Apuaaatus for Continuous ElectrochrQmatography B
TAKUYA R. SATO, WILLIAM P. NORRIS, AND HAROLD H. STRAIN Argonne National Laboratory, Chicago, 111.
Eleetromigration plus chromatography serves for the continuous resolution of mixtures of ionized solutes. As the separability depends upon the size of the apparatus, large electroehmmatographir!cells have been designed, with special mechanical supports and a device for the continuous addition of the solution of the mixture. This apparatus has been utilized for separating various ions such as radioactive calcium and phosphate and silver and chromate. T h e paths of the ions in the cell were determined by radioautographsa n d by the use of speeifio reagents. Complete or absolute separations of oppositely charged ions were obtained.
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TRAIK and Sullivan have recently described a widely sppliceble analytical or separatmy method that is adaptable to continuous operation. In this method, which serves for the resolution of mixtures of ionized solutes, the principles of chromatographic separation by flow of solvent were combined with those of differential electricalmigration (5). As the efficiency of this continuous electrochromatographic method depends, in part, upon the dimensions of the apparatus, larger cells have now been tested for the continuous resolution of mixtures of various ions. These larger cells have been utilized for separating mixtures of cations and anions and mixtures of carrier-free radioactive ions, and have a180 been employed for the coutinuous, complete-separation of the cations. of metals from phosphate, a separation that is often incomplete by chemical precipitation methods (1 ). With the larger electrochromatograpilie cells, the flow of the electrolyte or wash liquid wm frequently uneven, especially in the region of the electrodes. Maintenance of uniform flow of solvent in the cells has required the construction of qecial equipment for their support. Improvements have also been made in t.he technique for the addition of the electrolyte and the mixture to the cells.
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vessels Figure 3). F o r t e support of the 24inch cell, a cheap, simple, and cmvenient apparatus was oonstructed of plywood. Resembling an by 30 inches. inches, this support s u D D O r t was built at inclined table about 30 bv an angle of about 45' wifh with the vertiial, vertical, thereby permitting the use of lead weights on wood cleats for compression of the cell. OPERATION OF CELLS
Addition of Electrolyte. For onemtion of the electroohromsto-
APPARATUS
Electromigration Cells. The cells for continuous flow of solution and solvent and for tranverse Ron- of elect.rical current were constructed of industrial filter paper (Grade 320, 0.1 inch thick, Estou-Dikeman Co., Mt. Holly Springs, Pa.; or Filpaco paper, No. 046,The Filter Paper Go., Chioitgo, Ill.), which was out into the appropriate shape and size. Some of the cells were oonstmcted with a small compartment and wick a t the uppw center ( 5 ) (Fiymre 1): others were made vithout this oomuartment
kPgure 1. I'aper with W i c k for Use in by 24 Inches Wide
Le11 aa Inches 1all
Silver ions (left) separated from ohmmats i o o a (right) i o 1 iM ammonis
With the supporting electrolyte flon-ing through the electroohromstographic cell, electrical potential from an electronic rectifier (Figure 3) was applied to the electrodes continuously during the operation of the cell. Potentials varied from 200 to 500 volts, and the current varied from 60 to 200 milliamperes, depending upon the electrolyte itself. Addition of Solute Mixture. In the 12-inch elertrochromatographic cell with a single compartment a t bhe top of the paper 776
V O L U M E 24,
No. 8,
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M A Y 1952
ions in the electricd field was much more rapid than the m i g r e tion of phosphate ions. The flow rate of the electrolyte was 150 ml. per hour and about 30 minutes were required for the flaw of wash liquid from the top to the bottom of the cell. The time
5.6 hours, and 1.4-hours were recluired"to decontaminate the filter paper of the cell). The 6nal volume of Ca46 effluent was 75 ml. and of Pa204 effluent was 85 ml. The recovered activities were 15.4pc. ror Ca45 and 7.8 p ~for . Pa?
In another experiment, the paths of calcium and phosphate ions in the 12-inch cell were ascertained by the radioautograph reproduced in Figure 2. The radioautograph itself was prepared by drying the paper after removal from the cell followed by exposure t o the photographic film (Kodak No-Screen x-ray safety film, 14 by 17 inches). Because very mmll amounts of the radioactive ions were detectable and because t.he region between the paths of the ions was free of radioactive species, a nearly absolute (100%) separation of the ions must have been obtained, Similar results were obtained when higher concentrations of radioactive calcium ions were present in the mixture. Analogous separations wore also effected when 0.1 M acetic acid was employed as the electrolyte. Addition of inactive carrier ions of caloium and phosphate to the radioactive ions had little effect upon the separations &s long as the total concentration remained below ahout 0.01 to 0.03 M . At higher concentrations, the zones became much wider, and the space between the zones decreased rapidly. Other cations such as sodium also separated rapidly from phosphate in the electrochromittographic cell ( 1 ) .
Figure 2.
Paper for Use in 12-Inch Square Cell
(Figure l ) , the solute mixture was added through a fine hypodermic needle (No. 21) or through a, glass capillary tube inserted into the paper. Some hypodermic needles liberated large amounts of cupric ions to ammoniacal solutions; therefore they could not be used when copper interfered with the separations. Uniform flow of the solution of the mixture of ions was achieved by the use of an apparatus designed by Snyder, Lawrence, and Finkle for the continuous injection of very small quantities of solution into the tail vein of a rat (5). The ouemtion of this m"~
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This apparatus deliverid about 0.003 ml. per minute.^ With t.he 24inoh cell, the solution of the mixture was siphoned ~
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of mixture as well a i upon %he concentration of this solution. The wider the wick, the wider the initial zone of the mixture and the poorer the separations. The higher the concentration of the solution, the poorer the seprLrations, and the narrower the wick required for the formation of the initial zone.
Figure 3. S u p p o r t for 12-Inch Eleetrochromatographic Cell w i t h Pressure Applied to Front Plate
SEPARATIONS
Twelve-Inch Cell. With 0.1 M lactic acid 8.8 the electrolyte, the smaration of trace amounts of calcium ions from DhosDhate ions proceeded rapidly and quantitatively.
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The separation was made with carrier-free radioactive isotopes obtained from Oak Ridge National Laboratory. One milliliter of mixture of 15.6 pc. of C a W L and 7.9 pc. ofHaPa*Oldissolved in 1 N hydrochloric acid was continuously inieoted into the cell by the slow injector (5). With 8. potential of 300 volts and an electrical current of 150 milliamperes, the migration of calcium
Twenty-four-Inch Cell. When 1 M ammonia. was employed a8 the solvent and &s the electrolyte, silver chromate separated rapidly in the large electrochromtograp~c cell. After the cell had been operated for several hours, t h e paths of the silver and the chromate ions were located by spraying the moist paper with sym-dipheuylcmhride dissolved in 90% acetic
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ANALYTICAL CHEMISTRY
acid, silver yielding a black zone, chromate a violet-red zone. For spraying, the reagent solution was placed in an atomizer (De Vilbiss) operated with compressed gas at 5 to 10 pounds’ pressure. A typical separation is shown in Figure 1. In this separation, the silver chromate solution was 0.03 111. I t flowed into the cell at the rate of about 5 ml. per hour. The electrolyte flowed at about 270 ml. per hour, and about 1.7 hours were required for the flow of wash liquid from the top to the bottom of the cell. The electrical potential was 260 volts and the current was 55 ma. Safety Precautions. The high potential and large power output of the electronic rectifier (Figure 3) are a great hazard in the operation of the electrochromatographic cells. Care must be taken to avoid simultaneous contact with opposite sides of the moist cells or with the solutions flowing from the cell. Wires to the electrodes should be heavily insulated, especially where there is danger of contact with the cell support. DISCUSSIOK AND STRUCTURAL DETAIL
The separations illustrated by Figures 1 and 2 depend upon the delicate adjustment of many variable conditions such as electrical potential, the electrolyte and its concentration, the flow of the electrolyte, and the concentration and flow of the solution of the mixture. When all these conditions have been adjusted, continuous and complete separations of many ions may be effected. In conventional chromatography each zone of solute is contaminated by traces of less-absorbed solutes that form more rapidly migrating zones. In the continuous electrochromatographic method, by contrast, the ions follow separate paths through the cell; hence, the separation of ions by the continuous procedure should be absolute, as is indicated by the radioautograph in Figure 2. The separability of ions in the continuous electrochromatographic procedure is a function of their separability and sequence in one-way electromigration and in conventional chromatog-
raphy. As shown already, ions that separate to the same degree and in the same sequence by electromigration and by chromatography do not separate in the continuous method ( 5 ) . Ions that separate to the same degree but in reverse sequence should be readily separable in the continuous method. Scale drawings of the steel cell support and a wiring diagram and specifications of the electronic rectifier are presented in an unclassified document issued by the Atomic Energy Commission (Z), which contains a discussion of the effects of concentration of mixtures upon their separability, and includes a section on the selection of electrolytes. It also contains a discussion of the separability of mixtures by chemical precipitation, by conventional chromatography, and by continuous electrochromatography. independent methods that supplement one another. A part of this report concerns the nomenclature of separations based upon differential migration of solutes caused by flow of solvent and by flow of electrical current (4). ACKNOWLEDGMENT
The authors wish to thank Jane K. Glaser for preparing the photographs for this paper. LITERATURE CITED
(1) Sato, R.. and Norris, W. P., Quarterly Report, Division of
Biological and Medical Research, Argonne National Laboratory, ed. by A. AI. Brues, ANL-4531, 153-5 (August-October 1950)
(2) Sato, T. R., Norris, W. P., and Strain, H. H., Office of Technical Services, Department of Commerce, Washington, D. C., C. S. Atomic Energy Commission, Document ANL-4724 (1951). (3) Snyder, R. H., Lawrence, B., and Finkle, R. D., U. S. Atomic Energy Commission, MDDC-270, 1-7 (1946). (4) Strain, H. H., ASAL.CHEM.,2 3 , 2 5 (1981); 2 4 , 5 0 , 3 5 6 (1952). ( 5 ) Strain, H. H., and Sullivan, J. C., Zbid., 2 3 , 8 1 6 (1951).
RECEIVED for review September 4 ,
1951. Accepted January 17, 1952.
Automatic Determination of Radioactivity on Filter Paper Chromatograms LOUIS B. ROCICLAND’, JOSE LIEBERJIAN, AND bI.-iX S. DUNS Chemical Laboratory, Linicersity of California, Los Angeles, Calif.
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IRECT counting of radioactivity from isotopically labeled compounds separated on filter paper chromatograms is increasing in use for metabolism and other studies (1,3-5,9-11). Recently, Muller and Wise (6) have developed an automatic recording bet,a-ray densitometer nith which the radioactivity of labeled organic compounds may be determined with increased precision and economy of time. The automatic recording Geiger-Muller counter and automatic sample changer described in this paper were designed to determine the radioactivity of isotopically labeled compounds separated by the authors’ (6-8) small scale filter paper chromatographic techniques. The background counting rate is approximately one third that attained by Muller and Wise and the precision appears to be higher, especially for low-activity samples. DESCRIPTION AND OPERATION OF APPARATUS
K i t h minor alterations in the sample changer circuit, Scaling Cnit Model 163 (Nuclear Instrument and Chemical C o p ) has been used satisfactorily. The automatic sample changer was constructed for use with 1 Present address, Fruit and Vegetable Chemistry Laboratory, U. 9. Department of Agriculture, Pasadena, Calif.
Tracerlab equlpment including an SC-1-4 Autoscaler, an SC-4 Eagle preset counter, an SC-9 manual sample changer containing a TGC-2 Geiger-Muller (1.86 mg. per sq. em.) tube, an SC-5A printing interval timer, and an SU-38 laboratory monitor as well as with a General Electric photoelectric continuous pen recorder (Model 8CE 1 DPIB-2, D’Arsonval 2.5-0-2.5 pa.). The sliding platform of the manual sample changer was replaced by the parts shoivn in Figure 1. The new platform, e, was designed to permit movement of a brass bar, c, in small incrementa on a predetermined schedule governed by the radioactivity of the sample and the preset count. The scheduled movement of the bar (with attached filter paper chromatogram) under the slit, g, and the Geiger tube, b, is regulated by the dimensions of the ratchet (paired n-ith the slit) and by electronic actuation of the device shown in Figure 2. Paired slits and ratchets are employed interchangeably to vary the increment counted during a cycle from to 1 inch. As indicated in Figure 3, the apparatus is operated continuously and automatically by means of a series of switches, relays, and minor modifications of the stated commercial units.
The movement of the sliding bar, c, is synchronized with the sample number indicated on the printing interval timer and printed on the paper tape after a count is completed. The power supply is shut off automatically by means of the microswitch, k,
