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FACTORS AFFECTING the PRODUCTION of DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES of a PLASMA VOLUME EXPANDER DISSERTATION Presente

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FACTORS AFFECTING the PRODUCTION of DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES of a PLASMA VOLUME EXPANDER DISSERTATION Presente FACTORS AFFECTING THE PRODUCTION OF DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES OF A PLASMA VOLUME EXPANDER DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University MOSTAFA KAMAL HAMDY, B. Sc., M.Sc. i' The Ohio State University 1953 Approved by: 1 FACTORS AFFECTING THE PRODUCTION OF DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES OF A PLASMA VOLUME EXPANDER INTRODUCTION During the second World War, it was impossible to supply the demand for blood plasma. This stimulated a great number of investigators to study the problem of blood expanders with the aim of developing a synthetic or naturally occurring sub­ stance for this use. In general, a good plasma extender must be of the pro­ per molecular size, homogeneous, non-toxic, non-antigenic, soluble in dilute salt solutions, and neutral in reaction. It must also be stable so that it can be used under a vari­ ety of geographical and climatic conditions. With the potential possibilities of widespread casual­ ties, because of the use of atomic weapons, civilian defense authorities advise that to be properly prepared, a stock pile of at least one pint of plasma for every person in metropoli­ tan areas is needed. Without this stock pile it is estimated that all survivors of such an attack would have to be bled to furnish enough blood fluids for the injured. In addition to the advantages derived from a stable material for intravenous use, it would be possible to eliminate the danger of infect- 91 that the rate of degradation is directly proportional to the intensity of the ultrasonic vibrations, as presented in table 3 0 . Molecular weight distribution. A large amount of high molecular weight dextran B - % 1 2 was subjected to ultrasonic vibrations, under controlled conditions, to reach a value of O.2I4.-O.26 intrinsic viscosity”. The degraded polymer was subjected to molecular weight homogeneity test, as formerly described, using two liters of 2 per cent aqueous solution. Five main fractions (U^-U^) were obtained at the following isopropyl alcohol concentrations: 32.5, 37*5* 1+2.and 55>*0 per cents. Table 31 presents the data for the per cent yield and the intrinsic viscosity of the fractions, indicat­ ing that 91+.28 per cent of the degraded dextran are in the range of the proper intrinsic viscosity of a plasma extender, and that 9 2 .2 9 per cent of the fractionated polymer has al­ most the same intrinsic viscosity of the original degraded material. Further fractionation was carried out on fractions U-j_ and Ug* to separate each of them to the subfractions a, b, c, etc., and the data for percentage yields of the sub­ fractions (based on the original degraded dextran) and their intrinsic viscosity are recorded in table 3 2 , from which the following conclusions may be drawn. This degradation was carried out in Battelle Memorial In­ stitute, using a Radio Sonorator Model RS-2 . 92 TABUS 30 Effect of calculated energy output (intensity) of the ultrasonic'''* vibrations on the degradation of dextran B-512 Total period ^ sp/ c of energy output irradiation J-l-30 watts 630 watts . 0 min. 2.92 2.92 30 " 1.66 1.65 60 " 1.11+ 1.05 90 " 0.91+ 0.85 120 " 0.87 0.65 150 ” 0.72 0.50 18 0 11 0.54 0.1+2 210 " 0.50 0.38 Energy output ■ voltage input x current input watts k.v. m. amps 93 TABLE 31 Fractionation of two liters of a 2 per cent aqueous solution of ultrasonic depolymerized dextran B-512* W W Alcohol Weight of fo Notation 4\ ml. fractions yield of t n g. fraction 481.46 27.71 69.27 ui 0.260 108.00 4.59 II.49 0.255 121}.. 30 0.250 3.14 7.87 U3 11+4.30 1 .4 6 3.66 \ 0.240 275.00 1.99 0.190 0.79 US This dextran has an intrinsic viscosity value of 0 . 24- 0 . 2 6 . Percentage yield based on the original weight of degraded dextran. Intrinsic viscosity measurements were made at 25 C t o.o5 . TABLE 32 The molecular weight distribution of ultrasonic degraded dextran B-5 1 2 Fraction Subfraction % IPA yield ft] u i a 32.5 38.36 0.280 b 35.0 114-.62 0.275 c 37.5 5.99 0.270 d i+2.5 1.83 0.230 e 55.0 1.66 0.200 V2 a 37.5 6.25 0.270 b ll-O.O 3.16 0 .18 0 c 55.0 0.99 0 .1 5 0 Percentage yield based on the original weight of dextran before fractiohation. 95 (1 ) Most of the fractions were precipitated in the first and second subfractions. (2 ) The intrinsic viscosity for the majority of the subfractions, is in the range of the desired molecular size. (3 ) A narrow range in molecular weight distribution is present in the ultrasonic degraded dextran. Comparison of Degradation Processes In order to provide a good comparison of the various methods for the degradation of high molecular weight dex­ tran to the desired molecular size, the same batch of dex­ tran B-512 was used in this study. A 6 per cent aqueous solution of this dextran was hy­ drolyzed using 0 .10N hydrochloric acid at 5 5 -6 0 C in a wa­ ter bath until an intrinsic viscosity value 0 .1 9-0 .2 0 was reached. Another 6 per cent aqueous solution from the same batch was depolymerized by ultrasonic vibrations until an intrinsic viscosity of O.2I4.-O.26 was reached. The depolymer­ ization in both methods was followed by viscosity measure­ ments. The results are presented in table 33 &n<3- in figure 11, which indicate that the fall in viscosity was gradual in the case of ultrasonic degradation, in contrast to that of the acid hydrolysis, which fell rapidly. The third sam­ ple was degraded through autolysis (extended incubation of the fermented media). The autolyzed dextran had an intrin- 96 TABLE 33 Comparison of acid and ultrasonic depolymerization of the high, molecular -weight dextran Time of hydrolysis Relative viscosity of the 6% solution in hours Acid Ultrasonic hydrolysis depolymerization 0 .0 2 3 .0 0 2 3 .0 0 0.5 10.77 1 8 .7 0 1 .0 7.^6 16.15 1.5 6.15 1 3 .8 0 2 .0 5.38 12.30 2.5 I4-. 60 11.60 3.0 Ij.. 18 IO.J4.O 3.5 3.77 10.07 k-o 3.07 9 .2 0 1J-.5 2.75 8.77 5.0 ------ 8 .2 3 5.5 7.69 6.0 7 -J+8 6.5 7.23 7.0 7.07 7.5 6.92 8.0 6 .7 6 8.5 6.23 9.0 Viscosity measurements were made at 25 C. Figure II Comparison, between add and ultrasonic degradation on a high molecular weight dextran. so/af/0/2^. % 6 v/scoJ/'fy fo e / i ' / 2 3 4 S 6 7 Q 9 Time in hours. 98 sic viscosity of 0.33 ^ 0.05. Two liters of 2 per cent aqueous solution of each, de­ graded polymer were subjected to molecular weight homogene­ ity tests, as previously described. Table 3k- depicts the yield of the fractions and their intrinsic viscosity for the three different degraded polymers. Further fractionation was carried on some of these fractions to separate them to subfractions, a, b, c, etc. Intrinsic viscosity measurements were made on all the subfractions, while light-scattering ex­ periments for molectilar weight determinations were performed on some of the fractions and their subfractions. The results of these measurements are recorded in tables 18, 32 and 35 from which the following conclusions can be attained: (1 ) Acid and autolysis degradation lead to a very high polydispersed polymer. (2 ) Ultrasonic depolymerization of dextran results in a product which is more uniform in respect to molecular weight and is in the range of the value desired for use as a plasma volume expander. Integral distribution curves. The integral distribu­ tion curve provides a simple and direct means for comparing the mo'lecular weight or intrinsic viscosity distributions produced by the different procedures of degradation under investigation. Using the dry weights and the intrinsic viscosities of the subfractions obtained from fractional 99 TABLE 34 Fractional precipitation of two liters of 2 per cent aqueous solution of autolyzed, acid hydrolyzed and ultrasonieally depolymerized dextran B-512 % Autolyzed Acid hydrolyzed Ultrasonieally IPA % % degraded yield m i yield HI Jo yield A 32.5 49.52 0.48 — 69.27 0. 260 35.0 --- — 25.35 0.30 --- --- 37.5 14.52 0.36 --- — 11.49 0.255 4 0 .0 --- — 21.85 0.19 --- --- 4 2 .5 6.82 0 .27 --- — 7.87 0.250 45. o --- — 23.62 0.16 --- --- 47.5 5.87 0.18 --- — 3.66 0.240 5o.o 3.97 0 .1 4 10.25 0.11 1.99 0.190 55.0 — 5.50 0.09 --- ——— Sample not recovered. TABLE 35 The molecular weight distribution of acid hydrolyzed dextran B-512 Fraction % Subr % Wt.-ave. vr IPA fraction yield r m Mol. wt. 32.5 a 7.50 0.38 205,000 G1JL 35.0 b 9.22 0.30 127,800 40.0 c 6.05 0.22 69,000 11-5 .0 d 1.12 0.19 55,500 30.0 e ----- — a 55.0 f 7.50 0.16 36,000 G 2 37.5 a 13.50 0.28 111,000 5.0.0 b 1.12 0.21 63,000 1+2.5 c 2.25 0.18 46,000 1+5 .
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