A fascinating and essentially simple electroplating process known as polarography has in recent years been developed into an exquisitely sensitive method of chemical analysis. As a means of identifying chemically reducible constituents in compounds and measuring their respective concentrations, it surpasses spectrography in sensitivity and matches the analytical power of radiochemistry. Yet in its essentials the polarograph has changed little since its invention in 1925 by the Czechoslovakian chemist Jaroslav Hevrovsky. In its simplest form the instrument consists of a small electrolysis cell and a battery and volt-microammeter. Positive ions of substances in the electrolysis cell pick up electrons at the negative electrode of the cell; this neutralizes tile charge of the ions and transforms them into atoms. Hevrovsky observed that if the area of the cathode is very small, the voltage at which ions acquire electrons from it is characteristic of each substance and that the accompanying current varies in direct proportion to the concentration of the substance that is under analysis. To make an analysis he simply added the unknown substance to a solution of a substance known as a supporting electrolyte, gradually increased the voltage across the cell from zero and simultaneously measured the current. A graph made by continuously plotting the applied voltage against the current served both as a qualitative and a quantitative analysis of the unknown compound.

In spite of the simplicity and power of the method, polarography attracted little interest immediately following its invention, largely because characteristic voltages had been determined for only a few substances. But by 1940 both the theory and the procedural details had been reported in some 2,000 papers and a comprehensive treatise had been compiled by I. M. Kolthoff of the University of Minnesota and James J. Lingane of Harvard University ( see "Bibliography"). These publications laid a foundation of information for the explosive growth of the method in recent years. In 1959 Herovsky received the Nobel Prize for originating polarography, and today no chemical laboratory is considered complete with the instrument.

Commercial polarographs range in price from $500 to $2,000. But Sam Epstein, chief chemist of the Federated Metals Division of the American Smelting and Refining Company in Los Angeles, has designed an instrument that can be constructed at home for less than $75. It detects and measures substances diluted to .0001 mole per liter of solution. (One mole is equal to 6.02 x 10²³, atoms or molecules of any substance.)

"The principal distinction between the polarograph and other devices, such as pH meters, that employ electrolysis cells," Epstein writes, "is found in the negative electrode (cathode), which consists of a small drop of mercury that constantly renews itself as mercury flows from an elevated reservoir through a capillary tube. This arrangement continuously removes from the vicinity of the cathode the products of electrolytic reaction that would otherwise interfere with the analysis. The positive electrode, which has a large surface compared with that of the cathode, is most commonly a pool of mercury at the bottom of the cell or a saturated-calomel electrode, the construction of which will be described.

"The accompanying circuit diagram [Figure 1] indicates the basic simplicity of the polarograph. To measure the characteristic potential at which an oxidized substance is reduced, the electrolysis cell is first filled with supporting electrolyte; one of the most frequently used is an aqueous solution of potassium chloride. The electrolyte provides a conducting path through the cell but does not interfere with the reduction of specimen compounds because potassium ions decompose at a higher potential than do most other substances. Now suppose that a small amount of nickel is added to the electrolyte in the form of a salt and that a gradually increasing voltage is applied to the cell. The resulting current—consisting of electrons flowing into and charging the cathode in addition to a few electrons associated with the reduction of impurities in the electrolyte—will amount to only a fraction of a microampere until the voltage exceeds the potential at which nickel ion decomposes. In effect the junction between cathode and solution constitutes a high-resistance circuit. At a sufficient potential, electrons are transferred from the cathode to neighboring ions of nickel; the current increases to a maximum limited only by the rate at which nickel ion diffuses from the electrolyte to the surface of the cathode. The rate of diffusion is governed by the concentration of nickel ions in the solution. The difference between the residual current and the maximum, or limiting current is known as the diffusion current and is proportional to the concentration of nickel ion. Atoms of metallic nickel so reduced deposit on the cathode, as in conventional electroplating, and form an amalgam with the mercury.

"The decomposition potential is often difficult to determine precisely. It has therefore become standard practice to observe the slightly higher voltage that corresponds to the point on the graph midway between the residual current and the limiting current. The graph, incidentally, is conventionally referred to as the polarographic wave and the voltage at which reduction occurs as the half-wave potential [see Figure 2]. Tables of characteristic half-wave potentials for many elements and compounds can be found in most chemical handbooks.

"A centrifuge tube of approximately 40-milliliter capacity serves as the electrolysis cell, and the cathode is a capillary tube approximately 21 centimeters long, supplied by mercury from an elevated reservoir as shown in the accompanying drawing [Figure 4 ]. The flexible tubing between the reservoir and the cathode capillary should include a two-way stopcock for draining mercury from the apparatus after use. The bore of the capillary should not exceed .002 inch, and the lower end should be cut square so that it is perpendicular to the bore within five angular degrees. The height of the mercury reservoir should be adjusted so that mercury flows from the capillary at the rate of one drop every three or four seconds. The capillary should be clamped to operate vertically. Electrical contact between the mercury and the meter circuit must be made by platinum electrodes. The electrodes can consist of No. 36 platinum wire one centimeter long, sealed in the end of a quarter-inch glass tube and equipped with a copper lead, as shown at the right in the accompanying drawing [Figure 5]. Three platinum electrodes are required: one for the saturated-calomel electrode, another for an electrolysis cell employing a mercury anode and the third for connecting the negative lead of the metering circuit to the mercury reservoir.

"The saturated-calomel electrode and agar-potassium chloride bridges that couple it to the electrolysis cell consist of 40-milliliter centrifuge tubes and U loops of quarter-inch glass tubing, with chemicals and associated hardware as shown. The mercury, calomel paste, solid potassium chloride and saturated potassium chloride solution are prepared and placed in sequence in one of the centrifuge tubes. The relative proportions are not critical and can be judged from the illustration. Some care must be exercised, however, in preparing the agar-potassium chloride bridges, which protect the saturated-calomel electrode from contamination by the test solutions. To prepare the bridges, soak four grams of agar in a small beaker containing 100 milliliters of distilled water, preferably overnight. Then place the small beaker in a larger one of boiling water and heat until the agar is fully dissolved. Add 30 grams of potassium chloride and, with the beaker still in the boiling water, stir until dissolved. If necessary, add just enough water to dissolve the salt completely. Then invert the U tubes, fit the ends with short sleeves of rubber tubing, clamp and fill with the agar solution as shown in the accompanying drawing [Figure 6]. The agar solution must completely fill the bridge tubes and flexible sleeves, or air bubbles may be trapped when the tubes are inverted in the solutions. In spite of the best care, the agar eventually becomes contaminated, producing erratic results. To replace it, disassemble the apparatus, put the bridges in boiling water to melt the agar, rinse them thoroughly and refill.

"All mercury except that in the saturated-calomel electrode and the reservoir should be stored in narrow-mouthed polyethylene containers with screw caps. Work in a well-ventilated room without rugs or other fabric floor covering, so that mercury spilled accidentally can be cleaned up. Should mercury be spilled, collect as much as possible with a scoop made of sheet copper. The inner surface of the scoop should be amalgamated with mercury before use. The microscopic droplets remaining are recovered by sprinkling the area with finely divided zinc or copper moistened with a 10 per cent solution of hydrochloric acid. It is essential always to have these materials ready for action. Don't let mercury run down the drain! Even a seemingly small amount can poison the air in a small closed room. When a sizable amount of used mercury has been collected, it should be cleaned as described in the Handbook of Chemistry and Physics, but do not heat the material as recommended in the handbook. The polarograph requires two pounds of mercury of the grade used by dentists. [Most of the required components and chemicals can be bought locally from druggists and dealers in scientific supplies. Parts specially selected for the construction of Epstein's design can be ordered individually or in kit form from Henry Prescott, Main Street, Northfield, Mass.]

"Set up the completed apparatus as shown and fill the reservoir (filter funnel) about half-full of mercury. Switch the stopcock to feed the capillary with mercury and adjust the rate of How by increasing or decreasing the height of the reservoir so that a pinhead-sized drop forms and falls every three or four seconds. Close the stopcock and return the mercury to the reservoir.

"An interesting experiment for checking the apparatus and acquiring experience with the procedure is based on the detection of dissolved oxygen. The supporting electrolyte consists of dilute potassium chloride that has been shaken several minutes to assure that it contains an appreciable amount of dissolved air. Incidentally, the supporting electrolyte constitutes the bulk of the dissolved material in all test solutions. Its concentration, particularly if the substance under analysis is ionic, must be at least 35 times that of the substance under analysis and usually even more. When the ions of the supporting electrolyte outnumber those of the specimen by a ratio of this magnitude, the rate of migration of the ions under analysis through the electrolysis cell is negligible.

"The extreme sensitivity of the polarograph restricts the experimenter's choice of test materials to reagent grade chemicals. Distilled water must be used not only for making up test solutions but also for rinsing the apparatus between tests and preparing it for storage. Normally the strength of solutions is expressed in terms of molarity. One mole of a substance is equal in grams to the sum of the atomic weights represented in the formula of the substance. The atomic weight of potassium, for example, is 39.1 and that of chlorine 35.5. The sum of the atomic weights constituting potassium chloride, a compound consisting of one atom each of potassium and chlorine, is accordingly 74.6. One mole of potassium chloride therefore weighs 74.6 grams. Cadmium chloride contains one atom of cadmium (atomic weight, 112.4) and two of chlorine (atomic weight, 2 X 35.5). The sum is 183.4, and one mole of cadmium chloride weighs 183.4 grams. A one-molar solution (1M) of potassium chloride contains one mole, or 74.6 grams, of potassium chloride per liter of solvent. Similarly, a .001M solution of cadmium chloride contains .1834 gram of cadmium chloride per liter.

"To set up the introductory experiment, prepare a .1M hydrochloric acid supporting electrolyte containing dissolved air, place 35 milliliters in the electrolysis cell and open the stopcock. Readjust the height of the reservoir for the specified drop rate if necessary. Check the tip of the capillary tube and carefully remove any bubbles. Switch the meter to measure voltage and adjust the potentiometer for zero indication. Apply .1 volt to the cell and switch the meter to indicate current. Record both current and voltage. Increase the voltage to .2 volt, read the current and continue the procedure in steps of tenths of a volt, tabulating both current and voltage to a maximum of 1.5 volts. When switched to indicate current, the meter will oscillate from minimum indication, as a new drop of mercury begins to form, to maximum, just before the drop falls. The fluctuation of the current is induced by the periodically expanding area of reaction at the cathode. Record the maximum current. Plot the tabulations with voltage as the abscissa and current as the ordinate. A sharp rise in the graph .It about .1 volt indicates that dissolved oxygen was reduced to hydrogen peroxide. At 1.1 volts the hydrogen peroxide was reduced to water, as indicated by the polarographic waves in the accompanying graph [Figure 7].

"These polarographic waves from the reduction of oxygen are so pronounced that they interfere with the waves of most other substances. Hence oxygen must be removed from all solutions just before they are electrolyzed. This is accomplished by bubbling hydrogen or nitrogen through the electrolysis cell for five minutes. Connect a length of glass tubing to a source of hydrogen gas, immerse the tubing to within approximately one centimeter of the bottom of the electrolysis cell and pass a rapid but not violent stream of hydrogen through the electrolyte for five minutes. If compressed hydrogen is not available, one can put together a gas generator as shown in the accompanying drawing [Figure 8]. Test for oxygen removal by electrolyzing the solution. Tabulate the current and voltage and draw a corresponding graph. The residual current should persist to approximately 1.8 volts, the decomposition potential of potassium, as shown in the accompanying graph [Figure 9]. This indicates that oxygen has been completely expelled from the solution by the stream of hydrogen.

"Before setting up the next experiment close the stopcock, lower the electrolysis cell and clean it. Rinse off the capillary, agar and hydrogen tubes. Always repeat this procedure between analyses. Never expose the agar tube to air for more than a few minutes or it will be damaged by drying. When you have finished working with the polarograph, immerse the dropping-mercury electrode in distilled water and the agar tube in a container of saturated potassium chloride. They can be so stored for long periods.

"Next, replace the electrolyte with 1M potassium chloride solution that is also made .004M with respect to lead nitrate. Add one drop of concentrated hydrochloric acid, flush out the oxygen with hydrogen and electrolyze in steps of tenths of a volt. The plotted results should resemble the solid curve of the accompanying polarographic wave [Figure 10].

"This experiment demonstrates what has come to be known as a 'maximum,' an undesirable peak in the polarographic wave that appears at about .5 volt in this case Such maxima, which frequently interfere with an analysis, have not been fully explained, but they must be eliminated. This is done by adding a 'maximum suppressor' to the solution. Many substances are effective as suppressors, including gelatin. Dissolve .5 gram of plain gelatin in 100 milliliters of distilled water. (Place a small beaker containing the gelatin solution in a larger beaker of hot water and boil until the gelatin dissolves; heating the small beaker directly over a flame would damage the gelatin.) Add one drop of the dilute gelatin to the lead nitrate solution. Store the remainder for future use; it will keep about three months if refrigerated. Electrolyze the lead nitrate solution again and plot the results. This wave should be identical with thc first one except for the maximum, which will have vanished, as indicated by the broken curve. Remember this stratagem when maxima are encountered in future experiments, but never use more suppressor than absolutely necessary. Begin with a single drop and add one drop at a time until the maximum peak disappears. Too much suppressor will reduce the limiting current and thus introduce error.

"To demonstrate how the polarograph measures concentration, prepare solutions of cadmium chloride in three strengths: .001M, .005M and .020M. Make up a supporting electrolyte of .1M potassium chloride and add one drop of gelatin. Drive out dissolved oxygen before each electrolysis. Apply voltage in steps of tenths of a volt from .3 to 1 volt The plotted results should resemble the waves in the accompanying graph [Figure 11 ]. Plotting concentration against current results in a linear graph [Figure 12], which proves that the current increases in direct proportion to the concentration.

"To demonstrate the analysis of mixed substances, prepare a supporting electrolyte 1M with respect to both ammonium hydroxide and ammonium chloride and add one drop of gelatin. Then make up separate 100-milliliter solutions of 1M copper sulfate, nickel sulfate and zinc sulfate. Place about 20 milliliters of supporting electrolyte in the electrolysis cell, add two drops of copper solution, sweep out the oxygen and electrolyze between zero and two volts in steps of .1 volt, tabulating the results. Repeat after adding two drops of nickel sulfate and again after adding two drops of zinc solution. Having acquired experience with the method, the experimenter should be able to visualize the results while the analysis is in progress. A new wave appears for each added element as shown in the accompanying graph [above]. Observe the double wave for copper. It is a nice example of how the polarograph can be used in theoretical studies. The double wave is explained by the fact that cupric ion, which is characterized by a double positive charge (the atom of metallic copper has lost two electrons), is reduced in two stages. It first acquires a single electron and is thereby reduced to cuprous ion, which then acquires a second electron and is reduced to metallic copper.

''Although oxygen interferes with the polarographic analysis of most substances, its double wave can be used to determine the amount of gas present in solutions. For these experiments a reference electrode of mercury is most convenient. Remove and store the saturated-calomel electrode. (Remember to protect the exposed agar by immersing it in a saturated solution of potassium chloride.) The experiments to be made now involve biological materials. Use a 1 per cent solution (by weight) of sodium chloride as the supporting electrolyte. Place a small quantity of mercury in a clean centrifuge tube of 40-milliliter capacity and assemble the cell shown in the accompanying drawing [left]. For these experiments the electrolysis cell is filled completely and tightly stoppered, except for a breather tube of small bore. The oxygen content of solutions is proportional to the difference between current readings at .1 and 1 volt. The determination is only relative between solutions that differ in oxygen content. If the difference in current between .1 and 1 volt with solution A is higher, for example, than the difference in current with solution B, then A contains proportionately more oxygen than B. The absolute content of any given solution must be determined by chemical analysis. But experiments based on relative determinations alone can be fascinating.

"As an example, first electrolyze a cell of saline solution and plot the oxygen wave. Then add one milliliter of fresh chicken blood to the saline solution and make a second run. The second polarographic wave will indicate the amount of oxygen absorbed by the hemoglobin. The absorption will be found to differ when the blood of various animals is analyzed, a result that can be correlated with the differing structure of the red cells of the respective species.

"The analysis of a suspension of green algae can demonstrate the production of oxygen by photosynthesis. Fill the cell with a thick suspension of algae in 1 per cent saline solution. Let the material stand in darkness overnight and analyze for relative oxygen concentration. Then focus a strong light, such as the beam of a slide projector on the electrolysis cell for an hour and repeat the analysis. Compare the results. Turn off the light and make a series of runs. The concentration of oxygen will diminish gradually as the algae consume oxygen in the process of respiration. Expose the suspension to light transmitted by a red, green and blue filter respectively. Observe the resulting influence of wavelength on the efficiency of the photosynthetic process.

"Having completed these experiments, you are on your own. Like the microscope, the polarograph opens the way to a broad field of investigation. In recent years the instrument, with appropriate modifications, has been applied to the analysis of solutions in disciplines as widely separated as medicine and metallurgy. Hospitals routinely subject biological materials to polarographic analysis, and research workers in nuclear physics use the procedure for the analysis of transuranium elements produced in microgram quantities by cyclotrons."

Bibliography

POLAROGRAPHY. I. M. Kolthoff and James J. Lingane. Interscience Publishers, Inc., 1952.

PROGRESS IN POLAROGRAPHY. P. Zuman and I. M. Kolthoff. John Wiley & Sons, Inc.–Interscience Division, 1962.

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