Design and Use of a Refined Microelectropl BENJAMIN W. GRUNBAUM' Deporiment of Poihology, University o f California, School of Medicine, Son Francisco, Calif. PAUL L. KIRK School of Criminology, University o f California, Berkeley, Colif.
,With the improved precise microelectrophoresis apparatus described, eight 0.01- i o 0.1-wI.samples can b e subjected to electrophoresis simultaneously. Accurate positioning of precisely cut, multistrip papers produces an unusually high degree of reproducibility between patterns. The quality of the separations is tested and illustrated with ink dye, proteins of blood serum, and ferritin solution.
Z
ONE ELECTROPHORESIS carried out on
a porous medium, such as filter paper, has become a .highly useful technique (1, 4) which is inhexent.ly adapted to the analysis of samples in the range of 1 to 50 1.1. The method has also been adapted for application on single fibers (t?) or in thin liquid films adherent to a surface (6). The latter procedures allow application to microscopic samples comparable in size to the content of single biological cells. I n biology and clinical medicine there is an extensive need for a technique and equipment intermediate between the microscopic and conventional elecPresent address, Cancer Research Institute, University of California. Medical Center, San Francisco, Calif.
Figure 1.
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trophoresis. For example, fluids of the eye combine a very low protein content with small fluid volume which cannot be readily studied with available apparatus. I n criminalistics, there are also many uses for small-scale procedures for separating and identifying materials in samples containing much less than 1 y of the substance of interest. Previous equipment for continuous separation in this range (5) can be used only with a single sample, thus precluding direct comparison of multiple electrophoretic patterns. The apparatus described has several novel features: Very small samples can be processed; operation with single or multiple samples is clean-cut; also it has a new device for maintaining the paper fully suspended in air without sagging.
for the electrical fittings and paper holder. It is 23.5 cm. long, 9 em. wide, and 14 cm. high, including a detachable stand which supports the heavy base of the apparatus about 4.5 em. above the table top. On the base are two permanently fixed end vessels with three interior baffles in each to diminish the effects of electrode products. The electrodes
Microelectrophoresis assembly
ANALYTICAL CHEMISTRY
are sealed direc.., y..Lvus.. l.lyl..lvllu and are not removable. A leveling tube connecting the chambers is closed by a Teflon stopcock (Lab-crest, 80 G 2400, Fischer & Porter Co.). The paper holder, shown disassembled in Figure 2, is a completely new design. Its main member is an aluminum block (lower left, Figure 2) fitted on the hottom center with a setscrew, which is attached to a knife-edged crossbar on the top center. Two stainless steel crossbars are attached on the top, one a t each end, and machined to slip under the screwheads that hold them in place. The crossbars attach the second detachable member of the paper holder (middle, Figure 2). This consists of a stainless steel strip, 7 em. long and 2.85 cm. wide, bent upward on each end, carrying on the bent portions Teflon block supports through whicb are machined parallel grooves. The grooves are dimensioned and spaced so as to hold the paper snugly. The main strip of the detachable paper support is flexible so that it is bent upward in the center when the setscrew of the main block is advanced. This movement tilts the Teflon end blocks outward in equal arcs, which adjust the tension on the paper. This prevents sagging after the paper is wet. The Teflon blocks must he machined precisely to give uniform and reproducible tension on every part of the paper. Filter paper rectangles, 135 mm. long and 27 mm. wide, are prepared by removing seven parallel strips, 80 mm. long. This leaves eieht uarallel
Figure 2.
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Paper holder assembly
strips, 1.2 mm. wide and 80 mm. long. The paper design is not limited to the use of eight strips. By cutting the papers appropriately, the entire width of paper has been used, as well as two or four strips of correspondingly greater width. This allows a smaller number of larger samples to be studied than the maximum number of eight. A useful accessory, also illustrated (lower right, Figure 2) Thich serves to guide the sample application and support the strips while sample is applied, is constructed as follows: A piece of Plexiglas, 45 mm. long, 32 mm. wide, and 18 rnm. thick is machined so that it can slip under the paper strips on top of the support block. A suspended vertical portion at one end positions the sample guide at right angles t o the support block. The broad trough machined across the top leaves two narrow parallel ridges, one at each edge. These just clear the paper strips above them, but are close enough to give support when a pipet is applied t o the strip. The dimensions of the sample guide are chosen to allow either edge to be placed under any portion of the parallel paper strips. The entire apparatus is fitted with a tight cover that minimizes evaporation. It is designed to carry two prongs of a quadruple electrical attachment, the other two being carried by the base. Attachment to these prongs is possible onlv with the cover in n1n.e~. voltage may be applied to the unit by any conventional direct current power supply fitted with the proper Connecting attachment. That which is available with the duostat regulated power supply (Spinco Division, Beckman Instruments, Inc.) yields up to 500 volts and/or 50 ma.. 1
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TECHNIQUE AND APPLICATIONS
Sampling. The method of cutting the paper allows eight samples to be added successively and processed simultaneously under exactly the same conditions. Samples were applied by two different methods: Biological materials, such as blood and protein solutions, were added with calibrated fine capillary pipets. Sample volumes of 0.01 to 0.1 pl. were suitable. Larger samples flooded the paper and prevented sharp zone formation. With ink samples, a modification of the Spinco sample applicator was more efficient. Eight uniform samples could be applied simultaneously in an accurate straight line, even though the sample might dry before application. Ink samples were very suitable for studying the characteristics of the apparatus because of their known electrophoretic hphn.vior 1 0 . Protein' Samples, placed with capillary pipets, could be separated and the fractions located by staining. The block carrying the paper support was removed from the apparatus, and the support itself was removed. The paper could be processed completely without touching it or removing it from the support. A second paper support could be used for the next series of samples
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' l n e tnree sem Of typical multiple electrophoretic patterns (Figure 3) illustrate the degree of reproducibility that is obtained with identical samples on the same paper. Each paper contained a different black ink, eight samples of
Figure 3. Reproducibility of replicates of tt different block inks
which were applied to the strips of each paper. The bufferwas standard acetate mixture composed of 2 grams of sodium acetate and 0.5 ml. of glacial acetic acid per liter, diluted 10-fold with distilled water. Potentials applied were either 300 or 500 volts, corresponding to voltage drops in the strips on the order of 15 to 25 volts per em. Dye separations were obtained in 3 to 5 minutes with total currents on the order of 1 ma. Despite the rather drastic drop of potential, the strip-tostrip reproducibility was almost perfect; the eight different black inks tested were invariably identified without difficulty. Because originally it was intended to use this instrument to examine small samples of biological materials, some tests were made with pure proteins and blood serum, the latter being employed in eight duplicate strips. Samples of 0.2 pl. were added to each strip, although it was practical to use samples as small as 0.05 p1. The electrophoresis was carried out in 0.1M veronal buffer at p H 8.6 on Schleicher & Schuell 2043A paper. A current of 0.7 ma. was passed at 200 volts for 2 hours at room temperature. Early results showed a symmetrical distortion as a curved front on the parallel strips. This was corrected when the stopcock connecting the end vessek was opened. Although a small portion of the current was bypassed through this connection, the secondary effects due to ion accumulation at the electrodes and electroendosmosis on the paper were cancelled or minimized. The electrophoretic pattern of 1.5 y f horse spleen ferritin in 0.05 pl, per trip is shown in Figure 4 (ferritin is a iurified protein of ahout 500,000 moleclar weight). Good reproducibility of a ingle hand in each strip was produced 1 4 5 minutes a t 200 volts. The protein moved 25 mm. from the origin. Protein fractions on the order of 1 y or less can he easily identified with the above described electrophoresis apparatus. To identify and quantitate a certain fraction it is possible to remove the portion of paper containing . it and elute it for chemical analysis. Attempts were made to analyze the bromophenol bluestained paper strip by the Spineo Analytrol. However,
Figure 4. Electr( ferritin per 0.05 VOL. 32, NO. A, APRIL 1960
565
when a mask was inserted into the Analytrol to accommodate 1-mm. strips, the instrument was insufficiently sensitive. Wider strips or a more sensitive instrument should make this procedure practical. ACKNOWLEDGMENT
The authors thank Joseph Dorsa and Robert Palacios of the Research and
Development Laboratory for helpful suggestions in the design of the electrophoresis apparatus. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig, G., “Manual of Paper Chromatography and Paper Electrophoresis,”
Academic Press, New York, 1958. (2) Edstrom, J. E., Nature 172, 908 (1953).
( 3 ) Karler, A,, Brown, C. L Kirk, P. L., Mikrochzm. Acta 1956, 1585. (4) Strain, H. H., ~ ~ N A LCHEM. . 30, 620 (1958). (5) Turner, B. M., Mikrochim. Acta 1958, 305. (6) Brown, C . L., Kirk, P. L., Ibid., 1956, 1729.
RECEIVEDfor review April 29, 1959. Accepted November 9, 1959. Work supported in part by U. s. Public Health Service Grant C43341.
Precipitation of Crystalline Iron(lll) Oxide from Homogeneous Solution SIR: The preparation of granular or macrocrystalline precipitates from aqueous solution is generally limited to species with large lattice energies and relatively small hydration energies. Because multivalent metal ions possess large hydration energies and usually exist in solution as hydrolytic species [Fe(OH) +2, Fe(OH)i, etc.], the direct separation of most metal oxides from aqueous solution is often assumed to be precluded by the hydrophilic character of such oxides. However, there is in principle no limitation to the formation of macrocrystalline oxides in aqueous solution. Energetically, such oxides are stable and may be expected to form. Kinetically, the mechanism for their formation is usually sufficiently complex to prevent the separation of a granular product. We report the preparation, by direct precipitation from aqueous solution, of crystalline iron(II1) oxide as p-FeO.OH. The product is granular, easily filtered, and not peptized upon washing. The procedure appears to be the first preparation of the granular rather than the hydrous (gelatinous) oxide which has been effected rapidly a t ordinary temperatures and pressures. The primary significance of the procedure is the demonstrated ability to influence profoundly the relative rates of crystal nucleation and aggregation for the hydrous oxides. The separation of microcrystalline iron(II1) oxide by slow hydrolysis of concentrated iron(II1) solutions has been reported under a variety of conditions, but the precipitates are invariably hydrous and are finely divided and easily peptized upon washing. Recently Gayer and Wootner ( 3 ) reported the preparation, by slow hydrolysis of ferric nitrate solutions over a period of weeks, of a microcrystalline iron oxide which is not peptized upon washing. 566
ANALYTICAL CHEMISTRY
Precipitation from homogeneous solution is particularly advantageous for the hydrous oxides, but prior application of this method to the preparation of metal oxides has not alleviated the hydrous character of the precipitates. In the present procedure, the precipitations were performed by neutralizing in a suitable manner acidic iron(II1) solutions containing a suitable complexing agent. Precipitation from homogeneous solution invariably provided the most satisfactory method for the separation, and the following procedure is recommended. Five to 10 grams of urea are added to 100 ml. of a 0.001M iron(II1) solution containing 0.01M hydrochloric acid and 0.002 to 0.02121 N,N-dihydroxyethylglycine (DHEG). The solution is rapidly brought to boiling temperature, after which the iron precipitates in 1 to 15 minutes, depending upon the concentrations of the reagents. The solution is removed from the heat upon incipient precipitation of the oxide and filtered through porous glass or porcelain crucibles. The precipitate may be washed repeatedly without peptization and can be dried with alcohol or acetone. The crystalline product has been identified by x-ray analysis as the beta monohydrate of iron(II1) oxide, 8FeO.OH. Although a chloride medium appears to be essential for the formation of the 8-FeO.OH, the beta modification appears to be a distinct (though perhaps metastable) crystalline species (4). and the interplanar spacings and relative line intensities observed in this study agree well with those reported by Weiser and Milligan (8) and Kratky and Xowotny ( 5 ) . Visually the precipitates appear as clusters of hexagonal amber platelets whose diameters are 2 to 4 microns. The clusters are considerably larger, on the order of 20 to 60 microns in diameter. Because of occluded water,
the freshly prepared oxide usually weighs 25 to @yo more than pure FeO.OH. The pyrolysis curve is similar to that for the iron hydroxides (sic) (2) with the transition from pFeO.OH to a-FepOB occurring between 150’ and 185’ C. (8). Some of the conditions favorable to the preparation of the granular iron oxide have been investigated in order to provide information concerning the mechanism of the precipitation. The formation of a granular product is critically determined by the rate of formation of the primary nuclei, which is influenced primarily by the rate of change of p H during the neutralization. ilt room temperature, the hydrolysis of urea is slow, and a quantitative precipitation requires several days. The precipitation may be effected rapidly a t boiling temperature, but the product is more sensitive to hydration than a t lower temperatures, even though precipitates formed from homogeneous solutions are much less susceptible t o peptization than those formed by other procedures. By heating the solution rapidly from room to boiling temperature, a compromise between an initially slow rate of nucleation and a rapid aggregation (growth) of the nuclei is obtained which may permit an immediate quantitative recovery of the granular oxide. Often, however, the precipitation of the granular product is only 90 to 98y0 complete, and a quantitative separation may require aging at boiling temperature, which if prolonged may tend to peptize the precipitate. Apparently, it is the nucleation of the uncomplexed iron(II1) Thich determines the granular character of the precipitate. The complexing agent DHEG is a weak acid with pK2 = 8.10 (r), and studies on the stability of the iron(II1)-DHEG complex (1, 7‘) have indicated that in the p H range 2
