An improved electrolytic desalter

ANALYTICAL

BIOCHEMIS’MtY

13,

354-365

(1965)

SHORT COMMUNICATIONS

An

Improved

Electrolytic

Desalter’

The desalting of relatively large volumes of solutions containing “dialysable” proteins, peptides, and amino acids has been accomplished with the aid of a simple electrolytic cell. Blainey and Yardley (1) demonstrated that small volumes (3 ml) of plasma, urine, and cerebrospinal fluid could be desalted in an electrolytic cell equipped with ion-exchange membranes. The lossesof amino acids desalted at their isoelectric point were insignificant, and the resulting solutions could be applied directly to paper for chromatographic separation. The electrolytic cell described here is essentially identical to the cell described by Blainey and Yardley except for the increased dimensions and the use of mechanical stirring. Construction of the cell was facilitated by using a l-in. thick Plexiglas (Lucite) sheet from which three 6-in. squares were cut,. The center section of the cell was formed by cutting a S-in. square out of the 6-in. square, leaving a ?&in. thick frame. The FIG. 1. Cell assembly: (A) Anode compartment. (B) Sample compartment. (C) Cathode compartment. (D) Membrane support, cut from &,-in. Plexiglas (Lucite) sheet; 16 evenly spaced l-in. diameter holes cut in support. (E) Anion membrane, Permaplex A-20 anion-exchange membrane, purchased from Permutit, Ltd., London. (F) Cation membrane, Permaplex C-20 cation-exchange membrane, purchased from Permutit, Ltd., London. (G) Platinum electrodes, cemented at intervals to wall of compartment. (H) Electrical socket plug, color coded. (I) Polyethylene spigots. (J) Reciprocator. (K) Brass shaft, a&in. diameter. (L) Teflon bearing, %-in. diameter. (M) Brass coupling, s-in. diameter. (N) Plexiglas (Lucite) drive shaft, %-in. diameter. (0) Drive shaft support. (P) Stirring rods, +$-in. diameter Plexiglas (Lucite). (Q) Stirring motor assembly support. (R) Set screws. (S) Screw, ?&in. diameter. (T) Filling holes, %-in. diameter. (U) Slot holes for stirring rods. (V) Stirring motor assembly plate. (W) Fly wheel, 2-in. diameter. (X) Ball bearing, %-in. diameter, fastened % in. from center of fly wheel. (Y) Bodine motor, type KCl-23RB, 115 volts, 8.5 watts input, 56 rpm. (Z) Stainless-steel screws (12), %-in. diameter, 3%-in. length, and stainless-steel nuts (24). ‘This investigation Health of the Public

was supported Health Service

by grant funds from the (Grant No. AM-05851). 354

National

Institutes

of

SHORT

COMMCNICATIONS

355

356

SHORT

COMMUNICATIONS

outer sections of the cell were formed by cutting similar sections out, but leaving the l/,-in. thick outer wall of the cell intact. Maximum volumes of 400 ml of sample in the center cell compartment and 300 ml of dilute acid and base solutions in the outer sections were obtained. Platinum wire electrodes were fixed to the outer cell wall and connected to color coded plugs. Additional holes were drilled in the cell for the stirrer and for filling and emptying, and for the bolts used to assemble the cell. Membrane frames were prepared from ?&-in. thick Plexiglas (Lucite), and a like series of l-in. holes were cut in the frames to allow the solutions to have maximum exposure to the membrane. Membranes used in the cell were obtained from Permutit, Ltd., London, W4, and were as follows: Permaplex A-20 Anion Resin Membrane and Permaplex C-20 Cation Resin Membrane. The membranes were soaked in water overnight to allow stretching to the full 6-in. size. Assembly of the cell is illustrated in Fig. 1. The assembled cell is held secure by l/s-in. diameter stainless-steel screws and nuts as shown in

FIQ.

2. Assembled

cell.

357

SHORT COMMUNICATIONS

Fig. 2. A reciprocating stirrer was constructed from a Bodine motor, type KCl-23RB, and an eccentric ball-bearing drive. Once the cell is assembled, the three compartments must be kept filled with distilled water to prevent the membranes from deteriorating. The cell is prepared for use by washing the compartments several times with distilled water. The anode compartment is then filled with 0.2 N NaOH and the cathode compartment with 0.2 N H&O,. It is convenient to label the compartments with the solutions added to prevent errors in further use. The sample compartment is filled, or partially filled, with the sample. It is wise to adjust the amino acid, peptide, protein, etc. to its isoelectric point to minimize losses of the sample. Also, an approximate calculation of the salt concentration and the equivalents of acid and base in the electrode compartments will indicate whether the acid and base will need replenishment. In desalting solutions of bovine pancreatic ribonuclease in 0.01 M borate buffer, pH 8.0, containing 0.1 M NaCl, the direct-current power supply is adjusted to 100 volts output before connecting the cell. This setting should not be changed and, once the cell is connected, this voltage drops and the amperage rises to about 250 ma. After stirring the solution for about 3 hr, the voltage returns to nearly 100 volts and the amperage drops to less than one-tenth its original reading. The sample solution was found to be free of chloride ions by adding an aliquot to silver nitrate solution. The solutions are removed from the desalter via the polyethylene spigots and replaced with distilled water or fresh solutions. Only a few degrees of heating was observed in the solutions during desalting at room temperature. The desalting may be accomplished more rapidly in a cold room at a higher voltage. The exact best conditions depend upon the sample, the salt and its concentration, the surface area of the platinum electrodes, and the power supply. REFERENCE 1. BLAINEY,

J. D., AND YARDLEY, H. J., Nature

177,s

(1956). RAYMOND

Department of Biochemistry Emory University Atlanta, Georgia Received July 19, 1966

SHAPIRA