A Rapid Circulating Dialyzer A. R . TAYLOR, A. K. PARPART,
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R. BALLENTINE, Princeton University, Princeton, N. J.
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sis in the absence of oxygen if necessary. The gas pump is so constructed as to reduce to a minimum the possibility of surface denaturation. The solution to be dialyzed may be introduced and removed with great ease and very little loss. Samples may be removed for analysis a t any time during the dialysis. The apparatus may be made from glassware available in any laboratory, and a t slight expense. The details of construction and the dimensions of the dialyzer are shown in Figure 2.
REEING protein solutions or suspensions from salts is frequently a time-consuming operation, since provision is not always made for adequate stirring. Although a number of types of dialyzers have been discussed in the literature (I-4), none has all the advantages of the double continuousflow apparatus described here. A method for the rapid dialysis of protein solutions has been devised which combines the following advantages : Small quantities (25 to 150 cc.) of protein solution may be 98 per cent freed of concentrated salts in 9 hours (Figure 1). This is approximately four times faster than a recently described technique ( 2 ) . The method is also adaptable for larger quantities. The construction of the dialysis apparatus permits the use of concentrated salt solutions, without danger of changing the selectively permeable character of the dialyzing membrane either by stretching or rupturing i t through osmotic effects. The present technique does lead t o a slight dilution of the fluid being dialyzed, which in the authors’ experience has not been greater than 25 per cent even when half saturated ammonium sulfate is used. The entire dialysis apparatus can be put into a n ordinary electric refrigerator or other thermostat, and the dialysis carried out at a low or constant temperature. The washing fluid is supplied b y a reservoir outside the refrigerator. T h e thick rubber gasket on the door of the refrigerator permits the use of slightly flattened metal tubing in order to bring fluids in or out. The dialysis membrane (19-mm. cellophane tubing used in authors’ experiments) may be used repeatedly for new samples of the same protein solution. One membrane has been in continuous use for 7 weeks without any sign of leaks or deterioration. The solution to be dialyzed is continuously circulated by a gas pump mechanism It is thus possible to perform the dialy-
The fluid to be dialyzed is introduced through the opening of reservoir A . It then passes by means of a rubber connection down to the 5-mm. opening, B , from which it flows between the cellophane membrane, C, and tube D, which is closed off a t both ends and fits snugly inside the cellophane tube. The solution is thus exposed for dialysis in a thin cylindrical layer, about 1 mm. thick. This fluid can F then flow into tube F through the small (5-mm.) hole, E , and is kept circulating by means of nitrogen or M other gas admitted by tube H . Bulb G permits the gas to collect under the fluid in the upper arm of F until the pressure is sufficient to force this fluid over in one slug. This has proved better than continuous bubbling when protein-containing solutions are dialyzed, since it checks foaming and minimizes surface denaturation. The solution being L H dialyzed may be removed through Tthe side tube, J, which is closed by rubber tubing and a pinchcock. The outer jacket, K , of the dialyzer is sealed a t both ends by rubber stoppers, L. Either tap water or distilled water is admitted to this jacket through the side arm, M , from a reservoir which is set a t a height sufficient to give a rapid N flow of fluid without creating enough pressure to collapse the dialysis membrane. The outflow from the jacket occurs through tube N , which is of larger bore than the inflow tube. Numerous dialyses with solution containing 900 mg. per cent protein FIGURE2. DIALYSIS precipitated by one-quarter saturated ammonium sulfate have been APPARATUS found practically salt-free within 6 hours. To test the rate of loss of salt from the dialyzer, 100 cc. of a 50 per cent saturated solution of ammonium sulfate were put in and a t various times 0.5-cc. samples were removed by the side tube, J . The samples were analyzed for their ammonia nitrogen by the manometric micro-Kjeldahl method of Van Slyke (5). The results, plotted in Figure 1, show that 98 per cent of this salt was removed in 9 hours. The rate of outflow from the outer jacket was 75 cc. per minute during the first 3 hours and was cut to one third during the last portion of the dialysis.
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Literature Cited (1) (2) (3) (4) (5)
AMFIGURE1. DIALYSISOF 50 PER CENTSATURATED MONIUM SULFATE Per cent of ammonium sulfate remaining in dialyzer a t various times after start of experiment
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Aitken, J . Biol. Chem., 90, 161 (1931). Kunitz and Simms, J . Gen. Physiol., 11, 641 (1928). Northrup and Kunitz, Ibid., 9, 351 (1926). Schmidt, Biochem. Z., 225, 216 (1930). Van Slyke and Kugel, J . Biol. Chem., 102, 489 (1933)
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