US 2884367 A
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KARLER ETAL 2,884,367 MEANS FOR CONTROLLING CURRENT DENSITY DURING 7 -April 28, 1959 CONTINUOUS PAPER ELECTROPHORESIS 2 Sheets-Sheet 1 Filed may 10, 1954 INVENTORS ARTHUR KARLER A Lemme F. VECKARP NEYb E I ATToR April 1959 A. KARLER ETAL 2,884,367
MEANS FOR CONTROLLING CURRENT DENSITY DURING CONTINUOUS PAPER ELECTROPHORESIS Filed May 10, 1954, 2 Sheets-Sheet 2 INVENTORS ARTHUR KARLER m Leonmv F. VECKARV J mi itmwwl ATTORNEYS United States Patent MEANS FOR CONTROLLING CURRENT DENSITY DUlgNG CONTINUOUS PAPER ELECTROPHO- Arthur Karler, El Cerrito, and Leonard F. Deckard, Berkeley, Calif; said Deckard assignor to said Karler Application May 10, 1954, Serial No. 428,528
2 Claims. Cl. 204-299 This invention relates generally to the art of continuous separation of flowing materials under the procedures referred to as electrophoresis and chromatography, but also known in the art broadly as electromigration, electrochromatography, ionophoresis or ionography, and partition chromatography or adsorption analysis. This invention relates specifically to continuous-flow hanging-- curtain electrophoresis.
The principal object of this invention is to provide improved apparatus for effecting electrophoretic separation.
The specific object of this invention is to provide an improved apparatus having a more controlled current density, having less electrode reaction products, and having a higher capacity than has heretofore been the experience in the art without the deleterious temperature effects of the prior art.
The art of electrophorwis is theoretically known and is discussed in such publications as Partition Chromatography of Amino Acids With Applied Voltage by Gotfred Haugaard and Thomas D. Kroner appearing at pages 21352137 of Journal of American Chemical Society for June 1948; An Apparatus for Continuous Electrophoretic Separation in Flowing Liquids by Harry Svensson and Inger Brattsten, appearing at pages 401-411 of Arkiv for Kemi for November 1949; Ein einfaches Verfahren zur kontinuierlichen Trennung von Stofigernischen auf Filterpapier durch Electrophorese by W. Grossmann and K. Hannig appearing in Volume 37, page 397, of Die Naturwissenschaften for 1950; Analysis by Electromigration Plus Chromatography by Harold H. Strain and James C. Sullivan, appearing at pages 816-823 of Analytical Chemistry for June 1951; Continuous Electrophoresis and Ionophoresis on Filter Paper by E. L. Durrum, appearing in volume 73 of Journal of the American Chemical Society at pages 4875-4880, October 1951; and Apparatus for Continuous Electrochromatography by Takuya R. Sato, William P. Norris and Harold Strain, appearing in volume 34 at pages 776-778 of Analytical Chemistry, May 1952.
With an understanding of this art, further objects and advantages of the improved apparatus will become apparent from this description, the appended claims, and the accompaning drawings.
Referring to the drawings,
Fig. 1 is a perspective view of one embodiment of this invention taken from the left front, with some of the parts broken away for ease of understanding;
Fig. 2 is a cross-sectional view of the apparatus illustrated in Fig. 1 taken on the line 2-2 of Fig. 1;
Fig. 3 is a cross-sectional view of the apparatus illustrated in Fig. 1 taken on the line 3-3 of Fig. 1;
Fig. 4 is a fragmentary, longitudinal sectional view taken on the line 4--4 of Fig. 3;
Fig. 5 is a fragmentary, longitudinal sectional view taken looking downwardly on the line 5-5 of Fig. 2; and
Fig. 6 is a perspective and fragmentary sectional view looking downwardly on the line 66 of Fig. 1.
The illustrative environmental feature of this invention includes a free-hanging curtain 1 made of loose fibrous material. An excellent substance is a good grade of heavy filter paper similar to ordinary white blotting paper.
In the illustrated embodiment of this invention, the overall dimensions of this curtain are approximately 19 /2 inches by 19 /2 inches.
This hanging curtain 1 is secured in place by bending the upper edge 2 (see Figs. 2 and 3) over a glass plate 3 and clamping it by means of a companion glass plate 4. This sandwich structure so formed comprises the glass plates 3 and 4 on either side of the upper edge 2 of the curtain. The two glass plates are similar in size and shape and in the illustrated embodiment are inch thick, 2 inches wide and 25 inches long. They are clamped together at either end by any suitable means as, for example, rubber bands (see Fig. 1).
The lower edge 6 of the curtain 1 is provided with a plurality of toothlike serrations 7 forming drip points for the collection of the substances which have been electrophoretically fractionated upon the curtain. In the illustrated embodiment, 31 of such drip points are illustrated.
A plurality of collection tubes 8 are provided for recovering the substances fractionated by the apparatus. These collection tubes 8 are located below the lower edge 6 of the curtain 1 and each one of the series of collection tubes 8 is held in aligned registry with a companion drip point '7 in any convenient fashion, such as the rack 9.
Means are provided for furnishing a background electrolyte within which the substances to be fractionated are carried. As illustrated, an electrolyte reservoir 10 is provided for supplying electrolyte 11 to the curtain.
Means are provided for feeding the substance to be fractionated to the hanging curtain. These means include a vessel 12 in which the substance is placed. The substance is fed to suitable tabs, such as one of a plurality of tabs 13 located in the upper portion of the hanging curtain 1, but below the upper edge 2 of the hanging curtain. Means 14 are supplied for transferring the substance from the vessel 12 to the tab 13. These means may be either a capillary tube, as illustrated, a paper wick or any other desired means.
Means are provided for applying an electrical field longitudinally across the curtain. These means include a negative electrode 15 secured to the right edge of the hanging curtain 1 and a positive electrode 16 located at the left edge of the hanging curtain 1. These electrodes are connected to a source of direct current, as by means of wires 17.
The entire apparatus is securely mounted in a cabinet 18 provided with a transparent front face 19 for viewing the operation of the apparatus.
The foregoing environmental aspects have been generally known in the art for some time. Applicants have found that their improvements hereinafter described; used either singly or in combination, greatly increase the efficiency and capacity of .the hanging curtain electrophoretic apparatus.
Electrolyte feed Applicants have learned that the operation of the apparatus is greatly improved if the source of electrolyte is isolated from the curtain. This restricts the current flow to the curtain itself and prevents irregularities in current density upon the curtain.
In the illustrative example, a secondary curtain 20 is provided for feeding the electrolyte from the reservoir 10 to the separating curtain 1. This curtain 20 may be made of the same material as the separating curtain 1 or of a different material and like the curtain 1, it is provided with a series of drip points 21. These drip points may be cut the same as or differently from the drip points of the curtain itself.
The upper edge of the curtain 20 is placed between two rectangularly shaped Lucite or glass plates 23 and 24. The entire assembly is associated with the reservoir in such a manner that the upper edge 22 of the hanging curtain clips down into the electrolyte 11 in the reservoir 10 and so that the drip points 21 are located above and spaced from the upper edge 2 of the hanging curtain 1. The electrolyte 11 is carried out of the reservoir 10 by means of capillary attraction and drips from the points 21 upon the upper edge 2 of the free hanging curtain 1.
In this connection, the reservoir 10 is preferably equipped with a liquid leveling device, not illustrated, serving to maintain the level of the electrolyte 11 in the reservoir 10 at a uniform height, thereby establishing a steady flow of the electrolyte to the curtain.
Field current barriers To aid in the establishment of a uniform field across the curtain and to minimize stray fields, current barriers are provided at the upper end of the hanging curtain 1. In the illustrative embodiment, these current barriers consist of a series of slots 25 extending from the upper edge 2 of the hanging curtain down to lateral coincidence with the tab 13. In the illustrative embodiment, these slots are /3 inch wide and 4 inches long. It will become apparent that the establishment of these barriers tends to maintain the effectiveness of the electrical field in that area of the hanging curtain lying below the tab points 13.
Electrode wash Means are provided for washing the electrodes and for preventing migration of the electrode material to the curtain. These means avoid contamination or interference due to or arising from reaction products which are produced at the electrodes and migrate out into the curtain of the prior art apparatus.
In the illustrative embodiment, the electrode 16 (Fig. 4) is secured to the left edge of the curtain 2 by means of two plastic strips 26-27 and a series of clamps 28. The plastic strips 26 and 27, together with the curtain 1 and the electrode 6 form a sandwich structure (Figs. 4 and 6) which is secured by the plurality of clamps 28. A glass rod 29 located interiorly from, and spaced from, the electrode 16 is also secured between the plastic strips 26-27 and the curtain (see Fig. 4). This forms a passage 30 extending downwardly from the top of the cur-. tain to the bottom of the curtain and separating the electrode proper from the fractionating field of the hanging curtain. A capillary tube 31 (Figs. 1 and 3) extends downwardly from the reservoir 10 into the top of the passage 30 and supplies the electrolyte wash to the electrodes.
The bulk of the electrolyte washes down the electrode channel between the electrode and the glass rod barrier and comes off at the edge drip points.
The electrode wash for the negative electrode located at the right-hand edge of the curtain is identical with that just described.
Cooling means Means are provided for cooling the atmosphere Within the cabinet 18 in which the hanging curtain 1 operates. In the illustrative example these cooling means comprise a large metal tank 32 suspended from the top of the cabinet 18 (see Figs. 1 and 2). Dry Ice and an organic solvent are placed within this metal tank and tend to prevent an undue raising of the temperature at which the curtain operates. It will become apparent that other means, such as refrigeration coils within the interior of the cabinet, may be substituted for the metal tank 32 forming the specific embodiment of this invention.
A blower 33 (Fig. 2) is provided for circulating the cooled air within the cabinet 18 and for maintaining uniform temperature conditions.
Curtain stabilizer Means are provided for stabilizing the curtain. These means comprise a pair of rubber bands 34. One end of one of these bands is fastened to the lowermost clamp 28 on the right side of the curtain 1 and the other end is fastened to a screw eye 35 upon the right interior wall of the cabinet 18 (see Fig. 1). The other band is similarly arranged at the left side of the apparatus. These bands lend support to the lower end of the curtain 1 taking some of the strain ofi of the upper end and pre* venting the entire curtain from wavering in response to air currents within the cabinet.
Operation of apparatus In operating the apparatus, a supply of electrolyte 11 is placed in the electrolyte reservoir 10 and the electrolyte is permitted to flow down until the curtain 1 is completely saturated and a condition of flow equilibrium is established.
A portion of the electrolyte is also permitted to flow down the passages 30 for washing the electrodes 15 and 16. The tank 32 is then filled with Dry Ice and organic solvent and the blower 33 is turned on. Then a suitable source of stabilized or constant potential direct current is applied to the electrodes 15 and 16. As soon as the temperature and current conditions have reached equilibrium, the equipment is ready for use.
The sample of material to be studied is placed in the vessel 12 and fed to one of the tabs 13. The exact tab 13 to be used depends upon the nature of the sample and the extent of separation desired. The sample collection tubes 8 are placed in their rack 9 below the tab points 7 on the curtain 1 and fractions are collected until the entire sample is fractionated. The fractions are now ready for further fractionation, identification and study by known physical, chemical, biological and medicinal techniques.
While we have herein disclosed certain specific structural features, it should be understood that our invention is capable of modification and changes in structural design compatible with the embodiments herein disclosed without departing from the spirit and scope of the appended claims.
Having thus described selected embodiments of our invention, what we claim as new and desire to secure Letters Patent for is:
1. In an electrophoretic separator including a vertically disposed curtain having upper and lower horizontal edges, and vertical side edges; two elongated electrodes 1 longitudinally extending along and in electrical Contact with each side edge of said curtain; transversely extending electrolytic feed means for supplying an electrolyte to the upper edge of said curtain; and sample receiving means located below said electrolytic feed means; the improvement which comprises a plurality of horizontally spaced elongated non-conducting portions extending from said upper edge of said curtain through an electrolyte feeding zone to horizontal alignment with said sample receiving means; and means for washing with electrolyte vertical strips of said curtain located adjacent the right and left side edges of said curtain and located inwardly from said electrodes.
2. In an electrophoretic separator including a vertically disposed curtain having upper and lower horizontal edges, and vertical side edges; two elongated electrodes longitudinally extending along and in electrical contact with each side edge of said curtain; transversely extending electrolytic feed means for supplying an electrolyte to the upper edge of said curtain; and sample receiving means located below said electrolytic feed means; the improvement which comprises a plurality of horizontally spaced elongated non-conducting portions extending from 5 6 said upper edge through the electrolyte feeding zone FOREIGN PATENTS to horizontal alignment with said sample receiving means. 845938 Germany 13, 52
References Cited in the file of this patent OTHER REFERENCES UNITED STATES PATENTS 5 Strain et aL: Analytical Chemistry, vol. 23, No. 2,017,049 Jones Oct. 15, 1935 6, June 1951, pp. 816 to 8 3. 2,555,487 Haugaard June 5, 1951 2,566,308 Brewer Sept. 4, 1951