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; t h a t 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; t h a t zinc cannot be determined by weighing as zinc sulfide; and t h a t 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 t h e 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
ANALYTICAL EDITION
lune IS, 1941
381
The vitamin is eluted from the column by use of 2.5 oc. of a hot 25 per cent solution of potassium chloride, fnlloruing the procedure of Hennessy and Cerecedo (8). A 25 per cent solution of sodium chloride is equally effeotive, provided the Decalso oolumn has been previously activated with this reseent instend of ootassium chloride^ A ~ - c c . aliquot of this eluate is then pipetted into a separatov funnel for
.~ ~
~~
~
~~
~~~
~
able to u8e separatory funnels eqbipfed with No-Lub stopcocks for this purpose rather than those described by Henuessy and Cerecedo (a). To this 5-ce. aliquot is added 1 eo. of a freshly prepared solution containing 0.002 grain of potassium ferricysde and 0.45 gram of sodium hydroxide. Twentv cubic centimeters of isobutvl alcohoi are finally added and t i e separstory funnel is shaken for one minute in a mechanical shaking machine and then centrifuged. The lower &aueous layer is d r a m off. the isobutyl-alcohol layer is treated with 2
tion of the resulting thiochrome should he carried o& as quicklv
more than that eiven hv an eauivalent amount of distilled water.
volumetric flask and &.de uu to volume with di8tilled water.
a8
fractions. Following the sodium sulfate treatment, a suitable aliquot (15 ce.) of the l'obutyl alcohol solution containing the thiochrome i s
incubation oven'&ntaineYd a temGerat6re of 45' C. The tubes are ineuhated for 2 hours st this temperature with frequent. stirring. Following the incubation period, each stirrer is carefully washed down with 1 cc. of distilled water delivered from a measuring
before eieh measurement &th a standard quinine solution and the light intensity adjusted to the desired value by changing the setting of tlie iris diaphragm. The frequent checking of the instrument with quinine sulfate has been found useful, as the light 8ource used in it has only a limited life and the prolonged use of
e;: ai a 5
Gr cent
solution of clarase (prepared fksh daily
~ . - ~. .~~~~ ~~~~~~
at
~
..___ . . ..
.
.
~
~~~
~~~~~
~
~
~
~
~
~
~
5 mieroerams of thiamin is then u%etted out ofihd iupernataGt liquid &to a 5 h ~ beaker . cont&ng 5 DO. of 2 per cent acetic acid. The acetic acid solution containing the thiamin is heated just to bailing on an electric hot late and passed through a 5-cm. (2inch) column of activated (%p60-to 80-mesh Deealso, resting on a ~~
~~~~~~
thiamin chloride and to compari the galvanornet& reidinis of the unknown samples with that of theitstandard solution, rather than to depend entirely upon a. calibration curve for determining the concentration of thiamin in the unknown. The standard solution of thiamin chloride is prepared in 0.5 per cent solution of chlorobutanol a t a pH of 3.2 and is kept under refrigeration when
~
described in thr! pmcedure of Merck & Co. (7). Each ahaorption tube is attached by means of a one-hole rubher stopper to a 12.5.~~. elass suction flask as shown in"Fiaure 3 When t h e vitamin-containing solution reaches the bottom of the Tkmlso column. a eentle surtion is momentsril$ gppliedi io that a rate of How of approximately 1 oc. per minute ia obtained. After the vitamin solution has passed through, the beaker and the column are rinsed several times with hot distilled water and the washings are passed through the column. By testing the filtrate and washings for thiamin, this procedure was found to l e d to practically 100 ner cent, ahsorotion of the v i h ~~
FIQURE3.
SETUP OF ABSORPTION
TUBS5
382
INDUSTRIAL AND ENGINEERING CHEMISTRY
not in use. The chlorobutanol prevents mold growth and does not influence the fluorescence measurements. The cuvette in which the fluorescence measurements are to be made should always be placed in the same position in the fluorophotometer before each measurement, as the walls of the cuvette may vary in their light transmittancy. Cuvettes have also been found to vary among themselves in this respect. A “complete blank” is run daily for each series of determinations. This consists of 50 cc. of 0.04 N sulfuric acid, 10 cc. of 5 per cent clarase solution, and 6 cc. of distilled water. The same incubation and centrifuging procedure is applied to the blank as to the samples. A volume of aliquot is taken from the centrifuged blank corresponding to that used in each determination. Five cubic centimeters of the potassium chloride eluate from the Decalso column, through which the blank solution has been passed, are then oxidized under the same conditions employed for the samples under test, and its fluorescence is determined. The latter is the correction to be applied in arriving at the true thiamin value of each sample. In the case of the standard thiamin solution, only an “oxidation blank” correction need be applied, This is the fluorescence resulting from a mixture of 0.45 gram of sodium hydroxide, 20 cc. of isobutyl alcohol, and 5 cc. of distilled water. The blanks described do not cover the possibility that some fluorescent material other than thiamin may be eluted from the Decalso. il suggestion that this is not a serious contingency is contained in the comparative chemical and bioassays shown in Table T?I.
Critical Study of Steps i n Procedure
ENZYMATIC HYDROLYSIS. Lohmann and Schuster (6) have shown that a great part of the naturally occurring thiamin may be present in the form of its pyrophosphoric acid ester known as cocarboxylase. Although cocarboxylase is converted by alkaline ferricyanide to the pyrophosphoric ester of thiochrome, Kinnersley and Peters (5) have shown that this compound cannot be extracted from an aqueous solution with isobutyl alcohol. Therefore, it is necessary to hydrolyze the pyrophosphoric acid group from the cocarboxylase prior to oxidizing i t n-ith the alkaline ferricyanide. Hennessy and Cerecedo (8) employed an enzyme preparation prepared from beef kidney for this purpose. As the preparation of such material is tedious and time-consuming, the adaptability of other enzyme products was investigated. The use of takadiastase has already been mentioned ( 7 ) . Clarase, mylase, and a mylase with high phosphatase activity were also studied. Since one criterion of the suitability of an enzyme preparation is the completeness of the hydrolysis of cocarboxylase, this reaction was given careful study. In order to simulate as closely as possible the actual conditions which would be encountered in the enzymatic hydrolysis of an unknown sample, 20 cc. of a pure solution containing 10.47 micrograms of cocarboxylase were placed in an extraction tube to which were added 30 cc. of 0.04 N sulfuric acid. The solution was heated in the usual manner for 1 hour and a t the end of that time each stirrer was washed down with 5 cc. of distilled water and the tubes were cooled to room temperature. To each tube were then added 10 cc. of the enzyme solution. I n the case of takadiastase and clarase, a 5 per cent solution was used, but as the two mylase preparations were less soluble, a lower concentration of these enzymes was employed, as shown in Table I. All the enzyme solutions were prepared in a sodium acetate-acetic acid buffer as previously described, so that the cocarboxylase solution, after the addition of the enzyme preparation, would have a pH of 4.5. The tubes were incubated for 2 hours at 45” C. with frequent stirring. At the conclusion of the incubation period, each stirrer was washed down with 3 cc. of distilled water, a 5-cc. aliquot was pipetted out of the solution, and a determination was made of its thiamin content using the procedure previously described. Control solutions containing only the enzyme preparations were run parallel to the test solutions, so that a correction could be made for any small amounts of the vitamin possibly present in the enzyme preparations.
Vol. 13, No. 6
From Table I i t appears that either takadiastase, clarase, or the high-phosphatase mylase preparation can be employed for the hydrolysis of the cocarboxylase. Subsequent experiments have shown that, in most instances, a 2 per cent solution of clarase is as effective as the five per cent solution. As all the enzyme preparations are essentially diastatic, their activity toward cocarboxylase would appear to be due t o the presence of an associated phosphatase. The starch-splitting property of the enzymes, however, is of value in obtaining a clear extract of the cereal products. TABLE I. HYDROLYSIS O F COC4RBOXYLAsE BY ENZYME PREPARATIOKS Enzyme Solution Used 5% takadiastase 5Yc clarase 1% mylase (high phosphatase) 0.5% mylase (normal)
Yield of Thiamin in 5-Cc. Aliquot Theoretical Actual Micrograin Microgram 0.7i 0.77 0.77 0.77 0.7i 0.76 0 7i 0.56
Hydrolysis
70 100 100 97 73
IXFLUENCE OF AMOUST OF ALKALI ON OXIDATIONOF THIOCHROME. A b various amounts of sodium
T H I l M I N TO
hydroxide have been recommended for the oxidation of thiamin to thiochrome, a study of the effect of this variable was undertaken. Solutions of thiamin chloride (1 cc. = 1 microgram) at pH values of 0.4, 2.2, and 4.7 were used. The solution a t a pH of 2.2 was made up in 25 per cent potassium chloride to simulate the solution which would be obtained after the potassium chloride elution of the Decalso column. Solutions of sodium hydroxide were prepared, containing per cc. 0.001 gram of potassium ferricyanide, and 0.075, 0.15, 0.30, 0.45, and 0.60 gram, respectively, of sodium hydroxide. To 1-cc. quantities of each of these sodium hydroxide-potassium ferricyanide solutions were added 0.5, 1.0, and 2.0 cc. of the thiamin chloride solution (representing 0.5, 1.0, and 2.0 micrograms of the vitamin) and the resulting solutions were made up to 5 cc. with the 25 per cent potassium chloride solution. To each 5-cc. portion, contained in a separatory funnel, were added 20 cc. of isobutyl alcohol and the oxidation was carried out in the manner previously described. Table I1 shows that the amount of sodium hydroxide used plays a n important role in the oxidation of thiamin to thiochrome. The results indicate that l cc. of a solution of sodium hydroxide containing 0.45 gram of the alkali and 0.001 gram of potassium ferricyanide was required to obtain maximum oxidation of thiamin chloride solutions containing from 0.5 to 2.0 micrograms of the vitamin, irrespective of the pH of the solution. The oxidation, as indicated by the galvanometer readings, is not further enhanced by using an amount of sodium hydroxide in excess of 0.45 gram per cc. ox TABLE11. EFFECTOF AMOUNTOF SODIUMHYDROXIDE OXIDATION OF THIAMIN TO THIOCHROME NaOH Added, Gram 0.075 0.150
0.300 0 450 0.600 0.075 0.150
0.300 0.450 0.600 0.075 0.150 0 300 0,450 0 600 a
$H
of hiamin Solution 0.4 0.4 0.4 0.4 0.4 2.2 2.2 2.2 2.2 2.2
Galvanometer Readingsa Obtained from Indicated Amounts of Thiamin 1.0 microgram 2.0 micrograms 11.0 4.0 4.0 14.0 24.0 4.0 15.7 29.0 44.5 16.0 29.3 54.0 15.9 29.5 53.3 14.8 25.9 44.9 14.3 25.6 44.7 15.8 26.6 50.0 16.1 29.0 54.1 16.0 29.0 54.5
0.5 microgram
Obtained a i t h Pfaltz & Baurr fluurophotometer.
ANALYTICAL EDITION
June 15, 1941
383
I N F L U E ~ C E OF AMOUST OF POTASSIU~LI. FERRICY-~SIDE. After centrifuging and separating the aqueous layer, each solution was made up to contain 20 cc. of isobutyl alcohol. To each ~~~i~~ determined the arnouIlt of sodium hydroxide reseparatory funnel were then added 2 grams of anhydrous sodium quired in the oxidation of thiamin to thiochrome, the effect sulfate. the seDaratorv funnels lvere centrifuged. and a 15-cc. aliquot was pipktted i&o the cuvette of the fluorophotometer for a on the oxidation of varying the amount of potassium ferrifluorescent reading. cyanide was nest studied.
For this purpose, samples of the same thiamin chloride solutions were employed as for the study of the effect of alkali variation. Solutions of potassium ferricyanide were prepared containing per cc. 0.45 gram of sodium hydroxide and 0.0005, 0.001, 0.002, and 0.003 gram, respectively, of potassium ferricyanide. One cubic centimeter of each solution was then added to 0.5, 1.0, and 2.0 cc. of the thiamin chloride solution, representing 0.5, 1.0, and 2.0 micrograms of the vitamin, and the resulting solution in each case was made up to 5 cc. with the 25 per cent potassium chloride solution. After adding 20 cc. of isobutyl alcohol, the oxidation was carried out in exactly the same manner as in the sodium hydroxide study, with results shown in Table 111.
TABLE 111. EFFECT O F AMOCXT O F POTAsSIUM FERRICY.4NIDE Oh' OXIDATIOK O F THL4MIh' TO THIOCHROME IiaFe(CN,a Added Gram
pH of Thiamin Solution
Galvanometer Readings" Obtained from Indicated iZmounts of Thiamin 0 . 5 microgram 1 0 microgram 2 . 0 micrograms
0.000.5 0.0010 0.0020 0.0030
0.4 0.4 0.4 0.4
16.1 16.0 16.2 16 0
28.9 29.3 29.2 29.0
53.9 54.0 55.0 54.0
0,0005 0.0010 0.0020 0,0030
2.2 2.2 2.2 2.2
16.5 16.1 16 0 16.0
0.0005 0.0010 0.0020 0.0030
4.7 4.7 4.7 4.7
16 0 16.3 16.2 16.0
29.8 29.0 29.5 29.0 29.0 29.4 29.0 29.0
54.9 54.1 54.9 54.0 54.1 54.4 56.0 56.0
a
Obtained with Pfalts B Bauer fluorophotometer.
When the amount of sodium hydroxide is optimal, varying t h e amount of potassium ferricyanide has little effect on the oxidation of thiamin to thiochrome except in the case of the thiamin chloride solution at a pH of 4.7 containing 2 micrograms of the vitamin. I n general, i t may be concluded that 0.45 gram of sodium hydroxide and 0.002 gram of potassium ferricyanide will give a maximum oxidation of thiamin to thiochrome under any of the experimental conditions herein studied. From Tables I1 and 111, the conclusion mag also be drawn t h a t if these amounts of sodium hydroxide and potassium ferricyanide are employed, the presence of potassium chloride is without influence on the reaction. This is of importance in that it shows that the standard solution of thiamin chloride, used for comparison with the unknown sample, does not need t o be prepared in a 25 per cent solution of potassium chloride. INFLUENCE OF AMOUNTOF ISOBUTYL ALCOHOL. Having established the optimal amounts of soduim hydroxide and potassium ferricyanide t o be employed in the oxidation of thiamin t o thiochrome, a study was next undertaken to determine the effect of the amount of isobutyl alcohol used in the estraction of the thiochrome formed in the reaction. For this investigation, a solution of thiamin chloride (1 cc. = 1 microgram) prepared in 25 per cent potassium chloride was employed. To 0.5, 1.0, and 2.0 cc. of this thiamin chloride solution, representing 0.5, 1.0, and 2.0 micrograms of the vitamin, was added 1 cc. of the sodium hydroxide-potassium ferricyanide solution containing 0.45 gram of the alkali and 0.002 gram of
potassium ferricyanide. The resulting solution in each case was made up to 5 cc. with the 25 per cent potassium chloride solution. To each solution, contained in a separatory funnel were added either 13, 15, or 20 cc. of isobutyl alcohol and the separatory funnel was shaken for 2 minutes on a mechanical shaking machine.
TABLE IV. EFFECTOF AMOUSTOF ISOBUTYL ALCOHOL [On extraction of thiochrome formed in oxidation of thiamin by NaOHK3Fe(CK;)6] Galvanometer Readinga from Indicated Amount of Amounts of Isobutyl Alcohol Thiamin Chloride 13 cc. 15 cc. 20 cc. Micrograms 0.5 16.0 15.9 16.1 1.0 28.4 29.0 29.0 2.0 54.0 54 9 54.9
Table IV shows that 15 cc. of isobutyl alcohol gave a maximum estraction of the thiochrome formed from amounts of thiamin chloride varying from 0.5 to 2.0 micrograms. I n the authors' routine procedure, however, the use of 20 cc. of isobutyl alcohol has been found preferable, as this will allow a larger volume of liquid to be placed in the cuvette of the fluorophotometer for the fluorescence readings. The use of alcohols other than isobutyl alcohol for the extraction of the thiochrome is now being studied and will be reported in a subsequent paper. EFFECTOF DURA4TIOK O F SHAKING OK EXTRACTION OF THIOCHROME FORMED IK OXIDATION OF THIAMIN.For the study of this variable, the same thiamin chloride solution and the same experimental conditions were employed as in the foregoing. I n each instance, 20 cc. of isobutyl alcohol were used in the extraction and the separatory funnels were mechanically shaken for intervals of 1, 1.5, 2 , and 3 minutes. The results are given in Table V.
TABLE V. EFFECTOF DURATION OF SHAKING ON EXTRACTIOX OF THIOCHROME BY ISOBUTYL ALCOHOL Amount of Thiamin Chloride Micrograms 0.5 1.0 2.0
Galvanometer Readings for Indicated Intervals of Shaking l.5min. 2 min. 3 min.
1 min. 16.1 29.2 54.0
16.1 29.5 54.4
16.1 29.0 54.9
16.1 29.0 52.1
For amounts of thiamin chloride ranging from 0.5 to 1.0 microgram of thiamin chloride, the interval of shaking was without effect on the extraction of the thiochrome by 20 cc. of isobutyl alcohol. For solutions of thiamin chloride containing 2 micrograms, the shaking time should not exceed 2 minutes, as beyond t h a t time a decrease in the galvanometer readings occurred. Since in most routine analyses the concentration of thiamin chloride in the mixture being oxidized does not exceed 1 microgram, a 1-minute shaking mill prove sufficient. DEGREEOF COWERBIOKOF THIAMIN TO THIOCHROME. Having established the optimal conditions for conducting the oxidation of thiamin to thiochrome, the degree of conrersion of thiamin to thiochrome was studied. For this study, a solution of pure thiochrome was prepared in 25 per cent potassium chloride. Precautions were taken to protect the solution from light destruction prior to its measurement. Amounts of this solution theoretically correspondin to 0.5, 1.0, and 2.0 micrograms of thiamin chloride were introiuced into a separatory funnel, which was then shaken for 1 minute with 20 cc. isobutyl alcohol and 1 cc. of the sodium hydroxide-potassium
INDUSTRIAL AND ENGINEERING CHEMISTRY
384
ferricyanide mixture containing 0.45 gram of the alkali and 0.002 gram of potassium ferricyanide. The resulting solution was centrifuged, treated with anhydrous sodium sulfate, and its fluorescence determined in the usual manner. TABLEVI.
CONVERSION OF THIAMIN TO THIOCHROME
Thiamin Chloride and Theoretical Thiochrome Equivalent
Galvanometer Rea d i n g
Thiamin chloride (0.5 microgram) Thiochrome (0.38microgram) Thiamin chloride (1.0 micro ram) Thiochrome (0.77microgram? Thiamin chloride (2.0 micrograms) Thiochrome (1.54 micrograms)
16.1 24.3 29.3 44.2 54.4 81.0
Conversion
% 67 66
Vol. 13, No. 6
TABLEIX. THIAMIN CONTENT OF VARIOUSSUBSTANCES Thiamin Content or Range
Material
M icrograms/Gram 4.86-5.28 1.56 34.68 1,05-6.87 1.69 187.00 -3.s i
Fortified flour Barley Wheat germ Wheat Whey powder Yeast Skim milk powder Cocoa Green coffee Fresh lima beans Fresh broccoli Fresh string beans Fresh squash Frozen peas
0.84
2.10 2.97 1.26 0.93 1.47 1.37-4.32
67
The data of Table VI suggest that within the range of 0.5 to 2.0 micrograms of thiamin chloride, the degree of conversion of the thiamin to thiochrome is approximately 67 per cent. This is in confirmation of the results obtained by Ferrebee and Carden (1). However, the degree of solubility in isobutyl alcohol of the thiochrome formed in the oxidation of the thiamin is not known. It is possible that the low “conversion” values may in part be due to solubility relationships rather than to incompleteness of the oxidation itself. This point is under further investigation. COMPARISON OF CHEMICALRESULTS WITH BIOASSAY VALUES. The reliability of the present method has been judged by comparing the results obtained on certain food materials with those obtained by biological assay on the same samples, using a rat-growth method. The results of such a comparison are shown in Table VII.
THIAMIN CONTENTOF VARIOUSFOODPRODUCTS. The method has been successfully applied to various food materials, including cereals and fresh and frozen vegetables. Typical results obtained on various types of foodstuffs are given in Table IX.
Summary
A study of the thiochrome method for determining thiamin showed that, by the procedure developed, extraction and hydrolysis of the sample may be carried out in the same vessel, thereby eliminating any error due to transfer of the extract inherent in previous methods. For the enzymatic hydrolysis of cocarboxylase, the enzyme clarase has been introduced. From a study of the oxidation of thiamin to thiochrome, it was found that the most important factor was the amount of sodium hydroxide employed. One cubic centimeter of a solution of sodium hydroxide-potassium ferricyanide containing 0.46 gram of sodium hydroxide and 0.002 gram of TABLEVII. COMPARATIVE RESULTS OBTAINEDFROM CHEMICAL potassium ferricyanide gave an optimal oxidation of thiamin AND BIOASSAY PROCEDURES to thiochrome for solutions of thiamin chloride containing Thiamin Content 0.5 to 2.0 micrograms of the vitamin. These amounts from Materials Bioassay Chemical of sodium hydroxide and potassium ferricyanide gave an Micrograms/gram Micrograms/gram optimal oxidation of the thiamin to thiochrome, irrespective 3.27 Processed cereal product No. 1 3.15 1.12 1.26 Processed cereal product No. 2 of the p H of the thiamin solution or of the presence or absence 1.37 1.44 Frozen peas of potassuim chloride. The optimal conditions for the extraction of the thiochrome It is apparent that there is a close agreement between the formed in the oxidation of thiamin chloride solutions conchemical and biological assays and that the values obtained taining 0.5 to 2.0 micrograms of the vitamin have been found by chemical assay fall well within the range of the error into be a 1-minute mechanical shaking in the presence of 20 cc. herent in the biological method. of isobutyl alcohol. For amounts of thiamin chloride ranging from 0.5 to 2.0 TABLEVIII. PERCENTAGE RECOVERY OF THIAMIN ADDED~TO micrograms, the conversion of thiamin to thiochrome is approximately 67 per cent under the conditions studied in this VARIOUSSAMPLES paper. Amount of Added The method is in close agreement with biological assays, Thiamin Content Thiamin Thiamin Material of Sample Addeda Reoovered Reoovery and has been applied to various types of natural products, MicrogramslGrarn Micrograms Micrograms % ’ including grains and fresh and frozen vegetables. Processed cereal KO. 5 Processed cereal No. 6 Processed cereal No. 7 Processed cereal No. 8 Skim milk powder Wheat Frozen spinach Frozen peas Frozen broccoli 0
3.15
4.00
3.61
90.00
3.36
4.00
3.5s
89.50
1.56
8.00
7.96
99.50
0.30 3.36 3.90 1.26 2.97
6.00 2.00 2.00 2.00 2.00
0.84
2.00
5.80 1.98 2.00 1.92 1.86 1.86
96.00 99.00 100.00 96.00 93.00 93.00
Thiamin added to initial extraction mixture.
DEGREEOF RECOVERY OF THIAMINCHLORIDE ADDEDTO
FOOD PRODUCTS. I n order to determine the degree to which the vitamin was preserved throughout the determination, a study was made of the percentage recovery of thiamin chloride added to various samples with the results given in Table VIII. The recovery of added thiamin is seen to be 90 per cent or better.
Acknowledgment The authors are indebted to the Scientific Glass Apparatus Company, Bloomfield, N. J., which supplied the specially designed extraction tube, absorption tube, and separatory funnel, and to Merck & Co., Inc., which kindly supplied the cocarboxylase and thiochrome.
Literature Cited (1) Ferrebee, J. W., and Carden, G. A., J . Lab. Clin. Med., 25, 1320-4 (1940). (2) Hennessy, D. J., and Cerecedo, L. R., J . Am. Chem. Soc., 61, 17983 (1939). (3) Jansen, B. C. T., Rec. trav. chim., 55, 1046-52 (1936). (4) Karrer, W., and Kubli, B., Helv. Chim. Acta, 20, 369-73 (1937). ( 5 ) Kinnersley, H. W., and Peters, R. A., Biochem. J., 32, 697 (1938). (6) Lohmann, K., and Schuster, P., Biochem. Z.,294, 188 (1937). (7) Merck & Co., Rahway, N.J., “Determination of Thiamin Chloride by Fluorometric Method”, 1940.
