ANALYTICAL CHEMISTRY
810 dipicrylaminate. This is accompanied by erratic results for potassium. A series of solutions containing the sulfates of both potassium and zinc were prepared, and each solution was analyzed for potassium in the usual manner. The presence of zinc did increase the optical transmittance, but it appears that as the amount of potassium in the sample increases, more zinc ions can be tolerated without affecting the results for potassium. A safe and general rule in this instance is that only when the meight of zinc in a sample is greater than the weight of potassium need the zinc be removed or reduced in amount before proceeJing with the analysis for potassium. This rule has been followed in this laboratory and has given good results. Under such conditions no yellow precipitate is formed during the period the solutions stand before dilution. The zinc can be removed from the solution by precipitation with small amounts of a dilute sodium hydroxide solution. According to the thesis of Shapiro ( 7 ) , the yellow precipitate formed by the zinc ions would he dipicrylamine itself, and it is produced because the zinc salts hydrolyze, giving an acid solution. On this basis, the amount of yelloiv precipitate should be determined by the pH of the solution. However, it is a t once evident from Figure 2 that more than a pH factor is involved in the formation of this precipitate. If only pH were involved, then zinc sulfate solutions should have the same effect as acid solutions of the same pH. Honever, only a slight change in pH of the zinc sulfate solutions produces a large error in the potassium determination. This is in contrast to the effect of sulfuric acid solutions. When solutions containing only zinc sulfate TI ere treated with the lithium dipicrylamine reagent, a yellow-orange precipitate was formed, the amount of which increased n.ith increasing amounts of the dipicrylamine reagent. This precipitate was
but very slightly soluble in water. Its color was definitely different from that of plain dipicrylamine in the same solution. Kolthoff and Bendix (6) suggest that since the dipicrylamine reagent has an alkaline reaction, a cation such as zinc should yield a precipitate consisting of the hydrous oxide or some basic salt of zinc. There is definite evidence that this did occur. A close examination of the precipitate showed that there were two types of solid matter-the yellow or orange precipitate which may have been the zinc dipicrylaminate, and a smaller amount of a lighter colored, gelatinous material. This latter diswlved readily in dilute sulfuric acid and also in concentrated s3dium hydroxide solutions. This would indicate that this precipitate is a mixture of an oxide or hydroxide of zinc and some zinc dipicrylaminate. S o further study of this precipitate was made. ACKSOW LEDGMEKT
The xork reported here was carried out in connection xith a project sponsored by the Office of Saval Research. It is a pleasure to express thanks for this support. LITERATURE CITED
(1) Amdur, E., ISD. ENG.CHmr., A N ~ LED., . 12, 731 (1940). (2) Duval, C., Anal. Chim. Acta, 1, 105 (1947). (3) Kielland, J., Ber., 71B,220 (1938). (4) Kielland, J., Ger. Patent 704,545, Feb. 27, 1941. ( 5 ) Kohn, W., 2. anal. Chem., 128, 1 (1947). (6) Kolthoff, I. M., and Bendix, G. H., IND.ENG.C H m r . , A N ~ L . ED.,11,94 (1939). (7) Shapiro, LI, Ya., Zacodakaya Lab., 7, 790 (1935). (8) Sheintsis, 0. G., Ihid., 4, 1047 (1935). (9) Ibid., 8, 1198 (1939). (10) Winkel, d.,and Maas, H., Angew. Chem., 49, 827 (1936).
RECEIVED for review
October 18, 1952.
Accepted January 9, 1953.
Determination of Thiamine by the Thiochrome Reaction Application of Cyanogen Bromide i n Place of Potassium Ferricyanide MOTONORI FUJIWARA AND KIYOO XIATSUI Department of Hygiene, Kyoto L'niversity, h i o t o , Japan potassium ferricyanide and alkali reaction, which was T first proposed for the determination of thiamine in biological materials by Jansen (6) in 1936, is generally recognized as the HE
most useful reaction for converting thiamine to thiochrome, and is most commonly used for the determination of thiamine. I n 1949 Fujiwara (1) found that a blue fluorescent compound, "thiochrome," was produced on addition of alkali after thiamine was mixed with cyanogen bromide a t room temperature. This compound was proved to be thiochrome, melting point 227' C. (decomposes) by Matsukawa ( 6 ) , a staff investigator of the research laboratory attached to Takeda Pharmaceutical Industries, Ltl. The authors have studied the utilization of this reaction in the determination of thiamine in biological materials. in place of oxidation of thiamine with potassium ferricyanide and alkali. REAGENTS
Synthetic zeolite, prepared according to the method of Hennesqy ( 3 ) ; 25% potassium chloride in 0.1 S hydrochloric acid; isobutyl alcohol; 30% sodium hydroxide; anhydrous sodium sulfate; and cyanogen bromide solution. PROCEDURE
One and one half grams of activated zeolite are placed in an exchange tube which has an internal diameter of 7 mm. and a stopcock ( 2 ) . A finely pulverized sample, containing 3 to 5 micrograms of thiamine, is weighed and about 40 ml. of distilled water are
zddsd. This mixture is heated on a water bath at 80" C. for 1.3 minutes, being continuously stirred, after the pH is adjusted to 4.. After addition of about 3 ml. of 2% takadiastase solution and a few drops of toluene, the mixture is allowed to stand overnight in an incubator a t 38" C. Distilled water is added to make the total volume of liquid 50 ml. and the mixture is centrifuged a t high speed. A certain amount of clear supernatant solution, containing 1 to 2 micrograms of thiamine, is allowed to pass through the zeolite column a t the rate of about 1 ml. per minute after the pH has been adjusted to 4.5. The zeolite is washed with hot water, and then thiamine is eluted ivith 25 ml. of boiling potassium chloridehydrochloric acid solution. The eluate is made up to exactly 25 ml. in a graduated cylinder. A 5-ml. portion of the potassium chloride-hydrochloric acid eluate is transferred to a centrifuge tube, 3 ml. of cyanogen bromide solution are added and mixed, and 2 ml. of 30% sodium hydroxide are added and mixed. The thiochrome produced is extracted in 20 ml. of butyl alcohol in the customary manner. The blank is treated in the same way by omitting the cyanogen bromide. Fluorescence is measured in the Pfaltz & Bauer fluorophotometer according to the procedure of Hennessy and Cerecedo (4). CRITICAL STUDY OF STEPS IN PROCEDURE
Preparation of Cyanogen Bromide Solution. This reagent is prepared by adding an ice-cold 10% aqueous solution of potassium cyanide drop by drop to ice-cold saturated bromine water until it is decolorized. This reagent is stable for 3 hours a t room temperature (22" C.) as shown in Table I. Influence of Amount of Cyanogen Bromide Solution on Oxidation of Thiamine to Thiochrome. To decide the amount of
811
V O L U M E 25, NO. 5, M A Y 1 9 5 3 Table I.
Stability of Cyanogen Bromide Solution
Time after Preparation, Hour3 0 1 2 3 5 7 0
Galvanometer Readingsa 87 0 87 0 86 5 86 5 84 0 84 0
Thiamine content, 1 microgram.
Table 11. Effect of Amount of Cyanogen Bromide Solution on Yield of Thiochrome from Thiamine C S B r Solution Added. 1\11. n i
Galvanometer Readings
1.n
58.0 58.5 58.5 56.5 54.5
2.0 3.0 4.0 5 . 11
. 5 ~n ~
Thiochrome Yield, S 91 99 100 100 97 93
was carried out in the usual manner, The results are shown in Table IT'. The thiochrome yield is very slightly affected by the difference in thiamine content when 3 ml. of cyanogen bromide solution and 2 ml. of 30% sodium hydroxide solution are used for 5 ml. of thiamine solution. Influence of Temperature of Treatment with Cyanogen Bromide. A series of mixtures, each prepared by mixing 5 ml. of thiamine solution (potassium chloride-hydrochloric acid) containing 1 microgram of thiamine with 3 ml. of cyanogen bromide solution, was subjected to temperatures ranging from 3" to 80" C. for 5 minutes and then alkalized, Thiochrome \\'as extracted
Table V.
c.
3 20 30 50 30
cyanogen bromide solution required for oxidation of thiamine to thiochrome, the following study was undertaken. To a seties of thiamine solutions, each containing 1 microgram of thiamine in 5 ml., 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml. of cyanogen bromide solution were added and each solution was mixed with 2 ml. of 30% sodium hydroside. The thiochrome was extracted with butyl alcohol and fluorescence was measured in the usual manner. The results are shown in Table 11. There are no distinct differences in the intensities of fluorescence of thiochrome converted from thiamine when betn-een 1 and 3 ml. of cyanogen iiromide are used for 5 ml. of thiamine solution. Influence of Amount of Alkali in Oxidation of Thiamine to Thiochrome. After determination of the amount of cyanogen bromide required in the oxidation of thiamine to thiochrome, the effect on the osidation of various amounts of sodium hydroside was studied.
To a serie. of the mixtures, each prepared by mixing 5 ml. of thiamine solution (potassium chloride-h Tdrochloric acid) eontaining 1 microgram of thiamine with 3 m.! of cyanogen hromide solution, 1, 2, 3, and -1 nil. of 30% sodium hvdroxide solution were added. The results are shown i n Table 111. There are no distinct differences in the intensities of fluorescence of thiochrome converted from thiamine \\-hen sodium hydroxide solution used exceeds 2 ml. for 5 ml. of thiamine solution. Yield of Thiochrome from Various Amounts of Thiamine, A series of solutions (potassium chloride-hydrochloric acid) containing amounts of thiamine ranging from 0.1 to 5.0 micrograms, was mixed Kith 3 ml. of cyanogen bromide solution. Oxidation
Table 111. Effect of Amount of Sodium Hydroxide on Thiochrome Produced from Thiamine 30T0 XaOH Solution Added, 111. 1.0 2.0 3.0 4.0
Galvanometer Readings 79.5 85.0 83.0 86.0
Thiochrome Yield, So 94 100 100 101
Table IV. Production of Thiochrome from Thiamine Thiamine Content, y 0.1 0 . .5 1.0 1.0 3.0 5.0
Galvanometer Readings 14.5 72.5 146.5
30.0 91.5 152.5
Thiochrome Yield, % 100 in0 101 100 101 101
Table VI.
Thiochrome Yield, 70 100 100 100 99 96
Influence of pH of Thiamine Solution in Treatment with Cyanogen Bromide
pII of Thiamine Solution
Galvanometer Readings 92
93 0 93 0 92 0 86 0 72 0 1.0
7
13
Table YII.
I
Influence of Salts in Thiamine Solution
Concentrations of Salts in Thiamine Solution 10% NaCl Saturated S a C l Saturated KC1 2 5 7 , KCI-HC1
Thiochrome Yield,
n
1
i 5 6 8 10
10% KCl
Influence of Temperature Galvanometer Readings 79.0 79.0 79.0 78.0 ,75.5
Temperature,
Galvanometer Readings 74.0 74.0 73 .i 74.0 74 s
Thiochrome Yield
S
99 99 94 99
100
with butyl alcohol in the usual manner. Table V shows that variations in temperature from 3" to 50" C. had no effect on maximal production of thiochrome from thiamine. Influence of pH of Thiamine Solution in Treatment with Cyanogen Bromide. A series of solutions, each containing 1 microgram of thiamine in 5 ml., \\as prepared. These solutions [yere adjusted to pH 1, 4.5, 6, 7 8, 10, and 13, and each was mixed with 3 ml. of cyanogen bromide. Oxidation was cairied out in the usual manner. The results are shown in Table T I The thiamine is convwted to thiochrome nithout any interference a t pH from 1 to 7 , but hindrances oceur n hen the pH i;j above 8. Therefore, if 30% sodium hydroxide is added to the thiamine solution prior to treatment nith cyanogen bromide, practically no fluorescence of thiochrome converted from thiamine is found. This indicates that the character of the present reaction is entirely different from that of the potassium ferricyanide reaction. Influence of Salts in Thiamine Solution. Salts-potassium or sodium chloride-present in the thiamine solution do not affect the conversion of thiamine to thiochrome; 25% potassium chloride in 0.1 hydrochloric acid solution, which is commonly used for eluting thiamine from zeolite, does not interfere v-ith this reaction (Table VII). Influence of Cysteine Hydrochloride in Thiamine Solution. To clarify the influence of admisture in the thiamine solution on the process of oxidation of thiamine to thiochrome, the authors
812
ANALYTICAL CHEMISTRY
studied the influence of cysteine mixed n i t h the thiamine solution. A series of thiamine solutions, each containing 1 microgram of thiamine in 5 ml. together with varying amounts of cysteine hydrochloride was mixed n i t h 3 nil. of cvanogen bromide solution, and the oxidation w.s carried out in the usual manner. As the control, similar series was mixed with 0.1 ml. of 1yopotassium ferricyanide and 2 ml. of 30% sodium hydroxide, and thiochrome was extracted with butyl alcohol in the same manner (Table VIII)
Table VIII. Influence of Cysteine-Hydrochloride in Thiamine Solution upon Production of Thiochrome from Thiamine by Cyanogen Bromide and Potassium Ferricyanide Reactions Cysteine-HC1 Added, Alg, 0.5 1 0 3.0 5.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0
may safely vaiy over a relatively broad range (cf. Table 11). and the production of thiochrome from thiamine is not affected by the presence of any reducing component (cf. Table VIII). Because of these tv-o features this reaction affords a safe and reliable method for determining thiamine in both pure solutions and biological materials. The niai ked difference in specificity between the cyanogen bromide and potassium ferricyanide reactions is disclosed when heteropyrithiamine is oxidized, as shown in Table X-that is, while a green-blue fluorescence is produced by the latter, nonfluorescence is produced by the former reaction. This fact may be utilized for differential determination of thiamine and heteropyrithiamine ~ ~ - hthey r n are both present in one solution.
Thiochrome Yield, % C S B r react. &Fe(CS)6 react. 100 71 100 37 100 100 100 100 100 96 92 68 26 0
19 13 10 0 0 0 0 0 0 0
Table X .
Specificity of Reaction ~ i Concn., ~/bIl.
Substance Thiamine chloride Cocarboxylase Thiothiamine Heteropyrithiamine Nicotinamide Sicotinic acid lVl-Methylnicotinamide
0.2 0.2 0.2 0.2 2.0
0.2 0.2 2.0 0.2
Pyridoxal
Table IX. Sample Crine Polished rice Beef
Percentage Recovery of Added Thiamine Thiamine in sample,
Thiamine .idded,
Y %
Y
Y
%
15 120
2 2
35
2
1.90 1.96 1.88
95 98 94
-
82.0 0.0
31 . O R 1.0 0.0 0.0 0.0 2.0 0.0
81.0 0.0 9.5 17.0 0.0 0.0
?.?
9.J
0.0
a When oxidation is carried out a t a low temperature, very little thiochrome is produced.
Thiamine Recovered
The cyanogen bromide reaction proceeded undisturbed in spite of the presence of 30 mg. of cysteine hydrochloride in the solution, while the potassium ferricyanide reaction >vas greatly hindered. Recovery of Added Thiamine. The recovery of thiamine added to various samples was always more than 94“,, as shown in Table IX. Specificity of Cyanogen Bromide Reaction. The following compounds were tested: cocarboxylase, thiothiamine [N-(2’methyl 4’ - aminopyrimidyl 5’) - methyl - 4 - methyl 5 phydroxyethylthiazole-(2)-thione], heteropyrithiamine [3-(2’methyl-4’-aminopyrimidyl-5’)-methylpyridinium], nicotinamide, nicotinic acid, SI-methylnicotinamide chloride, and pyridoxal, in 5 ml. of potassium chloride-hydrochloric acid solution. The results are shoan in Table Y.
-
Galvanometer ~ ~ l Readings CNBr KaFe(CS)o react. react.
- -
ANALYTICAL RESULTS
The thiamine content of various biological materials, analyzed by the cyanogen bromide and alkali reactions, is presented in Table XI. DISCUSSION
In the common potassium ferricyanide oxidation method it is difficult to find the condition for obtaining the maximal yield of thiochrome from thiamine. S o t only does too little potassium ferricyanide give a low thiochrome yield, but an excess of potassium ferricyanide diminishes the yield, and the range of potassium ferricyanide that gives masimum yield is narrow. Besides, in routine analyses of biological materials some unknown component tending to reduce potassium ferricyanide might contaminate the thiamine solution, making it more difficult to find the optimal condition for oxidation in the potassium ferricyanide reaction. I n the cyanogen bromide reaction, on the contrary, the amount of cyanogen bromide that gives a maximum thiochrome yield
Table XI.
Thiamine Content of Biological -Materials Thiamine Content,
Materials Polished rice 1 Polished rice 2 Wheat Spinach Beef Leaves of Japanese radish Japanese tea Urine Feces Human blood 1 Human blood 2
7 %
160 360 290 140 20 ; :2
22 400 12 10
Teeri ( 8 ) stated that in his “thiamine and cyanogen bromide reaction” thiamine produced a colored compound ~1hen it reacted with cyanogen bromide a t an elevated temperature. But it is clear that this reaction is not applicable to the determination of thiamine. Though nicotinamide produces a green-blue fluorescent compound with cyanogen bromide and alkali when the reaction is carried out in a neutral medium and a t an elevated temperature, as Scudi ( 7 ) reported, it has no effect whatever when the reaction is carried out in the potassium chloride-h>-drochloric acid solution and a t room temperature, as shown in Table X. ACKNOWLEDGMEYT
The authors wish to thank Unichi Miura of Kyoto University and Taizo Matsukax a of Takeda Pharmaceutical Industries, Ltd., for valuable advice in this investigation. LITERATURE CITED
(1) Fujiwara, M., J . Japan. Biochem. SOC., 21, 200 (1949). (2) Fujiwara, >I,, and Shimizu, H., ilx.4~.CHEM.,21, 1009 (1949). (3) Hennessy, D. J., ISD. EX. CIIEM.,ANAL.ED.,13, 216 (1941). (4) Hennessy, D. J., and Cerecedo, L. R., J . Am. Chem. SOC.,61, 179
(1939).
( 5 ) Jansen, B. C. P., Rec. t r a . chim., 55, 1046 (1936). (6) Matsukawa, T., Report of Special Committee on Vitamin B investigation, Scientific Council of Japan, Vol. 29, p. 24, 1949. (7) Scudi, J. V., Science, 103, 567 (1946).
(8) Teeri, E., J . B i d . Chem., 173,503 (1948). RECEIVED for review June
24, 1949. Accepted January 8, 1953.
