Improved Apparatus for Zone Electrophoresis ARTHUR M. CRESTFIELD and FRANK WORTHINGTON ALLEN D e p a r t m e n t of Physiological Chemistry, University of California School of M e d i c i n e , Berkeley, Calif.
so that no hydrostatic flow occurs. Leakage of current has been detected when the siphon is left in place and merely clamped off. Therefore the siphon is removed after the initial liquid level adj ustment. The filter paper is cut with a single edge razor blade on a sheet of glass to produce as uniform an edge as possible, since structural variatjons in the paper affect the flow of liquid due to electroosmosis. Marking of the paper with pencil, as is the usual practice in paper chromatography (Z)] is not permissible, as the graphite particles act as secondary electrodes and hence cause pH variation as well as oxidation and reduction. The paper is wet with electrolyte by drawing it rapidly through the liquid in one of the buffer vessels and withdrawing it against the end of the glass plate to aid drainage of excess liquid. Rubber gloves should be used to ensure that no contamination occurs. As rapidly as possible, the fully wet paper is laid on the support plate, starting a t the center and taking care to prevent the trapping of small air bubbles between the paper and the glass. Any bubbles may later develop into hot spots and hence should be removed by sliding the sheet around on the plate or by starting over again. It is best to have the paper excessively wet rather than too dry, as drainage is faster than inflow and bubbles are easier to eliminate. After the paper is centered on the plate, polyethylene cover sheets are placed on the ends of the paper so as to estend about
.4n apparatus for electrophoresis on paper which features improved cooling, drj ing, and ease of nlanipulation is described. .4n evaluation of the apparatus show-s that the mobilities of charged substances in filter paper, as distinguished from those in free solution, may be determined with caffeine as an uncharged reference substance, easily detected by ultraviolet radiation.
T
HE rediscovery of filter paper electrophoresis bv Wieland
and Fischer ( 1 6 ) 11 years after the apparentlv unnoticed description of the technique ofaKonig ( 5 , 8 ) has stimulated a rapid development of experimental methods and applications. Investigations in this laboratory in which the resolution of certain proteins, nucleic acids, and nucleotides have been studied have resulted in the drvrlopment of an improved apparatuq. APPARATUS
I n order to study the movement of a charged substance through filter paper for the purpose of separating it from other species or of comparing its rate of movement with that of a known substance for qualitative identification it is neressary to provide a constant voltage distribution in the paper, to control and measure the f l o ~of liquid in the paper, and to localize the zones without distortion or movement during detection procedures. Published methods (3-5, 8-10] 15) for zone electrophoresis show a wide latitude in the efficiency with which these requirements are met. The apparatus shown in isometric form in Figure 1 resembles that of Kunkel and Tiselius (9) and has certain features which permit control over the foregoing listed requirements. Thus, the apparatus is provided with a top plate which is raised ' / I C inch above the paper by a spacer of plastic. This serves the double purpose of permitting an equilibration period under applied voltage before addition of the samples through a sliding panel in the top plate and allowing the removal of the top plate after completion of migration without distortion or movement of the zones. The evaporation of water from the electrolyte and condensation onto the top plate during the flow of current is prevented by a sprinkler system which sprays cooling water to the bottom of the supporting plate. The efficiency of this means of cooling is such that 0.10 to 0.25 watt per sq. cm. of paper may be applied. The ends of the filter paper are not cooled by the sprinkling system. Polyethylene film is laid in place to prevent evaporation at these positions. Upon completion of the electrophoresis, rapid drying of the paper with a minimum movement of the zones is achieved by application of hot water through the sprinkler. In cases where ultraviolet absorbing substances are undrr investigation] observation without interruption of the migration may be carried out in the darkened room by means of ultraviolet radiation applied through the 96% silica glass sliding panel in the top plate.
Figure 1.
OPERATIOV
Isometric drawing of apparatus for zone electrophoresis
Scale. 1 cm. of drawing = 15.0 om. of completed apparatus 1. 96% silica glass viewing panel ground to 1-mm. thickness and polished 2. Lucite mounting box for panel with silicone grease seal 3. Lucite top plate with opening for panel mounting box 4. Alternative top plate with ports covered by greased polyethylene film 5 . Spacer strip either glued or held to top plate with grease 6. Polyethylene film to cover paper ends 7. Filter paper sheet 8. Glass plate cut from '/ls-inch picture frame glass 9. Power supply lead makes contact through mercury 10. Glass tube with platinum wire sealed in bottom t o mount a piece of platinum gauze. Tube is filled with mercury 11. Slotted rubber stopper 12. Sprinkler 13. Adjustable center support for glass plate 14. Sheet metal retainer to prevent force of sprinkler system from shifting glass plate 15. Ring atand 16. Buffer vessel 17. Flange for three-point suspension 18. Adjustable foot for leveling 19. Polyethylene film for collection of cooling water 20. Mounting board
It is essential for the proper performance of the cooling system that the bottom surface of the supporting glass plate be free from grease. This can be assured by cleaning with alcoholic potassium hydroxide The top surface of the supporting plate is coated with a hydrophobic silicone film by rubbing with a Desicote soaked tissue (Beckman Instruments, Inc.). The excess is removed nith benzene and any residual hydrochloric acid produced by hydrolysis of the chlorosilanes is rinsed off uith nater. The edges of the buffer vessels are coated with a bead of silicone stopcock grease and the glass plate placed between them with about '/ainch overhang on each end. The grease prevents water leakage into the vessels and also prevents current leakage out of them The edges of the buffer vessels are made level, using a spirits level and the adjustable screw feet on the vessels. The sag in the plate is prevented by means of the adjustable center support and the plate leveling completed by means of the adjustable foot on &hemounting table. A siphon is used to equalize the liquid level in the buffer vessels
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422
V O L U M E 27, NO. 3, M A R C H 1 9 5 5 1 inch int,o the cooled region of the paper, and then the top plate is added and clamped in place with ordinary screw clamps. I n order to allow equilibration of the liquid content of the paper, the sprinkler is turned on and the voltage to be used is applied for from 30 to 60 minutes. A sheet of plastic film serves to collect the sprinkler water and prevent excessive splashing as well as to divert the water down the center well drain. KOpart of the apparatus is touched while the voltage is applied. After the equilibration period, the voltage is turned off and the . from Kirk-type samples are added in from 1- to 5 ~ 1 portions capillary pipets which may be inserted through ports in the top plate. The starting position is marked by means of an indentation made in the paper nith a sharp point. At any time the progress of ultraviolet absorbing samples may be observed by darkening the room and illuminating the zones with ultraviolet, radiation from a mineral light (Ultraviolet Products, Inc.). A yellow filter held in front of the e?-e eliminates disturbing reflections of the visible output of the lamp. This procedure is used whenever materials of unknoivn mobility are applied and whenever positions before drying are to be noted. \Then the migration is completed, the power is turned off, the sprinkler system turned off, and the cold n-ater drained from it. The ends of the paper are torn off to prevent the inflow of liquid which would occur because of evaporation after the top plate is removed. The top plate is removed and t,he paper is further cut off 1 inch back from the ends of the supporting plate, since this portion of the paper is not dried as fast as the remainder and any inequality in drying rate causes zone movement. Finally, hot mater is turned into the sprinkler. The paper dries in about 10 minutes and is ready for detection of the zones. Cltraviolet-absorbing substances are detected either bj- the direct visual method of Holiday and Johnson ( 7 ) or by the ult’raviolet radiation contact printing method of Markham and Smith ( l a ) , modified by affording better contact between papers by means of a nylon net stretched over a hemicylindrical surface and by exposure with an 8-watt germidical lamp (General Electric Co.). Proteins have been detected by means of the aqueous bromophenol blue method of Iiunkel and Tiselius (9). EV 4 L U 4 T I O S
In order to evaluate the prrckion and performance of the apparatus in terms of the constancy of the distribution of voltage in the paper and the control and measurement of the flow of liquid in the paper, studies of each requisite were undertaken. The first requisite was tented by direct measurement of the ‘voltage distribution. For this purpose a vacuum tube voltmeter !vas equipped n-ith probes. The probes were inserted through ports a t various intervals in the top plate. Measurements that were taken a t various points immediately after placing the \vet paper in the apparatus and applying the voltage showed a nonuniform field strength throughout the paper. The nonuniformity of field strength gradually disappeared during equilibration until a period of 30 to 45 minutes, wherein the field strength became constant. The period of 30 to 45 minutes applies only to the use of buffers from pH 3.0 to 9.0. .4 longer period is required for buffers of pH greater than 9.0. Voltage measurements that are taken rithin 5 em. of the anode side of the paper show a reduction under those in the remainder of the paper. This reduction increases with time but can be partially eliminated if polyethylene film is laid in place to prevent evaporation a t the ends of the filter paper. Complete elimination of the occurrence has not been found necessary, as no more than 5 cni. of the total 40 cm. of paper develop the nonuniformity. Similar ohservations have been made by Slater and Kunkel (13). The precision with ivhich the flow of liquid in the paper could be controlled and measured was determined by studies of the mobility of caffeine and of picrate ion. The pK1 of caffeine is 0.15 (6); hence, a net charge of zero above pH 3 is expected. The mobility of caffeine, then, should represent the rate of electroosmosis through the paper. The pK of picric acid is 0.8 (6). At any p H value greater than 3, picrate ion has one net charge and the mobility should be independent of the pH. Possible interaction of picrate ions with buffer ions as well as variations in electroosmotic flow which are known to be dependent upon the species of cation (1) were eliminated as variable factors by the use of only sodium ions with a total ionic strength of 0.10.
423 Table I.
Mobilities of Picrate Ions a t Different pII Yalues ~
PH
~
Ion
f Picrate i Iona ~ T o anode
~Caffeineb T o cathode
Sq. em. per volt per second X 106 3.6 4.6 6.8 Phosphate 8 3 9.2 Borate 7 5 Carbonate 8 9 3 4 9.9 Referred t o site of application as zero. b Electroosmotic rate. C Referred t o caffeine as zero.
Picrate IonC T o anode
12 3
A solution which contained both picric acid and caffeine was prepared by the neutralization to pH 7.0 of a solution of picric acid saturated a t room temperatures. This solution was finally saturated with caffeine. One-microliter samples of the solution were applied to the paper a t various time intervals. ilfter migration the final positions of the zone centers a? detected by ultraviolet radiation were marked before drying. This latter precaution was taken because earlier R-ork in this laboratory as well as certain reports in the literature (11) had shown movement of zones upon drying. Comparison of zone positions noted before drying the paper with those after drying showed that the hot water sprinkling system as introduced in this apparatus effectively eliminates the movement of zones during drying. Mobilities were calculated from the slope of a graph of movement 2s. time and the measured voltage distribution. Calculated mobilities were corrected for the difference in viscosity between the buffer and distilled water (14). The paper shrinks 1 to 2% upon drying. No correction for the factor was applied, as it is both small and consistent. The mobility data are presented in Table I The mean mobility of picrate ion referred to caffeine as zero mobility in buffers which range from pH 3.6 to 9.9 is 11.6 X 10-5 eq cm. per volt per second in Whatman S o . 1 filter paper Kith a variation coefficient
(5X 100) of 4 3%.
In 17 studies of the migration of
picrate ions in seven different buffers with a range from pH 1 to pH 9.9 the average range from the mean movement a t each pH value vias f 3 6% for a migration time of 100 minutes a t 18 volts per cm. Caffeine is an easily detected substance which can be used as a reference for the estimation of the rate of electroosmotic f l o ~ of liquid through the paper. The precision obtained is adequate for studies of the effects of the variables of solutions upon the mobility of a given substance. The comparison of substances of similar mobilities, as in qualitative analyses, is best conducted on one sheet of paper LITERATURE CITED
(1) Abramson, H. A., hloyer. L. S., and Gorin, LI. H., “Electrophoresis of Proteins,” p. 243, Reinhold, Xew York, 1942. (2) Balston, J. X., and Talbot, B. E., “Chromatography,” H.
Reeve Angel and Co., London, 1952. (3) Consden, R., and Stanier, W. AI., Nature, 169, 783 (1952). (4) Durrum, E. L., J . Am. Chem. SOC.,72, 2943 (1950). (5) Grassmann, W., and Hannig, K., Z. physiol. Chem., 292, 32 (1953). (6) Hodgman, C. D., ed., “Handbook of Chemistry and Physics,” 7th ed., Chemical Rubber Publishing Co., Cleveland, 1949. (7) Holiday, E. R., and Johnson, E. A., Nature, 163, 250 (1949). (8) Konig, P., Actus e trabelhos Terceiro Congr. Sul-Ameracano Chzm., Rzo de Janeiro e Sdo Paulo, 2, 334 (1937). (9) Kunkel, H. G., and Tiselius, A, J . Gen. Physzol., 35, 89 (1981). (10) hIcDonald, H. J., J . Colloid S e t , 6 , 236 (1951). (11) McDonald, H. J., Lappe, E. P., Marbach, R. H., Spiteer, R. H., and Urbin, M. C., Clin. Chemist, 5 , 35 (1953). (12) Rlarkham, R., and Smith, J. D., Biochem. J . , 45, 294 (1949). (13) Slater, R. J., and Kunkel, H. G., J . L a b . C l m . Med., 41, 619 (1953). (14) Ulich, H., T r a n s . Faraday SOL,23,388 (1927). (15) Wieland, T., and Fischer, F., ivaturwissenscha~ten,35,29 (1948). RECEIVEDfor review September 24, 1954.
Accepted November 24, 1954.
