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788
TABLE111. EFFECTOF IRONIN DETERMINATION OF OXIDE COPPER (Using 5 % HzSOr saturated with SOZ) Oxide Copper Calculated Found Chalcocite (78.46%.Cu) 1 chslcocite : 1 bornite Chalcoaite 4 4 F e a s FezOa 4% F e a s Fer(S0dr Chalcocite Chalcocite 15% F e a s Fe(SOr)a Mill feed,(3.12% Fe) 1 chalcocite : 1 mill feed
+ ++
%
70
Nil Nil Nil Nil Nil
0.15
1.80
1.86
0.07
0.07
2.11 7.02 3.59
equipment was removed with a magnet prior to the determination. It is evident from Table I11 that iron added to chalcocite in the form of sulfide or oxide has no solvent action on the copper sulfide mineral. I n fact, the quantity of copper dissolved in these cases is always slightly less when iron is present than where pure chalcocite is leached with the oxide copper solution. Where ferric sulfate is initially present, however, the addition of the leaching solution leads to immediate attack of the copper sulfide minerals by the ferric salt. It is probable under these conditions that ferric sulfate dissolves appreciable copper sulfide before the sulfur dioxide of the leaching solution reduces the iron to the ferrous state. A similar attack of
Vol. 14, No. 10
copper sulfides by ferric sulfate was observed when 3 per cent sulfur dioxide was used as a solvent for oxide copper.
Summary The use of 3 per cent sulfur dioxide as a solvent for oxide copper, recommended by standard analytical textbooks, has been found unsatisfactory, but the substitution of 5 per cent sulfuric acid saturated with sulfur dioxide has resulted under the same conditions in complete recovery of oxidized copper, except cuprite. I n the absence of ferric sulfate, solution of copper sblfides is negligible. Contrary to the references cited, cuprite cannot be determined by the sulfurous acid method for oxide copper, nor even by employing 5 per cent sulfuric acid saturated with sulfur dioxide. The presence of iron as sulfide or oxide does not lead to attack of copper sulfide, using 5 per cent sulfuric acid saturated with sulfur dioxide. If ferric sulfate is initially present, appreciable solution of copper sulfide occurs during the leaching period in spite of the presence of sulfur dioxide, and results for oxide copper are not reliable. Literature Cited (1) Low, A. H., Weinig, A. J., and Schoder, W. P., “Technical Methods of Ore Analysis”, New York, John Wiley & Sons, 1939. (2) Scott, W. W., and Furman, E,H., “Standard Methods of Chemical Analysis”, Vol. 1, New York, D. Van Nostrand Co., 1939.
Determination of Thiamine in Vegetables JAMES C. RIOYER AND DONALD K. TRESSLER New York State Agricultural Experiment Station, Geneva, N. Y.
The thiamine content of eight frozen vegetables has been determined by the bioassay, thiochrome, and fermentation procedures. When the sulfite cleavage modification of the fermentation method was used, the results were in good agreement with those by the thiochrome procedure. In the case of three vegetables the potency obtained by bioassay agreed with that determined by the thiochrome and fermentation methods, but the other vegetables gave somewhat lower values by the latter procedures. The suggestion is made that the animal diet may not have been complete in other factors.
R
ELATIVELY rapid methods for the accurate estimation of thiamine are needed both for the determination of the vitamin B1 content of fresh vegetables and for the study of changes which may occur during their storage, processing, and preservation. Bioassay methods cannot be used, since most fresh vegetables will not keep long enough to permit completion of the assays, and furthermore, even if they do not spoil, their composition is subject to change due to respiration. Hennessy and Cerecedo (4) have proposed a modification of the thiochrome method of determining thiamine ivhich appears to be applicable to vegetables. Schultz, Atkin, Frey,
and S.5Tilliams (1.2) have modified the original fermentation method of determining thiamine proposed by Schultz, Atkin, and Frey (11) to include a n estimation of the fermentationstimulating action of substances other than thiamine in the material under test and its subtraction from the total volume of carbon dioxide obtained. The work reported in this paper is a comparison of analyses by the thiochrome and fermentation methods of determining thiamine in frozen vegetables with those obtained by a growth bioassay procedure. The results should be useful in determining the relative merits of these rapid procedures and standard bioassays. Since it has been shown that in the case of carotene and ascorbic acid there is little or no loss of vitamin potency in storage a t -40” C. (5, 6, 13, I,$), this mode of storage seemed most feasible in the interim between harvesting and examination of the thiamine content by the three procedures. This supposition in the case of thiamine has been demonstrated a t this station (?’) to be correct. The vegetables used in this study-peas, broccoli, cauliflower, and spinach-were obtained from the station gardens, cleaned, sorted, blanched in boiling water, and cooled. The drained product was then packed in a cellophane-lined Peters type of carton and frozen in a Birds-eye multiplate freezer. The frozen asparagus, cut corn, string beans, and lima beans were obtained from commercial sources. I n Table I the varieties and blanching times of these vegetables are given. Although the blanching before freezing does not affect the comparison of the three methods of analyses, it does lower the total thiamine figure, since the blanching water extracts some of the thiamine from the vegetable. A study of the losses of thiamine from asparagus and peas during processing prior to freezing has been reported in another paper ( 7 ) .
ANALYTICAL EDITION
October 15, 1942
789
gus, snap beans, cauliflower, peas, cut corn, and spinach were TABLEI. VARIETIESAND BLANCHING TIMESOF VEGETABLES assayed during the period from February to April, while STUDIED broccoli and lima beans were assayed during the months of Blanching Time May and June. Because some time elapsed between the at 212' F., Vegetable Variety Min. two sets of assays, a second curve of response was determined when the broccoli and lima beans were assayed. These curves Peas Thomas Laxton 50 seconds Asparagus Mar Washington 3 are shown in Figure 1, curves 1 and 2 being for the first and Cut corn G o d n Cross 7.5 second response curves, respectively. Lowe's Champion 2 Snap beans Spinach King of Denmark 1.5 The curves of response were drawn by plotting the average Snowball Cauliflower 2.5 Henderson Bush Lima beans 3 gains in weight, as the ordinate, against the logarithms of Calabrese Broccoli 2.5 micrograms of thiamine, as the abscissa. B y dividing the corresponding thiamine units obtained by the grams of vegetable fed, the equivalent amount of thiamine in each gram Samples for assay by the three methods were finely ground of food was determined. in a food chopper at, -23.3' C. (- 10" F.). The samples for Two feeding levels of each vegetable were started, but it bioassay were shipped under dry ice refrigeration t o the nusoon became apparent that the supply of cauliflower was being trition laboratory of the State College at Amherst, Mass. rapidly depleted, so that the higher level was discontinued. All eight rats constituting the negative control group died Bioassay Procedure before the assay was completed, except two which remained The two most common methods for the bioassay of thiaalive though they showed polyneuritic symptoms. One animine are the growth and curative tests with rats (8). Since, mal of the group receiving 0.75 microgram of thiamine daily with the possible exception of peas, the materials to be asalso died. sayed were bulky, fibrous, and of low potency, it was necesI n Table I1 the results of the bioassay on the various vegesary to use the growth technique. tables are presented. The greatest variation in the thiamine content of a single vegetable, when fed at two levels, was in the case of peas, where the average value differed by 16 per cent from either assay. This divergence may be considered 90 as being within the range of error encountered in bioassays. f'
I
CURVE
I.'
*
'
MICROGRAMS OF
THIAMIN FED DAILY, LOGARITHMS
FIGURE 1. THIAMINE IN VEGETABLES
All the"anima1s used in this work mere raised in the rat colony of the Massachusetts State College and weighed from 35 to 50 grams at 21 to 28 days of age when placed on the diet recommended by Chase and Sherman (I). Within 28 days after the B1-free diet was started, the rats had definitely stopped growing or had begun to lose weight. This maintenance of or loss in weight was observed from the wei hts of the individual animals taken every other day over a perioi of a week and a half towards the end of the depletion period. The animals from the various litters were then selectively randomized into groups of eight, so that each group contained the same number of males and females with no more than one member from each litter in any one group. One of these groups received no thiamine at all-i. e., they were set aside as a negative control-while five groups were given daily 0.75, 1.5, 3.0, 4.5, and 6.0 micrograms of thiamine, respectively. The remaining groups were fed two levels of vegetables. The addenda were administered daily for a period of 28 days. All animals were weighed once a week,
It was not possible to obtain enough animals t o complete assays on all vegetables at one time and, therefore, aspara-
Thiochrome Determination The chemical methods for determining thiamine have been reviewed by Hennessy (8). A modification of the Hennessy and Cerecedo (4) procedure has been used by Conner and Straub (2) for the determinaton of thiamine in frozen spinach, peas, and broccoli, fresh lima beans, string beans, and squash. They found that there was an agreement between the bioassays and chemical analyses which was better than 12 per cent for the two cereal products and frozen peas tested, the results from the chemical method being in all three cases higher. Recently the Research Corporation Committee in conjunction with the Committee on Vitamin Fortification of the American Association of Cereal Chemists (0) suggested a procedure for the determination of thiamine which is simpler than the Conner and Straub method. The details of the modified method which was used in the authors' studies are given below: Twenty-five grams of vegetable are placed in the cup of a Waring Blendor and the tissues macerated in 150 ml. of 0.1 N sulfuric acid for 2 or 3 minutes. Then the Blendor cup is rinsed out with 260 ml. of 0.1 N sulfuric acid into a 500-ml. Erlenmeyer flask. The flasks are set in boiling water or on a steam bath. During the first 5 minutes the flasks are frequently agitated and then once every 5 minutes for one-half hour. The flmks are then cooled below 50" C., and 25 ml. of 2.5 J4 sodium acetate solution containing 0.5 gram of Clarase are added. (The Clarase is added t o the sodium acetate solution just previous to addition to the extract.) Incubation at 50" C. for 2 hours permits a complete hydrolysis of the thiamine phosphates. Throu h the addition of distilled water the volume of the extract is mafie up to 600 ml. and then, after thorough mixing, a portion is filtered. The first 10 ml. of filtrate are discarded and the rest is collected. The remainder of the procedure-that is, the base exchange purification and the production of thiochrome-was followed exactly as outlined by the committee. The percentage recovery of thiamine added to test samples of vegetables before extraction varied from 93 to 105. The thiamine contents of the eight vegetables as determined by the thiochrome method are presented in column 3 of Table 111.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLE 11. THIAMINE CONTENT OF VEGETABLES BY BIOASSAY Supplement Group I Thiamine
Amount Fe,d Daily Micrograms
Average Gain per Week Grams
...
0.00 0.73 1.50 3.00 4.50 6.00
7
30 49 70 73
AS INDIC.4TED
Thiamine per G r a m of Average Thiamine Supplement Value .. Micrograms Micrograms
...
...
... ... ...
... .... .... .... ....
..
...
2.00 2.23 0.83 0.69
2.11
0.53
0.53
. Grams Asparagus Snap beans Cauliflower Peas Spinach
1.00 2.00 2.00 4.00 4.00 0.50 1.00 3.00
5.00 C u t corn
2.00 3.00
.
40 67 33 47 41 41 54 31 49 43 60
4.22 3.06 0.52 0.63
1.15 1.24
0.76 3.64 0.52 1.20
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I n the case of frozen peas there is considerable difference between the results obtained by the three methods. The bioassay is the average of two rather widely different values and since there is no reason to discard the higher potency, it must be retained. Nevertheless, were the lower of the two bioassay values to be taken, the thiochrome, total stimulation and after stimulation and after sulfite cleavage fermentation results would only be 15, 5 , and 12 per cent, respectively, too low. There is the possibility that the animal diet was deficient in known or unknown members of the B complex other than thiamine and if these factors were present in the vegetable being tested, a higher response would be obtained than would normally be due to thiamine. I n general, the thiochrome and after sulfite cleavage fermentation values agree well. It is assumed that there are substances other than thiamine in vegetables which stimulate the rate of fermentation and therefore the sulfite cleavage procedure should be used.
Micrograms Group I1 Thiamine
0.00
1.60 3.00 4.50 6.00
... 33 62 74 86
...
... ... ... I
.
.
...
....
.... .... ...
Grams Lima beans Broccoli
1.00 3.00 2.50 5.00
Fermentation Method of Assay All the experimental work reported in this paper was completed before the paper of Schultz, Atkin, and Frey (10) appeared in the press. The fermentometer used was of the older type and consequently in all analyses double the quantity of reagents recommended by the above authors was used for each determination. However, the results obtained do not vary materially from those obtained -when the newer type of equipment is used. The amounts of fermentation stimulation expressed as micrograms of thiamine before and after sulfite cleavage are given in columns 4 and 5 of Table 111. From these data it will be seen that the total stimulation was always greater than that considered to be due to thiamine alone. This would seem to indicate that there are one or more substances in vegetables other than thiamine w-hich have a stimulatory effect on the rate of fermentation. The presence of this effect makes necessary the use of sulfite cleavage when determining the thiamine content of vegetables by the fermentation method.
Comparison of Results Obtained by Bioassay, Fermentation, and Thiochrome Methods The thiamine contents of the eight frozen vegetables as determined by the thiochrome and fermentation methods are compared in Table I11 with those obtained by bioassay. Taken as a whole, the thiamine contents of eight vegetables as determined by bioassay, thiochrome, and fermentation “after sulfite cleavage” methods are in good agreement. There is, however, a slight divergence in the case of cut corn, the after sulfite cleavage fermentation results being about 12 per cent higher. The thiamine content as derived from the total stimulation of gas production in the fermentation method gave results which in four cases out of eight were considerably higher than the bioassay. However, three vegetables, lima beans, cauliflower, and broccoli, gave thiamine values by the total stimulation method which were in agreement with the results of the bioassays.
TABLE 111. COMPARATIVE VITAMINB1 VALUES OBTAINEDBY BIOASSAY, FERMENTATION, AND THIOCHROME ~IETHODS Vegetable
Fermentation Before After sulfite sulfite Bioassay Thiochrome cleavage cleavage Micrograms of thiamine per gram
Pea0 Asparagus C u t corn Lima beans Broccoli Snap beans Cauliflower Spinach
3.64 2.11 1.20 1.17 1.06 0.76
0.53 0.52
2.60 2.04 1.17 0.89 0.88 0.64 0.43
0.51
0.55
0.72
2.83 2.52 1.50 1.10 1.09 0.87
2.69 2.12 1.31 0.90 0.81 0.63 0.39
0.55
Acknowledgment The authors are indebted to Carl R. Fellers and Mrs, Anne Wertz of the hlsssachusetts State College, both for the use of a laboratory and guidance and assistance during the conduct of the bioassays. The aid of Miss Katherine Wheeler of the New York State Agricultural Experiment Station in carrying out some of the chemical studies is also acknowledged.
Literature Cited Chase, E . F., and Sherman, H. C., J . Am. Chem. Soc., 53, 3506 (1931).
Comer, R. T., and Straub, G.J., 1x0.
[email protected].,ANAL. ED., 13,350 (1941). Hennessy, D. J., Ibid., 13,216 (1941). Hennessy, D. J., and Cerecedo, L. R., J . Am. Chem. Soc., 61, 179 (1939).
Jenkins, R. R., Tressler, D. K., and Fitzgerald, G. A,, Proc. Brit. Assoc. Refrig., General Conference on Refrigeration, 26 (1938).
Jenkins, R. R., Tressler, D. K., Moyer, J., and McIntosh, J.. Refrig. Eng., 39, 351 (1940). Moyer, J. C., and Tressler, D. K., Food Research (in press). Munsell, H. E., J . Am. .Wed. Assoc., 111, 927 (1935). Research Corporation Committee (in press). Schulta, A. S., Atkin, L., and Frey, C. N., IND.ENG.C H E M , ANAL.ED., 14, 35 (1942). Schulta, A. S., Atkin, L., and Frey, C. N., J . Am. Chem. Soc.. 59, 948 (1937).
Schulta, A. S.,Atkin, L., Frey, C. N., and Williams, R . R.. Ibid., 63, 632 (1941). Zimmerman, TI’. I., Tressler, D. K., and Maynard. L. A , , Food Research, 5, 93 (1940). Ibid., 6, 57 (1941). A BUXMYARYof p a r t of the thesis submitted in FebrualJ-.1942, by James C. Moyer t o the Graduate School of Cornel1 University in partial fulfillment of requirements for t h e degree of doctor of philosophy. Approved by t h e Director of the S e w York State Agricultural Experiment Station for publication a s Journal Paper No. 516, July 1, 1942. This work was supported largely by grants made by t h e General Foods Corporation and t h e Frosted Foods Sales Corporation of New York.
