1776
ANALYTICAL CHEMISTRY
Table V. Comparison of Chemical Assay for Vitamin BIZwith Theoretical Concentration and with Microbiological Assays T- h.e.l n_.
______
Sample Vitamin
I.
Blt
Hours
3.
4.
Same as 3
5.
Same as 3 + ascorbic acid 50 mg./nil.
6.
Vitamin BIZcrystalline+ ascorbic acid 50 ma./ml.
33.7
33.7 33.7 33.7 33.7
33.7 14.1 32.0 29.5 32.2 30.9
30.8
1
33.7 33.7 33.7 33.2 33.,
33.2 13.7 2.4 29.0 9.5
35.7 14.1 0.9 30.0 9 0
27.2 27.2 27.2 27.2 27.2
26.9 8.3 6.1 20.0 7.9
None
15.0
14.9
15 5
None
15.0
14.6
14 3
33.7 33 7 33.7 33.7 33.7
29 0 22.1 8.6 3.0 0.8
Iione
14.1
14.7
14.6
Kone
14.1
14.4
15.6
ACKNOWLEDGMENT
14.1
14.3
15.8
T h e authors gratefully acknowledge the aid of J. T. Stephenson and his associates in provid-
1 1
Kone 100” c. 100° C. UVb
UV
Kone 100’ C. 100’ C. UV UV
hone 1000 c. 1000 c.
YV LV 7. 8.
Same as 6 Vitamin BIScrystalline folic acid 3.5 mg./ml. Vitamin BI? cryntallinef vitamin A 6000 U/ml., vitamin D 5000 U/ml., vitamin E 5 mg./ml., Equiv. of a-toropherolin?O% propylene glyrol-water
+
9.
[
b
y/hl!.
Microbiological Assaya, -f/Jll.
Chemical Assay, -,/All.
..
crystalline in w a t w None (controll 1000 c. 1000 C. YVb UV
Vitamin Bn crystalline t thiamine IICl 3 mg./ml.. riboflavin 3 mg./ml., pyridoxine HCI 5 mg./ml. Ca pantothenate 5 mg./lni., nicotinamide 75 mg./ml., Vitamin Bu crystalline+ thiamine HCI 5 mg./ml., rihoflavin 2 nig./ml., nicotinamide 75 mg./ml,, ?;a p a n t o t h m a t e 2.5 mg./ml.. iiyridoxin? HC1 5 mg./ml.,
2.
retical Concentration,
1
None
.. 4 1 4
1 4 1 4
I 4 1 4
1
4 1 4
14.1
15.6
27.9 27.7
35.4
33,2
29 . 3 ~. .
.
19.8 7.3 2.7 < 0.1
Jlodification of method of Skeggs el al. (6). Exposure t o ultraviolet radiation from Hanovia mercury arc lamp a t distance of 1 foot.
_combinations with other vitamins is within a level of f 2 % . Partially decomposed samples exhitiit Pomewhat less precision of recovery, partly because of the lower absorbancy level of the eluate and partly because of the tendency for the decomposition products of some samples to break up portions of the column and cause small “channeled” areas. The comparisons with microbiological assays indicate a good approximation of potency evaluation to the extent that the precision of microbiological results will permit. eucept a t the lower
Table VI.
tion are encountered which in turn limit the quantity of sample that can be applied to the column. A practical limiting concentration mag be considered to be approximately 5 micrograms per m1.-a 20-ml. aliquot of which when eluted to a volume of 50 ml. and measured in 10-em. cells will have a theoretical absorbancy of 0.132. Lower concentrations of vitamin B,, can, of course, be measured with less precision. The conditions for separation and determination of vitamin BI2 which have been described here in detail are in a sense a special case of a rather flexible procedure. T h e technique of separation of vitamin B12from mixtures in which the other components are ionic in their behavior toward ion euchangers can be modified to fit a large number of combinations and concentrations, prsvided the proper changes are made in resin types and amounts and in the amount of buffer used to control the pH of the eluate. The extension of the procedure to vitamin BIZ concentration ranges below those indicated will depend to a certain extent on the utilization of more sensitive light absorption-measuring devices o r the use of lowvolume absorption cells having a relatively long light path.
Reproducibility of Hecovery of Vitamin R I iti ~ Synthetic Vitamin Mixtures
Average Absorbancy Sample KO.of rlnsays (550 mp) Rsnge Crystalline vitamin Bin aolution (control) 8 0.173 0.172-0.17.5 Vitamin BIZ with vitamin R complex 8 0.183 0.181-0.181 Vitamin Biz with vitamin B complex and ascorbic acid 4 0 179 0 . 1 7 8 M . 180 Vitamin B I with ~ vitamins .4, D, and E in propylene glycol-water mixture 4 0.176 0.1744.177 Vitamin B I with ~ folic acid 4 0.178 0.177-0.170 Vitamin BIZ with ascorbic acid 4 0.181 0.180-0.182 Column components: 7 om. of 1:l mixture of 2,5Y0 ISnSOi-regenerated I R 120 and 10% NaOII-regenerated I R A 400 overlying 8 cm. of 10% NaOHregenerated I R A 400
vitamin Bl2 potency levels. T h e capacities of the 5- and 10-cm. cells used in absorption memurements are approximately 14 and 34 ml., respectively; for this remon, minimum volumes for elu-
ing the microbiological assay results on the samples tested; the suggestions of W. H. Rix and S. W, Arnett in regard to the preparation of the various vit,amin mixtures are also greatly appreciated. LITERATURE CITED
(1) Boxer, G. E., and Rickards. J. C., Arch. Biochem., 29, 75-84 (1950). ( 2 ) Boxer, G. E., and Rickards, J. C., presented a t AAAS meeting, Cleveland, Ohio, December 1950. ( 3 ) Brink, N. G., Wolf, D. E., Kaczka, E., Rickes, E. L., Koniussy, F. R., Wood, T. R., and Folkers, K.. J . Am. Chem. Soc.. 71, 1854-6 (1949). (4) Fantes, K . H., Ireland, D. M., and Green, N., Biochem. J.. Proc., 46, xxxiv-xxxv (May 1950). (5) Jackson, W. G., Whitfield, G. B., and De Vries, W. H., J . Ant. C h m . SOC.,73,337-41 (1951). (6) Skeggs, H. R., Huff, J. W., Wright, L. D., and Bosshardt, D. K., J . Bid. Chem., 176, 1459 (1948). (7) U. S.Pharmacopoeia XIV, pp. G61-2. (8) Wetzel, N. C., Fargo, W.C . , Smith, I. H., and Helikson, J., Science. 110, 651-3 (1949). RECEIIE D April 18, 1951. Presented before the Division of Analytical Chemistry a t t h e 119th Meeting of the AMERICAN CHEMICALSocI?.‘rI. Clevcland. Ohio.
Correction In the article on “Lithium Aluminum Hydride as a Qualitative Test Reagent for Aromatic Nitro Compounds” [Nelson, L. S., and Laskowski, D. E., AKAL.CHEM.,23, 1495 (1951)], the last column of Table I should have been labeled “Limit of Detection, G./MI. x 104.”
