ANALYTICAL CHEMISTRY
516 lower part of its range. in particular, the coulometer should be of great usefulness, since accurate and convenient methods have not hitherto been available for work in this region. ACKNOWLEDGMENT
The authors would like to express their appreciation to the Surdna Foundation, whose financial support has made this work possible. LITERATURE CITED
(1) Campbell, 11938).
IT. E., and Thomas, U. B., Suture, 142, 253-4
(4) Greenwood, 1. A., Jr.. Holdam. J. V.. Jr., and AIacRrre, D., Jr., “Electronic Instruments,” h1.I.T. Radiation Laboratory Series, Vol. 21, New York, hlcGraw-Hill Book Co., 1946. (5) Lingane, J. J., J . Am. Chem. Soc.. 67, 1916-22 (1945). (6) Lingane, J. J., and Small, L. A,, ASAL. CHEM.,21, 1119-22 (1949). (7) Muller, R. H., and Lingane, J. J., Ibid.,20, 795-7 (1948). (8) Radio Corp. of America, “Tube Handbook,” HB-3, Vol. 2, Type 5651, Harrison, N. J., Radio Corp. of America. (9) Reilley. C. N., Bdams, R. N., and Furman, X . H., A s . 4 ~ . CHEM.,24, 1044-5 (1952). (10) Wise, E. N., Gillies, P. W., and Reynolds, C. A., J r . , Ibid., 25, 1344-8 (1953). (11) Zakhar’evski:, AI. S.,Khim. Referat. Zhur., 2, Xo. 4, 84 (1939). (12) Zakhar’evskii, M. S., Voprosy Pitaniyu, 7, 415, 180-8 (1938).
\----,-
(2) Campbell, W.E., and Thomas, U. B., Trans. Electrochem. Soc., 76, 303-24 (1939). (3) Francis, H. T., J . Electrochem. Soc., 93,79-83 (1948).
RECEIVED for review September 4, 1953. Accepted December 19, 1953 Presented before the Division of Analytical Chemistry a t the 124th Meeting of the .4XERICAS C H E ~ I I C A SOCIETT, L Chicauo, 111.
Electronic Controlled Potential Reduction or Oxidation Apparatus FREDERICK KAUFMAN, ELI OSSOFSKY, end HELEN J. COOK Ballistic Research laboratories, Aberdeen Proving Ground, M d . In the course of work on the reduction of nitrate esters, the need arose for a controlled potential device of rapid response and large possible electrolysis currents and cell potentials. An electronic instrument is described which fulfills these requirements. It is capable of delivering currents up to 7 amperes, cell potentials up to 150 volts, and has fast response, high stability, and sensitivity. Its principle of operation is as follows: The amplified difference between the desired and obtained cathode-calomel potentials controls the grids of a bank of 6AS7G power tubes. The tubes are in the electrolysis circuit and control the current. An unbalance potential of 1 to 2 mv. corresponds to a change of up to 1 ampere in electrolysis current. The instrument is also capable of controlling very small currents. Reductions of several inorganic and organic compounds at low concentrations are described.
E
LECTROLYTIC reduction a t controlled cathode potential has been the subject of a considerable number of investigations in recent years. Much of this interest aroae from polarographic work through efforts to reduce chemical species at high concentrations in the same specific and selective manner in which they are reduced at low concentrations a t the dropping mercury electrode. When successful, this technique is of great usefulness in preparative organic chemistry, in controlled reduction of metal ions, and in many other problems. Moreover, a basic understanding of the rate of large scale electrode processes is still very incomplete. Instruments of the type described here permit a study of the factors that affect the over-all rate of the electrolytic oxidation or reduction process, such as the electrode reaction, transport of solute by diffusion and convection, rate of stirring, etc. During the past few years, interest in this field has increased rapidly and a number of instruments for controlled potential electrolysis have been described (1-4, 6-11, 13). Even though several of these instruments provide good control, it seemed worth while to build another version of such an apparatus with the following properties in mind: 1. Completely electronic design, making possible instantaneous response 2. High possible cell potentials and currents large change of current per unit 3. High sensitivity-i.e.,
unbalance between desired arid obtained cathode-referenre electrode potential 4. Stability, ease of operation, and reliability Some entirely electronic instruments (3, 4 ) are among those described in the literature. The one described here is closely related to Hickling’s ( 4 ) in its basic regulating principle, but differs from i t in various ways as is shown. Its main disadvantages are low power efficiency and requirement of alternating and direct current power. Of these, the former is not serious in a research instrument and the latter is not too restrictive, since any direct current line voltage between 20 and 250 volts is suitable. DESCRIPTIOY AVD OPERATION O F I\STRU.MENT
A block diagram of the apparatus is shown in Figure 1. The small unbalance signal, e , represents the difference between the desired, eo, and obtained, ec, cathode-reference electrode potentials. It is fed into a direct current amplifier whose output, eo, controls the grids of a bank of power tubes (Type GASiG). The power tubes are in series with the electrolypis cell and control ~ electrolysis current. the f l o of Figure 2 shows the apparatus in greater detail as a controlledpotential reduction instrument, though it is easily modified for other uses as described below. The maximum obtainable electrolysis current is determined by the number of power tubes in parallel. This instrument was constructed with 27 tubes and could deliver about i amperes (0.25 ampere per tube). -4240volt direct current line eerved as power source in most of the work. While this ip well within maximum plate voltage of the
”,
CURRENT COWTROL TUBES
Q GRID
Figure 1.
-
Block Diagram of Apparatus
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
517
v
sv
3ooW 120 v
I
I
Figure 2.
amplified and impressed on the grids of the power tubes. The magnitude of e, the unbalance potential, determines how closely the desired reduction potential is actually obtained. It is an important quantity and was therefore both calculated and measured. The calculation was based on the equivalent circuit of an amplifier and expressed the change in unbalance potential, e, per unit change of electrolysis current as a function of tube charac-teristics, amplifier gain, and load resistance. This quantity turned out to be around 1 mv. per ampere change in current. I t was also measured directly using a LTillivac direct current vacuum
& U 40-2
Controlled Potential Reduction .4pparatm
RI. 1000-R voltage divider (Gcn. Radio 314-A) A.M. Recording ammeter (Esterline-Angus shunts) V . Calibrated voltmeter C. Saturated calomel electrode VI, V ? . . Vzi. 6AS7G douhle triodes
millivoltmeter
.
and L
tube, it meant that a t high electrolysis currents the maximum rated power dissipation (26 watts per tube) would be exceeded and the tubes damaged. -4suitable arrangement of six 300watt light bulbs in series with the plates served to decrease the plate voltage a t high currents. The instrument was also successfully operated using direct current line voltages between 20 and 250 volts without change in adjustments or procedure. For line voltages of 120 volts or below the lamp load is unnecessary. The direct current amplifier is a comparison amplifier as described in a monograph ( 1 2 ) with the minor change of having variable positive feedback, Ra. The wiring diagram of the amplifier is shown in Figure 3 and that of the power supply and all filament circuits in Figure 4. I t is operated with sufficient feedlmck to give a gain of about 10,000, although its output is stable up to 30,000. The output voltage of the amplifier varies from about 0 to +250 volts and ip balanred by four SO-volt B batteries on a voltage divider, Xn,so that the voltage impressed on the grids of the power tubes can swing from -250 volts to 0 volt. A 6H6 diode between t,he grid and cathode of the power tubes prevents the grid from ever heconling positive with respect to the cathode and drawing excessive grid rurrent. The output of the amplifier is initially adjusted with voltage dividers K, and Rq. I f c is first $et in such a way that the potential from e , to ground is in t,he region of greatest change per unit change of Ra. This assures that, the output potential lies in the region of maximum amplifier gain. Then, this voltage range is made to coincide with the uieful grid potential range of t,he 6ASTG by adjustment of Rf. At a line voltage of 220 volts, for example, this is from about - 120 to -30 volts. Actual operation of the instrument is very simple. The amplifier and power tuhe filament circuits are turned on. The desired cathode potential is set on RI and read on voltmeter, V. The cathode, anode, and calomel leads are connected to the cell and the main direct cwrrent power switch is turned on. Current begins t o flow and regulation is continuous. A current versus time record is conveniently obtained on the rerording ammeter, AM. PERFORMANCE DATA AND DISCUSSION
Since a change in thc electrolysis current brings about a corresponding change in the cathode-calomel potential, a feedback loop is established and the instrument is self-regulating. For any desired cathode-calomel potential, e D , set on R I , that current. I , flows through the cell which corresponds to an unbalance potential e z e c - e D (where ec is the actual cathode-calomel potential)
...
ex, K ZERO ADJUST
Figure 3 .
Direct Current .iniplifier
Filament heated, terminals 2 and 7 on power s u p p l y output plug
P-4080
C-1410 G-1410 G
sw-l
7K-10
-
t 480 V
F
6 '+ Tl8VOl
rievoi
Figure 4. Power Supply and Filament Circuit
ANALYTICAL CHEMISTRY
518
tube millivoltmeter, Model MV-l7B, and satisfactory agreement was found in all cases. It appears that the instrument is very sensitive, as a change of current of 1ampere is brought about by a corresponding change of the unbalance signal of 1 to 2 mv. This means that the actual calomel-cathode potential, ec, will not differ by more than 5 to 10 mv. from the desired potential, e D , even if the current increases or decreases by 5 amperes. Since e can be set equal to zero for a n y desired grid potential, eo-i.e., for any desired current-by the simple amplifier output adjustment previously described, it will remain negligible during any experiment.
10
0.1
0 VOLTS.VS SAT. CUOYEL
Figure 5.
Reduction of Cadmium(I1) at Large Mercury Cathode
Supporting electrolyte, 2M lithium chloride Point where E = -0.7 volt ia indicated on each curve Cadmium sulfate concentrations: 1. 7.99 x 1 0 - 4 ~ 2. 1.59 X 10-aM 3. 2.36 X 10-8M 4. 2.85 x i o - w 5. 7.68 x 10-aM 6. 1.14 x 10-ZM 7. 1.52 x 10-zM 8. 2.26 x IO-rM 9. 4.4 X 10-M 10. 7.68 x 10-wf 11. 0.405M
Stability of the instrument was tested in two ways. Long time stability was apparent from the recording ammeter traces which mere always smooth and free of irregularity. This implies stability for time intervals as long as or longer than the time constant of the galvanometer-about 2 seconds. For short time stability test, the cell potential, plate, and grid potentials were put on a Tektronix oscilloscope, Type 511 AD, and examined for alternating current components over a wide range of frequencies. No oscillations were found and only line voltage fluctuations and the effect of the stirrer on the calomel-cathode potential were able to be detected. OTHER USES
Any controlled potential instrument is easily adapted for several different uses. The necessary changes for some of them are listed here for the sake of completeness. Oxidation. Reverse polarity of battery B. Exchange input leads e,, e2 (see Figure 2) on the direct current amplifier and connect e* to anode rather than cathode. Controlled Cell Potential. The reference electrode is removed and its lead is connected directly to the anode. The desired cell potential can now be set on RI and will be held constant. Constant Current Operation. The calomel cell is removed and a stable precision resistor (about 0.33) is placed in series with the cell between the cell cathode and ground. The positive terminal of the small resistor is connected to the positive terminal of the battery B. Voltage Scanning. A simple voltage scanning device turned out to be a very useful feature of this instrument. It consisted of a large geared wheel mounted on the shaft of voltage divider R1 driven by a synchronous motor a t about l/g r.p.m. Different scanning rates could be obtained by changing the voltage of battery B. R, was also equipped with two variable stops activated
by microswitches and the synchronous motor was fitted with a reversing switch. These features combined with the multirange recording ammeter (ranges 0 to 1 ma. to 0 to 10 ampere9, in suitable steps) made the instrument a high current polarograph. APPLICATIONS
The instrument was used for a great many reductions at constant cathode potential. Inorganic reductions included mercury (II), lead (11), cadmium (TI), iron (III), copper (11), and ainc(I1) a t various concentrations. Organic reductions were carried out on maleic acid, several aliphatic mono- and polynitro compounds, several simple nitrate esters (6),and a few polynitrates. The quantity of electrical charge taken up was obtained by graphical integration of the i versus time record and, in some early experiments, by coulometric analysis. In all inorganic and most organic reductions, there was good agreement between experimental and theoretiral quantity of charge. The z versus time curve is strongly dependent on the rate of stirring and therefore a synchronous motor and gear arrangement was used to drive the stirrer a t a slow and constant speed (about 200 r.p.m,). At high currents, heating effects caused by the cell ZR drop are important and a large constant temperature bath did not eliminate them entirely in these experiments. Even with good constancy of stirring rate and temperature, the i versus time plot can differ greatly for different reductions. In most cases, the ratedetermining processes depend on the concentration of reducible material, and an exponential decay (first-order process) or similar de'crease of the current with time is observed. I n other cases, such as the reduction of Hg,++, the rate is independent of the concentration of reducible over most of the process. The current remains constant during that time and then falls sharply to zero. I n many reductions a t high negative cathode potential, the decrease of current which is due to the desired reduction is masked by the increasing liberation of hydrogen. Because of these complications, it was decided to study the effect of concentration using the voltage scanning device-i.e., obtaining high current polarograms a t a large quiescent mercury cathode. The results for cadmium sulfate solutions in 0.5M potassium sulfate solutions are shown in Figure 5 . As well-defined limiting current, i ~ is, obtained a t cadmium ion concentrations up to about 0.02111 and it is proportional to the concentration. A 30-fold increase in [Cd++] is accompanied by a decrease in i L / C of only 20%. This is in agreement with Hickling's findings (4)in similar
140t
:l'ol 100
VOLTS.VS.
Figure 6.
SAT CALOMEL
Reduction of Methyl Nitrate at Large Mercury Cathode
Supporting electrolyte, 0.9M sodium acetate, 0.5M acetic acid 1.0 is indicated on each curve Point where E = Methyl nitrate concentrationa: 1. Blank z. 4.41 x 1 0 - 4 ~ 3. 8.76 X 10-4M 1 7 2 Y in-aM i: i:zi x io-aM 6. 8.39 X lO-3M 7. 1.66 X 10-rM 8. 3.97 x 10-2M 9. 7.60 x lO-:M
-
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4 reductions. It is interesting that a value of Z’L/C = 12.5 milliamperes per millimole per liter was obtained with a cathode area of 42 square em. I n a regular polarogram of 0.001M cadmium(I1) with m = 2 mg. per second and t = 3.5 seconds, t d would be 7.0 pa The average surface area of the mercury drops is given by
&,“.
= 0.0051m2’3t2/3 = 0.0187 sq. cm.
Therefore, the current per unit electrode area in large scale experiments n a s 0.30 ma. per square cm. while in polarographic runs it would be 0.38 ma. per square cm. This means that the flux of reducible species is about equal in both cases and that the concentration gradients near the surface are similar. Similar experiments were run with nitromethane, e t h j 1 nitrate, and maleic acid in the presence of excess electrolyte. While there nas an indication of a limiting current a t very low concentrations in all cases, Z L was not too well defined and the current increased continuously. Figure 6 shows the nitromethane curves. i~ disappeared a t about l O - * X for nitromethane and a t even lower concentrations for ethyl nitrate and maleic acid. This may be due to irreversibility and low rate of the electrode reaction, accumulation of reduction products near the electrode, and contamination of the mercury surface. The low concentration runs are described here to emphasize the sensitivity and versatility of the instrument. With total currents as low as 10 ma. and correspondingly small changes in amplifier input and grid voltage, the i versus time graphs ‘sere entirely smooth and reproducible.
519
K i t h a line voltage of 220 volts, it is possible to have a large potential drop across the cell and this may become important in work with nonaqueous solutions. With cathode-calomel potentials as high as 8 volts in aqueous solutions containing only a trace of electrolyte, cell potentials of 150 volts a t currents of 3 to 4 amperes were obtainable. These applications will be expanded in future work. LITERATURE CITED
illlen. 11.J., ANAL.CHEM.,21, 178 (1949). Caldmell, C. W., Parker, R. C., and Diehl, H., IND.ENG.CHEM., ANAL. ED.,16, 5% (1944). Greenough, AI. L., Williams, W. E., Jr., and Taylor, J. K., Rev. Sei. Instr., 22,444 (19.51). Hickling, A , , Trans. Faraday SOC.,38,27 (1942). Kaufman. F., Cook, H. J. and Davis, S. >I., J . Am. Chem. SOC., 74,4997 (1952).
Lamphere, R. W., ANAL.CHEM.,23, 253 (1951). Lamphere, R. W. and Rogers, L. B., Ibid.. 22,453 (1950). Lingane, J. J., IND.ENG.CHEM.,ANAL.ED.,17, 332 (1945). Lingane, J. J., and Jones, S. L., A N ~ LCHEX., . 22, 1169 (1950). Miher, 0.W. C., and Whitten, R. K’.,Analyst, 77, 11 (1952). Penther. C. J. and Pompeo, D. J.. IND.ESG. CHEX..ASAL. ED.. 21,178 (1940).
“Vacuum Tube Amplifiers,” edited by G. E. Valley, Jr., and H. Wallman, p. 485, Sew York, AZcGraw-Hill Book Co.. 1948. Wehner, P., and Hindman, J. C., J . Am. Chem. SOC.,72, 3911 (1950). RECEIVED for review U a y 26, 1953.
Accepted January 4, 1954.
Determination of Adrenocortical Steroids in Mixtures ERICH HEFTMANN and DAVID F. JOHNSON National Institute o f Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda 74, Md.
Partition chromatography on paper is limited to relatively small amounts of substances. For conventional methods of identification and assay and for the determination of one compound in the presence of a large excess of others, column chromatography is preferred. A method for the separation of all six active adrenocortical hormones by partition chromatography on silicic acid columns is presented. Water is used as the stationary phase and a mixture of petroleum ether and progressively increasing amounts of dichloromethane is the mobile phase. Eluates are collected in an automatic fraction collector and assayed by ultraviolet spectroscopy. The sulfuric acid test serves as a method for identifying the fractions. Modifications of this method may be applied to biological extracts.
W
H I L E several methods for the separation of adrenocort,ical steroids by partition chromatography on paper are now available, there is still a need for a relatively simple procedure of quantitative analysis of hormone mixtures. The separation of adrenocortical steroids by column chromatography offers the advantage that relatively large amounts of mixtures can be handled, allowing the determination of one steroid in the presence of a large excess of another and making sufficient amounts of individual steroids available for conventional methods of identification and assay. Morris and TVilliams ( 5 ) have described the determination of individual adrenocortical steroids in blood by use of partition chromatography on Hyfio Super-cel columns with the solvent
systems toluene-ethyl alcohol-water and petroleum ethertoluene-ethylene glycol and subsequent polarographic estimation. Two methods of separating adrenocortical hormones by partition chromatography on silica gel columns have been reported. Katzenellenbogen el al. ( 4 ) have separated the acetates of the steroids, using dilute methanol or ethyl alcohol as the stationary phase and various mixtures of dichloromethane and petroleum ether a s the mobile phase. Haines ( 2 ) reported the separation of 10 mg. of each of various adrenocortical hormones on a column of 30 grams of silica gel impregnated with ethylene glycol. A series of solvent mixtures, containing increasing proportions of dichloromethane in cyclohexane, was passed through the column by means of an ingenious device, known as the “mechanical chemist.” I t occurred to the present authors that the same eflect could be produced by progressively increasing the dichloromethane concentration in cyclohexane, using an adaptation of the automatic dispenser of Donaldson et al. ( 1 ) . Using the solvent system of Haines, the two isomers A4-pregnene-llp,21-diol-3,20-dione (cor-
Table 1. Steroid
s S
B
E F
Recovery of Adrenocortical Steroids Amount Taken,
Bmount Recovered,
Y
Y
32 1 59 4 247.5 128 5 600 2 976 8
%
35 0 57 243 130 533 896
Recovery,
5 5 7 7 3
109 96 98 102 89 91
8 4 0 8
