Anal. Chem. 1988, 60,82-86
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Silver Composite Electrode for Voltammetry Steven L. Petersen and Dennis E. Tallman*
Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105
A composlte electrode fabricated from a unliorm mixture of silver and poly(chlorotrliluoroethylene) (Kel-F) powders Is descrlbed. The Kelsll (Kel-F/dlver) electrode, contahlng as little as 10% silver by volume, possesses high electrical conductlvity along wlth excellent mechanlcal strength and machlnablllty. Instructions for fabrkatlng the electrode are provlded and applications of the electrode to aqueous and nonaqueous voltammetry are cfemonstrated. Results of optical and scanning electron mkrbcoplc lnvestigatlons of the surface silver reglons of the composlte are also dlscussed. Compared to solld sllver electrodes, the advantages of the KeW electrode (and most likely of preclous metal composite electrodes In general) Include lower cost, lighter weight, and, on the tlme scale of many electroanalytlcal measurements, enhanced dmuslonal flux (current denstty) leadlng to currents exhlbltlng reduced tlme dependence.
A variety of approaches have been used over the years to fabricate new composite electrode materials. In some cases a porous conductor has been impregnated with an insulator, as in the fabrication of the wax-impregnated graphite (I)and epoxy-impregnated reticulated vitreous carbon (2) electrodes. In other cases a powdered conductor (usually carbon) is suspended in an insulating matrix consisting of either a viscous liquid, as in the fabrication of the carbon paste electrode (3), or a polymer, as in the fabrication of the epoxy/graphite (41, silicone rubber/graphite ( 5 ) ,polyethylene/carbon black (6), Teflon/graphite (7), and Kel-F/graphite (8) electrodes. Alternatively, bundles of carbon fibers may be suspended in an epoxy matrix, in either parallel (9,lO) or random (IO) fashion. Virtually all of the composite electrodes introduced to date have been based on carbon. We know of no previous work describing composite electrodes based on precious metals. In this paper we describe a new precious metal composite electrode fabricated by suspending finely divided silver metal in a matrix of poly(chlorotrifluoroethy1ene) (PCTFE), also known as Kel-F (3M Co.). The composite material, hereafter referred to as Kelsil, contains as little as 10% silver by volume and yet possesses high electrical conductivity. The Kelsil electrode exhibits electrochemical behavior typical of an ensemble of microelectrodes, including enhanced diffusional flux and diffusion current which is less time dependent. As a result, Kelsil may prove superior to solid silver electrodes in a variety of electrochemical applications including amperometric detection in ion chromatography (11)and flow injection analysis (12),anodic stripping voltammetry ( I 3 ) ,and reductive voltammetry of nitrate esters and nitroaromatics (14). Furthermore, precious metal composite electrodes, for which Kelsil may be considered a prototype, should find application where cost (Kelsil is ca. 90% polymer by volume) and/or weight (the density of Kelsil is ca. 30% that of silver metal) of electrode materials is an important consideration. In this paper we describe the fabrication of the Kelsil composite, examine the surface silver regions of the composite by optical and scanning electron microscopies, and present the results of several types of electrochemical experiments
performed at the Kelsil electrode. EXPERIMENTAL SECTION Reagents and Materials. All chemicals were reagent grade unless otherwise noted. Tetraethylammoniumperchlorate (TEAP, Eastman) and p-chloronitrobenzene (PCNB, Eastman) were purified by recrystallization. The 2,4-dinitrotoluene (DNT, Eastman) and o-dinitrobenzene (DNI3, Aldrich) were used without further purification. All aqueous solutions were prepared from Milli-Q water (Millipore Corp.). Acetonitrile (SpectrAR, Mallinckrodt) was stored over 3A molecular sieve (Union Carbide) to minimize water content. The silver used in the fabrication of the composite electrodes was AESAR 1-3 pm silver powder (99.9%, Johnson Matthey, Inc.). Silver disk electrodes were constructed from 16- and 20-gauge silver wires (General Refineries, Inc.). The PCTFE used in the fabrication of the Kelsil composite was Kel-F 81 powder (3M Co.). The electrodes used for voltammetry were prepared from Kel-F particles which passed a 200-mesh sieve and therefore had dimensions of 74 pm or smaller. A couple of electrodes were prepared from Kel-F particles having dimensions between 7 5 and 150 pm; these electrodes were used in microscopic comparisons with the electrodes prepared from the smaller Kel-F particles. Apparatus. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were performed by using an in-house constructed potentiostat of the standard difference amplifier controller design. Voltage waveforms for CV and LSV were generated by a PAR Model 175 universal programmer (Princeton Applied Research). Differential pulse voltammetry (DPV) was performed with a PAR Model 174A polarographic analyzer. Voltammograms were recorded on a series 2000 Omnigraphic x-Y recorder (Houston Instrument). The auxiliary electrode was a glass-sealedplatinum coil. For aqueous voltammetry the reference electrode was Ag/AgC1/3.5 M KCl which was isolated from the cell via a 0.1 M NaN03 salt bridge. The salt bridge contained 0.1 M NaOH for voltammetry in NaOH solution. For nonaqueous voltammetry the reference electrode was Ag/O.Ol M AgN03, 0.1 M TEAP in acetonitrile and was separated from the cell with a salt bridge containing the supporting electrolyte in acetonitrile. Isolation salt bridge solutions were replaced daily. Scanning electron micrographs of electrode surfaces were obtained on a Jeol Model JSM 35 scanning electron microscope. Photomicrographswere obtained on an Olympus Vanox-1 optical microscope. Procedures. Electrochemical measurements were performed at room temperature (22-24 "C) on 50-mL volumes of solution. Solutions were sparged with purified, solvent presaturated nitrogen for at least 15 min prior to measurement, and the solution was blanketed with nitrogen during measurement. Electrodes were polished prior to the recording of each voltammogram. For nonaqueous measurements, the electrodes were hand polished with a 0.3-pm alumina/water slurry on broadcloth polishing cloth (Mark V Laboratory), then polished on bare cloth, rinsed with water, rinsed with dry acetonitrile, and air-dried. For aqueous electrochemistry, the electrodes were polished with 1and then 0.3-pm alumina/water slurries, polished on bare cloth, rinsed with water, and air-dried. Polishing on bare cloth was found necessary to remove alumina which adhered to the electrode surface even after extensive rinsing. Sonication of the electrodes preceding and/or following polishing had no noticeable effect on their electrochemical behavior. Electrode Fabrication. Silver and PCTFE powders were mixed in the desired proportion with LC grade methanol and the slurry was sonicated to break up large silver agglomerations. Steel balls were added t o the mixture which was then rotated on a
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Flgun 1. Completed Kekil electrode assembly.
Fbtovap until the mixture was uniform. Heat was then applied to drive off the methanol, and the composite was further dried in an oven at 115 'C. Finally. the dry composite was again hall milled to ensure a uniform mix. A portion of the Kelsil powder was gently packed into a 6.4 m m diameter stainless steel die. The die was plaeed in a wcuum chamber and pressed under vacuum with CB. 150 Ih of force in a Carver laboratory press. The die was then removed from the vacuum chamber, placed hack in the press, wrapped with heating tape, and heated to ea. 290 O C under ea. 300 Ib of force. When the PCTFE had melted, as signaled by the onset of a pressure increase, the heating tape was removed, the force on the die was increased to ca.2wO Ih, and the die was quickly moled to ambient temperature with a spray of cool water. The Kelsil pellet was then removed from the die and the ends of the pellet were sanded successively with wet 220-, 400-, and 600-grit carborundum (Wetordry Tri-mite Paper, 3M Co.). Following a resistance check, a concentric hole was drilled into one end of the pellet and a copper wire was press fit into the hole to provide electrical contact. Resistance measurements with a metal probe indicated that this single point contact was sufficient to provide conduction to silver sites across the entire working surface of the pellet. A section of plastic soda straw was slipped over the copper wire and the end of the straw was fastened to the Kelsil pellet with tape. The straw was filled with Epon 828 epoxy resin (Shell Chemical Co.) containing 11% triethylenetetramine (Eastman) as the curing agent. This provided electrical insulation for the copper wire contact. When the epoxy had hardened, the straw was removed, the Kelsil end of the assembly was placed concentridy in a 3 cm section of 1cm diameter Teflon tubing (one end of which had been epoxied to a computer card), and the Teflon tubing was filled with Epon 828. This last step provided an insulating sheath around the outer circumference of the cylindrical Kelsil pellet. When the sheath had hardened, the Teflon tubing was cut away and the electrode assembly was further cured overnight in an oven at ea. IO "C. The finished electrode assembly was sanded with wet 2mgrit carhonmdum until the KeIsil face waa exposed and then with 400and 600-grit carhorundum. The electrode was then polished with a 1-pm alumina/water slurry on polishing cloth until sanding scratches were no longer visible under an optical microscope. A finished Kelsil electrode is shown in Figure 1. Silver disk electrodes were prepared by cleaning silver wire in dilute nitric acid, cutting the wire into 0.5-cm sections, and encasing each section in PCTFE hy using the compression-molding technique described above. The silver disk electrodes used here had diameters of 0.76-1.3 mm.
RESULTS AND DISCUSSION Physical Characterization. The silver content of the composite could range from a high of ca. 80 wt % to a low of ca. 35 w t %, the upper limit being governed by the mechanical stability of the composite and the lower limit by the requirement for sufficiently high electrical conductivity. The electrodes of this study were of two compositions, either 45.2 wt % silver (corresponding to 14.2 vol %) or 38.2 wt % silver (corresponding to 11.0 vol 70).As observed with Kelgraf
Flgun 2. Optical micrographs of two differentKeisii electrodes p r e pared from KeCF particles with dimensions of (a) under 75 pm and (b) 75-150 pm. The bars represent 100 pm.
composite electrodes (1.9, lower conductor content results in reduced active site sizes. The compositions used in this study reflect a compromise between minimizing the nominal active site dimensions and maintaining a suitable electrical conductivity. Since volume percent of silver in the composite closely reflects the percent of geometric area which is active silver, all further references to composition will be in terms of volume percent. I t is the size discrepancy between the Kel-F particles (nominally 100 pm in dimension) and the silver particles (nominally 1 Irm in dimension) which makes possible the fabrication of a conducting composite having such low silver content. During initial stages of fabrication, the small silver particles occupy the interstitial space between the larger Kel-F particles, forming a highly connected network of packed silver particles pervading the Kel-F matrix. During compression molding, this network apparently remains intact, yielding a rigid material with good conductivity and yet possessing many of the advantageous properties of the Kel-F polymer itself. For example, the physical strength and the machining characteristics of the 14.2% composite appear to differ very little from that of Kel-F itself. The electrical resistance of this composite was too low to measure with an ohmmeter. Using the four-probe method, we estimate the conductivity of the 14.2% composite to be 4400 mhos/cm. Optical micrographs of the surfaces of two Kelsil composite electrodes are shown in Figure 2. The view of this material under a binocular stereomicroscope is truly spectacular since one can peer into the three-dimensional network of subsurface silver through the largely transparent Kel-F matrix. Surface silver appears as the blackest regions of the micrographs (Figure 2) while subsurface silver exhihits a very bright rather sparkling appearance (the whitest regions of Figure 2). The rather hazy appearing regions of the micrographs are areas where one is peering deeper into the composite through transparent Kel-F. The surface silver regions appear much more homogeneous in size and in distribution than were the active regions of our earlier Kelgraf electrodes (15),a conse-
ANALYTICAL CHEMISTRY. VOL. 60. NO. 1. JANUARY 1. 1988
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quence of our tighter control of Kel-F particle size distribution in this work. As observed in our earlier work with Kelgraf (15),increasing the size of the Kel-F particles or increasing the silver content of the composite leads to an increase in average active site dimension. The electrode in Figure 2a was prepared from Kel-F particle8 with dimensions under 75 pm while the electrode in Figure 2h was prepared from Kel-F particles having dimensions between 75 and 150pm. Careful inspection of the optical micrograph of Figure 2 reveals that somewhat larger surface silver regions are ohtained when the larger Kel-F particles are used in the fabrication of Kelsil. The fraction of the geometric electrode area consisting of silver was computed hy a projection method. Approximately 200 silver sites were traced from a selected region of a projected optical micrograph. By the cut-and-weigh method it was determined that 13.4% of the geometric surface consisted of silver, in very good agreement with that predicted from the electrode composition of 14.2%. The aspect ratio of each of the silver regions WBS also measured and ranged from 1to 7, with an average of 2.4 and a standard deviation of 1.2, reflecting the elongated nature of the silver regions. Higher aspect ratios reflect a greater perimeter-toarea ratio for each silver region, a desirable feature for enhancing flux and, hence, current a t each region by the edge effect (16). The largest dimension of the silver regions averaged 44 pm while the dimension measured normal to and a t the midpoint of this largest axis averaged 20 pm. The geometric area of a 6.4 mm diameter electrode consists of ca. 4700 active regions or microelectrodes. Scanning electron microscopy (SEM) permits closer inspection of the surface silver regions of Kelsil. Figure 3a shows an SEM micrograph taken in the same region of the same electrode as the optical micrograph of Figure 2h. Under SEM observation, conducting surface silver appears gray while insulating Kel-F charges under the influence of the electron beam and appears white, except where Kel-F is in intimate contact with conducting silver in which case the charge is dissipated and these regions of Kel-F then appear void (or
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black). Very little surface silver appeara to be nonconducting or electrically isolated. A couple of very small such regions of silver. can be identified by comparing silver sites in the optical micrograph of Figure 2b (which reveaLs all surface silver regions, conducting and nonconducting) with the corresponding image8 in the SEM micrograph of Figure 3a (where nonconducting surface silver tends to charge and blend in with the surrounding Kel-F). For the most part, surface silver is confined to small conducting regions. A closer look a t one of these regions from Figure 38 is provided in Figure 3b, where the individual silver particles making up the region can be discerned. Electrochemical Characterization. The intent of this section is to demonstrate the utility of the Kelsil composite material as a working electrode for both aqueous and nonaqueous analytical voltammetry. In order to establish that the silver regions on the surface of the composite electrode electrochemically resemble the surface of polycrystalline silver metal, we have examined the potentiodynamic behavior of oxide formation and reduction a t the Kelsil electrode in aqueous alkaline solution, comparing this behavior to that ohtained at the silver disk electrode (17,18). Representative cyclic voltammograms for the formation and reduction of silver oxides in fresh 0.1 M NaOH are shown in Figure 4 for (a) Kelsil and (h) Ag disk electrodes. The highly peaked c h a r a h r of the waves is indicative of surface processes, with peaks 1 and 2 corresponding to the formation of Ag(I)-oxygen containing species, peak 3 to the formation of Ago, and peaks 4 and 5 to the reduction of Ago and Ag20, respectively (17, 18). The peak potentials, peak current densities, and shapes of the voltammetric curves of Figure 4 are quite similar, indicating little if any intrinsic difference between the surface silver regions of Kelsii electrodes and the surface silver of silver disk electrodes. Current densities a t Kelsil are based on the computed area of surface silver. The voltammogramsof Figure 4 were recorded on the tenth cycle of a repetitive potential sweep program and agree well with those reported by Teijelo et al. (17). As observed by other workers, the peak currents increase with subsequent m s and eventually stabilize, a behavior attributed largely to an increase in the degree of surface roughness of the hase metal upon repetitive cycling (17,18).We observed that hy the tenth cycle the peak currents were still increasing slightly, hut at a very slow rate. The similarity of the current densities a t the KeLsil
ANALYTICAL CHEMISTRY, VOL. 60, NO. 1, JANUARY 1, 1988
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and Ag disk electrodes (agreement to within ca. 7%) suggests a similar degree of surface roughness is established at these two electrodes upon repetitive cycling of the potential. In order to assess the reproducibility of Kelsil electrode surfaces, linear sweep voltammetry was performed on DNT and PCNB in 0.5 M KC1 at pH 10. Under the conditions employed here, these compounds undergo a four electron per nitro group reduction to the corresponding hydroxylamines (14, 19). Fine and Miles found that all three meta isomers of DNT show a single eight-electron reduction wave a t Ag under the conditions stated above, with chloride solutions providing the sharpest waves (14). Voltammograms of saturated aqueous solutions of the individual nitroaromatics (concentrations were determined by optical absorbance) were obtained from -0.1 to -0.9 V vs. Ag/AgCl at a scan rate of 50 mV/s. Ten scans, each separated by a 1-min pause with gentle stirring, were recorded for each new surface of a given electrode, and this was repeated for five to ten new surfaces on each electrode. Typical voltammograms for DNT are shown in Figure 5 for (a) Ag disk and (b) Kelsil electrodes. While the voltammograms obtained at Ag disk electrodes exhibit normal peak-shaped waves, those obtained at Kelsil electrodes have a sigmoidal or nearly sigmoidal shape, characteristic of the steady-state current observed at microelectrode ensembles under appropriate conditions of scan rate and microelectrode dimensions and separations (20). The small second wave in the voltammogram of DNT at Kelsil (barely discernible but present in the Ag disk voltammogram) was not present in background scans nor was it removed upon further purification of the DNT by recrystallization. Since a single reduction wave for DNT is expected under our experimental conditions (14),the origin of this small wave remains a mystery. The voltammograms obtained for PCNB (not shown) also exhibited near-steadystate behavior at the Kelsil electrode. Enhancements in current density for several different Kelsil electrodes relative to a Ag disk electrode averaged 1.6 and 2.1 for DNT and PCNB, respectively. The reproducibility of the Kelsil active surface appears to be comparable to that for Ag disk electrodes, with peak/limiting currents having a relative standard deviation (RSD) of ca. 5% upon repeated resurfacing of a single Ag disk or Kelsil electrode. The variation in the peak/limiting current for a given electrode surface in a single solution was ca. 1% (RSD) a t both Ag disk and Kelsil electrodes. The limiting current .for two different Kelsil electrodes prepared from the same batch of composite powder agreed to within 5 % . Kelsil electrodes are also suitable for voltammetry in nonaqueous solutions. The cyclic voltammetry of PCNB and
Figure 6. Differential pulse voltammograms of 2.0 mM p-chloronltrobenzene In acetonitrile containing 0.20 M TEAP at (a)an 11.0% (v/v) Kelsil electrode of dlameter 6.4 mm and (b) a Ag disk electrode of diameter 1.3 mm. Scan rate = 2 mV/s, modulation amplitude = 25 mV, and pulse frequency = 2 Hz.
DNB in acetonitrile at Kelsil electrodes exhibits chemically reversible behavior, corresponding to reduction to the radical anion on the forward sweep and subsequent oxidation back to the parent compound on the return sweep (19). Again enhancements in current density are observed, averaging ca. %fold higher for three different Kelsil electrodes at a scan rate of 50 mV/s. In all cases the enhancement in current density a t Kelsil electrodes decreases with increasing scan rate, the expected behavior for a microelectrode ensemble (20). One example of nonaqueous voltammetry at the Kelsil electrode is provided in Figure 6, showing the differential pulse voltammogram of 2 mM PCNB in acetonitrile. Peak shapes and peak potentials are quite similar for voltammograms obtained at Kelsil (Figure 6a) and Ag disk (Figure 6b) electrodes, reflecting similar degrees of electrochemical reversibility at these two types of electrode materials. The differential current density measured at the peak maximum is 2-fold higher at the KeKi electrode. Background currents are also significantly higher at the Kelsil electrode (Figure 6a), although the analytical utility of the peak does not appear to be compromised. This increased background is believed to be capacitive in origin, perhaps reflecting the micron dimension roughness of the silver regions of the Kelsil electrode. The effects of electrochemical pretreatment on the double layer capacitance of Kelsil electrodes are under investigation and will be reported in a future communication.
CONCLUSIONS Kelsil is one of the first precious metal composite materials to be fabricated as an electrode for electroanalytical measurement. This work demonstrates the feasibility of our approach for producing new, lightweight, low-cost, readily machinable precious metal electrode materials which may find application in many areas of electrochemistry, including energy generation and storage, bulk electrolysis, and electrosynthesis. For analytical applieations, the enhanced current density at Kelsil composite electrodes may result in improved limits of detection for certain electroanalytical measurements, depending on the time scale of the measurement. Perhaps the most promising analytical application of Kelsil will be for electrochemical detection in flowing streams, where significant enhancements in convective-diffusion current are anticipated (21, 22). Future research will explore the advantages of mercury-plated Kelsil electrodes for reductive mode electrochemical detection in liquid chromatography and flow injection analysis.
ACKNOWLEDGMENT The authors thank Christian Oseto for assistance in obtaining the optical micrographs. The authors are also grateful to 3M Co. for supplying the Kel-F used in this work.
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LITERATURE CITED (1) Morris, J. B.; Schernpf, J. M. Anal. Chem. 1959, 3 1 , 286. (2) Sleszynski, N.; Osteryoung, J.; Carter, M. Anal. Chem. 1984, 56, 130. (3) Aderns, R. N. Electrochemlstry at Solid Electrodes; Marcel Dekker: New York, 1969;pp 26-27, 280-2133, (4) Anderson, J. E.; Tallman, D. E. Anal. Chem. 1978, 48,209. (5) Nagy, G.; Feher, 2s.; Pungor, E. Anal. Chlm. Acta 1970, 52,47. (6) Arrnentrout, D. N.; McLean, J. D.; Long, M. W. Anal. Chem. 1979, 51, 1039. (7) Klatt, L. N.; Connell, D. R.; Adarns, R. E.; Honigberg, I. L.; Price, J. C. Anal. Chem. 1975, 47,2470. (8) Anderson, J. E.; Tallman, D. E.; Chesney, D. J.; Anderson, J. L. Anal. Chem. 1978, 50, 1051. (9) Caudill, W. L.; Howell, J. 0.; Wightrnan, R. M. Anal. Chem. 1982. 54,
2532. (10) Nacernulli, L.; Gileadi, J. J. Appl. Electrochem. 1982, 12, 73. (11) Rocklin, R. D.; Johnson, E. L. Anal. Chem. 1983, 55,4. (12) Eggll, R.; Asper. R. Anal. Chlm. Acta 1978, 101, 253. (13) Stojek, 2.; Kublik, 2. J. Elechoanal. Chem. 1977, 7 7 , 205. (14) Fine, D. A.; Miles, M. H. Anal. Chim. Acta 1983, 153,141.
(15) Weisshaar, D. E.; Tallman, D. E. Anal. Chem. 1983, 55, 1146. (18) Oldharn, K. B. J. Electroanal. Chem. 1981, 122,1. (17) Teijelo, M. L.; Vilche, J. R.; Arvia, A. J. J. Nectroanal. Chem. 1984, 162, 207. (18) Hepel, M.;Tornkiewicz, M. J. Electrochem. Soc. 1984, 131, 1288. (19) Kernula, W.; Krygowski, T. M. I n Encyclopedia of Electrochemistry of the Elements Bard, A. J., Ed.; Marcel Dekker: New York, 1979;Vol. XIII;Chapter XIII-2. (20) Reller, H.; Kirowa-Eisner, E.; Gileadi, E. J. Electroanal. Chem. 1984, 161, 247. (21) Tallman, D. E.; Weisshaar, D. E. J. Li9. Chromafogr. 1983, 6 , 2157. (22) Cope, D. K.; Tallman. D. E, J . Nectroanal. Chern. 1986, 205, 101.
RECEIVED for review June 25,1987. Accepted September 22, 1987. This work was supported by the U.S.Department of the Interior/Geological Survey through Grant No. 14-080001-G1441 administered through the North Dakota Water Resources Research Institute as Project No. 6582.
CORRESPONDENCE Highly Selective Protein Separations with Reversed Micellar Liquid Membranes Sir: The separation and purification of proteins continue t o be areas of great interest and intense investigation. This area of separation science has received impetus from the expanding fields of biotechnology and genetic engineering (1-10). Over the last decade, liquid chromatography (LC) has emerged as an important technique for the analysis of biologically important molecules. It has been used for separating closely related as well as disparate proteins, determination of structural form, peptide mapping, and so on (11,12). The various theoretical and empirical shortcomings of LC have been documented as well ( 7 , 8 , 1 3 ) .From a practical standpoint, LC is a fine analytical tool for the analysis of whole proteins but is often inadequate for quantitation and preparative scale separations. Some of the problems result from the nature of the solute (i.e. the protein) while others result from the nature of the technique (i.e. LC). For example, problems of protein denaturation and irreversible adsorption are well-known and much work has been done to minimize these (7-13). Also, LC is a batch process, which requires large amounts of support and mobile phase for moderate-sized preparative separations. An ideal system for the preparative separation of proteins would be compact, have the efficiency and selectivity of LC, the ability to operate in a continuous mode and easily handle large quantities of protein with little or no denaturation. Currently no such technique or system exists. In the late 1970s Luisi and co-workers (14,15)and Menger and Yamada (16)demonstrated that proteins could be solubilized in nonpolar organic solvents without adversely affecting their conformation or enzymatic activity. This was done by using certain reversed micelle forming surfactants. Recently there have been attempts to utilize reversed micelles in the separation and purification of proteins. Goklen and Hatton took advantage of this phenomenon and demonstrated that significant amounts of protein could be separated and purified with no denaturation by using solvent extraction with reversed micelle containing organic solvents (17, 18). The apparent selectivity of this method was impressive as was the ability to maintain the activity of the protein.
Membrane-based separations are easily configured to operate in a continuous, preparative-scale mode. Unfortunately, most membrane techniques lack the selectivity necessary for many important separations including that of proteins. In this work we examine reversed micellar liquid membranes and show that they can be used to efficiently separate proteins while maintaining selectivitiescurrently found in better-known methods. Problems of denaturation during isolation and purification often are minimal. EXPERIMENTAL SECTION Materials. Aerosol OT (AOT), also known as sodium dioctylsulfosuccinate,was obtained from American Cyanamid Co. The proteins cytochrome c (horse heart), myoglobin (horse heart), and lysozyme were obtained from Sigma and used as received. Bovine serum albumin (BSA) was obtained from Advanced Separation Technologies. HPLC grade water and n-hexane were obtained from Fisher Scientific. Methods. A schematic of the membrane chamber used in the protein transport studies is shown in Figure 1. The chamber was filled with 4.0 mL of 0.1 M NH,OAc (adjusted to the desired pH) and different concentrations of KCl (from 0.05 to 0.20 M). The liquid membrane (0.2 mL) was injected into the connecting tube (Figure 1)with a syringe connected to a 5-cm piece of microbore Teflon tubing. The reversed micellar liquid membrane was made by dissolving the desired amount of AOT in n-hexane and presaturating this with the protein solution to be separated. Each experiment began by adjusting the KCl concentration as desired and placing 10 mg of protein in one side of the chamber (henceforth referred to as the mixture side). The protein concentrations on the receiving side were determined by UV spectrophotometry, using a Perkin-Elmer 559 instrument. BSA and lysozyme were quantitated at 280 nm and cytochrome c and myoglobin were quantitated at 408 nm. Equilibration times of 24 h were used in this study. More intensive data on transport rates and large scale competitive separations are to be presented in a more extensive future article. Both UV and circular dichroism (Jasco 20) spectrophotometry were used to monitor all proteins for conformation changes and denaturation. RESULTS AND DISCUSSION There are a t least three basic phases that must be consid-
0003-2700/88/0360-0086$01.50/0C 1987 American Chemical Society
