1 to 10 or even 1 to 8, sample to solvent, can be safely employed with motor stirring during extraction a t 70” to 75” C. Digestion with clarase was carried out overnight a t 45’ to 48’ C. I n view of the fact that digestion was carried out and volume made up in calibrated 250-ml. centrifuge bottles it was found convenient to clear digests by centrifuging after vigorous shaking with 5 ml. of reagent grade chloroform which took the fat to the bottom. That the chloroform had no effect on the adsorption of thiamine (or riboflavin, 11) or its subsequent determination by fluorescence was shown by comparison of values with filtered portions of identical digests not exposed to chloroform. HENNES6Y BASE-EXCHANGE. Before being used in this study all samples of Decalso had been tested by placing 5 micrograms of buffered synthetic thiamine hydrochloride on 7- to &cm. columns. Recoveries were always 95% or better with the adsorp tion and elution techniques employed. All adsorptions and most elutions were carried out a t room temperature. However, in the spring of 1943 samples of Decalso were obtained from which complete elution could be accomplished with 25 ml. of 25% potassium chloride41 N hydrochloric acid only if the latter was used a t practically boiling temperature. The experiment reported in Table V was done with such a sample of Decalso. HARRIS THIOCHROME REACTION. Harris (14) states that an excess of ferricyanide is to be avoided to prevent a partial destruction of the thiochrome formed and variable effects on nonspecific fluorescent substances. I n attempting to determine the correct amount of ferricyanide to add, by applying the Wang and Harris (34) criterion of “yellow color lasting more than 30 seconds” some difficulty was encountered because of the faint brownish ellow color of the digests which was accentuated by the prefminary addition of alkali and because additional drops of ferricyanide progressively increased the readings. (Throughout this study the amounts of 2% potassium ferricyanide used per reaction were 0.03 to 0.08 ml., varying from 1 to 3 dro s depending on the size of drop and on the digest.) Frequentb, one additional drop used when the excess was questionable increased the “thiochrome” value as much as 10%. Since the authors have never observed a decrease in readings with larger excesses of ferricyanide, even though thiochrome itself is known to be unstable under these conditions, it would seem as though beef digests con-
116
ANALYTICAL EDITION
February, 1944
tain some material, other than thiamine, which can add to the reading with this direct oxidative treatment. Such a possibility finds some support in the observation that very large excesses of ferricyanide, such as 2 ml. of 2% potassium ferricyanide, cause increases in fluorescence up to 40% of the thiochrome value a8 determined by the usual Wang and Harris criterion. STANDARDIZATION. For all the Harris determinations and for the first group of Hennessy experiments (1 through 5, Table I ) standardization was done with “external standards”--that is, crystalline thiamine in proper diluent, reacted by itself. Later Hennessy experiments (6 through 9) were based on reading of crystalline thiamine added to th-e unknown eluates, “internal standards’’ (84). Although occasional internal standards read just as high or higher than “external” ones the average internal standard readings were about 6% lower than the average of external standards reacted in this series. Hence, the Hennessy values of the first group of experiments (samples 1 through 5, Table I) could be corrected upward by about 6% to be on the same basis as the second group. Fluorescence readings of “external standards” over several months averaged: for Hennessy, using 15 ml. of isobutanol, 10.8* 0.22 galvanometer units per 0.1 microgram; for Harris, using 2 ml. of methanol, 10 ml. of isobutanol, and 2 ml. of ethanol, 12.35 * 0.37 units per 0.1 microgram. For both, a quinine working standard containing 0.0135 mg. of quinine sulfate per 100 ml. of 0.1 N sulfuric acid was set a t 60 on thP Coleman electronic photofluorometer (Model 12). DISCUSSION OF RESULTS
KO doubt, pronounced differences in thiamine content of various beef samples are to some extent responsible for the variations found in reported values and for the variations in values obtained by the Harris thiochrome technique in the authors’ laboratory and theTexas laboratory. Moreover, if the Harris technique measures some additional fluorescent material produced in the oxidation reaction, besides thiochrome, variations in the occurrence of such another factor would also contribute to differences between samples analyzed in the two laboratories by this method. The authors sought t o raise their values on beef by digesting with papain because Harris stated that papain removes an inhibition (present in animal tissues) t o hydrolysis of cocarboxylase by takadiastase. If this is true, papain digests ought to show higher thiochrome valuw than acid-clarase digests by both the
Table
1.
117
Harris and Hennessy methods, but as results recorded in Table I indicate, the authors have not once obtained higher values by either Harris or Hennessy thiochrome technique on papain-clarase or papain-takadiastase digests. However, they cannot dismiss the possibility of an influence on Harris values related to the method of digestion because hlclaren, working in the authors’ laboratory, made four papain-clarase digests of rib sample 5 (Table I) on which they obtained an average Harris thiochrome value of 1.54micrograms per gram as contrasted to the 1.15 micrograms per gram (see table) for the acid clarase digests made on the same day, whereas the Hennessy values were in agreement for the two. The only possible fact to which this variation in Harris values can be ascribed is the use of a different papain, for McLaren used a Difco preparation, whereas the authors have used Parke, Davis preparations from two different lots, both of which have shown strong proteolytic activity in that they dissolved the beef almost completely. It is possible that the higher Harris thiochrome results are related t o the presence in some papain samples of another enzymatic activity other than the proteolytic, The authors hope t o investigate this further. According to McIntire and Elvehjem ($0) the thiochrome values on pork muscle are the same whether digestion is done with papain-clarase or acid-clarase. Moreover, in the acid-clarase digests, the adsorption and the direct oxidation techniquesgive the same thiochrome value. In attempting t o discover the reason for the difference in values with and without adsorption the first line of attack used was one suggested by Dr. Elvehjem-applying sulfite blanks to the Harris values obtained on the beef digests. The sulfite treatment done a t room temperature overnight a t pH 5 t o 5.5 (37) was more satisfactory than the short treatment a t boiling temperature (SI) because with the latter new fluorescent material was formed which added to the reading after ferricyanide treatment. However, the oxidized fluorescence values left after sulfiting a t room temperature were variable, sometimes so low that if applied on the Harris values as blanks, they would leave thiamine values higher than the Hennessy by 10 to 407& whereas in one test the residual values were so large as to reduce the Harris values far
Thiamine Values on Beef
(Showing variation in discrepancies between Harris and Hennessy thiochrome results) Length of Fresh or Sample C u t and Agin Frozen Storage No. and Type Volume NO. Grade Perlo3 Period of Digests Adsorheda Days M1. Raw frozen 96 days 4 , HzsOr clarase 100 Rib, packer’s grade 4 21 4, papain clarase 100 Raw frozen 72 days 2, HnSOi clarase 50 Rib, packer’s grade 4 8 (heifer) 4, papain clarase 80 Whole round, packer’s 11 Raw fresh 100 Raw frozen 21 days 6, HzSO4 clarase grade 3 (heifer) 75 Raw frozen 34 days 5 , &SO4 clarase Raw fresh 3, HzSO4 clarase 75 Chuck, packer’s grade 4 Cooked fresh 3, HzS04 clarase 75 3 (steer) Rib eye ? Raw fresh 3, HnSO4 clarase 70 Outside round, U. 5. 5 100 Raw frozen 7 days 4,HzSOI clarase grade commercial (steer) Raw fresh 4, HzSO4 clarase 100 Rib, U. 5. grade good 9 100 Raw frozen 41 days 2 HzSO4 clarase 2: papain takadiastase 100 Raw frozen 74 days 2, HzSO4 clarase 50 Outside round, U. ,S. 6 grade commercial (heifer) Liver
a
?
Raw fresh
1, H&OI clarase
1, papain clarase 1, HC1 takadiastase
35 35 20*
9
Harris Thiochrome Hennessy On filtrates Thiochrome On from adon digests sorption Eluates Y/Q. Y/O. r/a. 1.29 0.67 0.64 1.30 0.72 0.61 1.22 .. 0.78 1.48 1.55
y/g.
%
0.65 0.69 0.44
101 113 56
0.49 0.46 0.58 0.69
0.51 0.52 0.44 0.33 0.21 0.57 0.34
53 50 40 67 47 98 49
0.44 0.36 0.42
0.77 0.90 0.85 0.91
0.26 0.29 0.19 0.25
34 24 22 21
..
2.65 2.70 2.93
0.33 -0.08 -0.07
14 0 0
0.62
..
1.55b
..
0.82 0.66 1.15 1.03
0.34 0.26
1.03 1.19 1.04 1.16
2.88 2.62 2.82
Difference between Harris and Hennessy Values Percentage of Hennessy value
..
0.48
..
..
0.97 1.03 l.llb
Beef concentrations were between 0.035 and 0.040 gram per ml. in all digests except starred one of sample 8, 0.133 gram per ml. and starred one of sample
9. 0.061 gram per ml.
Crystalline thiamine added t o two of the five digests included in these averages was equivalent to 0.71 y and 0.63 y per gram of sample and the recoveries based on the sample averages here given were by Harris 97 and 98%, respectively, and by Hennessy 100 and 87%, respectively. b
INDUSTRIAL A N D ENGINEERING CHEMISTRY
118 Table
II.
Fluorescence b y Harris Thiochrome Technique on Sulfited Beef Digests” Sample 4 Sample.3 8/25/42 Di‘bts Sample 7b 7/28/42 D i g h Raw Cooked 1/10/43 Dige’sts Sulfited Sulfited Sulfited Sulfited Sulfited Sulfited
Digest No. 1 2 3 4 5 6
7/31
8/3
8/28
8/28
1/18
1/27
Y/Q.
7/g.
Y/Q.
Y/Q.
r/o.
Y/P.
0.21 0.19 0.00 0.18 0.09 0.13 0.13
0.23 0.33 0.36 0.33 0.25 0.21 0.28
0.20 0.15 0.26
0.08 0.16 0.24
0.19 0.23 0.00 0.34
0:37 0.65 0.60
..
..
.. .. ..
.. .. ..
..
..
Vol. 16, No. 2
backs for many days and one had extreme paralysis and the whirling syndrome on the 20th day and died on the 21st day. The filtrates from which the concentrates were prepared had been obtained by passing portions of digests containing about 6 micrograms of thiamine through 7- to 8-cm. (1.0 to 1.3 grams) columns of Decalso. These conditions are near the “preferred” for cereals as given in the directions by the Research Corporation Committee (30),but apparently they were not the optimum for the adsorption of thiamine from the beef digests. To obtain further evidence about the efficiency of adsorption in relation t o various concentrations and various volumes of one
Av. 0.20 0.16 0.25 0.64 Harris minus sulfite blank 1.42 1.27 0.62 0.50 0.87 0.58 Hennessy 1.03 0.49 0.45 0.88 6 Calculated as y of thiamine per gram of beef. b Digests 1 and 2 were acid clarase, 3 and 4 were papain-clarase.
below the Hennessy. The erratic results with repeated sulfite treatments of the same digests on different days and the variability between the reveral digests of one sample are shown in Table 11,which repeats some Harris and Hennessy values given in Table I. Because there is such variation the authors felt insecure in assigning too great significance t o sulfite blanks, for it seemed that neither the specificity of sulfite for destruction of thiamine only, nor the prevention of formation of a new interfering oxidized fluorescence could be depended upon when measuring such fluorescence in beef digests. Since Wang and Harris (34), following McFarlane (M), suggest the use of hydrogen peroxide after the ferricyanide treatment to destroy interfering fluorescent substances in urines, milk, and yeast extracts, the authors made a few comparative tests on beef digests with 5 drops of 5 % hydrogen peroxide per test as Harris uses it and also with 1 ml. of 30% hydrogen peroxide as McFarlane and Chapman direct for extracts of grass, wheat germ, yeast, and flour. As Table I11 indicates, peroxide treatment did not destroy any of the fluorescence present after oxidation of a beefdigest aliquot. Since the difference between the two thiochrome values on the same digest of beef muscle is more than equaled by the values obtained when applying the Harris thiochrome technique to filtrates from adsorption (Table I), the determination of whether or not such filtrations contain any appreciable thiamine activity seemed to be the best approach to the problem, Using such filtrates for rat assay WBS suggested by R. R. Williams. The completion of a conclusive experiment of this kind waa frustrated by the fact that three samples of beef bought in large quantity for this purpose (samples 6 , 7, and 8) showed less actual difference, expressed as micrograms per gram, in the thiamine, values obtained by the two method* of assay than most earlier samples. I n fact, no real difference beyond experimental error was found between the two values in sample 8 and in a beef liver sample, No. 9, which was tested with the thought that this tissue might display more discrepancy between the two techniques. Such low differences brought about problems in the preparation of concentrated filtrate to be fed to rats. The authors felt that to be tested for activity the filtrate fed each day must represent a t least 1 microgram of thiamine difference between the two thiochrome values. For sample 7, with which a limited rat experiment was carried out, this meant that filtrates from adsorption needed to be concentrated so that 1.2 ml. of final preparation represented 4 grams of the original beef. The concentrate was tube-fed to 20 rats in four groups, as shown by the chart of average weight changes (Figure 1). Although the results cannot be considered as conclusive because of the small number of animals used, they indicated that there was a slight thiamine activity in the filtrate from the adsorption. This was further substantiated by the fact that, whereas Jhree out of five of the negative control anunals, Group D, died on the 17th, 18th, and 20th day of the supplementing period, among five anim d s on the experimental supplement, Group C, none had died on the 20th day. In this latter group all showed extremely hunched
Figure 1.
Average Weight Changes in Rats
In a 2Oday supplementing period: A . 4 rats received daily 1.2 mi. of concentrate treated with NamSOI to desboy thiamine, and I Y of nvtblline thiamine in 1 0 % alcohol. 8. 6 rats received daily 1.2 mi. of concentrate wlth crvskliine thiamine added before concentration In an amount eauivalent to Ir Der 1.2 ml. of concentrate. C. 5 rats received daily 1.2 mi. of concentrate being assayed for thiamine activity. D . Started with 5 rats, received daily 1.2 mi. of concentrate treated with NaSOs.
Table
111.
Digest NO.
Effect of Hydrogen Peroxide in the Harris Thiochrome Reaction on Beef Digests (Sample 3) 7/28/42 Digests NO 5 drops HzOr 5 % H20?
Table
8/10/42 Digests NO 1 ml. HzOz 30% H:01
IV. Hennessy Thiamine Values
(In relation to total amount of thiamine placed on a Decalso column. Obtained in several experiments on a raw beef round, sample 8.) Beef Thiamine Recovery of ConoentraVolume Placed on Thiamine Crystalline tion .4dsorbed Columna Determined Thiamineb G/d. M2. Y 7/Q. % 25 1.07 1.27 ... 0.0332 25 1.20 1.29 102 0.0373 0.0421 25 1.36 1.28 ... 40 2.12 1.29 ... 0.0412 50 2.15 1.18 100 0.0332 25 2.20 1.22 96 0.0682 0.0705 25 ?.28 1.28 97 0.0373 50 ...41 1.26 88 A r . 1 . 2 7 i 0.017 (omitting 5th) 2; 3.16 1.19 ... 0.0982 75 1.25 91 3.60 0.0373 75 4.07 1.19 ... 0.0421 1.19c ... 25 4.29 0.1333 25 4.29 1.25e ... 0.1333 50 4.39 1.11 .. . 0.0682 4.72 1.16 100 0.0366 1.16 ... 1.81 100 0.0373 1.11 ... 4.88 100 0.0378 1.19 ... 100 5. Oti 0.0393 .
.
I
4 Computed by use of highasr value, 1.297 per gram. ever obtained on this sample. b One or 2 y added to another column with same quantity of beef digest. c Made with 0.05 .V HCI, all others with 0.1 1v HaSOd.
February, 1944
ANALYTICAL EDITION
119
concentration, the experiments presented in Tables I V and V were carried out. Table V. Hennessy Thiamine Values (Obtained on one digest of raw beefa with accompanying Harris values on filtrates from adsorption at The two beef samples used in these exseveral levels. Beef concentration in digest: 0.0551 gram per mi.) periments were higher in thiamine content Thiamine Placed Hennessy Computed Harris Decalso than the rib used in the rat experiment Volume on Column Thiamine Recovery Loss in Values on Column of Di est From Added Values Found of Added Adsorption Hennessy by about 35y0 in the case of sample 8 N0.b bdsor%ed beefe crystalline for Beef Thiamined Determination’ Filtrates (Table IV), and about 6% in the case of MZ. 7 7 Y/B. % Y/O. 7/0. the “heel of round” (Table V). Hence, 1 25 1.35 .. 0.98 .. 0 2 25 1.35 0.94 0.04 o:i4 the proportion of interfering material to 3 25 1.35 2.0 .. 95 4 25 1.35 2.0 02 o:i4 o:ia thiamine might have been appreciably 5 25 1.35 .. 0.93 .. lower in the former at least. Neverthe6 25 1.35 0.87 .. o:io o:i3 less, the results on this show that the 7 25 1.35 2.0 .. 92 0.19 0.18 8 25 1.35 2.0 .. 98 .. .. trend of thiamine values is toward lower 9 50 2.70 ., 0.81 . . 0.18 0.32 values as larger amounts, up t o about 7 10 50 2.70 0.82 11 50 2.70 2.b .. 99 o:i7 0:26 micrograms of thiamine, are placed on 12 50 2.70 2.0 .. 104 .. .. the 7-cm. (1.0 to 1.3 gram) Decalso 13 75 4.05 .. 0.72 0.26 0.23 column; that the gradation toward lower 14 75 4.05 0.76 .. 15 75 4.05 2.b 98 0:23 0:26 values is not absolutely consistent with 16 75 4.05 2.0 .. 98 .. .. increasing amounts adsorbed; but that 17 100 5.40 .. 0.70 .. 0.27 0.35 18/ 100 5.40 0.73 when adsorption was in the range of 1 t o 19 100 5.40 2.0 .. 47 0147 0156 2.5 micrograms the average of seven 20 100 5.40 2.0 .. 73 0.34 0.35 values, 1.27 =t0.017 micrograms per gram 21 150 8.10 0.66 0.32 0.38 22 150 8.10 0.76 0.22 0.40 (omitting one which was in less good 23 150 8.10 2.0 .. 59 0.42 0.40 24 150 8.10 2.0 39 0.37 0.51 agreement) is appreciably higher than a “Heel of round” cut. most values obtained with adsorption of b Lengths of Nos. 1-4, inclusive, tiere 13 t o 14 cm. (2.2to 2.4 grams of Decalso); all others 7 to 8 cm. (1.1 to 1.3 grams). larger amounts. I n the range of 3 t o 5 e CompGed by use of highest Hennessy value 0.987 per gram. micrograms per column there is some d Based on average of two values on beef a1one)obtained a t corresponding level of adsorption, except columns 19.20.23, and 24,where recovery was based on single determination with corresponding elution overlapping of values with the first group, volume. See f. e Based on highest Hennessy ralue and same plus addpd thiamine. but most of the ten determinations in this I Volumes of elutions with boiling hot KCl-HCI were 25 ml. in all exrept: 18, 40 ml.; 20, 40 ml.; range were 6 to 9% lower than the above 22, 30 ml.; 24, 50 ml. average and two were almost 13% lower. With 6 t o 7 micrograms placed on a Table VI. Comparison of Thiamine Values b y Yeast Fermentation column the values were 12 to 28% lower. and Thiochrome Methods In the experiment with the “heel of round” sample (Table V) Schultz and possible variations due to sampling and extraction were elimiHennessy Frey Yeast Harris nated, for all adsorptions were done from one large digest. Also, Fermentation Sample Date Thiochrome Thiochrome standard recovery determinations were carried to higher levels Y/O. Y/Q. Y/B. 3, raw frozen 8/10/42 1,53 1.11 of adsorptions and Harris thiochrome determinations were 1:22 8/31/42 included on a t least one of every pair of adsorption filtrates t o 8/25/42 4,raw fresh 0.82 0.49 discover whether there was any inverse correlation of these 0:so 4,raw frozen 8/31/42 .. with the decreasing Henneasy values on eluates. 0.45 0.66 4, cooked fresh 8/25/42 0134 4, oooked frozen 8/31/42 The downward trend in Hennessy values was even more rapid 0.91 0.91 7, raw frozen 2/12/43 1.16 in tliis sample, being 10 to 15% a t the level of 2.7 micrograms of 1.22 1.19 8, raw frozen 2/12/43 1.34 beef tliiitmine per column, and 18 to 23% at 4.05-microgram level. The decrettse was not, hon-ever, correspondingly greater above 5 micrograms, averaging around 20 t o 25% but reaching an extierne of 31% in one determination with 8.1 micrograms per colbe equally good in more concentrated digests-that is, in those umrl. However, the recovery of an added 2 microgrms of syncontaining 0.07 to 0.09 gram of beef per milliliter-as in those thetic thiamine per column was decidedly reduced when the which are from one half to one third as concentrated. Therebeef t1ii:rmine was above 5 micrograms as compared to the recovfore the more practical technique would seem t o be adsorption of eries a t intermediate levels. The latter, in fact, appear to be small volumes, 20 to 25 ml., of digests which contain from 1 to misleading in their highness when based on beef values obtained 2.5 micrograms in the total volume adsorbed. a t the same level of adwrption \\-here apparently there was 10 to There is no obvious reason for the relatively lower differences 207, loss of beef thiamine. between Harris and Hennessy values found in the last samples There is no doubt that the amounts of thiamine as measured of rib and round as compared to those in the earlier ones. by the Harris thiochrome reaction carried out in the adsorption It would seem that there must be variation in the occurrence of filtrates show a n upward trend as Hennessy values become lower, that factor which is responsible for the discrepancies between the and though the correlation is not perfect, this finding makes it fluorescence readings obtained after direct ferricyanide oxidation more conclusive that the major loss a t hinher levels is in the adof digests and that following adsorption. Since there is no sorption. Physical interference with photofluorometry a t reason to believe that adsorptions were any more complete in higher levels is ruled out by the fact that “internal standards” the Hennessy assays of samples 7 and 8 than in earlier ones, such added t o eluates are used for calculations of all Hennessy values, an explanation cannot be taken as the reason for the two values high and low, and such internal standard readings average about coming closer together. Besides, in most adsorptions of the the same in aliquots of cluntes from the 1:irger adsorptions as in first five samples the amounts of thiamine placed on a column those from the smaller one?. were under 2.7 micrograms, reaching as high as 4.0 and 4.5 in the Obviously, then, in beef digests, interfering material is present case of only a couple of the adsorptions of the second experiment in sufficient amount so that it is necessary to adsorb under 2.5 on sample 3. Hence, throughout this series, a t worst, adsorptions micrograms per column containing 1 t o 1.3 grams of Decalso, to could be considered t o be 10 to 20% too low and thus not so poor be assured of 90 to 100% adsorption. The extraction seems t o
120
INDUSTRIAL AND ENGINEERING CHEMISTRY
as t o explain large proportions of the discrepancies betareen the two thiochrome values. While making the attempts just described to obtain proof for the validity of either the direct oxidation or the adsorption technique for the thiochrome method, the authors thought that a comparison of values on the same samples by the yeast fermentation method might lend support to one of the thiochrome techniques. They were fortunate to find Dr. Schultz in Dr. Frey’s laboratory willing to make such determinations. His results are tabulated in Table VI with the corresponding Hennessy and Harris values on frozen samples taken from Table I. As may be seen, his are, in several instances, somewhat higher than the Hennessy values but they are decidedly closer to the Hennessy than to the Harris values in those cases where there is a large difference between the latter two. LITERATURE CITED
(1) Am. Assoc. Cereal Chem., Cereal Laboratory Methods, 1941. (2) Andrews, J. S., and Nordgren, R., Cereal Chem., 18,686 (1941). (3) Arnold, A.,and Elvehjem, C. A,, Food Research, 3,367 (1938). (4) Baker, A. Z., and Wright, M.D., Biochem. J., 29, 1802 (1935). (5) Ibid., 32,2156 (1938). (6) Booher, L. E., and Hartzler, E. R., U.S. Dept. Agr. Tech. BUZZ. 707 (1939). (7) Booth, R. G.,J . SOC.Chem. Id.,59, 181 (1940). (8) Christensen, F. W., Latzke, E., and Hopper, T. H., J . Agr. Research, 53,415 (1936). (9) Conner, R. T.,and Straub, G. J., Cereal Chem., 18, 671 (1941). (10) Conner, R. T., and Straub, G. J., IND.ENQ.CHEM.,ANAL.ED., 13,380 (1941). (11) Ibid., 13,385 (1941). (12) Cowgill, G. R.,“Vitamin B Requirements of Man”, Oxford University Press, 1934. (13) Daniel, E. P., and Munsell, H. E., U. S. Dept. Agr. Misc. Pub. 275 (1937). (14) Harris, L.J., and Wang. V. L., Biochem. J.. 35, 1050 (1941).
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(15) Hennessy, D. J., and Cerecedo, L. R., J . Am. C h m . Soc., 61,179 (1939). (16) Johannson, H., and Rich, C. E., Cereal Chem., 18,473 (1941). (17) Kemmerer, A. R., and Steenbock, H., J . Biol. Chm., 103, 353 (1933). (18) Lane, R. L.,Johnson, E., and Williams, R. R., J. NutTition, 23, 613 (1942). (19) McFarlane, W. O.,and Chapman, R. A., Can. J . Research, 19, 136 (1941). (20) McIntire and Elvehjem, University of Wisconsin, personal
communication.
(21) McIntire, J. M., Schweigert,B. S., Henderson, L. M., and Elvehjem, C. A,, J . Nutrition, 25, 143 (1943). (22) MacKinney, G.,reference made in ( 6 5 ) . (23) McLaren and Cover, Texas State Experimental Station, per-
sonal communication.
(24) Merck and Co., Rahway, N. J., Mimeograph, revised June 6, 1941. (25) National Cooperative Experiment Station Project, Committee onvitamin Assay Methods,Report, Mimeograph (June4,1942). (26) Pyke, M. A.,Biochem. J., 31, 1958 (1937). (27) Ibid., 34,330 (1940). (28) Ibid., 34, 1341 (1941). (29) Pyke, M.A,, J. Soc. Chem. Id.,58,338 (1939). (30) Research Corporation Committee,subcommitteeon fluorometric method, D. J. Hennessy, Chairman, Report; Coleman Electric Co., Technicul BUZZ.T-108,1941. (31) Roscoe, M. H., Biochem. J., 25,2050 (1931). (32) Schultz, A. S., Atkin, L., Frey, C. N., and Williams, R. R., J . Am. Chem. Soc., 63,632 (1941). (33) Waisman, H. A,, and Elvehjem, C. A., “The Vitamin Content of Meat”, Minneapolis, Minn., Burgess Publishing Co., 1941. (34) Wang, Y. L., and Harris, L. J., Chemistry &Industry, 1942,‘27. (35) Westenbrink, A. G. K., and Goudsmit, J., Entymologia, 5,!307 (1938). (36) Widenbauer, F.,Klin. Wochschr., 18,1613 (1939). (37) Williams, R. R., and Spies, T. D., “Vitamin B1 (Thiamin) and Its Use in Medicine”, p. 161,New York, Macmillan Co., 1939. THISstudy was supported by a grant made through the Kational Research Council by the Sational Live Stock and Meat Board.
Constant-Level Feeder for Continuous Evaporation in the Determination of Total Solids M. C. S C H W A R T Z
AND
F. L. G A Y L E ,
Louisiana State University, Baton Rouge, La. i
T
HE apparatus shown in the figure was developed for use in the determination of total dissolved solids in water, and built to
the authors’ specifications by the Scientific Glass Apparatus Company. An apparatus was desired which could be assembled and started quickly; the apparatus herein described has proved successful in a number of actual tests. The evaporator maintains a constant level of liquid in the evaporating dish, D, for continuous evaporation. The volumetric flask, A , is filled to the mark with the solution to be eva orated, B is put inglace, and the unit is then turned over as iiustrated. The liqui rises in B to height E and then ceases t o flow. C and D, the evaporating dish, are added. The solution is siphoned over through C and stops flowing when the level in D reaches a height equal to E. The siphon can be started by blowing in the vent of B. As evaporation proceeds, more liquid flows over, maintaining the original level in the dish. Any slight amount of liquid finally left in C or B can be emptied manually. The evaporation can be carried out on a water bath, steam bath, or electrical hot plate. Under certain conditions, glass in C, in contact with the liquid in the evaporating dish, will be undesirable when extremely pure liquids are evaporated. In such instances resistant glass, Vycor, fused quartz, or metal tubing (suitable for the liquid) can replace conventional Pyrex tubing. ACKNOWLEDGMENT
The authors wish to thank L. J. Lassalle of Louisiana State University for permission to publish the material in this paper. CONTRIBUTION from the Water Teohnology Laboratory, Engineering Expenment Station, Louisiana State University, Baton Rouge, Le.
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