Chemical Method for Determining Vitamin B,, G. 0. RUDKIN, JR., AND R. J. TAYLOR Chas. Pjiser and Co., Znc., Brooklyn, N. Y .
The new chemical method for determining vitamin& was devised to replace the more costly and less accurate biological assay, which uses the accelerating effect of the vitamin on the growth of certain types of microorganisms. The chemical method of assay approaches the biological method in sensitivity and excels it with respect to speed and accuracy. It enables one to determine vitamin B12 in fermentation broths and crude concentrates with a reliability not hitherto achieved by other published methods of analysis. Vitamin B,?is an important growth factor widely distributed in minute quantities. A red metal-containing pigment, vitamin BI2 stimulates the formation of red corpuscles in the human bod) and plays an important role in human and animal nutrition. The search for new sources of vitamin BI? as well as studies relating to the best methods for evaluating the vitamin in human and animal nutrition will be greatly accelerated by this new chemical analysis.
Apparatus. Cary recording spectrophotometer. Any spectrophotometer giving good resolution may be used. Separatory funnels, 500-ml. Centrifuge tubes Transfer pipets In a well ventilated hood solid sodium cyanide is added to a sample of the unknown containing approximately 200 micrograms of vitamin B12,a t a concentration of not less than 1 microgram per ml., so that the final concentration of cyanide is 1%. The sample is stirred to dissolve the sodium cyanide and the p H is adjusted to 9.5 to 10.0 with 10% sodium hydroxide solution if necessary. This is allowed to stand for 5 hours a t room temperature for complete conversion of the vitamin Blz variants to the dicyanide complex. Solid sodium sulfate (20y0 w./v.) is added and dissolved. The p H is further adjusted with sodium hydroxide to p H 11.0 to 11.5 and the aqueous solution is extracted three times with onetenth volumes of benzyl alcohol.
P
UBLISHED methods for the chemical determination of vitamin BIZ include the colorimetric determination of hydrogen cyanide liberated from vitamin Blz by photolysis ( 2 ) and the determination of hydrolysis products such as 5,6dimethylbenzimidazole ( 1 ) and a red pigment ( 5 ) formed by acid treatment of vitamin BIZ. While capable of assaying vitamin B12 under favorable conditions, these methods have not been found suitable for the analysis of broth and crude fermentation extractives. The method described here is based on the difference between the visible spectrum of vitamin Blr and thc spectrum of the dicyanide complex formed in solutions containing excess cyanide ions ( 3 , 4). The absorption curves for crystalline vitamin Bl2 and its dicyanide complex are shown in Figure 1. The difference 54. in the two spectra is a maximum a t 582 mp with Utilizing this difference for the analysis of vitamin Blz automatically corrects for much of the interfering absorption encountered in crude preparations. However, it is still impracticable to determine vitamin Biz directly in fermentation broths because of the low level of vitamin BIZ activity and the high level of colored impurities even in the red region of the spectrum. In order to effect a concentration of activity and a partial purification, a highly selective solvent extraction of the vitamin BIZ dicyanide complex with benzyl alcohol is incorporated in the present method. The extraction eliminates more than 90% of the extraneous absorption in the red region of the spectrum and a t the same time effects about an eightfold concentration of vitamin BIZ, so that reliable readings on a spectrophotometer may be obtained. The effectiveness of the solvent extraction step is illustrated by the absorption curves shown in Figure 2. From the difference in curves 2 and 3 at 582 mp, the concentration of vitamin B12 can be readily calculated.
WAVE LENGTH IN MILLIMICRONS
Figure 1.
BIZ 1. Dicyanide complex, 81 micrograms per ml.; 1 % cyanide, pH 11.0 2. Vitamin BE, 81 micrograms per ml., pH 6.0
sodium
T o the combined benzyl alcohol extracts one-half volume of chloroform is added and the solvent phase is extracted three times with one-tenth volumes of Jvater. The aqueous phases are made up to 25 ml. To a 10-ml. aliquot of the water extract, 2 inl. of a 10% sodium cyanide solution is added. To another 10-ml. aliquot, 2 mI. of a 12.5’35 potassium dihydrogen phosphate solution is added to adjust the p H to 5 to 6. The optical density of each solution is measured a t 582 mF in a 2-cm. cell. The vitamin B12 assay is calculated from the difference, ( A E ) o b s d , in the optical densities at 582 mp. AEiTm = 54 for crystalline vitamin Bu. ( A E ) o b s d . x 6/5 x 25 x 1.03 Total vitamin BIZ in sample = 0.0054 X 2 (cm. cell) where 1.03 is extraction factor. ~
EXPERIRZEKTAL
Reagents. Anhydrous sodium sulfate Benzyl alcohol, reagent grade Chloroform Sodium cyanide, solid and 10% solution Potassium dihydrogen phosphate, 12.5% solution Sodium hydroxide
Absorption Spectra of Crystalline Vitamin
RESULTS
The method described was applied to solutions of vitamin varying in purity from broth to pure vitamin Bl:. As a further check on its accuracy, crystalline vitamin B12 was added to crude solutions and the recovery determined. Table I presents the data obtained. B12
1155
ANALYTICAL CHEMISTRY
1156 Table I.
1 2
3 4 5
Preparation Crystalline vitamin B1;,7?5 y (25 y/ml.) Crystalline vitamin Bir (1 y ( d . 1 Fermentation broth A 216 Y. Bir Fermentation broth B 775 y. Bi, Fermentation broth C
+ +
6 Carbon eluate A 775 Y Bin 7 Carbon eluate B 775 Y BIZ Carbon eluate 8 C 9 D 10 E 11 12 13 a
Recovery of Vitamin Biz
Solid concentrate A B
+ +
Jlicrobiological Assay,a L. leichmanzi, *,/Liter
...
Recovery,
%
97.5
756 y (1)
95.6
201 (10)
98.6
933 (60)
776 (10)
213
1470 ( 6 ) 1545 (6) -,/&ll.
1040 (2) 1220 (4) +,/lIl.
784
101
3 6 . 4 (6)
43
(3)
790
101
30 2 (6)
4 2 . 6 (3)
798
103
4 1 . 7 (6) 3 6 . 7 (6) 5 0 . 5 (6) -,/Gram
5 3 . 5 (2) 30 (4) 81 (4) y/Gram
.. .. ..
630 (6) 665 (6) 560 (6) Number of replicate determinations in
C
ReChemical covery of Assay," ddded Biz, Y ?/Liter
590 (3) 5 5 2 (3) 509 (4)
... ... . ..
tion coefficient ( K = 30 at 1 microgram per ml.) achieved in extracting the dicyanide complex from a 20% sodium sulfate solution into benzyl alcohol. If the mtraction is carried out a t pH 11.0 to 11.5, spectacular color improvement is achieved, apparently by elimination of yellow phenolic pigments. Figure 3 shows that the absorption curves obtained after solvent extraction of a crude concentrate compare favorably with the curves obtained from pure vitamin BIz (Figure 1 ) . Equally favorable distribution ( K = 30) was obtained for the re-extraction into water with the aid of chloroform. Based on these distributions and the volumes mentioned above, a calculated recovery of 97% is achieved over the solvent step. Pure vitamin Bizsolutions and crude concentrates enriched with knon-n quantities of vitamin BIZhave yielded recoveries that agree well with this figure.
... ... ...
parentheses.
DISCUSSION
The analytical method described here furnishes a rapid, reproducible, and accurate procedure for determining vitamin BIZ. Baaed on ten separate analyses of each of three samples, the standard deviation for this method is 5%. In these laboratories the standard deviation for the L. leichmannii microbiological method is 15%. .4n indication of the reliability of the chemical method is the efficient recovery of crystalline vitamin BIZ added to broths and concentrates.
WAVE LENGTH IN MILLIMICRONS
Figure 3. Absorption Spectra of Crude Vitamin Biz Concentrate 1. Dicyanide complex after benzyl alcohol extraction, pH 11.0 2. Vitamin BIZ after benzyl alcohol extraction, pH 6.0
After the solvent extraction purification step, both the vitamin BITand vitamin B12 dicyanide spectra are determined a t 582 mp. Lsing the Cary spectrophotometer, it was found that the difference betxeen the two spectra (Figure 1) was a maximum a t 582 mp, Although the peak of the dicyanide complex occurs a t 578 mp, there is less likelihood of error in the spectrophotometer The wave length of readings a t 582 mp, the maximum the maximum varies slightly lvith different spectrophotometers. Errors up to 10% may easily be introduced unless the instrument is calibrated against solutions of crystalline vitamin BIZ. *4 means of checking the effect of cyanide a t p H 11.0 on the spectrum of impurities in the vitamin BIZ concentrates is afforded by the fact that a t 530, 545, and 558 mp the absorption curves of vitamin BIZ and its dicyanide cross each other. It would be expected that any effect of pH or cyanide on the impurities would be manifested at these points, which are close enough to the 582 mp Tyave length to serve as a control. In the authors' experience negligible or no differences have been noticed.
WAVE LENGTH IN MILLIMICRONS
Figure 2.
Effect of Solvent Extraction on Absorption Spectra of Vitamin BIZ
1. Vitamin BIZbroth, 1040 micrograms per liter 2. Dicyanide complex after benzyl alcohol extraction, 8320 micrograms per liter, pH 11.0 3. Vitamin Bn after benzyl alcohol extraction, 8320 micrograms per liter, pH 6.0
The large excess of cyanide used in the method shifts the equilibrium almost quantitatively in favor of the dicyanide complex ( 3 , 4 ) ,as well as accounting for other cyanide complexing reactants which may compete for cyanide in crude vitamin Biz solutions. The reaction time of 5 hours is necessary for the conversion of all vitamin BIZvariants to vitamin BIZ. The vitamin Bla dicyanide complex is stable a t p H 9.5 to 11.5 for periods up to 24 hours when standing in diffuse light a t room temperature. In order to concentrate dilute solutions of vitamin BI, (1 microgram per ml.), advantage is taken of the favorable distribu-
ACKNOW'LEDGMENT
The authors wish to thank William Boyd for technical assistance. The biochemical assays were carried out under the direction of Roger Ilersey and the spectrographic data were determined under the direction of John Means. LITERATURE CITED
(1) Boxer, G. E., and Richards, J. C., Arch. Biochem., 29, 75-84 (1950).
(2) Ibid., 3 0 , 3 7 2 - 4 0 1 ( 1 9 5 0 ) . (3) Cooley, G., Ellis, B.,'Petrow, V.,Beaven, G. H., Holiday, E. R., and Johnson. E. A,. J . Pharm. Pharmacol.. 3 . 2 7 1 (1951). (4) Conn, J. B., Xorman, S. L., and Wartman, T. G., Sczence, 113, 658 (1951).
(5) Fantes, K. H., and Ireland, D. M., Biochem. J., 46, xxxiv (1950).
RECEIVED for review February 28, 1952. Accepted April 15, 1952. Presented before t h e New York Section, AMERICAN CHEMICAL SOCIETY,Xew York, N. Y..February 8, 1952.
