NOVEMBER 15, 1940
ANALYTICAL EDITIOK
of Paris. The improvements in the technique permit the observation of cellular detail in the electrographic pattern produced b y the chloride ion on a silver chromate gelatin emulsion.
Acknowledgment The writer wishes to express his gratitude to R *T' 'Ox for his kind cooperation in the design and construction of the electrographic apparatus and accessories described in this re-
703
port. The writer is also indebted to R. F. Sigrelli for photographic assistance and to Alexander Gobus of Lucius Pitkin, Inc., for the photomicrographic reproductions.
Literature Cited (1)
Clarke, B. L., and Hermance, H. W., IND. ENQ.CHEM.,Anal. Ed.,
(2)
GIazunov, A., and K~ivohlavy,J., z. physik. Chem,, 161, 373
9, 292 (1937).
(1932). (3) Jirkovsky, R. J., Chem. ~ i s t y2, 5 , 2 5 4 (1931).
A Pyrex All-Glass Microelectrophoresis Cell DAVID R. BRIGGS Division of Agricultural Biochemistry, University of Minnesota, Minneapolis, Minn.
T
HE all-glass modification of the Sorthrup-Kunitz
microelectrophoresis cell (7) as described by Abramson (3) has proved very satisfactory in the measurement of migration velocities of particles suspended in aqueous media. It is not easy to construct, however, and its general use has been seriously curtailed on this account. I n certain cases, too, the danger of contaminating the liquid in the flat part of the cell by stopcock lubricant or electrode liquor may be of importance. A cell designed by Buzach and described by Bull (4) eliminates this latter difficulty but introduces others due to the fact that this cell is constructed by cementing the parts together. A recent discussion of the various modifications of microelectrophoresis cells can be found in a review article b y Abramson ( I ) . The microelectrophoresis cell and accompanying electrical hookup described here were primarily designed for use in measuring the migration velocities of particles suspended in organic liquids. The entire assembly may be used as well with aqueous systems. The simplicity of design and use and the relative ease of construction of the cell, together with the novel arrangement employed for determining the field strength during the measurement, seem to warrant publication of a description of this apparatus.
Design of Cell Figure 1 is a line drawing of the cell. The flat part, A , of rectangular cross section, is constructed from polished Pyrex glass slides, 25 X 75 X 1 mm. (1 X 3 inches X I mm.), obtained from the Corning Glass Company. Two such slides are ground
at the ends t o the shape shown in A' and are arranged in a parallel position with a spacer sheet of thickness equal to the depth desired in the finished cell (0.8 to 1.0 mm.) employed to hold the slides apart. (Sheet aluminum has been found usable for this purpose, but a slide of Pyrex glass similar to that used in forming the top and bottom of the cell is much better. A sheet of mica of the proper thickness should serve the purpose very well, since it would be resistant to heat and would not conduct heat away so rapidly as to cause strains to appear in the Pyrex slides.) The assembly may be supported in a vise with one long edge horizontal and extending well above the point of support. Careful adjustment of the distance between the slides (using a micrometer) at this point in the preparation will assure a minimum of deviation from parallelism of the top and bottom surfaces in the finished cell. The spacer sheet should extend about a third of the distance to the top edge of the assembly. A rod of Pyrex glass, approximately 2 mm. in diameter, is laid along the top edge of the assembly and melted on to the edge of the slides with an oxygen-gas flame (using a hand torch). The seal is extended along the ground ends of the slides. When the spacer sheet is removed the sealed edge supports the slides parallel to each other, By reversing the position of the slides in the sup ort, the other edge can be sealed in like manner. To the holes Eft at cither end of the flat cell is sealed a Pyrex tube of 8-mm. diameter. The slide which is to be the top of the flat cell is heated at the points of seal and blown to smooth connections with the tubes. The flat cell with its connecting tubes should be annealed carefully (by bringing it slowly to an incipient red heat in a muffle furnace and allowing it to cool slowly). The end parts of the complete cell, which should be prepared se arately and then sealed to the connecting tubes of the flat ceE, consist of the electrodes, B and B', the stopcocks, C, and, on one side, the funnel, D, through which the liquid will be introduced into the completed cell. Pyrex standard taper (So. 1) stopcocks, C, with I-mm. bore, present ample contact area to prevent leakage past these points when used either with or without lubricant. Each electrode is constructed from a Pyrex standard taper (12/30) ground-glass joint. The outer part, B , is sealed in, as illustrated, to form an integral part of one of the end parts of the cell. The inner part, B', may be sealed off at a point several millimeters below the joint and blown into the shape shown. The large ground contact area of the joint will prevent leakage at this point whether lubricant is used or not, and when the two parts of the joint are forced firmly together no other precaution need be taken to prevent the inner part from dropping out. I n order to use the cell with organic liquids i t is necessary to be able to remove the tvorking electrodes and change their contents easily, and to get the liquid into the flat part of the cell without danger of contamination from the electrodes or from stopcock lubricant. These requirements are met completely in the design as shown without introducing any trouble from leakage.
70 1
INDUSTRIAL AND ENGINEERING CHEMISTRI-
hottom of the bulb on B' t o make contact with R few drops of mercury placed therein. A solution of mercuric nitrate in the lower bulb of the electrode makes this a good reversible elec-
VOL. 12, NO. 11
++DC-C
them when desired without disturbing the contents of the lower bulb. In aqueous systems a half-saturated solution of potassium nitrate is used to fill the upper chamber of B' before putting the inner part of the ground-glass joint in place. In any case the solutions used in the electrode chamber must have a density
used on the stopcocks and 011 the ground-glass joints of the electrodes, since there exists no possibility of thereby contaminating the liquid in the flat part of the cell. Fresh liquid can be introduced and passed into the cell a t any time nithout danger of contamination. d hollow glass stopper with pinhole opening a t side may be used on D to prevent eyaporation a t this point. The complete cell must be mounted rigidly in a support n-hich can be set on the microscope stage in the proper position for observations. While the thickness of the glass slides (1.0 mm.) makes for greater ruggedness of the completed cell, it also requires that the objective used on the microscope assembly be of long working distance, since it is necessary to be able to focus a t all depths of the cell, when it is filled with the suspension upon which migration velocity measurements are to be made. 4s Moyer (6) has emphasized, it is desirable to obtain as high a degree of magnification as possible, since this narrow5 the depth in the cell in which particles will be in focus and thereby increases the accuracy with which their motion, at any given depth, can be determined. If the volume depth of the liquid in the cell is approximately 1 mm. it is possible to use a X 2 1 objective with a working distance of 1.6 mm. (in air). If, however, it is desired to use the X 4 0 water-immersion objective of working distance 1.9 mm. recommended by hloyer, the cell should be constructed with a depth of about 0.8 mm. This depth is, of course, determined by the thickness of the spacer sheet clamped between the slides during the construction process. I n either case, only a small excess in Tvorking distance is left when the microscope is focused on the bottom of the cell when it is filled with the liquid, and in order to measure the actual depth of the cell, when it is empty, another objective of longer working distance must be used. For a cell having a depth of 1 mm., the ratio of width to depth is about 25; for one having a depth of 0.8 mm. this ratio would be over 30. A t such values, the velocity of the particles measured a t the 0.21 or 0.79 levels should, according to the theories of Smoluchowski ( 8 ) and Komagata ( 5 ) , be the true migration velocities of the particles. hleasurements were made a t these levels in a cell 1.05 mm. in total depth, using human erythrocytes in 0.67 M phosphate buffer of pH 7.35 as standard particles, A mobility of 1 . 3 3 ~per second per volt per cm. (at 25' C.) was observed, which agrees well with values given by Abramson (2).
r
B
i l
Figure 2 shows the electrical setup required when organic liquids of lon- specific conductivity are used in the cell. When aqueous solutions are used, the ammet'er method of measuring the current' passing through the cell can be employed as the basis for calculating the field strength within the cell. The principle of the setup shown in Figure 2 is similar to one using an ammeter, except that an electron tube voltmeter is employed to adjust to a definite value the voltage drop across a known resistance, T , which lies in series with the electrophoresis cell in the working circuit. The working current in the circuit containing the cell can then be calculated by Ohm's law. This arrangement has the advantage that only a change in r will be required to make possible its use with liquids of any conductivity value. The electron tube voltmeter shown is the simplest possible type and costs very little to construct. The manner of use is as follows: With a battery of known voltage, S, in the grid circuit of the tube, the current in the plate circuit, measured by the 10-15 milliammeter, D, is adjusted to some convenient scale reading by the two 10 V potentiometers, z y. Using a double-pole double-thropv mitch. A , a part, r , of the resistance in the working circuit containing the electrophoresis cell, T , is substituted for S. By adjusting the potentiometer, C, which lies across the direct current voltage supply, the reading on D is brought to the same value as when S is present in the grid circuit. The potential drop across r then is equal to the standard voltage, S. The voltage drop per centimeter in the electrophoresis cell is calculated from the equation
+
E=---. s AXr where E = potential drop in the electrophoresis cell (volts per em.) S = voltage of the standard cell (volts) A = area of cross section of electrophoresis cell (sq. em.) = specific conductivity of liquid in electrophoresis cell (ohms-') and T = the known resistance in working circuit across which the standard voltage drop is obtained (ohms) By varying r , any desired value for E can be obtained. In practice, it is simpler to substitute a succession of known resistances in the circuit at r until a convenient rate of migration of the particles is observed in the cell and then to calculate the value of E by the above equation. (With each change in r the position of C must be changed to bring reading at D back to that given when S is substituted for r . ) The degree of accuracy for this method of measuring E , using the parts as shown in Figure 2, is of the order of 1 to
NOVEMBER 15, 1940
ANlLYTICAL E D I T I O S
705
2 per cent. Further refinements leading to a greater sensi-
Acknowledgment
tivity of the voltmeter could increase to almost any desired value the accuracy with which the field strength could be estimated. Switch B is used to reverse the polarity of the electrodes in the electrophoresis cell without affecting any other part of the circuit. B y occasionally comparing the reading a t D when first X then r is connected into the grid circuit, variations in the applied voltage a t C can be made to maintain constant the value of E during the course of the measurement of migration velocity. The electrophoretic properties of particles suspended in alcohols of specific conductivity approximating 10-8 mho, and of particles suspended in aqueous solutions of specific mho, have been measured conductivity of the order of with equal facility with this apparatus.
The author is pleased to acknowledge the aid of Ralph Hossfeld in the construction of the flat part of the electrophoresis cell.
Literature Cited (1) Abramson, H. A,, Ann. A:. Y . Acad. Sci., 39, 121-16 (1939). (2) Abramson, H. A , “Electrokinetic Phenoniena”, page 257, S e w York, Chemical Catalog Co., 193.1. (3) Abramson, H. A , , J . Gen. Physiol., 12, 469 (1929). (4) Bull, H. B., J . Phys. Chem., 39, 577 (1935). (5) Komagata, S., Researches Electrot&. Lab. T u k ~ 348 , (1933). (6) Moyer, L. S., J . Bact., 31, 531 (1935). (7) Northrup, J. H., and Kunita, XI.. J . Gen. Physiol., 7 , 729 (1925). (8) Smoluchowski, XI., in Graetz “Handbuch der Elektriaitit und des Magnetismus”, Vol. 11, p. 36G, Leipzig, B a r t h , 1921. PAPERNo. 1822, Scientific Journal Series, 2Iiiinesota Agricultural Experiment Station.
Microhydrogenation Apparatus ARTHUR N. PRATER AND A . J. HAAGEN-SRIIT California Institute of Technology, Pasadena, Calif.
A
LONG with the development of general microanalytical
procedures for organic compounds, i t has been necessary t o produce more specialized methods. One such method that has assumed great importance in structure determination is the quantitative measurement of the amount of hydrogen consumed in a microhydrogenation. For investigations on plant hormones and other physiologically active substances in the authors’ laboratories i t became necessary to construct a n apparatus for microhydrogenation. There have been several devices of this type described by various investigators (2-6, 8, 9) which have one or more of the following objectionable features: The apparatus must be operated in a
sensitiue thermostat, incomplete temperature compensation is provided, and a large, fragile, glass coil or d lubricated ground joint is used to connect the reaction and measuring systems. The apparatus described here is in part a combination of the desirable features of the earlier designs referred to above, and is so constructed that the entire reaction and measuring system is shaken as a unit, eliminating the necessity of having ground joints which must be able to rotate freely and yet be absolutely gas-tight. The apparatus has been in use for the past year, and has given satisfactory results on a number of compounds. Complete temperature compensation is pro-
FIGURE 1. TOPVIEW OF APPARATUS
