Anal. Chem. 1986,58,2549-2554
2549
Rapid Scanning Coulostatic Liquid Chromatography/Electrochemistry in Dual Electrode Cells R i c h a r d K. Trubey' a n d Timothy A. Nieman*
Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
Rapid scannlng coulostatlc Instrumentation was employed at one working electrode of a series dual-electrode flow cell while conventlonal flxebpotentlal amperometry was employed at the other electrode. A flow cell was designed that permtts compatlble operation of a coukmtat and a potentlostat as they slmultaneously control potentials of electrodes In close physical proxlmlty. A circuit for mlnlmlzlng electronlc crosstalk was Implemented. Use of the coulostat downstream of an electrode held at flxed potentlal enabled voltammograms to be obtalned for both Injected species and specles produced at the upstream electrode. For the hydroqulnone/l ,Cbenzoqulnone system, satisfactory voltammograms were obtalnable for 100 pM solutlpns ( 2 nmol Injected). The analytical detectlon limit was 16 pM (0.3 nmol Injected). Appllcatlon to a thiol/dlsulflde mixture was lnvestlgated. The feaslblllty of coulostatlc detectlon upstream of an electrode under potentlostat control was also studled. Significant voltammetrlc lnformatlon was found to exlst at the downstream electrode. Thk lnformatlon, however, Is somewhat convoluted by translt tlme between working electrodes If that tlme Is slow relative to the duration of the scan.
Liquid chromatography combined with electrochemical detection (LCEC) has played a prominent role in the rapidly expanding applicability of high-performance liquid chromatography (HPLC) to the needs of modern chemical analysis. Several excellent reviews attest to the considerable utility of this approach (1-5). In recent years, investigators have enhanced the capabilities of LCEC either by including more than one working electrode in the detection cell and holding their potentials constant or by scanning the potential at a single working electrode. The former strategy has been commonly employed in three configurations: parallel-adjacent, parallel-opposed, and series. Such dual-electrode approaches have been reviewed recently (6). Methods of effecting potential scans have been based chiefly on square wave voltammetry (7),staircase voltammetry (8),or coulostatic methods mimicking voltammetric scans (9-11). Tensammetric detection (12),semiintegral electroanalysis (13),semidifferential electroanalysis (14), and ac voltammetry (15) have also been utilized. The primary motivation behind most of these advanced LCEC strategies is the desire to create a detector better able to deal with the stringent demands imposed by the complex samples commonly encountered. Dual-electrode cells often provide much-needed selectivity enhancements. For example, parallel-adjacent designs allow simultaneous detection of hard-to-electrolyze species a t one potential and of easily electrolyzed solutes a t a less extreme potential. Parallelopposed and series configurations can be made highly selective for reversible or quasi-reversible species. Addressing other Present address: E. I. du Pont de Nemours & Co., Agricultural Chemicals Division, Building 402, Experimental Station, Wilmington, DE 19898. 0003-2700/86/0358-2549$01.50/0
analysis problems, users of scanning LCEC detectors enjoy many of the established advantages associated with conventional scanning detectors such as scanning UV absorbance detectors. Strengths of such detectors include the facilitation of reliable peak identifications and the capability of obtaining considerable qualitative information about the sample very rapidly. In addition, data obtained from scanning LCEC detectors may be plotted as a series of chromatograms, each corresponding to a different electrode potential. Thus, it is sometimes possible to resolve coeluting peaks in the potential domain. In work presented here, the approach of scanning electrode potentials is applied to a dual-electrode cell having the series configuration. Potential scans may be effected at the downstream electrode while the electrode potential upstream remains fixed. Voltammetric information is then obtainable on species produced as a result of reaction at the upstream electrode. Likewise, if the electrode potential is swept at the upstream electrode and the potential downstream is held constant, selected products of reaction at the upstream electrode may be monitored at the downstream electrode. A coulostatic instrument is used at one electrode to sweep electrode potentials by means of application of a staircase type potential wave form. Several reports of coulostatic LCEC have already appeared (9-11). The coulostatic method is potentially superior to a potentiostat-based approach in two major respects. Use of a coulostat prevents the electrode double layer charging current from interfering with faradaic current measurements, a concern a t high scan rates; also, because measurements are made at open circuit, there is no iR drop error, so high-resistance solutions can be employed (16). This paper describes results of a successful combining of dualelectrode concepts with the potential scan approach. Enhancements in LCEC detection capabilities are presented and discussed. EXPERIMENTAL SECTION Instrumentation. The electrochemical flow cell employed (Figure 1) was constructed from commercially available components (Bioanalytical Systems (BAS), West Lafayette, IN). Two 3.2 mm diameter glassy carbon electrodes were imbedded in a block of Kel-F (BAS MF1000). Holes for the flow inlet and for waste were drilled and tapped (size 10-32) to accept standard HPLC high-pressure fittings. The bottom half of the cell was also constructed from Kel-F. Here, either two 3.2 mm diameter gold disks (BAS MF1002) or both a gold disk and a glassy carbon disk (BAS MF1006) were located. Holes were drilled and tapped (size 1/4-28) to accept two reference electrodes (Ag/AgCl, BAS RE-3). The glassy carbon electrodes on top served as counter electrodes and were separated from the working electrodes underneath by a 0.13 mm thick Teflon spacer (BAS TG-SM). An oval, which defined a narrow channel for the flow stream, was cut out from this gasket. The distance from the upstream electrodes to the downstream electrodes (close edge to close edge) was 0.6 mm. The figure indicates the coulostat controlling electrodes located downstream of electrodes under control of a potentiostat which was a standard LCEC amperometric detector (BAS Model LC-4B);the reverse arrangement was also utilized. Some work was performed with gold working electrodes that were modified by the addition of triple distilled mercury to create a 0 1986 American Chemical Society
2550
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Waste
II
f
50nA
k
RE
-
LkZ-lJk-iu
POTENTIOSTAT
Mrninq (b)
C
F w e 1. Duaklectrode flow cell. Black electrodes are glassy carbon; white electrodes are gold. The letters r, c, and W refer to the ref-
erence, counter, and working electrodes.
(a) Figure 3. Chromatograms demonstrating effectiveness of crosstalk reduction circuit. Coulostat parameters: 100 ms hold time; 50 ms integrate time: 1000 ms pause time. (a)Without circuit. (b) Wth circuit.
Figure 2.
Schematic diagram of crosstalk reduction circuit.
Hg/Au amalgam (17). Glassy carbon was not used as a working electrode because of the high and variable background current that we find under conditions of rapid scanning; this current is presumably a result of the highly active surface chemistry commonly associated with carbon electrodes (18). The microprocessor-controlled coulostat was constructed inhouse and has been described previously (9). The coulostat software repeatedly applied a staircase potential wave form (with 10 equally spaced potential steps) throughout the duration of a chromatographic run. Data were obtained that could be plotted as either chromatograms or voltammograms. It should be noted that the flow cell (Figure 1)can be divided into two distinct three-electrode electrochemical cells having two separate paths for current flow. Six electrodes are necessary because the coulostat and the potentiostat must operate independently of each other. This can be contrasted to the more familiar means of imposing potential control at two working electrodes as carried out using a biopotentiostat (6); with that type of instrument only one counter electrode is used, and the working electrodes are linked together electrically. The great dissimilarity in design between a coulostat and a potentiostat does not permit such an arrangement. Crosstalk between the electrodes in the flow cell was dealt with by placement of the circuit shown in Figure 2 between the potentiostat current output and the strip-chart recorder input. The coulostat increments a BCD counter (TTL 7490) midway through each potential step. A digital comparator (TTL 7485) compares this counter output to a four-bit number determined by a row of four SPST switches. When the two numbers are in agreement, a monostable (TTL 74122) is triggered, causing a logic HI output for 1 ms. This causes the sample-and-hold amplifier (National Semiconductor LF398) to sample for 1ms every time the coulostat holds at the potential step which corresponds to the manual switch setting.
Chronoamperometric experiments were performed with a cybernetic potentiostat (BAS Model 100). The HPLC system was comprised of an Altex Model l l O A pump, a silica precolumn (Whatman 4391-103),an Altex Model 210 injection valve (20 pL sample loop), a Whatman guard column, an SSI high-pressure in-line filter (Rainin 05-0149),a Rainin C-18 10.0 cm long reverse-phaseanalytical column having 3 pm particles (Rainin 80-200), and the electrochemical flow cell. The system was assembled entirely with stainless steel tubing. The mobile phase was continuously sparged with helium to maintain oxygen-free conditions. Nitrogen was passed through samples for removal of oxygen. Reagents. All solutions used water obtained from a Continental/Millipore Milli-Q (Bedford, MA) reagent grade water system. Standards for HPLC were dissolved in the appropriate mobile phase. Hydroquinone and 1,4-benzoquinonesolutions were prepared daily, stored in dark containers, and refrigerated. Stock solutions of thiols and disulfides were prepared weekly and refrigerated. Dilutions were prepared daily. Hydroquinone and 1,4-benzoquinone were purchased from Aldrich (Milwaukee,WI). Cysteine was obtained from Nutritional Biochemicals (Cleveland, OH). Homocysteine, glutathione (both forms), and N-acetylcysteine were purchased from Sigma (St. Louis, MO). All other chemicals were reagent grade or better and were used as received without further purification. Chromatography. The mobile phase for the hydroquinone/ 1,4-benzoquinone studies consisted of 60% (v/v) 0.20 M Nac104/0.005 M trisodium citrate and 40% (v/v) CH3CN (Burdick & Jackson, Muskegon, MI, Distilled in Glass grade). The pH was adjusted to 4.9 with concentrated HC104. The mobile phase for the thiol/disulfide separations consisted of a mixture containing 0.1 M monochloroacetic 'acid (adjusted to pH 3.0 with NaOH pellets), 0.001 M sodium hexyl sulfate, and 1% (by volume) CH30H. Mobile phases were degassed with stirring under vacuum prior to use. The flow rate during chromatography was 1.0 mL/min.
RESULTS A N D DISCUSSION Crosstalk. Because of the proximity of the two pairs of counter and working electrodes in the flow cell, crosstalk is to be expected. Figure 3 shows the need for and the effectiveness of the crosstalk reduction circuit of Figure 2. Figure 3 presents chromatograms obtained with the potentiostat when an injection of 100 p M hydroquinone was made. The potentiostat enforced a potential of 0.75 V a t the upstream gold electrode. At the downstream gold electrode, the coulostat scanned from 0.35 to 0.80 V in 50-mV steps. When the potentiostat current output was connected directly to a
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
2551
c
0.8
UPSTREAM
S" 0.4 HYDROQUINONE
0.0
I
1.0
0.8
0.6
I 0.4
I 0.2
I
L
Il1
I
0.0 -0.2 -0.4
E (VI
Voltammograms obtained with coulostat downstream. Coulostat parameters: 100 ms hold time; 50 ms integrate time; 3000 ms pause time for scan A and 1000 m s pause time for scan B. Figure 4.
strip-chart recorder, the hydroquinone peak was seen superimposed on the crosstalk response. On an expanded time scale, the crosstalk can be seen to be a very reproducible staircase, similar to the potential wave form imposed by the coulostat. If the potentiostat current output was instead connected to the input of a properly timed sample-and-hold amplifier, the output of this amplifier produced a chromatogram that was essentially unaffected by crosstalk. For this work a switch setting (for the digital comparator) of one was employed. This means that when the coulostat held the potential corresponding to the first step in the scan (in this case, 0.35 V), the potentiostat output would be briefly sampled. For all other steps in the scan the output leading to the chart recorder remained at the same level. Only when the coulostat again held the downstream electrode at 0.35 V would the chart recorder level be updated. Thus, in the absence of an electroactive solute in the cell, a flat base line will be recorded, because even though the potentiostat output is continually changing due to crosstalk, that crosstalk noise is very reproducible. It should be noted that this crosstalk is a purely electronic phenomenon and should be distinguished from currents of an electrochemical nature. It should also be noted that this electronic crosstalk occurred whether the coulostat or a potentiostat was used to step the potential at one electrode in close proximity to a fixed-potential electrode. Coulostat Downstream, One Solute. To demonstrate the feasibility of potential scanning at an electrode downstream of another electrode held at a fixed potential, the well-characterized hydroquinone/ 1,4-benzoquinone system was utilized in a cell with two gold electrodes. Hydroquinone and 1,4-benzoquione react quasi-reversibly on gold (19). Reference voltammograms were obtained by using the potentiostat alone at the downstream electrode. Ten injections of 250 pM hydroquinone were made into the 40% acetonitrile mobile phase, followed by ten injections of 250 p M 1,4benzoquinone. For each injection the working electrode was held at a different potential. The normalized peak heights are plotted as a function of potential in Figure 4. From these plots a potential of 0.75 V was selected on the basis of being a potential where conversion of hydroquinone occurs at close to the diffusion limited rate while still being at a point where a fairly low background current level is found. The other voltammograms presented in Figure 4 were obtained with the potentiostat enforcing a potential of 0.75 V at the upstream electrode; both hydroquinone and 1,4-benzoquinone would then be present at the downstream electrode. With the coulostat scanning from 0.35 to 0.80 V, a voltammogram for the oxidation of hydroquinone was produced. Likewise, upon scanning from 0.15 to -0.30 V, a voltammogram for the reduction of 1,4-benzoquinone was generated. The coulostatically generated voltammograms agree quite well with the reference voltammograms. It should be noted that the ref-
8-0 Minutes
I , 4 - BENZOQUINONE
-y-
T
5 y
Flgure 5. Chromatograms obtained with coulostat downstream. Coulostat parameters: 100 ms hold time; 50 ms integrate time; 1000 ms pause time. (01
(bl
E (VI
E (VI
Replicate injections of 100 pM hydroquinone: (a) hydroquinone detected downstream; (b) 1,4-benzoquinone detected downstream. Coulostat parameters: 100 ms hold time; 50 ms integrate time; 1000 ms pause time. Flgure 6.
erence voltammograms required 20 separate injections but the coulostatically generated voltammograms required only two injections (one for hydroquinone and one for benzoquinone). Figure 5 shows the chromatograms obtained from the combined coulostat-potentiostat experiment. In both instances a peak is generated upstream due to hydroquinone. The peak shown underneath is coulostat data for a potential of 0.80 V. Typically the downstream hydroquinone peak under diffusion-limited conditions is abut 70% of the size of the peak upstream. Also shown is the peak for the detection of 1,4-benzoquinone at a potential of -0.30 V. This peak is typically 30% of the size of the upstream peak. This is consistent with the results of fixed potential series dualelectrode experiments (20). These chromatograms provide additional evidence that the coulostat is detecting electrochemically generated 1,4-benzoquinone; injected 1,4-benzoquinone is eluted 30 s after hydroquinone, but the coulostat detects 1,4-benzoquinone exactly during elution of hydroquinone, as expected. Detection capabilities were explored over the concentration range 50-1000 pM. The voltammetric detection limit is the lower limit at which reliable voltammetric information is obtainable. Experimenta similar to those previously described were performed. Again, upstream, the potentiostat held the gold working electrode at 0.75 V; downstream, hydroquinone in either of two redox forms could be detected with the coulostat. Figure 6 shows voltammograms for replicate injections
2552
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table I. Detection Limits for Coulostat Downstream (for S I N = 2)
t
T
detection limit detection mode species detected
u , nA ~
concn,b pM
electrons,c pmol 0
coulostat
hydroquinone
60
27
1100
1,4-benzoquinone
12
16
640
2
6
4
8
scanning E = +0.80 V
coulostat scanning E = -0.30 V
coulostat fixed hydroquinone potential E
12
5.5
220 I
= +0.80 V
coulostat fixed potential E
f
0
1,4-benzoquinone
5
5.5
220
= -0.30 V
potentiostat hydroquinone upstream E = +0.75 V
0.2
0.06
2.4
Standard deviation of the base line taken as one-fourth of the peak-to-peak base line noise. *Detection limit in terms of hydroquinone concentration injected. Detection limit in terms of number of electrons related to amount injected (Le., this value equals the product of the concentration detection limit, the sample loop size, and the number of electrons per molecule). of hydroquinone at the 100 FM level (corresponding to 2 nmol injected). Characterization of hydroquinone in its original redox state is still done with ease a t this concentration. However, considerable noise is appearing in voltammograms for hydroquinone as converted over to 1,4benzoquinone. This concentration would be a reasonable voltammetric detection limit if Eli2’sare desired with an uncertainty of less than 50 mV. These same data can be used to determine analytical detection limits as well. For this type of calculation, only the coulostat data for potentials on the diffusion limited plateaus would be used. Table I contains results of these experiments. Hydroquinone can be detected in its injected form a t a potential of 0.80 V; it is detected as l&benzoquinone a t a potential of -0.30 V. The analytical detection limit for hydroquinone in its original form was expected to be lower than that for the opposite redox form based on the limited conversion that takes place at the upstream electrode. However, the noise seen on gold at 0.80 V is about a factor of 5 greater than that observed at -0.30 V, increasing the detection limit accordingly. Table I also shows data resulting from use of the coulostat in a constant potential mode. The coulostat either enforced 0.80 V for detection of hydroquinone directly or held -0.30 V for detection of 1,4-benzoquinone produced upstream. The detection limits are significantly improved compared to the scanning mode, due to a significant reduction in noise. This phenomenon has been observed previously (8-10). Finally, Table I presents data obtained with the potentiostat at the upstream electrode. The signals will be greatest at this electrode since it is the first one a solute comes into contact with; in addition, the noise is very low here because of the stability of the electrode environment imparted by performing a constant potential experiment. Consequently, an enhanced signal-to-noise ratio is found, yielding reduced detection limits. This work successfully demonstrates an entirely new capability in terms of LCEC. With this detector, voltammograms are obtainable from both the species as injected and also as converted electrochemically. This implies quite a powerful means of qualitatively characterizing samples, based on both chromatography and electrochemistry. Even though quantitative information is available a t the downstream electrode, the much greater signal-to-noise ratio present up-
I
2
I
I 4
I
I
6
I
I
J
8
Minutes
Figure 7. Chromatograms of thiol mixture: (1) cysteine, (2) homocysteine, (3) glutathione (thiol),(4) acetylcysteine, (5)glutathione (disulfide);(a)upstream electrode off (b) upstream electrode h e 6 at -1.0 V. The thiols were 200 pM; the glutathione (disulfide)was 500 bM. Coulostat parameters: 100 ms hold time: 50 ms integrate time: 600
ms pause time. stream warrants selection of chromatograms produced there as the basis for quantitation. Coulostat Downstream, Mixture. A mixture of thiols and a disulfide was selected for testing the capabilities of combined coulostat-potentiostat detection. Such compounds are present in a wide variety of biological systems; their detection and characterization can be crucial to the understanding of a wide variety of processes. At present there are few methods for determinations of both classes by one procedure. However, LCEC approaches have had considerable success in this area. In particular, series detection using dual gold amalgam electrodes allows considerable flexibility in simultaneous determinations of a wide variety of thiols and disulfides ( I 7). At the upstream electrode, disulfides are reduced to the corresponding thiols. Downstream, thiol detection occurs based on the oxidation of mercury in the presence of such compounds. A mixture of glutathione in both thiol and disulfide forms plus the thiols cysteine, homocysteine, and acetylcysteine was used for this work. The chloroacetic acid mobile phase was used. The potentiostat held the upstream electrode at -1.0 V. The downstream electrode experienced a potential scan under coulostat control covering -0.30 to 0.15 V. With the upstream electrode switched off, the chromatogram corresponding to data a t 0.15 V is plotted in Figure 7a. Only the four thiols are detected. Figure 7b shows the downstream chromatogram for 0.15 V when an injection is made with the upstream electrode on. The additional peak arose from glutathione (thiol) generated at the upstream electrode. Voltammograms for both situations are depicted in Figure 8. It is seen that for the thiols, the potential of the upstream electrode has no impact on the shape of the voltammogram observed downstream. For glutathione, the shape of the voltammogram observed downstream is the same whether the thiol is injected or the disulfide injected and converted to the thiol at the upstream electrode. Reference voltammograms were obtained as described earlier in this paper for each species. They overlapped quite well with the voltammograms obtained with the coulostat. A chromatogram is unavailable in this particular analysis at the upstream electrode due to an extremely high background current level, making disulfide detection via the downstream electrode essential. This work points to the general utility of scanning at a downstream electrode. The additional voltammetric information associated with the additional peak found when the upstream electrode is on adds a high degree of certainty in
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986 CYSTEINE
2553
HOMOCYSTEINE
t-
z
W
a a
2 V
E (V) GLUTATHIONE
ACETYLCYSTEINE
POTENTIAL+ o.oslaio 10.15~ o . z ~ ( o . z s ~ o . ~~oo~.o~. o~ ~s o . ~ s ~ o . s o ~ UPSTREAM (V)
Chronoamperometric response downstream showing detection of ferricyanide generated upstream by the coulostat. Coulostat parameters: 200 ms hold time; 600 ms integrate time; 1000 ms pause time. Flgure 9.
E (V)
E (VI
Voltammograms for thiol mixture, all conditions the same upstream electrode as for Figure 7: (0)upstream electrode off; (0) on; (A)upstream electrode on (disulfide injected). Flgure 8.
its identification. An additional benefit specific to analyses such as this where electrode-fouling species are determined is that the voltammograms may be deterimed from one injection with less likelihood of fouling the working electrode than when multiple injections are made as in the conventional method of obtaining a hydrodynamic voltammogram via LCEC. Coulostat Upstream. Work with the coulostat downstream of an electrode held at a fixed potential indicates that the scanning experiment is much noisier than a constant potential experiment. In addition, using the coulostat in a fixed potential mode results in significantly more noise than when the same potential control is performed continuously by a potentiostat. Therefore there would appear to be some utility to reversing the coulostat-potentiostat arrangement, i.e., the coulostat would be used to scan the potential a t an electrode located upstream of a fixed-potential electrode controlled by a potentiostat. Consider the situation where the coulostat is scanning and an electrochemically reversible species is present in the cell. Because of the stepwise nature of the coulostat scan, a series of concentration profiles of the solute in two redox forms would be established at the upstream electrode. Molecules would then be brought into contact with the downstream electrode by convection and diffusion processes. If the electrode downstream is poised to detect the species in the form that is being generated a t the upstream electrode, a signal will arise at the downstream electrode that reflects the rate a t which such production is occurring upstream. Thus, at the downstream electrode, voltammetric information is available pertaining to the solutes in their original form. With this arrangement, though, this information is obtained in the low noise environment of a fixed-potential detection. Thus, one could generate voltammograms of species eluting from the LC column by scanning the potential a t an upstream electrode through the appropriate potential region to oxidize (or reduce) the eluting species and detecting via
0
a10
020
0.30
OAO
On%
060
E (VI
Voltammograms based on downstream current vs. upstream potential. Data shown for two different upstream scan rates. Flgure 10.
reduction (or oxidation) at a downstream electrode at fixed potential. Although the voltammogram would be scanned rapidly, the measured current would be free from charging current interference. This idea of using two working electrodes to generate a voltammogram is similar to use of a rotating ring-disk electrode with the potential scanned at the disk and held constant a t the ring. However, the approach we describe with series electrodes in a thin flow cell allows easy application to detection in flow injection or HPLC. To demonstrate the upstream scanning mode, a solution of 0.8 mM Fe(CN)64-in 0.02 M NaC104 was flowed through a cell containing two gold electrodes. Upstream, the coulostat scanned from 0.05 to 0.50 V, which covers the potential region where (Fe(CN)64-is oxidized on gold. Downstream, the potentiostat was operated in a chronoamperometric mode; current was recorded as a function of time at a fixed potential where Fe(CN)63-would be easily reduced (-0.20 V). The BAS-100 potentiostat (instead of the LC-4B) was used because it allowed data acquisition with minimal filtering. Figure 9 depicts results obtained with a relatively slow coulostat scan rate of 62 mV/s (800 ms per step, 1000 ms between scans). The response obtained with 0.2 m NaC104 alone is indicated as the background. Superimposed is the additional reductive response when Fe(CN)63-has been generated upstream. As expected, no Fe(CN)63-is detected until step number four (upstream potential of 0.20 V) is reached. The time for steady state to be achieved within each step is approximately 500 ms. If the background corrected downstream current response at the end of each step is plotted as a function of the upstream potential of that step, the voltammogram of Figure 10 results.
2554
Anal. Chem. l90& 58,2554-2557
Data analysis downstream is fairly simple if the concentrations within the cell are brought to steady state during each step in the coulostat scan. Convolution of the chromatographic or voltammetric data would occur if scans are performed too rapidly for such steady state to be established. If the experiment is repeated but with a faster scan rate of 333 mV/s (150 ms per step, 1000 ms between scans), the data downstream are not synchronized well with the scan. The voltammogram is shifted in the positive direction by several steps, corresponding to several hundred milliseconds. It would be expected that once the upstream electrode achieves a new potential, the time required for establishment of steady state in the cell would be approximately the time needed for species to flow across both electrodes (far edge to far edge distance). This distance is 0.70 cm, and the average linear velocity of solution through the flow cell is 2.6 cm/s, based on volume flow rate and cell geometry. This corresponds to a time of 270 ms. This time is certainly consistent with the observed time of about 500 ms. This work demonstrates that in LCEC experiments it is quite realistic to generate voltammograms of reversible species by use of series electrodes, with potential scanning at the upstream electrode and fixed potential detection at the downstream electrode. With the present cell, constraints of the chromatographic time scale conflict with requirements of establishing steady-state conditions. Further work would be required to deal with convolution of the voltammetric information if faster scan rates are considered. It would be desirable to design a flow cell with smaller electrodes and much less dead volume to speed attainment of steady state to conform with chromatographic requirements.
CONCLUSION The work with the coulostat controlling an electrode potential scan downstream of an electrode held at fixed potential indicates considerable utility to this approach. Considerable qualitative information is available simultaneously with the capability of obtaining good quantitative information under low noise amperometric conditions. Although coulostatic control should be advantageous in certain situations, the principles illustrated here are believed to have a wide applicability with respect to other scanning technologies. Applications of scanning upstream are realistic but would benefit from further investigations into the fundamentals of dual-electrode behavior under such conditions. Also, instru-
ments capable of amperometry downstream that are both fast in response and are of a low noise design are just now becoming readily available. Despite the difficulties in approaching this detector strategy, this work indicates a sound basis for the collection of voltammetric information in this fashion. Although our work employed a coulostat to achieve scanning a t the upstream electrode, one should be able to use a conventional potentiostat; since detection would be done only at the downstream fixed-potential electrode, charging current interference is not an issue. Registry No. Glutathione, 70-18-8; cysteine, 52-90-4; homocysteine, 6027-13-0; acetylcysteine, 616-91-1; hydroquinone, 123-31-9; 1,4-benzoquinone, 106-51-4; glutathione disulfide, 27025-41-8.
LITERATURE CITED Kissinger, P. T. Anal. Chem. 1977, 4 9 , 447A-456A. Rucki, R. J. Talanta 1980, 2 7 , 147-156. Stulik. K.; Pacakova, V. J . Electroanal. Chem. 1981, 729, 1-24. Palmisano, F.; Zambonin, P. G. Ann. Chim. (Rome) 1984, 7 4 , 633-671. (5) Stulik, K.; Pacakova, V. CRC Crit. Rev. Anal. Chem. 1984, 74, 299-351. (6) Roston, D. A,; Shoup, R. E.;Kissinger, P. T. Anal. Chem. 1982, 5 4 , 1417A-1434A. (7) Osteryoung, J. G.; Osteryoung, R. A. Anal. Chem. 1985, 5 7 , 101AllOA (8) Caudill, W. L.; Ewing, A. G.; Jones, S.;Wightman, R. M. Anal. Chem. 1983. 55. 1877-1881. (9) Barnes, A. C.; Nieman, T. A. Anal. Chem. 1983, 5 5 , 2309-2312. (IO) Last, T. A. Anal. Chem. 1983, 5 5 , 1509-1512. (11) Last, T. A. Anal. Chim. Acta 1983, 155, 287-291. (12) Bond, A. M.; Jones, R. D. Anal. Chim. Acta 1983, 152, 13-24. (13) Brillmyer, G. H.; Lamey, S. C.; Maioy, J. T. Anal. Chem. 1975, 4 7 , 2304-2306. (14) Stastny, M.; Volf, R.; Benadikova, H.; Vit, I . J . Chromatogr. Sci. 1983, 21, 18-24. (15) Trojanek, A.; DeJong. H. G. Anal. Chim. Acta 1982, 747, 115-122. (16) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980; pp 270-276. (17) Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8-12. (18) Panzer. R. E.; Elving, P. J. Nectrochim. Acta 1975, 2 0 , 635-647. (19) Gunsingham, H.; Fleet, B. Analyst (London) 1983, 706.316-321. (20) Roston. D. A.; Kissinger, P T. Anal. Chem. 1982, 5 4 , 429-434 (1) (2) (3) (4)
RECEIVED for review December 24, 1985. Resubmitted June 23, 1986. Accepted June 23, 1986. This research was supported in part by a Biomedical Research Support Grant (NIH RR7030), a grant from Dow Chemical Co., and a grant from the United States Environmental Protection Agency (Through Cooperative Agreement CR 806819 to the Advanced Environmental Control Technology Research Center, University of Illinois).
CORRESPONDENCE Simultaneous Separation of Inorganic Anions and Cations on a Mixed Bed Ion Exchange Column Sir: Recent studies have demonstrated that inorganic oxides, such as silica and alumina, are useful ion exchanger (IE) stationary phases for the liquid chromatographic (LC) separation of inorganic analyte ions ( I , 2). Furthermore, certain inorganic oxides have favorable isoelectric pH values and by adjustment of mobile phase pH they can be used as either anion or cation exchangers ( 3 ) . During studies of the properties of alumina as an anion (2, 4 , 5 ) and cation exchanger ( 4 , 6, 7) it was apparent that under certain conditions IE separations of anions and cations could be carried out si-
multaneously ( 4 , 5 , 7,8). These experiments also suggested that a mixed bed of anion and cation exchanger beads packed into a column should also be useful in this kind of application. One major advantage of this latter type of column is that both the anion and cation exchange capacity is determined by the exchangers used while for alumina its capacity is determined by the mobile phase pH. Mixed bed IE is well-known but has been used primarily in water treatment for electrolyte removal. Recently, other workers have attempted to simultaneously determine analyte
0003-2700/86/0358-2554$01.50/00 1986 American Chemical Society
