1146
ANALYTICAL CHEMISTRY
tions suggested here. This low yield obviously was a result of extensive decomposition of the relatively acid-sensitive ketohexose since the hydrolyzate was very dark in color. I n conclusion, therefore, the concentration of dextran in solution can be determined by total acid hydrolysis, provided a correction is made for concurrent destruction of D-glucose. The method is not affected by differences in the structure and properties of the dextran. The total hydrolysis procedure, of course, is not a method for the specific determination of dextran in the presence of other polysaccharides-for example, starch gives the same yield of reducing power as dextran. I n the use of total acid hydrolysis as a step in the characterization of preparations thought to be dextrans, allowance must be made for the fact that an acid labile sugar such as fructose will be largely destroyed under the conditions of hydrolysis. Thus small amounts of combined fructose, present either as a levan contaminant or as units in a mixed polymer with D-glucose, may escape detection. T h e presence of larger amounts of fructose would be indicated b\darkening of the hydrolysis mixture and the detection of a t least traces of fructose in the hydrolyzate. ACKNOWLEDGxMENT
The authors wish to thank Carl S. Wise for assistance in the running of paper chromatograms and anthrone determinations and Allene Jeanes and Carl A. Kilham for samples of dextran and levan. LITERATURE CITED (1) Daker, W. D., and Stacey, hI., Biochem. J . (London), 32, 1946 (1938). (2) Dimler, R. J., Schaefer, W. C., Wise, C. S., and Rist, C . E., ANAL. CHEM., 24, 1411 (1952). (3) Forsyth, W. G. C . , and Webley, D. M.,Biochem. J . (London), 4 4 , 4 5 5 (1949).
Fowler, F. L., Buckland, I. K., Brauns, F., and Hibbert, H., Can. J . Research, B15, 486 (1937).
(4)
(5) Gronwall, A., and Ingelman, E., Acta PhysioL. Scand., 7, 97 (1944). (6) Ibid., 9, 1 (1945). (7) Hassid, W. Z., and Barker, H. A., J . Bid. Chem., 134, 163 (1940). (8) Hehre, E. J., Ibid., 1 6 3 , 2 2 1 (1946). (9) Hehre, E. J., and Sugg, J. Y . ,J . Ezptl. Med., 7 5 , 3 3 9 (1942). (10) Jeanes, Allene, Haynes, W. C., Wilham, C. A., Rankin, J . C. and Rist, C. E., Abstracts of Papers, p. 14A. 122nd Meeting of the . ~ M E R I C A NCHEMICAL SOCIETY, September 1952. (11) Jeanes, Allene, and Wilham, C. A., J . Am. Chem. Soc., 72, 2655 (1950). (12) Jeanes, Allene, Wilham, C. A , , and Miers, J. C., J . Biol. Chem., 176, 603 (1948). (13) Jeanes, Allene, Wilham, C. A., and LMiers, J. C., unpublished
research. (14) Jeanes, Allene, Wise, C. S., and Dimler, R . J., ANAL.CHEni., 23, 415 (1951). (15) Klevas, S., Saensk Kern. Tidskr., 5 6 , 2 6 2 (1944). (16) Kobayashi, T., and Tsukano, Y . , J . Agr. Chem. Soc. J a p a n , 25, 424 (1951-2). (17) Lampitt, L. H., Fuller, C. H. F., and Goldenberg, S . , J . Suc. Chem. I n d . (London). 66. 117 (1947). (IS) Lampitt, L. Fuller, C. H. F.; Goldenberg, X., and Vine. II., J . Sci. Food Agr., 1 , 371 (1950). (19) Peckham, G. T., Jr., and Engel, C. E., J . Assoc. O$lc. A g r Chemists, 36, 457 (1953). (20) Perquin, L. H. C., Antonie van Leeuwenhoek. J . hficrobiol.Srrol., 6, 227 (1939-40). 121) Pirt. S. J.. and Whelan. W. J.. J . Sei. Food A m . . 2 . 224 (1951). ( 2 2 ) Seifter, S., Dayton, S.,Novic, B., and Mintwyler, E., Arch. Biochem., 25, 191 (1950). (23) Somogyi, 11..J . Bid. Chem., 160,61 (1945). (24) Stacey, M., and Swift, G., J . Chem. Soc., 1948, 1555. (25) Stacey. AI,, and Youd, F. R., Biochem. J . (London), 32, 1913 (1938). (26) Sugg, J. Y., and Hehre, E. J., J . Immunol., 43, 119 (1942). 127) Swanson. M.A , . and Cori. C. F.. J . Bid. Chem.. 172. 797 11918,. (28) Wolfrom, 11.L., Lassettre, E. N., and O’Seill, 4 . N., J . Am. Chem. Soc., 73, 595 (1951).
g.,
RECEIVED for review February 27, 1954. Accepted April 26, 19.54. Presented before the Division of Carbohydrate Chemistry at the 125th Lleeting of the VERICA CAS CHEMICAL SOCIETY, Kansas City, \Io., March 1984.
Radioactive Tracer Assay for Vitamin Btn and Other Cobalamins In Complex Mixtures F. A. BACHER, A. E. BOLEY, and
C. E. SHONK
Merck & Co., lnc., Rahway, N. J. \-itamin BIZcan be assayed in complex mixtures ranging from fermentation products to vitamin capsules, by the purification and concentration of the vitamin. Other cobalamins can be determined after conversion to vitamin B I ~ .Radioactive vitamin BIJis used as a tracer to determine recovery through the various extractions necessary for purification; the amount of purified vitamin BIZis determined spectrophotometrically. A combination of purification operations can be selected to fit each type of sample. Samples containing 100 y of vitamin Blz at concentrations as low as 0.1 y per ml. can be assayed. In a series of difficult assays a standard deviation of *4.3% was found.
T
HE availability of vitamin BIZ (cyanocobalamin) labeled
with cobalt-60 ( 2 , 4 ) has led to the development of methods which permit the determination of cyanocobalamin in mixtures from which its quantitative isolation is impossible. I n view of the wide application of these tracer methods, this paper stresses useful techniques rather than detailed analytical procedures.
The methods of extraction and purification, when applied t o a variety of fermentation products and other mixtures, yield aqueous solutions sufficiently pure for spectrophotometric determination of cyanocobalamin. Additional identification tests can be applied, although the combination of extractions usually required for adequate purification appears t o have high specificity for cyanocobalamin. While purity and identity of both tracer and pioduct are essential t o a tracer assay, adequate characterization can be achieved without isolation of crystalline cyanocobalamin, as the most telling criteria-Le., absorption spectrum ~ i i dbenzyl alcohol-JTater distribution-are applied in solution. .Is it is impracticable to make a suitable tracer for each of the large group of cobalamins, these materials can best be assayed by conversion t o cyanocobalamin and subsequent assay, using the radioactive cyanocobalamin tracer. The term “readily convertible” 18 applied here t o substances which can be converted t o cyanocobalamin by the procedure recommended below. An assay for total cobalamins can be made which includes cyanocobalamin plus readily convertible substances, expressed as cyanocobalamin. The conditions necessary for converjion t o cyanocobalamin depend on the nature of the sample. Although it is
1147
V O L U M E 26, NO. 7, J U L Y 1 9 5 4 difficult t o evaluate the completeness or the efficienc). of conversion t o cyanocobalamin, the best conditions are those that result in the highest value for total cobalamins. Conditions for this conversion are recommended below; the probability of finding a method which will produce more cyanocobalamin from the kinds of samples examined t o date (short of incubation with Bl?producing microorganisms) appears t o he small. For determination of cyanocobalamin in a sample, the essential steps are addition and equilibration of a k n o x n amount of tracer, separation of cyanocobalamin from interfering suhqtances, and measurement of the amounts of cyanocohslaniin and of tracer. For determining tot,al cobalamins, a conv(1rsion step i p necessar>+ and tracer is usually introduced in the course of thie operation. The tracer is introduced as a n aqueous solution of purified cyanocobalamin containing radioactive cobalt. Purity of the tracer-i.e., absence of radioactivity except in the form of c?-anocobalamiri-is essential for an accurate assay and is normally assured by the precautions taken in the preparation of the tracer. The purity may also he tested by assaying a standard solution of vitamin B12. Equilibrat,ion of the cyanocobalamin tracer with the cyanocobalamin of the sample is simple for homogeneous solutions, but testing of various equilibration times may he required when adsorbents are present. To determine total cobalamins, the logical point of introduction of tracer is after completion of the conversion t o cyanocobalamin. In practice, all cobalamins, including tracer cyanocobalamin after its addition, are in equilibrium in a solution containing escess cyanide, Stability studies have shown negligible decomposition of cyanocobalamin under the conditions that are used between the introduction of the tracer and the first separation. The extraction and purification methods are described below 8.3 unit operations, each of nhich has been shown to he highly effective in separating certain kinds of impurities from cyanocohalamin solution. S o t all operations need be applied t o every sample. Although combinations of these methods are sufficient for a wide variety of samples, other operations may be suhrtitutrd if necessary for adequate purificat.ion or convenience. The amount of cyanocobalamin is measured on a purified aqueouc solut’ion. Absorbance ratios, determined at the time of the ppectrophotometric measurement, are usually sufficient to characterize the final extract. The criteria are as follow: AbPorption maxima appear near 361 and 548 mp and a minimum near 430 m p ; the ratio of the absorbance a t 361 mp t,o t,he absorbance at 515 mp falls between 3.0 and 3.5 (best value 3.24): and the ratio of the absorbance a t 548 t o the absorbance at, 430 nip falls bet!$-een 2.4 and 3.1. If the presence of a substance with absorption spectrum nearly identical with vitamin Bl? is suspected. countercurrent distribution between benzyl alcohol and water can be applied either for further purification or as an additional criterion. The distribution coefficient of vitamin B1.L a t 25“ C. i? 1.2 (lwterbenzyl alcohol). The recovery of radioactivity is determined by counting a portion of the purified aqueous solution and an appropriate
aliquot of the original tracer solution. The amount of cyanocobalamin in the original sample (or in the converted sample a t the time of tracer addition) is computed by dividing the total amount of cyanocobalamin in the final purified solution by the fraction of the original radioactivity present in the same solution. S o correction is necessary for the amount of tracer cyanocobalamin added, as in all of the 1% ork reported here the specific activity of the radioactive tracer has been great enough to permit an adequate counting rate with addition of a negligible quantity of cy;inocobalamin. UVIT OPERATIONS
d procedure for the assay of a complex mixture is compoPed of the unit operations found necessary or desirable t o accomplish purification and, in most cases, concentration. The combination of unit operations will vary with the nature of the material t o he assayed and with the sample size required to provide an adequate amount of vitamin Bl2 in the final extract. The minimum amount of total cobalamins required for an assay is about 100 y ; the minimum concentration easily handled is about 0.1 y per ml. T>.pical materials and assay procedures which have been found satisfactory are shown in Table I. Examples of assays of a fcrmentation broth and a fermentation broth concentrate are shown in Table 11. Table 11. -4ssays for Total Cobalamin i n a Fermentation Broth and a Fermentation Broth Concentrate Sample size Final extract volume Tracer recovered A361 A430
A 5%
b (cell length) Aaal/Asao drrolA4ao Distribution constant, waterbenzyl alcohol
Concentration of original sample
Fermentation Broth 49.5 ml. 0 . 7 ml. 31 8% 1 265 0.151
0.400 2 5cm. 3 16 2 64
...... 1.51 y/ml.
Oral Grade Solids 4 . 7 7 mg. 6 . 0 ml. 60 9% 0.548 0.070 0.180 1 0 cni. 3 04
2 57 1 3
54 8
y/tng.
Assay procedure (unit operations) 1 Conversion and addi- Conversion and addition of tracer tion of tracer 2 Zinc defecation Cresol-butanol extraction 3 Acid-rresol-butanol Resin column treatextraction ment 4 Resin column treatMeasurements ment 5 >Ieasiirernents
The unit operations are designated by titles that briefly describe the operation. For ready adaptation t o any procedure, the xmounts of the reagents are indicated in parentheses in terms of the volume of the starting material in each operation. .4s used below, the term “extraction” implies transfer of the vitamin Blz t o the extracting phase. “Kash” means that the vitamin B,,remains in the original phase, _ _ _ . ~ _ _ -_ _ impurities _ passing into the washing phase which is disTable I. Typical llaterials and Assay Procedures carded. Comments conccrnFermentation Feed Liquid Oral Grade ing the use and performance Material Broth Supplement Concentrate Solids Vitamin Capsule of the operations accompany Approx. conrn. 1 *,/nil. 20 Y/B. 15 Y/ml. 5 mg./e 5 -y/capsule the descriptions. Sample wt. ..... 2 5 g. .... 100 me. SO capsules Volume 500 nil. 500 ml. ‘73 mi. 2 3 nil. 500 ml. .-I stock tracer solution of Siieeested Iprocedures vitamin B12 is prepared by di1 Conversion and addiConversion and addition Preparation of aqueous suspention of tracer of tracer sions (Waring Blendor) luting a solution of radioactive 17 Zinc defecation Cresol-butanol-Zepliiran Addition and equilibration of vitamin Bl, (available at a extraction trarer level of 4 to 6 microcuries per 5 3 .4cid-cresol-butanol exResin column treatment Ficin treatment traction ml.) to the radioactivity 1rveI 4 Resin column treatMeasurements Cresol-butanol extraction desired, as determined by the ment vaunting equipment t o be used. I) Measurements Resin column treatment 6 Measurements A drop of cresol is added to the tracer solution asa preservative. . ___ -. ~
~
~~~~~~
1148 Addition and Equilibration of Tracer. For the determination of cyanocobalamin, a known volume of tracer solution is added to the sample solution (or solids slurried in water) prior to any operations that might destroy a portion of the vitamin B12and prior to any separations. If the sample is a homogeneous liquid, equilibration may be considered nearly instantaneous. If the sample contains materials which may adsorb cyanocobalamin, equilibration should be demonstrated-for example, it could be shown that the same “highest assay value” is obtained by extension of the time allowed for equilibration. Desorption methods, such as heating and addition of various ions, may be used to aid in more rapid equilibration, so long as they do not cause large losses of vitamin BIZ. Conversion and Addition of Tracer. A sample of known volume (or the slurry of a known weight of solid) is placed in an Erlenmeyer flask large enough to contain the sample in case of excessive foaming. Boiling beads are added, and sufficient sodium nitrite and potassium cyanide are added to make their respective concentrations 0.5 and 0.2Y0 (w./v.). The p H is adjusted to approximately 4, using dilute acetic acid. The flask is placed on a hot plate, in the hood, and boiled for 5 minutes; a defoaming agent (such as capryl alcohol or a silicone defoamer) is used when necessary. The flask is removed from the hot plate and a known amount of tracer solution added. After thorough mixing, the sample is allowed t o stand for 2 minutes. Formaldehyde solution, 3 i 7 , of the sample volume), is added and the sample is again mixed thoroughly and is ready for the next operation. Formaldehyde is used t o eliminate excess cyanide ions, thus making i t possible t o perform later operations outside of a hood. This conversion procedure is more easily applied than other procedures (and other conversion conditions) giving the same assay values. Zinc Defecation. The p H of the aqueous solution or suspension is adjusted to approximately 4. Zinc acetate dihydrate (10 grams for each 100 ml. of solution) is added; the mixture is stirred until the zinc acetate dissolves. The p H is then adjusted to about 8.5 with 5N sodium hydroxide, with stirring during addition of the base. The solids are removed by centrifugation or by filtration, using large amounts of filter aid (Super-Cel). The centrifuge cake or filter cake should be washed well with water. (In practice, the filtrate volume from a 500-ml. sample will be about 800 ml.) Defecation with zinc removes many materials which form emulsions and gelatinous precipitates with cresol. It also aids in the removal of solid material which is difficult to remove by simple filtration or centrifugation. Cresol-Butanol Extraction. An aqueous solution (1.0 volume) is extracted with cresol-carbon tetrachloride (0.1 volume), and the aqueous phase is discarded. The cresol-carbon tetrachloride solution is a mixture of equal volumes of cresol and carbon tetrachloride. Carbon tetrachloride (0.05 volume) is added to the cresol-carbon tetrachloride extract and the resulting solution is washed with water (0.1 volume). Butanol (0.08 volume) and carbon tetrachloride (0.08 volume) are added to the cresol solution, and the vitamin B12 is extracted with water (2 X 0.05 volume). Extractions involving total volumes of over 200 ml. are conveniently carried out either in separatory funnels or by decantation of the waste phase and final separation of the phases by centrifugation. Total volumes of less than 200 ml. are most easily handled in centrifuge bottles or tubes; either phase is removed with a hypodermic syringe equipped with a &inch needle. Some form of the cresol extraction is generally used as an initial extractive operation because it affords considerable purification and great reduction in volume, the latter immeasurably facilitating handling of t’he sample. Repetition of an extraction is of much less value in purification than is a series of different extractions. Several other extractions and modifications of the fundamental cresol-butanol extraction are described below. Acid-Cresol-Butanol Extraction. An aqueous solution (1.0 volume) is extracted with cresol-carbon tetrachloride (0.1 volume). The cresol solut,ion is washed with 5Ar sulfuric acid (0.05
ANALYTICAL CHEMISTRY volume). Butanol-carbon tetrachloride (0.1 volume) is added t o the cresol solution. The resulting solution is extracted with 5N sulfuric acid (2 X 0.02 volume). The acid solution is in turn extracted with cresol-carbon tetrachloride ( 2 X 0.005 volume). This combined cresol extract is washed first Rrith a near-saturated solution of dibasic sodium phosphate which is 0.001,V in potassium cyanide ( 2 X 0.005 volume) and then with water (0.005 volume). Carbon tetrachloride (0.01 volume) and butanolZephiran (a cationic surface active agent made by WinthropStearns, Inc.) (0.02) are added t o the cresol solution and the cyanocobalamin is extracted with water (2 x 0.002 volume). The butanol-Zephiran reagent is composed of 1 volume of Zephiran (12.87, solution) dissolved in 9 volumes of butanol. I n strongly acid solution the distribution characteristics (and absorption spectrum) of cyanocobalamin are changed. The phosphate-cyanide n-ashes are used t o ensure complete reversion to the norma1 form of cyanocobalamin after the acid extraction step As cyanocobalamin is not completely stable in 5 S sulfuric acid, this operation should be carried past the point of the phosphate washes within a time span of an hour. Zephiran holds in the organic phase many impurities that otherwise n ould be transferred t o the extracting water phase. Cresol-Butanol-Zephiran Extraction. An aqueous solution (1.0 volume) is extracted with cresol-carbon tetrachloride (0.2 volume). The cresol extract may be washed with 5N sulfuric acid (0.2 volume), followed by washes with a near-saturated solution of dibasic sodium phosphate which is 0.001N in potassium cyanide (2 X 0.1 volume) and with water (0.1 volume). The cresol solution is diluted with carbon tetrachloride (0.2 volume) and butanol-Zephiran (0.4 volume). The cyanocobalamin is extracted with water (2 X 0.1 volume). This extraction is useful in vorking with small samples (10 t o 40 ml.) where additional concentration is not necessary. Butanol or Butanol-Zephiran Extraction. An aqueous solution (1.0 volume) is washed v,-ith butanol or butanol-Zephiran (0.2 volume). Sodium sulfate (0.2 gram per ml.) is then added and butanol or butanol-Zephiran (4 X 0.2 volume) is used to extract. The combined butanol extracts are extracted with water (3 x 0.1 volume). Butanol is used rather than other organic solvents in which vitamin B,z is more soluble because the distribution of vitamin B,, between butanol and water greatly favors the water phase; hence extraction from butanol is very easy and permits considerable concentration of the vitamin BIZ. As an aqueous solution of vitamin BIZ may be washed with butanol with only a slight loss of vitamin B12, excellent purification is often afforded by the wash with butanol preliminary to salting the vitamin B1, into butanol. Dicyanide Complex-Butanol Extraction. An aqueous solution (1.0 volume) is washed with butanol (1.0 volume). Sodium sulfate (100 mg. per ml.) and potassium cyanide (6.5 mg. per ml.) are added and the p H is adjusted t o 8, if necessary. The solution is alloved t o stand 15 minutes, then extracted with butanol (3 X 0.3 volume). The butanol solution is then extracted with 0 . 1 S acetic acid (3 X 0.2 volume). The dicyanide (purple) complex, formed in this operation, is more easily salted into butanol than is cyanocobalamin. Dicyanide Complex-Zephiran Extraction. The dicyanide complex is formed in aqueous solution (1.0 volume) by making the solution 0.lN in potassium cyanide, adjusting the p H to about 8, and allowing to stand for 15 minutes. Butanol-Zephiran (2 x 0.3 volume) is used for extraction. The butanol solution is extracted vith 0.1S acetic acid (2 X 0.1 volume). I n the presence of Zephiran the dicyanide complex is easily extracted into butanol. When the p H is loivered, the dicyanide complex reverts to cyanocobalamin. Butanol-Zephiran Wash. Butanol-Zephiran is used to wash an aqueous solution. Acid-Butanol Wash. An aqueous solution is adjusted to a pH of 2 t o 4 and washed with butanol. These washes are typical of many that may be used profitably to remove impurities.
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.) T o 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 (1) 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, 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
