Fractionation of Partially Hydrolyzed Dextran MORRIS ZIEF, GEORGE BRUNNER, AND JOSEPH METZENDORF J . T.Baker Chemical Co., Phillipsburg, N , J .
I
N ORDER to prepare clinical dextran of suitable molecular
weight distribution, an efficient fractionation procedure is required. The importance of fractionation cannot be overemphasized, because closely fractionated dextran of varying average molecular weight is of interest for improved types of clinical dextran ( I ) , hydrodextran (IO),and dextran sulfate (6),and the preparation of depot forms of various drugs (7, 8). This paper presents a simplified procedure for following solvent fractionation based on specific gravity measurements. The success of the method when applied to large scale production of dextran suggests that other polymeric materials can be fractionated similarly. HYDROLYSIS OF NATIVE DEXTRAN
Gronwall and Ingelman (6) amplified .the original work of Colin and Belval (3) on the effect of acid concentration and the temperature of hydrolysis on the time required to attain various stages of degradation. More recently Wolff and others (9) described hydrolytic conditions for producing optimum yields of clinical dextran and demonstrated that the hydrolysis could be represented as a zero or a firsborder reaction. Upon hydrolysis of dextran at pH 1.9 and 100" C. the relative viscosity reaches 4.0 within 1 hour. The rate of hydrolysis can then be moderated by adjusting the hydrolysis to pH 2.2 with alkali. When this technique is coupled with measurement of the viscosity of aliquots and immediate neutralization of the hydrolyzate to pH 6.0 to 6.9 when the desired viscosity is attained, neutral hydrolyzates with practically identical relative viscosities are attained. Triple fractionation of aliquots hydrolyzed for various periods of time and determination of molecular weights of these fraction's by light scattering in the laboratory indicated that the optimum relative viscosity of the neutral hydrolyzate is 2.5 a t 25" C. for a 5% dextran concentration. In replicate hydrolyses carried out as described above on a plant scale, the average relative viscosity of the neutral hydrolyzates was 2.5 f 0.05. Each hydrolyzate, therefore, represented a similar polymer distribution. The constancy of the molecular weight distribution was the basis of the subsequent fractionation procedure,
volume, the actual methanol content is closer t o 47%. A 2% error in the alcohol content seriously affects the molecular distribution and, in turn, the yield of clinical dextran. The methanol can be removed by boiling the solution of the dextran sirup, but complete removal by this method is neither practical nor necessary. An analytical method based on the specific gravity of dextran solutions was developed which eliminates errors due to residual alcohol and simplifies the problem of preparing clinical dextran. Six closely fractionated dextran samples were obtained by double fractionation of dextran hydrolyzates with methanol at 25' C. The properties of the fractions are shown in Table I. Five per cent solutions of these samples were prepared and stirred for 5 minutes with various volumes of methanol in a bath held at 25' & 0.1' C. The specific gravities of the resulting mixtures, after standing at 25" C. for 2 hours, are recorded in Table 11. At 35% methanol by volume the specific gravities of all samples are roughly equivalent.. This is to be expected, as all the fractions are completely soluble at this alcohol concentration. At the 45% methanol level, however, substantial changes have already developed. With increasiig molecular weight of the dextran fractions the specific gravities decrease. At 45% methanol concentration all of sample A is soluble; in the progression from B through F, more and more material of high molecular weight settles out as a sirup. Consequently, supernatant F represents a more dilute dextran solution than any other fraction. At 51% methanol by volume most of the dextran appears in the sirupy precipitate in every case, and all the supernatants represent dilute solutions of dextran. As the dextran concentration of the supernatant is extremely sensitive to changes in the molecular weight distribution of a particular fraction, specific gravity measurements offer a relatively simple analytical tool for controlling the fractonation of a polymer like dextran. At 45% methanol the variations from 0.9467 to 0.9376 suggests that isolated specific gravity values of an unknown sample
Table I.
FRACTIONATION OF DEGRADED DEXTRAN
Properties of Degraded Dextran after Two Fractionations
Intrinsic Viscosity Units, 250 0.13 B 0.16 C 0.20 D 0.20 E 0.23 F 0.28 (I Determined by light scattering.
Because investigations of the fractionation of hetero-disperse polymers have clearly shown that the efficiency of a fractionation is greater the more dilute the supernatant solution ( 4 ) ,dilute solutions of dextran have received considerable attention. I n the present work methanol was adopted as the solvent of choice for the precipitation of closely fractionated dextran from aqueous solution. In the fractionation of dextran from a neutral hydrolyzate with methanol the crude dextran precipitates as a sirup. After the sirup has been separated from the supernatant solution, it is dissolved in water and refractionated. The sirup precipitated from a 5% dextran solution in the range 44 to 51% methanol imbibes water and methanol from the supernatant solution; approximately 33% of the volume of sirup is methanol. Upon dissolution of the sirup in water, the bulk of the methanol remains in the final solution. If the alcohol content of the solution is disregarded and the final 5% dextran solution is adjusted for 45% methanol by
Sample A
c.
Weight-Average Molecular Weight4 25,000 39,000 60, oon 66,000 101,000 200,000
Table 11. Specific Gravities of Aqueous 5% Dextran-Methanol Mixtures (Specific gravity of supernatant, 25/25' C. 5% solutions of samples) MeOH,a VOl. Control % A B C D E F (Water) 35 0.9648 0.9648 0.9684 0.9654 0.9640 0,9516 45 0.9467 0.9461 0.9433 0.9409 0.9386 0.9376 0.9353 50 0.9275 .... . O.QZO9 0.9263 51 0.9250 0.9230 0.9201 0.9222 0.9246 Per cent methanol by volume was calculated without regard for volume contraction on mixing dextran solutions with methanol.
.
119
INDUSTRIAL AND ENGINEERING CHEMISTRY
120
::jo/
0
35
,
g
%METHANOL
Figure 1.
BY
2
VOLUME
Specific gravity method. Batch 142
Fractionation of 5% dextran solutions at 25' C.
i,
0 % 1.0-
v
w
2 , " 0 v1
0.0-
u z
,
I 40
35
-
Experimentally d e t e r m i n e d values plotted in Figure 3 illustrate that K increases linearly in the range 20 to 50y0 methanol. In the range 17.5 to 25% methanol dextran concentiations 005 i x r e obtained optically. For 30 40 50 this range a K value of 0.05 LT as % MeOH employed. The volume contraction-composition relationship of Figure 3* for the methanol-water system a t 5 % aqueous dextran25" C. has been reported (2). methanol mixtures From the concentration of ~ dextran in the Supernatant, the fraction of polymer in the aqueous phase of the supernatant wa8 calculated. The last column in Table I11 shows that at 50% methanol 2.12/5.00 or 42% of the original polymer remains in the supernatant. At 45% methanol 4.29/5.0 or 85.8% of the original polymer stays in solution. Fractionation between the ranges 45 to 50% methanol by volume, therefore, affords a 40% yield of crude clinical dextran. The quantities of crude dextran and polymer of l o i ~molecular weight in the hydrolyzate are approximately the same. As the efficiency of fractionation is greater in the lower than the high molecular weight range (4), this type of molecular weight distribution in the hydrolysate Tvas chosen for the preparation of a closely fractionated product. In the neutral hydrolyzate of a 5% dextran solution approximately 2% sodium chloride (based on the weight of dextran) id present. When crude dextran is precipitated from the hydrolyzate, K I
0.97-J
y,
Vol. 48, No. 1
2
Figure 2.
I
I
50
45
M E T H A N O L BY V O L U M E
Optical rotation method.
,
Batch 142
Fractionation of 5% dextran solution at 25' C. 1. Neutral hydrolyzate; 11. Crude dextran; III. Clinical dextran
Table 111. Fractionation of a Neutral Hydrolyzate at 25" C." Co,
cannot be correlated with methanol content. R i t h hydrolyzates containing a reproducible molecular weight distribution, however, it has been found that determination of the actual alcohol concentration is superfluous because the specific gravity is a reliable index of alcohol addition. The data shown in Table I11 were collected for a typical hydrolyzate. From accurately determined methanol concentrations, the relationship between specific gravity of the supernatant and methanol content was plotted in Figure 1. From 35 through 44% methanol, dextran was completely soluble; a t the 44% level the first sirupy precipitate settled out. In all such fractionations the specific gravity varies linearly with the percentage of methanol in the range of complete solubility of dextran. The break in the curve indicates precipitation of dextran. If the dextran concentration of the supernatant is plotted against the percentage of methanol, as in Figure 2, similar curves are obtained. Fractionation can be controlled by polarimetric or specific gravity measurements. The simplicity in adapting the Mohr-Westphal balance to plant control and the accuracy of the measurements preclude the use of a polarimeter. In the calculation of dextran concentrations of the aqueous dextran-methanol system at 25' C., the volume contraction on mixing must be taken into consideration. This contraction variea with the per cent methanol. Hence, the value for dextran concentration determined polarimetrically is high. A factor K must be subtracted from the calculated concentrations. In any waterdextran-methanol mixture, K can be calculated as followa: observed rotation in 1-dm. tube concentration of dextran in original aqueous aolution, CA gram/100 cc. X = per cent methanol by volume prior to mixing 199' = specific rotation of dextran = =
or K
= 0.502a
- C~(l.0- X/100)
Concentration of Dextran,
+ (m
Specific Supernatant I?$~ernatant I n aqueous 5 Cc. Water, - 0 . 0 5 ) component of RleOHa, Gravityc, Vol. 70 25/26' C. a, Degrees supernatant 3.35 3.26 35 0.9672 5.02 3.10 41 0.9565 5.12 3.02 3.00 2.92 42 0.9548 5.03 2.95 2.86 43 0.9526 5.02 2.75 44 0.9510 2.66 4.75 2.45 45 0.9473 2.36 4.29 2.05 46 0.9437 3.63 1.96 1.80 47 0.9405 3.21 1.70 1.55 48 0.9371 1.46 2.81 1.15 2.12 1.06 50 0.9314 Control 1,000 (mater) a Relative viscosity of hydrolyzate = 2.51 (25' C.): hydrolyzate contaius 5.05% dextran and 0.13% a s h (2.6% b y weight of dextran). b methanol prior t o mixing. C Determinations were carried out after solutions had been standing for a minimum of 1 hour i n bath.
5.02% 84IU Y t 15% N-CL 5.05% 8 4 E I Y + 2
%
N-CL
505%04 I E Y + L % NeCL
5.10%84
0.8 3 0+32
El
/
Y
5.07% 8 4 Z I Y
91
+ I Yo NwCL
0.920 ,9829 I
35
I
%
Figure 4.
I
43
40
I
50
MeOH
Effect 0f sodium chloride on fractionation of dextran
121
INDUSTRIAL AND ENGINEERING CHEMISTRY
January 1956
practically all of the sodium chloride remains behind in the supernatant. Upon refractionation of the crude polymer, a solution devoid of ash is therefore being treated with methanol. The effect of sodium chloride on the fractionation of dextran, therefore, was investigated. Figure 4 shows that 1%salt increased the specific gravity by 0.0005 unit. Concentrations of 2 and 4% salt increased the specific gravity by 0.0010 unit above the 1% salt curve; 15% salt raised the level 0.0020 unit above the 4% curve. However, it is more important to note that 1%higher methanol concentration was required to initiate precipitation.
weight distribution is found to be distinctly higher than that of the corresponding neutral hydrolyzate. Initiation of precipitation consequently begins a t lower methanol content. The steeper slope in the critical range 44 to 50% methanol is representative of a more closely fractionated material. The supernatant at 50% methanol now contains 0.44/5.00 X 2 X 100 or 17.6% of the crude dextran. Curve I11 for the double fractionated product parallels curve I1 closely. After stud of Table 11, Figure 1, and the previous literature (9), neutral &drolyzates and 5% crude dextran solutions were fractionated in the specific gravity range 0.9480 to 0.9301. Light scattering molecular weight data for such fractions indicated that the product was a closely fractionated material dangerously low for clinical injection (weight-average molecular weight 50,000; highest IO%, 90,000; lowest lo%, 25,000). Gradual. e1:vation of the specific gravity limits and correlation of these limits with the resulting molecular weights by light scattering evolved the following optimum values for 5% dextran solution: ( I ) neutral hydrolyzates, 0.9494-0.9328; (11) crude dextran, 0.9494-0.9322. LARGE SCALE FRACTIONATION
2
0.949
ul
0
I
I
20
40
GALLONS
MLTHAN OL
Methanol was added with agitation to a 5% dextran solution until the methanol content was approximately 40% by volume. As illustrated in Figure 5, the observed specific gravity of the solution a t 25" C. was plotted at 0 abscissa. Three small increments of methanol (12 gallons each) were added, aliquots were withdrawn after each addition, and a straight line was plotted from the recorded values. The quantity of methanol required was then determined by extrapolation to the designated end point, 0.9494. After addition of the required amount of alcohol with agitation and settling for 4 hours a t 25' C., the supernatant was decanted into another vessel. A similar graph was constructed for the precipitation of crude dextran; in this case methanol was added to approximately 48% by volume and the specific gravity of the supernatant was plotted at 0 abscissa. Extrapolation of the straight line plot again simplified the problem of correct alcohol addition. Similar procedures were repeated during subsequent fractionations.
I
60 ADDED
Figure 5. Large scale fractionation of neutral hydrolyzate-Batch 142 I
SPECIFIC G R A ~ I T Y 25/25:C.
EFFECT OF TEMPERATURE ON FRACTIONATION 3'5
a0
%
Figure 6.
45
;0
METHLNOL
Effect of temperature on fractionation of
5% dextran solutions-Batch 142 clinical
In normal practice hydrolyzates contain a maximum of 4% salt. The 15% salt curve (NaCl/dextran = 0.15) was determined because solutions for intravenous injection prepared from 6% clinical dextran in physiologic saline contain a similar sodium chloride-dextran ratio. At 15% salt content the increased ionic strength of the solution effectively inhibits the precipitation of dextran. At salt concentrations encountered in the fractionation of neutral hydrolyzates, the ionic strength of the solution exerts very little effect on the precipitation of dextran. The presence of eodium chloride, however, is highly desirable in order to aid the coagulation and settling of dextran precipitates. Consequently, 2% sodium chloride (on basis of dextran) was added to 5% aqueous solutions of crude dextran prior to normal fractionation. The bulk of the sodium chloride remained behind in the supernatant when the clinical dextran was separated.
-
In the three fractionations described in Figure 1, curve I re resents the only dextran solution containing sodium chlori& Preliminary experiments showed that the salt content of I cannot be responsible for the precipitation at lower methanol concentration typified by curves I1 and 111. The shift in the precipitation limits is best clarified by examination of Figure 2. When the crude fraction of the neutral hydrolyzate that precipitates in the range 45 to 50% is iso!ated, 1.1/5.00 X 2 X 100 or 44% of the original dextran remains in solution. Approximately 12% of the material of higher molecular weight precipitating between 43 and 45% methanol is also removed. Upon resolution of the crude dextran in 5% aqueous solution, the average molecular
A problem encountered only during the separation of fractions of high molecular weight was "overshooting" the end point. Inspection of Figure 6 reveals that a t 25" C. the end point 0.9494is no longer on the linear portion of the curve. The end point obtained by extrapolation is 0.0010 unit above the true value. Consequently, the precipitate of high molecular weight fraction becomes significantly larger and the yield of useful dextran diminishes. This error was eliminated by measuring the specific gravity a t an elevated temperature. At 30" C. the curve is linear a t 43.3% methanol. It is relatively simple to employ the extrapolation method for the 30" C. curve in the laboratory to add the requisite amount of methanol to the vessel in the plant, agitate, and cool the plant fractionation to 25" C. A specific gravity value of 0.9470 determined a t equilibrium at 30" C. is equivalent to an extrapolated value of 0.9494 a t 25' C., as indicated by Figure 6. LITERATURE CITED
(1) Boyd, A. M., Fletcher, F., and Ratcliffe,A. H., Lancet 264, 59 (1953). (2) (3) (4)
Carr, C., and Riddick, J. A,, IND.ENG.CHEM.43, 692 (1951). Colin, H., and Belval, H., Compt. rend. 210, 517 (1940). Cragg, L. H., and Hammerschlag, H., Chem. Rem. 39, 79
(5)
Gronwall, A. J. T., and Ingelman, B. G. A,, U. S. Patent 2,437,518 (March 9, 1948). Ricketts, C. R., Biochem. J . 51, 129 (1952). Smirk, F. H., Lancet 263, 695 (1952). Thulin, K. E., Swed. Patent 121,752 (March 25, 1948). Wolff, I. A., Mehltretter, C. L., Mellies, R. L., Watson, P. R., Hofreiter, B. T., Patrick, P. L., and Rist, C. E., IND. ENQ.
(1946). (6) (7) (8) (9)
CHEM.46, 370 (1954). (10) Zief, M., and Stevens, J. R., J . Am. Chem. SOC. 74, 2126 (1952).
R ~ C E I V Xfor D review March 29, 1955.
ACOBPTED August 27, 1956.
