Anal. Chern. 1983, 55, 1877-1881
An interpretation of eq 24 is that the slope of In lzi with respect to xAis (PAI P A C x C ) ; i.e., the "effect" of XA depends upon xC. Similarly, in eq 25 the slope of In k , with respect to xc PACxA);Le., the "effect" of xc depends upon xk is (Pa Substituting an estimated parameter value for, say, aniline 0 . 6 5 1 ~ ~for ) the xA effect, and (-0.996 + gives (1.356 0 . 6 5 1 ~ for ~ ) the xc2effect; similar effects are seen for the other solutes. In each case, the presence of one surfactant already adsorbed on the surface appears to cause a greater increase (or a lesser decrease) in In k , when the other surfactant becomes adsorbed. However, as seen in Figures l and 2, in. creased adsorption of one surfactant is usually accompanied by decreased adsorption of the other surfactant. Thus, there seems to be a type of "self regulatory feedback" associated with this parameter. The possibility exists that the net effect of the PAC parameter is zero and that it may be removed from the model. Elimination of the PACterm from the model expressed by eq 23 gives a total sum of squares of residuals only slightly larger than before (0.358 vs. 0.304). The values of the remaining parameter estimates change only slightly (35% in the worst case). Thus, whatever the real phenomenological interpretation of the parameter PAC, it does not appear to be a necessary parameter t o adequately model the systems described in this study. Registry No. Sodium octanesulfonate,5324-84-5;octylamine, 111-86-4;aniline, 62-53-3; phenylethylamine, 64-04-0;benzenesulfonic acid, 98-11-3; chromotropic acid, 148-25-4.
+
+
+
ian
(3) Knox, J. H.; Laird, G R. J . Chromatogr. 1976, 122, 17-34. (4) Knox, J. H.; Jurand, J. J . Chromatogr. 1976, 125,89-101. (5) Tomlinson, E.; Jeffeiries, T. M.; Riley, C. M. J . Chromatogr. 1970, 159,315-358. (6) Bidlingmeyer, B. A. J . Chromatogr. Sci. 1980, 18,525-539. (7) Hearn, M. T. W. I n "Advances in Chromatography"; Glddings, J. C., Grushka, E., Cazes, J., Eds.; Marcel Dekker: New York. 1980; Vod. 18, pp 59-100. (8) Bidlingmeyer, B. A.; Deming, S. N.; Price, W. P., Jr.; Sachok, B.; Petrusek, M. J . Chromatogr. 1979, 186, 419-434. (9) Kong, R. C.; Sachok, B.; Deming, S. N. J . Chromatogr. 1980, 1961, 307-316. (10) Lucassen-Reynders, E. H. I n "Progress in Surface and Membrane Science"; Cadenhead, D. A., Danielli, J. F., Eds.; Academic Pres!s: New York, 1976; Vol. 10, pp 253-360. (1 1) Corklll, J. M.; Goodman, J. F.; Harrold, S. P.; Tate, J. R. Trans. Farfitday SOC. 1967, 63, 247-256. (12) Nakamura, A.; Muramatsu, M. J . Colloid Interface Sci. 1977, 6;?, 165-171. (13) Rosen, M. J. "Surfactants and Interfacial Phenomena"; Wiley: New York, 1978. (14) Stranhan, J. J.; Denning, S. N. Anal. Chem. 1962, 5 4 , 2251-22513. (15) Locke, D. C. J . Chromatogr. Sci. 1974, 12, 433-437. (16) Lucassen-Reynders, E. H.; Lucassen, J.; Gibs, D. J . Colloid Interface Sci. 1981, 81, 150-157. (17) Box, G. E. P.; Hunter, W. G.; Hunter, J. S. "statistics for Experiments. An Introductlon to Design, Data Analysls, and Model Building"; Wiley: New York, 1978. (18) Natrella, M. G. "Explerimentel Statistics, National Bureau of Standards Handbook 91"; US. Govt. Printing Office: Washington, DC, 1963. (19) Mendenhall, W. "Introduction to Linear Models and the Design and Analysis of Experimlents"; Duxbury: Belmont, CA, 1968. (20) O'Neill, R. Appl. Statist. 1971, 20, 338-345. (21) Rodakiewicz-Nowak, J. J . Collold Interface Sci. 1982, 85,586-591. (22) Weast, R. C., Ed. "CRC Handbook of Chemistry and Physics", 56th ed.; CRC Press: Cloveland, OH, 1975; p D-147. (23) Rappoport, Z.,Ed. "CRC Handbook of Tables for Organic Compound Identification", 3rd ed.; CRC Press: Cleveland, OH, 1967; p 438. (24) Weast, R. C., Ed. "CRC Handbook of Chemlstry and Physics", 56th ed.; CRC Press: Clieveland, OH, 1975; p D-150.
LITERATURE CITED (1) Wittmer, D. P.; Nuessle, N. 0.; Haney, W. G., Jr. Anal. Chem. 197!i, 4 7 , 1422-1423. (2) Sood, S. P.; Nuessle, N. 0.; Haney, W. G., Jr. Anal. Chem. 1876, 4 8 , 796-798.
RECEIVED for review July 26, 1982. Resubmitted March '7, 1983. Accepted June 16,1983. This work was supported iin part by a grant from1 Chevron Research Co.
Liquid Chromiat ogra phy with Rapid ScanninCJ EIect roc hemicaI Detection at Carbon Electrodes W. Lowry Caudill, Andrew G. Ewing, Scott Jones, and R. Mark Wightman* Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Rapld scannlng electroclhemical detectlon has been Investlgated with channel-type! electrochemical flow cells whlch utllize glassy carbon and carbon flbers as working electrode materlals for use with high-performance llquld chromatography or flew injection analysis. Normal pulse voltammetry, back-step corrected nornnal pulse vottammetry, and stalrcase voltammetry were investigated for their utllity with rapid scanning electrochemlcal detectlon. Staircase voltammsgrams, which have beeln subtracted from the background slgnals, were found to give the most useful results. The vottammograms, which tare acquired In flowing streams, can facllltate ldentiflcatlon in sample unknowns. The chromatographic detectlon llmlts are approximately 3 orders of magnitude hlgher than those obtained with amperometric detectlon. However, the technique of HPLC with rapid scanning electrochemical detectlon provides a more complete Identlflcatlon than HPLC with the amperometrlc (dc), pulse, or dual electrode detectlon modes.
High-performance liquid chromatography with electro0003-2700/83/0355-1877$01 .B0/0
chemical detection (LCEC) has become a widely used technique for trace organic analysis. Amperometric (i.e., dc applied potential) is the most common detection mode because it offers high sensitivity (femtomole), requires relatively simple instrumentation, and provides considerable selectivity through judicious selection of the applied potential (I, 2 ) . Improved potential selectivity can be obtained with the use of dual electrodes operated amperometrically ( 3 ) . In the flow cell, the two working electrodes can be placed in parallel, in series, or geometrically opposed in the flow cell and usually are operated at different potentials (4). The increase in selectivity obtained with this approach arises because the differences in the electrochemical properties of various compounds can be exploited (4). Another mode of detection which has been investigated involves potential pulse techniques ( 2 , 5 , 6 ) .Pulse techniques are attractive because the detector response is relatively insensitive to flow rate a t short pulse times (is), because pulse techniques can also be used for increased selectivity and because pulse methods can be used for electrode cleaning (7). However, the use of the pulse mode has not been widespread because of reported higher detection limits which have been attributed to interference by electrode charging currents. 0 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Despite the advantages of all of these techniques, none provides complete voltammetric information. This can only be provided by coupling rapid scan voltammetry with the conditions required by high-performance liquid chromatography (8). This technique can provide a more complete identification of each peak: peak retention time and a characteristic voltammogram for each peak. The voltammogram can facilitate identification (or class identification) of sample unknowns. Further, the characteristic voltammogram is obtained with only one chromatogram in contrast to the time-consuming generation of hydrodynamic voltammograms by LCEC with the method of repeated injections at a series of applied potentials. The technique also offers immediate identification of coeluting peaks if the coeluting components possess different half-wave potentials and, thus, offers immediate selectivity because the chromatogram can be plotted a t the lower half-wave potential to remove the interference. From an electrochemical point of view, the technique provides the capability of obtaining voltammograms of small amounts (picomole) and/or small volumes (50-100 pL) of sample. In addition, the technique generates voltammograms of multicomponent samples without prior separation procedures. In this report, a technique is described which facilitates rapid scan voltammetry with LCEC in channel-type, carbon-based, detectors. Staircase voltammetry with background subtraction provides low picomole detection capabilities with either a glassy carbon electrode or an array of carbon fibers (9). Although the detection limits are higher than those in the amperometric mode, they are considerably lower than those obtained with semidifferential voltammetry for HPLC detection a t a glassy carbon wall-jet electrode (IO).
EXPERIMENTAL SECTION Reagents. All solutions were prepared in doubly distilled H20 with reagent grade chemicals. Dopamine (DA), 3,4-dihydroxybenzylamine hydrobromide (DHBA), homovanillic acid (HVA), epinephrine (E), 5-hydroxyindoleaceticacid norepinephrine (NE), (5-HIAA), and 3,4-dihydroxyphenylaceticacid (DOPAC) were obtained from Sigma Chemical Co. (St.Louis, MO), and potassium ferrocyanide (K,Fe(CN),) was obtained from Mallinckrodt, Inc. (St. Louis, MO). Standard solutions for chromatographic analysis were prepared in perchloric acid (0.1 M) and diluted to the desired concentrations. Standard solutions for flow injection analysis were prepared in potassium chloride (1.0 M, pH 3.0) for K,Fe(CN), and in a deoxygenated physiological buffer (pH 7.4, see flow injection analysis section for details) for DA and were diluted to the desired concentrations. Liquid Chromatography Apparatus. The liquid chromatography system consisted of a constant flow reciprocating pump (Model 396 Instrument Mini Pump, Milton Roy, Riviera Beach, FL), a 50-rL loop injector (Model 7010, Rheodyne, Berkeley, CA), and a 25 cm X 4.6 mm Biophase (5 pm particle size) reversed-phase column (Bioandytical Systems, West Lafayette, IN). A stainless steel tube (1m X 5.0 mm i.d.) between the pump and loop injector served as a pulse dampener. The mobile phase for the separation of DA, DHBA, HVA, NE, E, 5-HIAA, and DOPAC contained citric acid (0.1 M), sodium hydroxide (0.14 M), sodium octylsulfate (0.9 mM, Fisher Scientific, Pittsburgh, PA) (pH 4.1), and acetonitrile (4.2%, HPLC Grade, Fisher). The flow rate was 1.4 mL/min. For the analysis of the rat caudate nucleus, the mobile phase contained citric acid (0.05 M), sodium acetate (0.1 M), sodium octylsulfate (1.2 mM) (pH 5.0), and acetonitrile (10%). In this case the flow rate was 1.0 mL/min. Flow Injection Apparatus. The flow injection system consisted of a constant flow syringe pump (Model 940, Harvard Apparatus Co., Millis, MA) and a Teflon rotary valve loop injection (Type 50, Rheodyne Inc., Berkeley, CA). Teflon tubing (0.08 mm i.d.) and Teflon couplings (Altex Scientific Inc., Berkeley, CA) were used throughout the system. The loop injection (loop volume = 1.0 mJ, or 0.5 mL) was used to introduce the solutions through a 3.5 cm length of tubing. The mobile phase for K,Fe(CN), was potassium chloride (1.0 M, pH 3.0). For the analysis of DA, the
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multifiber detector: (A) normal pulse vobmmetrlc wave form, sampling time (a ) = 34 ms; (B) current vs. time response for the oxidation of K,Fe(CN), (1.0 X M, 1 mL injected) in potassium chloride (1.0M, pH 3.0)plotted at 0.8 V vs. SCE; (C) voltammograms of K,Fe(CN), (dots) and background (circles): (D) background subtracted voltammogram of K,Fe(CN),. A.
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Figure 2. Flow injection analysis with back-step corrected normal pulse voltammetry at the multifiber detector: (A) back-step corrected normal pulse voltammetric wave form, forward step tlme ( a )= 92 ms, reverse step time ( b ) = 108 ms, and sampling time (c) = 34 ms; (B) current M, 1 mL vs. time response for the oxidation of K,Fe(CN), (1.0 X injected)In potassium chloride (1.0M, pH 3.0)plotted at 0.8V vs. SCE: (C) voltammograms of K,Fe(CN), (dots)and background (circles):(D) background subtracted voltammogram of K,Fe(CN),.
mobile phase was a physiological buffer (pH 7.4) which contained HEPES (20 mM, Sigma), potassium chloride (5 mM), sodium chloride (150 mM), magnesium sulfate (1mM), glucose (10 mM, Sigma), and calcium chloride (2.4 mM). The flow rate for all flow injection analysis experiments was 1.0 mL/min. Electrochemical Apparatus. The potential wave forms were generated and the data acquired with an Apple 11+ computer with a DAS-5 A/D and D/A interface (Data Acquisition Systems, Inc., Cambridge, MA) and a Mountain Hardware clock (Mountain Computer, Inc., Scotts Valley, CA). The acquisition program was designed for rapid operation by storing all voltammograms acquired during a single chromatogram in computer memory without the need for disk storage until after the chromatogram is complete. The data retrieval program allows for the averaging and/or subtraction of voltammograms. A low noise potentiostat was employed with a 10-ms time constant that was especially designed for low current measurements in the pulse mode (11). For normal pulse voltammetry, 50-mV steps with 50-ms step times were employed (Figure 1). The current was sampled during the last 34 ms of the forward potential step. Voltammograms were obtained at a scan rate of 500 mV/s and at a repetition rate of 3 s. For normal pulse voltammetry with back-step correction (a method for removal of charging current at carbon fiber disk electrodes) (II), 50-mV potential increments were employed with a forward step time of 92 ms and a reverse step (Le., back-step) time of 108 ms (Figure 2A). The current was sampled on the forward step beginning at 58 ms for 34 ms and also on the reverse
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
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Flgure 3. Flow injection analysis with staircase voltammetry at the multlfiber detector: (A) staircase voltammetric wave form, sampling time (a ) =: 34 ms; (B) current vs. time response for the oxidation of K,Fe(CN), (1.0 X M, 1 mL injected) in potassium chloride (1.0 M, pH 3.0) plotted at 0.8 V vs. SCE; (C) voltammograms of K,Fe(CN), (dots) and background (circles);(D) background subtracted voltammogram of K,Fe(CN),.
step beginning at 58 ms jfor 34 ms. In the computer program, the measured current of thke reverse step was then added to the measured current of the forward step. Voltammograms were acquired at a scan rate of 200 mV/s and at a repetition rate of 6 s. For staircase voltammetry, 50 mV potential steps with 100 ms step times were employed (Figure 3A). The current was sampled over the last 34 ms of each step. Voltammograms were obtained with a scan rate of 500 mV/s and with a repetition rate of 3 9. Electrochemical detection was accomplished with glassy carbon (Tokai CC-20, Atomergic, New York) or an array of carbon fiber disks as the working electrode (9). The electrodes were polished to a mirror finish with the use of 600 grit sandpaper, followed by polishing on a felt cloth with 5, 0.3, and 0.05 pm alumina (Fisher), successively. 'The flow cells have been described previously (12,13). The area of the glassy carbon working electrode was 0.46 cm2 and the spacer thickness was 51 pm giving a cell volume of 2.3 pL. The carbon fiber array electrode consisted of 100 disks, each of 5 p m radius, and was designed to minimize diffusional crosstalk (9). A spacer thickness of 0.43 mm was employed with the array giving a cell volume of 20 pL. All electrochemical measurements were made with respect to a saturated calomel reference electrode (SCE). Tissue Preparation, Adult male Sprague-Dawleyrats were decapitated and the brain was removed rapidly. The caudate nucleus was dissected immediately, weighed, homogenized in 1.5 mL of perchloric acid (0.1 M), and subjected to sonication (Kontes Micro-Ultrasonic Cell Disrupter, Vineland, NJ) for 30 s. Subsequently, the protein was precipitated by centrifugation (Microfuge B, Beckman Instruments, Palo Alto, CA) at lOOOOg for 4 min. The supernatant was collected and chromatographically analyzed.
RESULTS AND DISCUSSION Voltammetry during Flow Injection Analysis. Various pulse techniques were examined for scanning in flowing streams, because pulse techniques can be used to discriminate against charging currents (14). The wave forms were tested with the multifiber array electrode. This electrochemical cell has a calculated time constant of 0.2 ps, and thus, in itself, is more immune to problems of charging current than electrodes of conventional size. The results obtained for the flow injection analysis of K3Fe(CN), with normal pulse voltammetry are shown in Figure 1. The current at 0.8 V from each voltammogram is plotted as a function of time to provide the temporal response of the flow injection system (Figure 1B). Voltammograms that were acquired in the limiting current region of the K,Fe(C:N), exhibit little faradaic information (Figure IC). Voltammograms of the mobile phase (i.e.9the background in this experiment) obtained before the flow in-
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M DA (circles; current scale for voltvoltammograms for 51.0 X ammogram, 63 PA) and for 1.O X lo-' M DA (solid line; current scale for voltammogram, 112 PA).
jection experiment are similar in shape. Subtraction of tlhese two sets of voltammograms yields the voltammogram characteristic of K3Fe(CN), (Figure 1D). To investigate the source of the current observed in the mobile phase, the technique of back-step corrected normal pulse voltammetry was employed (see Experimental Section) (Figure 2A). This technique was developed in our laboratory to remove the charging current at carbon fiber disk electnodes from the measured signal (11). This technique requires longer pulse times than normal pulse voltammetry to prevent distortion of the faradaic data. Although a considerable reduction of charging current is obtained (Figure 2C), the voltammogpm from the limiting current region of the flow injection experiment cannot be identified clearly as K3Fe(CN),. However, subtraction of the background voltammogram from this voltammogram, a($described above, leads to a well-defined hydrodynamic voltammogram (Figure 2D). The significant background currents present after back-step correction, which removes charging currents, demonstrate that other interferences exist which distort the voltammograms. These could include the presence of oxidizable species on the electrode surface or impurities in the solution. Staircase voltammetry with the background subtraction method was also examined with flow injection analysis. Because each potential pulse has the same amplitude, more uniform charging currents are obtained throughout the scan (15). The results in Figure 3 demonstrate that very similar results are obtained with this technique and normal pulse voltammetry with back-step correction. However, staircase voltammetry can be used at scan rates twice those used for back-step correctled normal pulse voltammetry. As will be shown, this parameter is important for obtaining voltannmograms of compounds separated by liquid chromatography. The limits of voltammetric detection were examined with the multifiber detector and staircase voltammetry. The flow injection analysis current w. time response and voltammogram of 5.0 X lo-' M DA are shown in Figure 4. Although the voltammogram is noisy, it is readily identified as DA. Thus, it is apparent that detection limits are sacrificed over those typically observed in our laboratory (nanomolar) in the amperometric mode (16),but additional molecular information is obtained. The detector was found to give a linear response to DA over 2 ordlers of magnitude.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
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Liquid Chromatography with Potential Scans at the Multifiber Detector. Figure 5A shows a chromatogram for the separation of NE, DOPAC, E, DHBA, 5-HIAA, DA, and HVA (1 nmol of each component) that was obtained with staircase voltammetry a t the multifiber detector. The chromatogram was plotted at 0.7 V. However, because of the computer data acquisition, the chromatogram can be plotted at any of the potential step values from 0.0 V to 1.0 V. Each point on the chromatogram corresponds to one complete staircase voltammogram. A complete scan is obtained in 2 s with a repetition rate of 3 s. The corresponding voltammograms (Figure 5B) were obtained by subtraction of the mobile phase voltammogram obtained before the peak (Le., the background voltammogram) from the voltammogram at the uppermost point of each chromatographic peak for a sample component. Figure 6A shows the chromatogram of a homogenate of the rat caudate nucleus. The chromatogram is plotted at 0.7 V and possesses a peak a t 8 min which corresponds to DA. The subtracted voltammogram of this peak is shown in Figure 6B along with the standard subtracted voltammogram of DA (500 pmol). Analysis of the chromatogram and the voltammograms indicates with high probability that the peak is DA. The chromatogram and voltammogram of the homogenate correspond to 117 pmol of DA and indicate that approximately 2 mg of caudate tissue is needed for this analysis. Liquid Chromatography with Potential Scans at Large Area Electrodes. Since residual current, rather than charging current, appears to be the major impediment to voltammetry in flowing streams, the viability of a conventionally sized, glassy carbon working electrode as a rapid scanning detector in a flow cell was investigated. The larger electrode area causes an increase in the RC time constant of the detector, but crude calculations suggest the value should still be less than 2 ms. This value should be small enough to permit staircase voltammetry under the conditions described previously. Figure 7 shows the subtracted voltammograms of NE, DOPAC, E, DHBA, 5-HIAA, DA, and HVA (1 nmol of each component) that were obtained following chromatographic separation (under the same conditions as those utilized with the multifiber detector). These well-defined voltammograms were compared to their respective hydrodynamic voltammograms
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Figure 7. Chromatographlc analysis with staircase voltammetry at a glassy carbon detector (see Figure 5 for peak identifications)(1 nmoi of each component): (A) chromatogram plotted at 0.7 V vs. SCE; (B) background subtracted voltammograms for each peak.
that were generated by repeated injection of a standard solution a t different applied potentials. Both techniques generated virtually identical voltammograms. Attributes of HPLC with Rapid Scanning Detection. Chromatographically, HPLC with rapid scanning electrochemical detection possesses several attractive features. The technique offers two modes of identification: peak retention time and a characteristic voltammogram for each peak (Figures 5 and 7). A voltammogram for each electroactive component in the multicomponent sample can be generated simultaneously with separation. It requires small volumes (50 p L in these examples) or small amounts (less than nanomole) of sample. Further, noise from flow fluctuations is reduced
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
metric detection limit requires an identifiable voltammogram whereas the chromatographic detection limit requires only a peak with a signal to noise ratio of two. These chromatographic detection limits are approximately 3 orders of magnitude higher than those typically obtained with either detector in the dc mode (9).
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Flgure 8. Same chromatographic analysis as in Figure 7 except that the chromatogram is plotted at 0.55 V vs. SCE.
because the detector should be flow independent with these short step times (6). The main advantage of this method, however, is the capability of class identification (Figures 5 and 7). The voltammograms of NE, DOPAC, E, DHBA, and DA are very similar since their common electroactive functionality is a catechol. A second electroactive class can be seen with the voltammogram of the indole, 5-HIAA. This selectivity is further emphasized with the voltammogram of the methoxyphenol, HVA. In the chromatogram with glassy carbon detection (Figure 7A), DA and HVA coelute. However, analysis of the voltammograms of DA and HVA indicates that if the chromatogram were plotted at 0.55 V instead of 0.7 V, DA can be detested selectively in the presence of HVA (Figure 8). Because of computer data acquisition, the chromatogram is simply replotted at 0.55 V to achieve a satisfactory resolution of DA and HVA. This example emphasizes the selectivity of the rapid scanning technique since the chromatogram can be replotted at various potentials, without repeating the experiment, to enhance the separation of sample components. While increased molecular information is obtained, there are disadvantages to this technique. The detection limit at which an identifiable voltammogram can be obtained during chromatography for the multifiber and the glassy carbon detector appears to be 50 pmol. While this is higher than that obtained amperometrically with carbon electrodes, this voltammetric detectioin limit is comparable to that obtained with scanning techniques at mercury electrodes (8), and it is a 200-fold improvement over that obtained previously at glassy carbon (10). It appears that the problems apparent with carbon electrodes are different than those that are confronted at mercury electrodes, since it has been shown that scanning and dc detection limits are similar at mercury (8, 16). The improvement in comparison to other results at carbon electrodes arises from the utilization of background subtraction. The limits of detection are not due to the subtraction of the signals to generate the voltammograms since sufficient dynamic range is available with the analog to digital converter. Instead, the major noise source appears to arise from the electrochemical cell when operated with the low time constant (10 ms) required for the potentiostat. The chromatographic detection limits (i.e., the chromatograms plotted at one plotential during scanning experiments) are also higher than those obtained amperometrically. For example, the detectioin limits at 0.7 V for NE (calculated a t a signal to root mean square noise ratio of two) are 26 pmol and 19 pmol for the glassy carbon and the multifiber detectors, respectively. The chromatographic detection limit is lower than the voltammetric detection limit because the voltam-
CONCLUSIONS High-performance liquid chromatography with rapid scanning electrochemical detection at carbon electrodes offers several advantages over high-performance liquid chromaitography with amperometric, pulse, or dual electrode detection. From a chromatogriaphic view, the technique offers two modes of identification. The voltammogram, which is obtained with only one chromatographic analysis, can facilitate identification (or class identification) in sample unknowns. Further, the technique can provide immediate identification of coeluting peaks and indicate the potential where selective detection can be achieved. Although the technique possesses a higher detection limit than the amperometric mode, in many instances the detection limit is not the limiting factor in analysis and, thus, the rapid scanning detection may be advantageous. Since the voltammograms can be obtained with conventional amperometric detectors, lower detection limits can easily be obtained by merely operating the detector in the dc mode. Thus, the rapid scanning detection and dc detection can operate in a complementary fashion. From an electrochemical point of view, HPLC with rapid scanning detection provides the capability of obtaining voltammograms of small amounts (picomole) or small volumes (50-100 1L) of sample. The technique also generates voltammograms of multicomponent samples without prior separation from only one chromatogram. Registry No. Dopamine, 51-61-6; 3,4-dihydroxybenzylamine-HBr, 16290-26-9; homovanillic acid, 306-08-1; norepinephrine, 51-41-2;epinephrine, 51-43-4; 5-hydroxyindoleacetic acid, 54-16-0; 3,4-dihydroxyphenylaceticacid, 102-32-9;carbon, 7440-44-0. LITERATURE CITED Mefford, I. N. J . Neuroscl. Methods 1981, 3 , 207-224. Kisslnger, P. T. Anal. Chem. 1977, 4 9 , 447A-456A. Blank, C. L. J . Chromatogr. 1976, 117, 35-46. Roston, D. A.; Shoup, R. E.; Klsslnger, P. T. Anal. Chem. I982!, 5 4 , 1417A-1434A. Fleet, B.; Little, C.J. J . Chromatogr. Sci. 1974, 12, 747-752. Swartzfager, D. G. Anal. Chem. 1976, 4 8 , 2189-2192. Hughes, S.;Johnson, D. C.Anal. Chim. Acta 1981, 132, 11-22. Sarnuelsson, R.; O'Dea, J.; Osteryoung, J. Anal. Chem. 19801, 5 2 , 2215-2216. Caudlll, W. L.; Howell, J. 0.; Wightrnan, R. M. Anal. Chem. 198:!, 5 4 , 2532-2535. Stastny, M.; Volf, R.; Benadlkova, H.; Vlt, I . J . Chrornatogr Sci. 1983, 21, 16-24. Ewlng, A. G.; Dayton, M. A.; Wightrnan, R. M. Anal. Chem. 198'1, 53, Wlghtrnan, R. M.; Palk, E. C.;Borrnan, S.; Dayton, M. A. Anal. Chem. 1978, 50, 1410-1414. Caudill, W. L.; Wlghtrnan, R. M. Anal. Chim. Acta 1982, 141, 269-278. Bond, A. M. "Modern Polarographic Methods In Analytical Chernistry"; Marcel Dekker: New York, 1980. Stefani, S.;Seelber, R. Anal. Chem. 1982, 5 4 , 2524-2530. Sarnuelsson, R.; Osteryoung, J. Anal. Chim. Acta 1981, 123, 97-105.
RECEIVED for review April 29, 1983. Accepted July 1, 1.983. This work was supported by NSF (CHE 81-21422). R.M.W. is an Alfred P. Sloan Fellow and the recipient of a Research Career Development Award (K04-NS-356).
