Precipitation of Zinc Sulfide from a Solution of Ammonium Citrate and Citric Acid S. A. COLEMAN AND G. B. L. SMITH, Polytechnic Institute of Brooklyn, Brooklyn, N. Y
T
H E Raring method for the separation of zinc, published in 1907 (11), was developed as the result of an extensive study of the analyses of zinc ores, conducted by the Committee on Uniformity in Technical Analysis of the AMERICAN CHEMICAL SOCIETY( 2 ) . This latter report indicated that the precipitation of zinc as zinc sulfide was the only satisfactory method available for the final separation of zinc. This procedure has been studied extensively, so that our knowledge of the conditions under which zinc sulfide may be precipitated quantitatively is as complete as it is for any analytical method available to chemists. OF ZINCSGLFIDE FROM A SOLUTION OF TABLE I. PRECIPITATION AMMONIUM CITRATE A N D CITRIC ACID (0.4259 gram of ZnSOd taken)
Excess of 1 M Citric
Acid Used
MZ.
25 26 30 30 40 40 40 40 50 50 .50 50
ZnzPzO7 Found Gram
ZnSOi Calcd. Grana
0.4017 0.4028 0.4020 0.4018 0,4021 0.4026 0.4023 0.4023 0.4009 0.4012 0.4023 0.4023
0,4259 0.4258 0.4263 0.4265 0,4253 0.4256 0.4255 0.4265 0.4259 0 4256 0.4260 0.4265 0.4262 0.4262 0.4247 0.4250 0.4262 0.4262 AV.
Deviation from Mean
MQ. 0.0 -0.1
+0.4 +0.6 -0.6 -0.3 -0.4 10.6 0.0
-0.3 t O . l t0.6 +0.3 +0.3 -1.2 -0.9 t0.3 +0.3
0.4259
The Waring method was studied critically, and was modified by Fales and Ware in 1919 (Q), with the result that the present Raring-Fales-Ware method for the separation of zinc is an elegant procedure, and the one most generally employed today. Long experience with the separation of zinc as zinc sulfide has shown that the quantitative precipitation (for analytical purposes) must be effected from an acid solution, pH 2 to 3, and that any buffer solution which will maintain the reaction within this range is satisfactory. Weiss ( l a ) precipitated zinc sulfide merely from a 0.01 N solution of sulfuric acid but Lundell, Hoffman, and Bright (8) and Jeffries and Swift (6) suggested a “sulfate-hydrosulfate” buffer. Mayr (9) regulated the acidity with a solution of chloroacetic acid and sodium acetate. The Waring method as modified by Fales and R a r e employs formic acid and ammonium formate as a buffer, and in addition they add citric acid to form a “citrate complex” with iron. Citric acid is of moderate strength, PKal -3.06, PKaz -4.74, and PKa3 -5.40, somewhat stronger than formic acid, and therefore the statement of Smith (10) that “a mixture of citric acid and ammonia in suitable proportions would serve not only to keep iron in solution by complex formation, but also as a buffer” would seem to be reasonable. The investigation upon which this paper is based was undertaken in order to test the validity of the above statement. The experimental results presented herewith prove conclusively that the T. B. Smith statement is entirely justified.
Accordingly, the Waring-Fales-Ware method may be simplified by eliminating the use of formic acid and ammonium formate.
Experimental The studies reported here have been planned to parallel as closely as possible the work of Fales and Ware, although pH determinations have not been made. Zinc solutions used were standardized by the Waring-Fales-Ware method, but zinc was determined by weighing as zinc pyrophosphate (6). This method of determination was also employed in all the studies, in order to ensure comparative results. Series of experiments were conducted, following the technique of Fales and Ware, but the solutions were buffered with ammonium citrate and citric acid, instead of with solutions of formic acid and ammonium formate. STAXDARD SOLUTIONS. -4 solution of zinc sulfate was prepared by dissolving 20 grams of pure zinc oxide in dilute sulfuric acid. This was neutralized with ammonium hydroxide, made slightly acid with sulfuric acid, and was then diluted t o approximately 2.5 liters. The solutions were standardized as stated above, making at least six determinations. In the standardization of one solution eight results were obtained; 25-ml. aliquots were taken and the zinc was weighed as zinc pyrophosphate, and these results were calculated to zinc sulfate: 0.4261, 0.4255, 0.4256, 0.4259, 0.4261, 0.4258, 0.4257, 0.4264 gram of zinc sulfate; mean, 0.4259 gram of zinc sulfate, standard deviation from the arithmetic mean of a single determination, 0.3 mg. (7 parts in 10,000); standard deviation of the arithmetic mean, 0.1 mg. (2.3 parts in 10,000). The solutions of interfering elements were not standardized, but were prepared by dissolving weighed quantities of the pure substances, ferrous sulfate, manganous sulfate, aluminum sulfate, nickel sulfate, and cobalt oxide, and diluting to definite volumes. These solutions were prepared so that 20 ml. of the iron solution contained 0.19 gram of iron and the others contained 0.17 gram of the element. REAGENTS.Formic mixture was prepared according to the directions of Fales and Kenney ( 3 ) . Citric acid was 1 M (200grams per liter). Ammonium sulfate, 200 grams per liter; ammonium thiocyanate, 200 grams per liter. TABLE11. SEPARATION OF ZINC FROM IRON Expt.
Fe Taken Gram
ZnzPzO7 Found Gram
ZnSO4 Calcd. Gram
Difference
MO.
Excess 1 iM citric acid, 25 ml.; 0.4766 gram of ZnSO4 taken 1 0.049 0.4501 0.4768 1-0.2
2 3 4 9 10
0.049 0.098 0.098 0.147 0.147
0.4494 0.4817 0,4814 0.4517 0.1512
0.4761 0.4785 0.4782 0.4785 0 4780
-0.5 f1.9 +1.6 +1.9
+1.4
Excess 1 A 4 citric acid, 40 ml.; 0.4259 gram of ZnSOd taken 5 0,098 0,4008 0.4246 -1.3 6 0,098 0.3662 0.3773 Result 7 0.098 0.4025 0.4264 discarded +0.5 8 13 14 17 18
0.C98 0.196 0.196 0.343 0.343
0.4026 0.4026 0.4026 0.4039 0.4028
0.4265 0,4265 0.4265 0.4279 0.4264
+0.6 +0.6 4-0.6 t2.0 4-0.5
Excess 1 M citric acid, 50 ml.; 0.4766 gram of ZnSO4 taken for Nos. 11 and‘ 12, 0.4259 gram of ZnSO4 for Nos. 15 and 16
37’I
378
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 13, No. 6
Discussion of Results
TABLE111. SEPARATION OF ZINC FROM IRON BY WARINC~FALESWAREMETHOD A m r o s r m CITRATEAND CITRICACIDAS BUFFER. Of the (Determinations run in conjunction with Nos. 11, 12, 15, and 16 in Table 11) eighteen results reported in Table I, all are apparently within ZnSOh Fe ZnzPzOt ZnSO4 the limit of the experimental error of the Waring-Fales-Ware Taken Taken Found Calcd. Difference method. The arithmetic mean of the eighteen determinaGram Gram Gram Mg. Gram tions is 0.4259 gram of zinc sulfate, which is the same as 0.4766 0.196 0.4501 0.4765 +0.2 0.4766 0.196 0.4506 0.4773 +0.7 that found in the eight determinations carried out in stand0.4259 0.343 0.4040 0.4280 +2.1 ardizing the solution by the Waring-Fales-Ware procedure. 0.4032 0.4271 0.4259 0.343 $1.2 The standard deviation of a single determination from the arithmetic mean of these eighteen determinations is 0.5 mg. TABLEIv. SEPARATION O F ZINC FROM MAUGANESE, ALUMINUM, or 12 parts in 10,000. The standard deviation of the arithAND NICKEL metic mean is 0.12 mg. or 2.8 parts in 10,000. When we (Excess 1 A4 citric acid, 50 ml.) compare these deviations n-ith those of the determinations Element Added, ZnSOr ZnrPtOl ZnSO4 made in standardizing the solution, there is no significant 0.17 Gram Taken Found Calcd. Difference difference in the results obtained by the Waring-Fales-Rare Gram Gram Gram Mg. method and those obtained by using the citrate-citric acid 0.4259 0,4022 Manganese 0.4261 +0.2 -0.3 0.4259 0.4018 0.4256 buffer. Treatment of these data by analysis of variance also 0.4090 0.3861 0.0 0.4090 0.4090 -0.3 0.3858 0.4087 shows that the two methods do not give results which are +0.4 0.4024 0.4259 Aluminum 0.4263 significantly different. +0.2 0.4022 0.4259 0.4261 +0.2 0.3863 0.4090 C ,4092 It is also evident that there is considerable latitude in the +0.3 0.4090 0.4093 0.3864 -0.2 0.4019 0.4259 Nickel 0.4257 amount of excess citric acid .ivhich may be added. Indeed, -.0.5 0.4259 0.4016 0.4254 in this series the amount of excess citric acid does not have a 0.3865 $0.4 0.4090 0.4094 -0.2 0.4090 0.3859 0.4088 significant effect upon the results. This conclusion is proved by analysis of variance in this series. We are therefore justified in concluding that ammonium citrate and citric acid used serve together as a satisfactory buffer for regulating the acidity The standard solution of zinc sulfate BUFFERSOLUTIONS. (25 ml.) was transferred t o a 500-ml. Erlenmeyer flask and 6 M of solutions from which zinc sulfide is precipitated quantitaammonium hydroxide was added until a slight permanent tively. precipitate of zinc hydroxide formed. Citric acid (25 ml. of 1 M solution) was added and neutralized with ammonium hydroxide, using methyl orange indicator, and this was followed by 25 ml. of the solution of ammonium sulfate. The desired volume of TABLEV. SEPARATION OF ZINC FROM COBALT citric acid was now added, and the total volume was adjusted to (0.17 gram of Co taken) 200 ml. In experiments conducted with solutions of zinc sulfate 20% Solution of ZnzPnOr ZnSOr only, 1he precipitate was allowed to stand over-nightbefore filterNHdSCX Found Calod. Difference ing, but when interfering ions were present the precipitate was 1. Gram Grank Mg. separated by filtration as soon as the supernatant liquid was clear, usually within one hour. Excess 1 Jf citric acid, 50 ml.; 0.4259 gram of ZnSOi taken The first series of experiments OUTLINE O F EXPERIMENTS. was conducted in order to establish the optimum proportions of cilric acid and ammonium citrate necessary for the precipitetion of zinc sulfide. After the neutralization of the 25 ml. of 1 M citric acid, 15-, 20-, 25-, 30-, 40-, and 50-ml. portions of 1 M citric acid in excess were added, and zinc sulfide was precipitated. The results of these experiments are tabulated in Table I. A detailed study of the separation of zinc from iron was then made in the second series of experiments (Table 11). Table I11 presents some results of the separation of zinc from iron by the standard Waring-Fales-Ware procedure. The third series of experiments shows the application of the new technique to the separation of zinc from manganese, aluminum, and nickel (Table IV). The separation of zinc from cobalt was studied in considerable detail. Ammonium thiocyanate was used instead of ammonium sulfate for salting out zinc sulfide, and in addition, for minimizing the postprecipitation of cobalt sulfide by forming a thiocyano complex ion with cobalt. The results of these experiments are presented in Tables V and VI. In Table VI1 is recorded a series of experiments planned to show whether or not zinc sulfide can be dried on a Gooch crucible and weighed as such. The results reported clearly demonstrate that this procedure is not reliable. In the final series of experiments, the new method is applied to the determination of zinc in three zinc ores, Sational Bureau of Standards Samples 2A and 113, and a Polytechnic Institute student sample. The last sample was analyzed by a sophomore student in the senior author’s class in quantitative analysis, The results of these experiments are tabulated in Table VIII.
a a
25 25 50 50 75 75 50 50
0.4127 0.4136 0.4037 0.4088 0.4031 0.4026 0.3893 0.3870 0.4014 0.4009
0.4372 0.4381 0.4315 0.4331 0.4270 0.4265 0.4124 0.4100 0.4252’~ 0.4247b
f11.3 $12.2 5.6 7.2 1.1 0.6 -13.5 -15.9 -0.7 -1.2
+ + + -+
Excess 1 M citric acid, 40 mL; 0.4782 gram of ZnSOl taken 0.4790 0.4522 +0.8 50 -0.5 0.4777 50 0.4509 Results 0.4822 50 0.4552 discarded 0.4721 Results 50 0.4557 discarded 0.4770b -1.2 0.4503 50 0.4771b -1.1 0.4504 50 Excess 1 M citric acid, 50 ml.; 0.4782 gram of ZnS04 taken of 1st two experiments, 0.4090 gram of ZnSOi taken for other experiments 0.4787 0.4519 50 0.4775 0.4507 50 0.4120 0.3890 50 0.4139) 0.3907 50 0.4127 0.3896 50 0.4137 0.3905 50 0.4123 0.3892 50 0.4110 0.3880 50 0.4090b 0.3861 50 0.4088b 0.3859 50
1
1
4
b
25 mi. of ammonium sulfate employed instead of ammonium thiocyanate.
K O cobalt present.
SEPAR.4TION O F ZINC FROM IRON. The second Series Of experiments consists of a detailed study of the separation of zinc from iron. The citrate-citric acid solution not only serves to regulate the acidity, so that the solubility product of ferrous sulfide will not be exceeded, but also reduces the concentration of the ferrous ion by forming a ferrous citrate complex ion or molecule. Table I1 indicates that there is
ANALYTICAL EDITION
June 15, 1941
some contamination of zinc sulfide with iron, and this increases with the concentration of iron in the solution. If we consider the last two sets of results, the standard deviation of a single determination from the results according to the Waring-Fales-Ware standardization is 1.O mg. or approximately 2.5 parts per 1000. These results are high. Table I11 reports four results obtained by following the WaringFales-Ware method. These results deviate from the standardization values in the same way and the order of magnitude is the same. The standard deviation is 1.25 mg. for a single determination. Fales and Ware (4) found that some iron contaminated the precipitate of zinc sulfide if iron mere present in the solution from which the separation had been effected. It is concluded, therefore, that the new technique is just as effective as the ~~7aring-Fales-Ware technique in separating zinc from iron. SEPARATION OF ZINC FROM MANGANESE, ALUMINUM, AND XICKEL. The separation of zinc from manganese, from aluminum, and from nickel is surprisingly satisfactory. Of the twelve results reported in Table IV, only one deviates by more than one part in 1000. We may conclude, therefore, that these separations are as nearly perfect as the techniques warrant. SEPARATIONOF ZINC FRolrI COBALT. A completely satisfactory method for the separation of zinc from large amounts of cobalt has not been developed. Apparently the contamination of zinc sulfide with cobalt sulfide is due to postprecipitation similar to the contamination of copper sulfide with zinc sulfide ( 7 ) . Caldwell and Moyer ( 1 ) employed acrolein for minimizing the postprecipitation of cobalt sulfide. A study is made in the series of experiments reported in Table V of the use of ammonium thiocyanate as a reagent to minimize the postprecipitation of cobalt sulfide. Ammonium thiocyanate acts also to salt out zinc sulfide. Using a 20 per cent solution of ammonium thiocyanate, the contamination with cobalt sulfide is reduced considerably. It appears that 50 ml. of the 20 per cent solution of ammonium thiocyanate are as much as can be used safely. However, in all cases, cobalt sulfide can be seen as a contaminant, and it appears after zinc is almost completely precipitated. In the six determinations (bracketed) toward the bottom of the table, we find a mean value of 0.4126 gram of zinc sulfate, or a constant error of 3.4 mg. (9 parts in 1000). The standard deviation from the arithmetic mean of a single observation is 0.95 mg. (2.3 parts in lOOO), and the standard deviation of the arithmetic mean is 0.4 mg. (1 part in 1000). Certain determinations reported here appear satisfactory, and it was thought that the time of precipitation was an important factor in the amount of cobalt sulfide present in the
TABLE VIII. Sample
379
ANALYSIS OF ZINCORESBY USE OF CITRICACIDCITRATEBUFFER Zn Found
Deviation from Mean
Deviation from Reported Result
%
%
%
E. S. B. S. 2A
30.41 -0.03 -0.12 30.41 -0.03 -0.12 30.47 +0.02 -0.06 -0.02 -0.07 30.46 U. 8. B. 9. 113 60.98 +0.04 -0.12 60,89 -0.05 -0.21 Student sample 19.19a -0.04 b 18.48" Result rejected 0.00 19.23a 19.28a +0.05 a Ana1,yseg made by, Aaron Wexler, Polytechnic '42, sophomore in course in quantitative analysis. b Value used in checking students' results, 19.17.
precipitate. Table VI demonstrates conclusively that this postulation is valid. Thiourea and thiophenol do not prevent the postprecipitation of cobalt sulfide. Further studies to establish optimum conditions are projected. WEIGHINGZINC AS ZINC SULFIDE. The possibility of filtering zinc sulfide on a tared Gooch crucible, drying a t 110" C., and weighing directly, was tested in the experiments summarized in Table VII. Zinc sulfide was precipitated from solutions of zinc sulfate by t'he Waring-Fales-TTTare method and by the ammonium citrate-citric acid procedure, the zinc was weighed as zinc pyrophosphate, and the results were calculated as zinc sulfate (columns l and 2, Table VII). Zinc sulfide which had been precipitated by the Waring-FalesWare method was filtered onto Gooch crucibles and weighed. The results are given in column 3, and in column 4 the zinc sulfide is calculated to weight of zinc sulfate. I n column 5 are given results in terms of zinc sulfate of two precipitates of zinc sulfide (first and third items in column 3) which were converted to zinc pyrophosphate. We conclude, therefore, that it is not permissible to weigh zinc as zinc sulfide by the technique described here.
Analysis of Zinc Ores Samples of three zinc ores were analyzed by the procedure developed as a result of t'hese studies.
The zinc ores are dissolved and silicic acid, copper, cadmium, and other metals of the hydrogen sulfide group are removed in the usual way. The solution of zinc is placed in a 500-ml. or 750-ml. Erlenmeyer flask and 6 M ammonium hydroxide is added until the precipitate of zinc hydroxide just fails to dissolve. Citric acid (25 ml. of 1 N solution) is added and made neutral t o methyl orange by adding 6 X ammonium hydroxide. An excess of citric acid (50 ml. of 1 -11 solution) is now added and also 25 ml. of a 20 per cent solution of ammonium sulfate (50 ml. of a 20 per cent solution of ammonium thiocyanate are used instead of ammonium sulfate if cobalt is present). The volume is adjusted TABLEVI. EFFECTOF TIMEOF PRECIPITATION ON CONTAMISA- to 200 ml., the solution is heated t o 60°, and air is replaced with TION OF ZINCSULFIDE WITH COBALT SULFIDE hydrogen sulfide. The heating of the solution is continued until a temperature of 95' is attained, and the exit tube is closed. The (20% solution of NH4SChT taken, 50 ml.; 0.17 gram of Co taken) solution is allowed to cool and become saturated with hydrogen Z n ~ P ~ O ~ ZnSO4 ZnSO4 Time of sulfide under the pressure of the hydrogen sulfide from the generTaken Pptn. Found Calcd. Difference ator, and is agitated frequently during a period of 20 to 40 minGram Min. Gram Gram ivg. utes. The precipitate is allowed to settle, separated by filtration, 0.4095 20 0.3906 0.4138 +4.3 and washed with a 0.1 M solution of citric acid saturated with 0.4095 20 0.3907 0.4139 t4.4 hydrogen sulfide. Zinc may now be determined by any of the 0.4098 45 0.3924 0.4157 +6.2 0.4095 45 0.3933 0.4166 +7.1 standard methods. In these analyses the zinc is weighed as zinc pyrophosphate ( 5 ) . TABLE VII. WEIGHIXG ZISC AS ZINCSULFIDE An examination of the results of the analyses as recorded in ZnSOd Found ZnS, Converted Table VI11 shows that the method described above is very Waring-Fales- Citric acid ZnS ZnS Calcd. t o Zn?P*O,, Ware method method Found as ZnSO4 Calcd. as ZnSOa satisfactory.
Gram 0.4242 0.4249 0.4238 0.4246 A r . 0.4244
Gram 0.4249 0.4243
....
.... 0.4246
Gram 0.2685 0.2681 0.2670 0.2665
Gram 0,4449 0.4442 0.4423 0.4415
Gram 0.4249
....
Conclusion
....
The studies described in this paper demonstrate that in the Waring-Fales-Ware method for the separation of zinc the formate-formic acid buffer is not necessary. since a mixture of
0.4242
INDUSTRIAL AND ENGINEERING CHEMISTRY
380
ammonium citrate and citric acid serves not only t o keep iron in solution but as a n efficient buffer solution; that the results obtained by the use of citric acid-ammonium citrate as a buffer are comparable to those obtained by the Waring-FalesWare method and have a precision of 1 part in 1000; that zinc cannot be determined by weighing as zinc sulfide; and that 50 ml. of a 20 per cent solution of ammonium thiocyanate serve to salt out zinc sulfide and to minimize the postprecipitation of cobalt sulfide.
Acknowledgment The authors are pleased to acknowledge the assistance of Frank Wilcoxon, Boyce Thompson Institute of Plant Research, Yonkers, Ti. Y., who has given advice regarding statistical treatment of certain of the data. Literature Cited Caldwell, J. R., and Xloyer, H. V., J . A m . Chem. Soc., 57, 2372
(1)
(1932).
Committee on Uniformity in Technical Analysis, Report of Subcommittee on Zinc Ore Analysis, Ibid.. 26, 1648 (1904).
(2)
Vol. 13, No. 6
(3) Fales, H. A., and Kenney, F., “Inorganic Quantitative Analysis”, p. 332, New York, D. Appleton-Century Co., 1939. (4) Fales, H. A., and Ware, G. M., J . A m . Chem. SOC.,41, 487 (1919). (5) Hillebrand, W. F., and Lundell, G . E. F., “Applied Inorganic Analysis”, pp. 334-5, New York, John Wiley & Sons, 1929. (6) Jeffries, C. E. P., and Swift, E. H., J . A m . Chem. SOC.,54, 3219 (1932). (7) Kolthoff, I. M., and Pearson, E., J . Phys. Chem., 36, 549 (1932). (8) Lundell, G. E. F., Hoffman, J. I., and Bright, H. A., “Chemical Analysis of Iron and Steel”, p. 388, New York, John Wiley & Sons, 1931. (9) Mayr, C., 2.anal. Chem., 92, 166 (1933). (10) Smith, T. B., “Analytical Processes”, pp. 233-4, London, Edward Arnold and Co., 1929. (11) Waring, W. G:, J . A m . Chem. SOC.,29, 262 (1907). (12) Weiss, G., “Uber die quantitative Bestimmung und Trennung von Zink und Nickel”, Dissertation, Munchen, 1906. PRESENTED before the Division of Physical and Inorganic Chemistry a t t h e 100th Meeting of the American Chemical Society, Detroit, Mich. Abstract of the thesis submitted by S. A. Coleman, Department of Health, City of New York, to the Graduate Faculty of the Polytechnic Institute of Brooklyn for the degree of master of science in chemistry in June, 1940.
Determination of Thiamin by the Thiochrome Reaction H. T. CONNER AND G. J. STRAkUB Central Laboratories, General Foods Corporation, Hoboken, N. J .
B
ECAUSE of the time and cost involved in the biological
assay of thiamin, several microbiological and chemical methods have been proposed for its determination. Of the chemical methods, the thiochrome procedure first proposed by Jansen (3) has received a considerable amount of attention. This method is based on the measurement of the fluorescence produced by thiochrome formed by the oxidation of thiamin with potassium ferricyanide in a n alkaline solution. The original method of Jansen has been modified by Karrer and Kubli ( d ) , and more recently by Hennessy and Cerecedo ($), who introduced the use of a synthetic zeolite, Decalso, for absorbing the thiamin, and thereby separating it from substances interfering with the reaction. They also were the first to employ a sensitive photoelectric instrument (the Pfaltz &- Bauer fluorophotometer) for measurement of the fluorescence. The work described in this paper attempts to define more exactly than has been done previously the optimal conditions for carrying out the thiochrome procedure, as well as to suggest some improvement in the equipment recommended for the determination.
Description of Method Generally a 3- to 5-gram sample is used for the determination. The sample should be in a finely pulverized state and representative of the material being analyzed. In the case of fresh ve e tables, it has been found convenient first to freeze with so& carbon dioxide, then grind in an ordinary meat grinder, and weigh a representative sample of the ground frozen vegetable. Grinding and weighing are conducted in a refrigerated room held below freezing. With materials of high fat content, it may be necessary first to extract a neighed amount of the material with ether in a Soxhlet extractor and then carry out the analysis on the fat-free residue. The weighed sample is placed in a specially designed extraction tube of about 75-cc. capacity, to which 50 cc. of 0.04 N sulfuric acid are added, giving a liquid t o solid ratio of about 10 to 1. The
pH (1to 2) of this extraction mixture is optimal for the extraction of the vitamin and sufficiently acid to prevent its destruction. The extraction assembly is illustrated in Figures 1 and 2. The extraction tube is attached by means of a ground-glass joint, A , to a small glass water condenser, B , through which extends a glass stirrer, C, attached to a small electric motor with a variable rheostat. The heat required for the extraction may be furnished by a microburner, electric hot plate, or hot water bath as shown in Figure 2. Before any heat is applied the mixture should be thoroughly stirred to prevent charring of the sample. The extraction is carried out at the boiling point of the mixture for approximately one hour with continuous stirring. Although for certain samples a shorter period of extraction may be employed, an hour’s extraction will ensure complete solution of the vitamin. At the conclusion of the extraction, each of the stirrers is carefully viashed down with 5 cc. of distilled water delivered from a measuring pipet and the tubes are cooled t o room temperature by placing them in running water. As the pH of the solution following the extraction usually lies between l and 2, it is necessary to increase the pH to the range of 4 to 5 in order to obtain optimal conditions for the enzymatic hydrolysis of any cocarboxylase present in the extract. Although Hennessy and Cerecedo (2’) employed 1 Ai sodium hydroxide for this purpose, it was felt that the use of this reagent might lead to a loss of thiamin due to the development of a local area of high alkaline concentration before FIGURE1
