V O L U M E 2 6 , NO. 7, J U L Y 1 9 5 4 Resin Column Treatment. The Amberlite resins I R 120 and IRA 400 may be used in the manner described by Marsh and Kuzel(3). For use with a tracer, however, certain modifications of their procedures may be made. The resin column is composed of a 10-cc. bed of Amberlite I R 120 overlaid with a 10-cc. bed of Amberlite IRA 400. The columns should be flushed with 10 to 15 ml. of water immediately before use. After use, the resins are discarded. An aqueous solution (5 to 10 ml.) is passed through the resin column, a t a flow rate of about 1 ml. per minute. The eluate is collected in a graduated tube; collecting tubes are changed when the red color of cyanocobalamin first appears in the eluate. Collection is stopped when the color becomes faint or disappears in the eluate. (.4pproximately 9 ml. of eluate will be collected before color appears; this portion may be discarded. Most of the vitamin BlQ will come through in the next 7 to 10 ml.) To the eluate is added 0.5 ml. of 4% acetic acid-0.0004 N potassium cyanide solution. The pH is measured and, if not in the range 4 to 8, is adjusted with dilute acetic acid or ammonia water. The use of the resin column affords an excellent final purification operation, after satisfactory concentration and partial purification. The pH of the eluate is controlled by the use of acetic acid rather than an inorganic buffer, so as t o avoid large residues in the preparation of the eluate for radioactivity determinations. The spectrum of cyanocobalamin is. not altered by the presence of acetic acid. Ficin Treatment. Aqueous suspensions of gelatin as encountered in the assay of capsules form a curdy precipitate when ex-
1149
tracted with cresol-carbon tetrachloride. These suspensions may be made amenable to extraction with cresol-carbon tetrachloride by adding a small amount of ficin (a proteolytic enzyme) to the suspension a t a near-neutral pH, and allowing 0.5 to 1 hour for the gelatin to be destroyed. Measurements. The quality of the extract and the amount of vitamin BIQ in the extract are determined by measuring the absorption a t 548, 430, and 361 mp. If the criteria listed above are not satisfied, a cresol-butanol-Zephiran extraction followed by a resin column treatment often gives the necessary additional purification. If the criteria are satisfied, radioactivity measurements are made and the assay values calculated. The standard deviation of a series of 18 duplicate fermentation broth assays waa *4.3%. Detailed procedures for application to specific products and a comparison of results of this method with other methods are being published elsewhere ( 1 ) . LITERATURE CITED
Chaiet, L., Miller, T., and Boley, A. E., J . Agr. Food Chem., in press. ( 2 ) Chaiet, L., Rosenblum, C., and Woodbury, D. T.. Science, 111, (1)
601 (1950).
( 3 ) Marsh, 121. hI., and Kurel, Ii. R., AN.~L.CHEM.,23, 1773 (1951). (4) Rosenblum, C., and Woodbury, D. T., Science, 113, 215 (1951). RECEIVED for review March 17, 1954. Accepted May 20, 1954.
Physicochemical Characterization of Clinical Dextran JOHN A. RIDDICK, EMORY E. TOOK, JR., ROBERT L. WIEMAN’,
and
ROBERT H. CUNDIFFZ
Commercial Solvents Corp., Terre Haute, Ind.
Acid-hydrolyzed dextran has proved an acceptable blood volume expander when fractionated to the proper molecular size. Because it is used intravenously, careful attention must be given to its chemical and physical characteristics. Some tests were made using standard analytical procedures; for others, the nature of dextran required the modification of existing methods or the development of new procedures. Analytical methods were studied for all of the tentative physical and chemical specifications for clinical dextran solution. A careful study was made of fractionation procedures for determining molecular weight distribution. Weight average molecular weights were determined by light scattering photometry and the results compared with those obtained hy viscometry. Both fractional precipitation with methanol and light scattering photometry were adapted for routine control. Fractional precipitation, for characterizing the end fractions of clinical dextran, can be run routinely if temperature and methanol concentration are carefully controlled. Light scattering photometry has proved the most satisfactory means of determining the weight average molecular weight. A routine procedure has been developed which can be usd by any competent technician.
environment. Jeanes and coworkers (15)have shown that, when a particular dextran is discussed, the producing organism and general cultural conditions must be defined. The dextran produced by an organism is generally referred to aa native dextran. The term “dextran” in this paper refers specifically to the material produced from sucrose by Leuconostoc mesenteroides, Yorthern Regional Research Laboratory Strain B-512. The native dextran is purified, subjected to acid hydrolysis, and fractionated to the proper molecular size with methanol for clinical use.
Table I. Tentative Physical and Chemical Specifications for Clinical Dextran Solution Dextran grams/100 ml. Sodium hhloride, gram/100 ml. Buffering capacity, ml. DH
mg./100 ml.
LOW 10% fraction Color
5.7-6.3 0.85-0.95
