gether as in this example. Finally, the window areas are masked with narrow strips of Scotch tape, the lower inlet tube is plugged and the assembly is dipped repeatedly into a thin acetone slurry of Eccobond 51 black epoxy resin (Emerson and Cuming, Inc., Canton, Mass.) until a black deposit has been built up. After cure, the masks are removed and small rectangular Plexiglas spacers are sealed onto the broad borosilicate glass faces of the cell to make the finished cell wide enough to fit correctly into the spectrophotometer cell compartment. If solutions of thiosulfate, cyanide, and ammonium hydroxide, which attack silver salts, are not to be encountered, the quartz windows can be sealed to the borosilicate glass in a more elegant fashion. First, the ground surfaces of the borosilicate glass body are coated with Liquid Bright Platinum No. 05 (Hanovia Liquid Gold Division, Engelhard Industries, Inc., East Newark, K. J.) and fired at 1025’ F. in a furnace
or lehr. The quartz windows are masked with tape and coated on the masked side with the platinizing solution. After the coat has dried, the masks are carefully removed and the windows are fired a t 1200’ F . A thin layer of a thick slurry of silver chloride in glycerine-water (50/50) is applied to the platinized borosilicate glass body. The windows, platinized side toward the borosilicate glass body, are then carefully put in place. In place of the silver chloride slurry, a “gasket” of silver chloride can be cut from sheet silver chloride (A. D. Mackay, Inc., New York 38, N. Y.). The assembly is then heated in the oven a t 840’ F. for 10 minutes and the cell is then allowed to cool slowly to room temperature. Masking of the outer surface of the windows and blackening of the cell is conducted as in the first method. The cell with the longer (straight) exit tube shown in Figure 2B is a finished assembly; windows have been
epoxy bonded. The shorter cell, assembled by the silver chloride technique, is complete with the exception of plastic spacers. Flow characteristics for two commercial cells are compared in Figure 3 with that obtained for cells described in this paper. The silver chloride-platinized glass technique finds general use for sealing together glasses with the same or differing coefficients of expansion ( I ) ; seals are vacuumtight (1 X 10-lo Torr). Sickel, nichrome, copper, alumel, and tungsten wires can be sealed directly into holes drilled in glass with silver chloride as sealant ( 2 ) . LITERATURE CITED
(1) Eichenbaum, A. L., Norman., F. H., Sobol, H., Rev. Sci. Znst. 35, 1056 (1964). (2) McCandles, H. E., private communi-
cation, RCA Laboratories, Princeton, N. J., 1964. Trade names referred to in this paper do not imply endorsement of commercial products.
A Scanning Potentiostat K. I. Wood, Division of Protein Chemistry, C.S.I.R.O., Wool Research Laboratories, Parkville N2, Melbourne, Victoria, Australia HE PRESENT apparatus was deT s i g n e d to provide controlled electrical conditions for studying the electrolytic reduction of groups such as disulfides in peptides and proteins ( 2 ) . Reduction could be effected either directly a t the mercury cathode surface or indirectly in the presence of a trace amount of carrier-e.g., thiol-which is continuously regenerated to the fully reduced state by electrolysis. The apparatus has a scanning feature for automatically producing a currentvoltage curve. This enables progress of preparative reduction to be quickly checked by pushing a button, rather than by taking an aliquot from the potentiostat cell and using a separate polarograph for estimations. The cell used, Figure 1, was similar to that of ikfeites (3) having a conical 500-ml. cathode compartment containing mercury of about 40 sq. cm. surface area, A glass stirring propeller is used to agitate the mercury cathode surface as vigorously as possible without causing detached droplets to be formed. This rapid and efficient stirring keeps the thickness of the diffusion layer to a minimum, permitting increased currents to flow, which expedites the electrolytic process. As a result of this stirring, the internal resistance of the cell is not steady and its value fluctuates in phase with the stirring motion. Potentiostats described in the literature have the disadvantage of being slow in response where a reversible
442 *
ANALYTICAL CHEMISTRY
ANODE-7 MOTOR
a
S. E.
- n
b
Figure 1 . Cell and basic circuit
CELL POTENTIAL POTENTIOMETER
DC.AMP: TO
motor has been used to control the power output from a rheostat or Variac, or inefficient in supplying large currents if entirely electron tube operated. Those where the output is controlled by power transistors ( I , 4, 6)
appear to offer the best solution to the requirements of high current handling capacity and an adequately rapid rate of correction. The present potentiostat is entirely line operated and transistorized through-
VOL. 37, NO. 3, MARCH 1965
443
out, capable of supplying currents up to 1 ampere, and a maximum of 50 volts is available when cell current is small. Figure 1 represents the basic setup. The current flowing from anode to cathode through the cell is controlled to maintain a constant preset value of potential between the mercury cathode and a standard calomel electrode (S.C.E.) immersed in the solution. This cell is used both analytically and preparatively. For the examination of a sample, a 6-r.p.m. synchronous motor coupled by a cork friction clutch, rotates the cathode potential potentiometer. The range covered by this potentiometer for the present work was chosen to be +0.5 to -3.0 volts but a reversing switch was included to extend the positive range should the need arise. The cathode potential is scanned and the applied current, which is controlled by the potentiostat, is simultaneously plotted with a 10-mv. strip chart recorder having a chart speed of 12 inches per minute. The resulting current-voltage curve may be examined for plateau indicating reducible sites. For preparative electrolytic reduction, the cathode potential corresponding to each plateau may be read from the curve, enabling the apparatus to be set to electrolyze a t a desired cathode potential. This allows selective reduction to be carried out, as only those sites where the half-wave potential is exceeded will be reduced. ELECTRONICS
The current applied to the cell from the voltage doubler power supply is controlled by two parallel-connected germanium power transistors type ADZ12, mounted on air convection cooled heat sinks of 80 square inch surface area each. Housing of the electronic gear in two separate units and grouping the power supply components and these heat sinks together, allowed the controls and input amplifying stages to be operated remotely from undesirable heating effects and hum pickup. The power transistors are driven by the output from a high gain d.c. amplifier comprised of 5 transistors. The two input transistors type BCZll connected as a “long-tailed pair,” were closely mounted in tight fitting, silicone smeared holes in a brass block to minimize the temperature difference between them. The BCZll fed from this input pair was also mounted in this brass block to increase its thermal inertia. The operating conditions of the d.c. amplifier input were chosen to give good voltage sensitivity while drawing least current from the standard calomel electrode. 444
*
ANALYTICAL CHEMISTRY
This was achieved by an initial setting up procedure which ensured that the point where zero input current flowed was about in the middle of the controlled cell voltage range. As shown in the circuit diagram Figure 2, a switch labelled Operate, Set 1, Set 2, and two potentiometers are provided for this purpose. These are located at the rear of the control unit and by successive adjustment of potentiometers Set 1 and Set 2, the cell voltage reading is brought to 25 volts on dummy load with the switch in each Set position. A measured input current required to turn the cell voltage from midscale down to zero and up to the maximum of 50 volts on dummy load was less than lo-* ampere of appropriate polarity. The overall gain of the system was such that a change from plus to minus 300 pv. was sufficient to change the cell voltage from zero to 50 volts under these conditions. To achieve stable, oscillation-free operation, the two phase-correcting networks and amplifier gain were adjusted for optimum on the completed setup. This was necessary as the cell forms part of the feedback loop and extremes of experimental conditions within the cell were explored for possible instability with a high gain, broadband oscilloscope. The circuit values were chosen for maximum safe gain for the cell used and no untoward tendency to instability has been shown over a 2l/2 year period. If a reduction of gain is necessary for some other experimental conditions not yet encountered, a series resistance can be added externally to the S.C.E. input lead. The power supplied to the cathode potential potentiometer is regulated by 2 stages of Zener diodes operated under conditions to give low temperature coefficient. Some care in transformer placement and shielding of components was necessary here to prevent hum being injected into the input from this floating supply. This was achieved satisfactorily provided the active and neutral line leads were not reversed. A neon indicator was included which glows to indicate the correct connection. Total ripple voltage from all sources between S.C.E. terminal and cathode under typical experimental conditions was less than 100 p v . peak to peak. The power supplied to the transistor amplifying stages is also regulated by Zener diodes and use is made of 1.5-volt cadmium-nickel electrolytic stabilizers to provide convenient low impedance voltage tappings. For reasons of economy, PNP germanium rather than silicon power transistors were used for the output stage and an additional small power supply was included to cancel their leakage current. By this means the
cell current may be reduced to zero, but to prevent any reverse current being applied to the cell during part of the Auto. Scan cycle, a series power diode is included in the anode current lead. To protect the power transistors in the event of an overload, a limiting circuit is employed which prevents a current greater than the maximum permissible from being exceeded. The current limit potentiometer is set so 1ampere cell current causes about 600 mv. to be fed to a silicon transistor which then conducts and prevents any further significant increase in current through the power transistors. Cell current is measured on a multirange meter protected against overload by a power diode, which includes a Log position ( 1 ) to enable a small residual end current to be detected after a large initial current without recourse to range changing. The output to operate a 10-mv. recorder is taken from a voltage divider across this meter. STABILITY
The electrical zero drift of the potentiostat system was assessed in the following way. The cathode potential potentiometer was set at 1.2 volts and an accurately measured input voltage from an adjustable source of 1.2 volts nominal, with an internal resistance of 2 kohms, was connected between the S.C.E. and cathode terminals. This input voltage was adjusted for a reading of 25 volts on the cell volts meter with a 500-ohm dummy load connected between anode and cathode terminals. Variation with time from this set reading of 25 volts was corrected by re-adjustment of the applied input voltage. Once the apparatus had come to temperature equilibrium within a n airconditioned laboratory, the maximum equivalent input drift variation was less than + l mv. for periods of several hours. COMPONENT PARTS
This apparatus was constructed with components readily available in Australia. Operation from a 117-volt, 60-cycle line however, could be achieved by choosing the 6-r.p.m. motor and transformer primaries to suit this line instead of 240-volt, 50-cycle as shown. Wire wound resistors and potentiometers were used in most instances for stability reasons rather than their 3watt rating. Where carbon resistors have been used they are of 1-watt rating and are labelled 1TYon the circuit diagram. Standard filament-type transformers were used for the power supplies. Some suggested American-type re-
placements for the Philips semiconductors used are &s follows---ehange ADZ12 to Motorola 2N174A, B C Z l l to Amperex 2N2617, OC74 to Motorola 2N1496, OA210 to RCA 1N3193, and BYZ12 to RCA 1N1614. Type 2N337 was manufactured hy Texas Instruments Inc. and type 125.6 hy International Rectifier Corp. Cadmium-nickel stabilizers type
15BC20 made hy L'Accumulateur Etanche, Belgium, would be available from Gould National Batteries h e . , St. Paul, Minn. ACKNOWLEDGMENT
The author thanks P. J. Beck for his assistance in the construction of the apparatus.
LITERATURE CTlED
~ ~ lM,l T,, ~ J ~ ,~ H, ~c,, Fisher, ~ ~ D. J., ANAL.CHEM. 31, 488 (1959). (2) Leach, S. J., Meschen, A,, Swsnepoel, 0.A., Rioehemislry, in press. (3) Meites, L., ANAL.CHEM.27, 1116 (1)
(1955). (4) Staicopoulos, D. N., Reo. Sei. I s l r . 32, 176 (lgF1), ( 5 ) Wadsworth, N. J., ~ ~ 85, 673 ~ (1960).
Powdered Sugar Adsorbent for Detection of Chlorophyll Components in Thin Layer Chromatography Marvel-Dare Nutting, Martin Voet, and Robert Becker, Western Utilization Research and Development Division, Agriculturol Research Service, U. S. Department of Agriculture, Albany, Calif. 94710
I
of an investigation of chlorophyll pigments in green vegetables (8) a simple microtechnique was needed for detection, identification, and proof of the purity of various chlorophyll components in amounts of microgram. Spectrophoto5 X metric determinations were satisfactorily made on 5 pg. per ml. However, an amount of contaminant too small to cause a change in the shape of the visible range absorption spectrum might go undetected if masked hy a component of greater concentration. Mangold (6) in 1961 suggested that "all adsorbent materials used in chromatography may also be applied in TLC." Since sugar columns had been successful for the separation of pigments in large N THE COURSE
amounts for many years and the available methods of glass strip (I, 3,4, 7) and glass plate (2, 6 ) TLC showed promise, various trial runs were made to determine a usable procedure. A mixture of confectioners powdered sugar containing 3% starch could be mixed with isopropanol 4:3 w./w. and after standing tightly covered overnight formed a smooth paste. This paste was applied to glass plates 20 X 20 em. with any of several commercial spreaders or a simple aerosol sprayer to give a satisfactory thin layer 0.25 to 0.35 mm. thick. After application of the adsorbent, the plates were dried a t 40' C. for one hour which produced a stable film that did not flake off appreciably on handline. The adsorbent
was then scribed into 10 to 14 strips 1 to 2 em. wide which produced strips similar to the ridged plates of Gamp et d (2). A tongue depressor whittled to a blunt end 2 mm. wide was used as a scraper by guiding along a straightedge laid across a 2- X 2-inch block on either side of the plate to completely and cleanly remove the adsorbent without scratching the glass. Each strip was spotted 2.5 em. from the bottom of the plate with 2 to 10 pl. of 100% acetone or ether solution containing a concentration of 5 to 15 X pg. (Solutions must be anhydrous when spotted on the sugar plates.) The solvent is allowed to rvaporate from the spots for 15 minutes and the spots are developed in a closed tank with solvent consisting..of 5% acetone in ikellysolve B, v./v. for 3'j2 hours. Figure 1 shows a plate which had been potted with known solutions of pheoihytin a and pheophytin b, drveloped n upward direction and subsequently photographed under ultraviolet light. The advantages of this method are its simplicity of preparation and t.he use of x ' --zdily ,r* available, inexpensive adsorbe n t which allows detection of very small amounts of pigment material. An amount of contaminant which failed to change the shape of the visible range . . absorption spectrum of the classical curve was discernible as a separate spot on a sugar plate when viewed under ultraviolet light. Strips 6 and 11 on Figure 1 were spotted with 1.0 X lo-' pg. of purified pheophytin a and showed one pink fluorescent hand which had traveled with the blue solvent front hut was not distinguishable on a black and white print. Strips 1 and 13 contained no pigment material and were blue on the solvent front under ultraviolet. Strips 4 and 8 were spotted with 2 X pg. of pheophytin b which contained a trace of pheophytin Q. Strips 3, 7, 9, and 10 illust,rate various stages of purification of crude material repre. 0
Figure 1. unknown
Thin layer powdered components
sugar
plate of known and
Spotted, dried, and developed 4 t h nrccndlng 5% acetone In Skelly solve B. v.[. for 3l11 hours. Photogrophtakenunder ultraviolet light, 80-sec:ond expofwe
VOL. 37. NO. 3, MARCH 1965
445
