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Anal. Chem. 1988, 6 0 , 258-263
(18) Nagy, 0.; @xhardt, G. A.; Oke, A. F.; Rice, M. E.; Adams, R. N.; Moore, R. E., 111; Szentlrmy, M. N.; Martin, C. R. J . Elecfroanal. Chem . Inter&&/ Electrochem. 1985, 788, 85-94. (19) Thackeray, J. W.; WrlQhton, M. S. J . Fhys. Chem. 1986, 9 0 , 6674. (20) Natan, M. J.; Mallouk, T. E.; Wrighton, M. S. J . Phys. Chem. 1987, 97, 648. (21) Natan. M. J.; W n g e r , D.; Carpenter, M. K.; Wrlghton, M. S. J Phys Chem. 1987, 9 1 , 1834. (22) Thackeray, J. W.; Whlte, H. S.: Wrlghton, M. S. J Phys. Chem. 1985, 89, 5133. (23) BBlanget, D.: Wrighton, M. S. Anal. Chem. 1967, 59, 1426. (24) Guadalupe, A. R.; Abru’ia, H. D. Anal. Chem. 1985, 57, 142. (25) Wler, L. M.; Guahlupe, A. R.; Abruira, H D. Anal. Chem. 1985, 5 7 , 2009. (26) Guadalupe, A. R.; Wier, L. M.; Abruha, H. D. Am. Lab (Fairfieid, Conn.) 1986, 78(8), 102. (27) Guadalupe, A. R.; Abru‘ia, H. D. Anal. Lett. 1986, 79, 1613 (28) Wlghtman, R. M. Anal. Chem. 1881, 53, 1125A. (29) Felndengen, L. E.; Kasperek, K. In Trace .€/ement Analytlcal Chemlsfry In Mdklne and Blobgy; Bratter, P., Schramel, P., Eds.; Walter de Gruyter: Berlin, 1980. (30) U,T.-K.; Vallee, E. L. I n Modern Nufritlon In Heaith and Disease, 6th ed.; cbodhart, R. S., Shils, M. E., Eds ; Lea 8 Feblger: Rlladeiphla, PA, 1960. (31) Membrane Tansport of Calclum; Carafoil, E., Ed.; Academic: London, 1962. (32) The Roton and Caklum Pumps: Azzone, G. F., Avron, M., Metcalfe, J. C.. Quagkrieilo, E., Silipfondi, N., Eds.; Elsevier: Holland, 1978. (33) Hokr, M. Transport Across Bldoglcal Membranes; Pitman Advanced Publ[sMng Program: London, 1981. (34) Ion and Enzyme Electrodes ln Bbbgy and Medlclne; Kessier, M ,
(35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45)
Clark, L. C., Lubbers, D. W., Sliver, I. A,, Simon, W.. Eds.; University Park Press: Balmore, MD, 1976. Glass Microelectrodes; Laveilee, M., Schanne, 0.F., Hebert, N. C., Eds.; Wiley: New York, 1989. Ewing, A. G.: Withnell, R.; Wightman, R. M. Rev. Sci. Instrum. 1981, 52(3), 454. Factor, A.; Heinshon, G. E. J . Polym. Sci., Part B 1971, 9 , 289. Abruha, H. D.: Bard, A. J. J . Am. Chem. SOC. 1981, 703, 6898. Dlehl, H. Calcein, Calmagite,and o ,o’Dihydroxyazobenzene. Tihimetric , Coiorlmetrlc and Fluorometric Reagents for Calcium and Magne slum: G. F. Smith Chemical Co.: Columbus, OH, 1964. Close, R. A.; West, T. S. Taianta, 1960, 5 , 221. Herrero-Lanclna, M.; West, T. S. Anal. Chem. 1963, 3 5 , 2131. Mendes-Bezerra, A. E.; Stephen, W. I. Analyst (London) 1969, 9 4 , 117. Scarpa, A.: Brinley, F. J., Jr.: Dubyak, G. Biochemlstry 1978, 7 7 , 1378. Latimer, G. W., Jr. Taianta 1968, 75, 1. Orion Research Analytical Methods Guide, 1965.
RECEIVED for review April 23,1987. Accepted October 6,1987. This work was supported by the National Science Foundation, Dow Chemical Co., Eastman Kodak Co., and Honeywell, Inc. H.C.H. acknowledges support by a fellowship from the Aerospace Corp. H.D.A. acknowledges support via the Presidential Young Investigator Award Program of the National Science Foundation and the Alfred P. Sloan Foundation.
Amperometric Detection of Catechols in Capillary Zone Electrophoresis with Normal and Micellar Solutions Ross A. Wallingford and Andrew G. Ewing* Department of Chemistry, The Pennsyluania State University, University Park, Pennsylvania 16802
A recently Introduced system for lnterfaclng caplllary zone eietrophords wlth ott-cdumn amperometrlc detectlon Is
demonrtratrdonsmaMorecdumswllhnormalandmlcellar e d u t h The u18 of emaWer h e r dlameter columns with a carbodlbw electrochemlcal detector allows for Increased reparation efflbkncy and decreased detectlon Ilmits. On a 26-~tn4.d. column, separation effklencles of greater than 400 OOO theofdcd plater and subfemtomole detection limits have been obtalned for several catechols. I n order to enhance the selectlvlty for nonionic solutes, mlcellar solutlons were employed to aHow separatlon of solutes based on partllionhg into the micdles. The electrochemistry of neutral and catknlc catechols In mlcdlar sokrwone was examlned In order to characterlze the usefulness of electrochemlcal detectlon wHh micdlar sokrtkrw. Amperometrk detectlon wlth mfcellar sdutlars Is demonstrated for a mixture of catechols on a 52-gm-1.d. column. Unknwn amounts of catechol detected were less than 20 fmol when using mlcellar solutlons.
Zone electrophoresis in capillaries was introduced by Mikkers et al. in 1979 ( I ) and was refined by Jorgenson and co-workers (2-4) in the early 1980s. Capillary zone electrophoresis (CZE) has developed into a highly efficient means of separating very small amounts of ionized compounds. Separation efficiencies of greater than lo5theoretical plates are often obtained with this technique. CZE has been shown to be an extremely powerful liquid-phase separation technique
(2-6);however, this method is limited to separations of charged molecules. In order to extend the advantages of CZE to neutral compounds, a technique known as micellar electrokinetic capillary chromatography (MECC) has been developed. First introduced by Terabe et al. (7,8), this method employs buffers to which surfactants have been added at concentrations above their critical micelle concentration (cmc) (9,lO). Two distinct phases, an aqueous phase and a micellar phase are formed within the column. Each phase migrates at different velocities toward the electrode having the same charge as the micelles. The micellar phase electrophoretically opposes the electroosmotic flow of the aqueous phase; however, the strength of the electroosmotic flow leads to a net migration of the micellar phase in the same direction as electroosmosis. The micellar phase moves much slower than the aqueous phase and is therefore termed a pseudostationary phase. Furthermore, nonionic solutes appear to partition into the micelles. Retention based on partitioning of solutes between the aqueous and micellar phases results in solute zone velocities between those of the two phases. Perhaps the largest obstacle preventing the widespread acceptance of both CZE and MECC lies in the area of detection. Small column dimensions and extremely small solute zone widths demand detectors with minimal dead volume in order to preserve the efficiency inherent in these techniques. In addition, detection methods must not disturb the potential field across the column. Based on these limitations, spectroscopy-based detectors capable of detection within the ca-
0003-2700/88/0360-0258$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 60, NO,
f.
Flgure 1. (A) Schematic of CZE system with electrically conductive A, buifwr resarvok; E, separation capikly; C. &at& capnlary. (E) Top Ww of amperomeW detection system: A. column; E, porous glass ioint assembly; C. Plexiglas block; D. carbon-fiber working electrode; E. microscope slide; F. micromanipulator; G. reference
m:
electrode port. pillary itself have been predominant. These detectors include arc-lamp fluorescence ( 2 4 , I I , 12), laser fluorescence (13-16). and UV absorption (7,8,17-23). Laser fluorescence is the most sensitive on-column detection mode available for CZE and MECC, providing suhfemtomole detection limits (15). W absorption detection, although more versatile, has poorer detection limits. Recently, two off-column detection methods have been demonstrated for use with CZE. Smith and co-workers (24) have employed an electrospray interface to couple CZE with on-line mass spectrometric detection. This system is unique in that no cathodic buffer reservoir is used. Instead, electrical contact is made directly to the solvent in the column through an electrospray needle at the column outlet. Our laboratory has recently demonstrated CZE with on-line electrochemical d e w i o n (25). Isolation of the electrochemical detector from the applied electrical field was accomplished with off-column detection following a conductive low-deadvolume capillary connection (Figure la). In this system, a conductive joint is made near the cathodic end of the column by fracturing the column and encasing the fracture in porous Vycor glass. When submersed in a buffer reservoir dong with the cathode, the separation or driving potential can he selectively applied across the first segment of capillary. The strong electroosmotic flow generated in this first section of capillary serves to force solvent and anal* zones past the joint and through the second section of capillary to the detector. Electrochemical detection can then be performed at the end of the second segment of column without severe interference from the applied separation field. This system represents a departure from conventional CZE and MECC apparatus where both ends of the column are immersed in buffer reservoirs to facilitate the application of a potential across the column. Electrochemical detectors in liquid-phase separations are desirable due to their selectivity and high sensitivity. As CZE and MECC develop further, an important application for these methods will be in the area of biological analyses where their low-volume capabilities may he utilized. For such separations, extremely sensitive detection methods are mandatory. Electrochemical detection has been shown to be among the most sensitive detectors available for capillary chromatography, with detection limits as low as 20 amol being reported (26). Laser fluorescence detection can approach these de-
3. FEBRUARY 1. 1988
259
tection limits; however, in most cases derivatization of solutes is necessary to produce adequate sensitivity. Electrochemical detection affords sensitive detection of many biologically important molecules without derivatization. In addition, electrochemical detection allows the use of smaller diameter columns without losses in sensitivity (26, 27). In this paper, we address three aspects of electrochemical detection in capillary zone electrophoresis with normal and micellar solutions. First, we report the use of electrochemical detection with 26-pm-i.d. capiuaries for CZE separations. Here we demonstrate the ability to efficiently couple columns of extremely small inner diameter, extending both separation efficiency and detection limits. Second, the voltammetric behavior of several catechols at a carbon-fiber electrochemical detector and in micellar solutions is presented. Finally, from these data we present the strengths and limitations involved in interfacing electrochemical detection to MECC. EXPERIMENTAL SECTION Electrokinetic Apparatus. The apparatus used for electrokinetic separations is shown in Figure l a and is similar to that described previously (25). Fused-silica capillarieswere obtained from Polymicro Technologies (Phoenix, AZ). The capillaries were filled with buffer and sodium dadecyl sulfate (SDS) solutions ria a helium-pressurized solvent reservoir at a pressure not exceeding 8 pig. A SpeUman high-voltape dc power supply was used to apply the potential field for separations. A Plexiglas hox equipped with an interloek system protected the operator from accidental contact with the high-voltage end. Construction of the Porous Joint. The electrically conductive joint was fabricated as described previously (25) with the following exceptions. After a section of capillary was cut to the required length, the polyimide coating was removed from the last 2-3 cm of the column. A 2-cm length of porous glass capillary (Coming, no. 7930) was placed over the exposed end of the column with approximately 1cm of the column protruding past the end of the porous capillary. This section of the column was then cemented (Dum Cement, Devcon Corp.) onto a 3-cm-longsegment of glass microscope slide, keeping the end of the porous g h s flush with the end of the slide. The porous capillary was shortened to 1cm hy crushing from each end toward the center. This section of porous capillary was then moved to the side of the exposed mea The exposed column was scored with a diamond-tipped glass cutter at a location of 1.5-2.5 em from the end. Under a microscope, the column was fractured at the scored region with the tip of a scalpel blade. The two segments of column were then positioned to form a tight joint with the best possible alignment of the inner bores. With practice, excellent alignment of the bores can be obtained. The porous capillary was then carefully moved into position over the fracture. The ends of the porous capillary were then sealed with epoxy (Devcon, %Ton) and the assembly was mounted in a plastic reservoir. Many buffers and micelle solutions react with certain epoxies causing them to deteriorate. Devcon 2-Ton epoxy appears to he stable in all solutions used to date. Electrochemical Detection. Electrochemical detection was performed with 10-rm-diameter carbon fiben protruding 0.2-0.5 mm from drawn glass capillariesas the working electrodes. The procedure for construction of these electrodes was detailed in a previous publication (25). A top view of the detection apparatus employed is shown in Figure lh. The end of the detection capillarly was positioned into the center of a 0.25-in.-diameter cell in the Plexiglas block. The end of the microelectrode was manipulated through the opposite slot in the Plexiglas block and into the end of the detection capillary with a micromanipulator (Oriel Corp.) while viewing under a microscope. A sodium-saturated calomel reference electrode (SSCE) was placed into a second reservoir in the Plexiglas block, which was connected with the cell. The cell was filled with an electrolyte solution of either 0.1 M KCI or 0.01 M phosphate buffer. Use of KCI solution in the cell when employing SDS solutions resulted in precipitation of SDS at the end of the column. Potential control between the reference and working electrodes was accomplished with a mercury cell and a voltage divider. Oxidation currents were amplified with
260
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a Kiethley 427 current amplifier and recorded on a strip-chart recorder and a Hewlett-Packard 3392A integrator. Linearity and detection limits were determined while employing a locally constructed second-order Bessel filter (28),having a time constant of 500 or 700 ms. Cyclic Voltammetry. Cyclic voltammetry was performed with a three-electrode locally constructed potentiostat at a scan rate of 100 mV/s and with a time constant of 10 ms. The electrochemical cell consisted of a 30-mL vial. A platinum wire was employed as auxiliary electrode. Chemicals. Catechol, 4-methylcatechol, and norepinephrine were obtained from Sigma. 4-Ethylcatechol was obtained from Lancaster Synthesis. All analytes were prepared from 0.01 M stock solutions in 0.1 M perchloric acid. Sodium dodecyl sulfate was obtained from Aldrich. Phosphate buffer (pH 6.95-7.0) was prepared by diluting appropriate amounts of sodium phosphate dibasic (Mallinckrodt) and sodium phosphate monobasic (Fisher) in doubly distilled water. MES buffer (2-morpholinoethanesulfonic acid) was obtained from Sigma and was adjusted to the proper pH with solid sodium hydroxide. All chemicals were used as received.
RESULTS AND DISCUSSION Electrochemical Detection Interface. The major problem encountered when attempting to couple CZE or MECC with electrochemical detection concerns isolation of the detector from the large separation potentials applied. Failure to electrically isolate the detector leads to extremely high noise levels which can be several orders of magnitude greater than the Faradaic currents measured. In our system (Figure la) this isolation has been accomplished by incorporating an electrically conductive joint composed of porous Vycor glass (29, 30) near one end of the column. The conductive joint allows the separation potential to be applied over only the first section of capillary (separation capillary). Detection via a carbon-fiber electrode inserted into the end of the second segment of capillary (detection capillary) can then be performed without severe interference from the separation potential. The detection end is a t a potential near ground but cannot be identical. This condition is evident in a background noise level that is proportional to the separation potential applied. In the work reported here, electrochemical detection was performed amperometrically. A potential was applied between the working and reference electrodes with a Hg cell and a voltage divider. This sytem was adopted in place of the three-electrode potentiostat used previously (25) for two reasons. First, it has been reported that two-electrode electrochemical systems generally produce lower noise levels than their three-electrode counterparts (31). Second, the Hg cell is better isolated electrically from any effects of the highvoltage power supply. This isolation was found to be necessary in order to eliminate occasional transient current spikes with this system. Amperometric Detection in Small-Bore CZE. A major concern with coupling off-column detectors to capillary columns is band broadening resulting from the interface and detector. The band broadening aspects of the coupling system presented here have been discussed in detail previously (25). The major sources of added band broadening concern the length of the detection capillary and the precision of the bore alignment. The volume of buffer contained within the detection capillary undergoes frictional forces with the capillary walls and creates a back pressure, which resists the electroosmotic flow generated in the separation capillary. The back pressure created appears to distort the normally flat flow profile of the electroosmotic flow, thus causing broadening of solute bands while they are in the separation capillary. In addition, some band broadening is also associated with the time the solute bands spend in the detection capillary under laminar flow conditions. Both of these effects can be mini-
A
\
~
1
0
I
I
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I
B
100 pA
I
4
I
I
I
,
6 8 TIME (minl
I
,
IO
,
,
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,
,
14
Flgure 2. Electropherogram of catecholamines with electrochemical detection on a 26-prn4.d. column: 0.02 M MES buffer (pH 6.05); injectlon, 2 s at 5 kV; separation potential, 25 kV (2 PA); electrode potential, 0.7 V vs SSCE; A, dopamine; B, norepinephrine; C, epinephrine; D, catechol.
mized by employing short detection capillaries. We have found that detection capillaries of less than 2.5 cm in length produce minimal band-broadening contributions. We have obtained superior results with capillaries having inner diameters as small as 26 pm in CZE separations. Figure 2 shows an electropherogram of three cationic catecholamines and a neutral catechol on a 26-pm4.d. column with an 87.9-cm separation capillary and a 1.8-cm detection capillary. The extremely small peak widths in Figure 2 are notable, especially for the cations, which might be expected to electrostatically interact with the column walls leading to band broadening ( 4 , 5 ) . The efficiencies calculated from the peak half-widths are 432000 for dopamine, 375 OOO for norepinephrine, 361 OOO for epinephrine and 408 000 for catechol. These efficiencies represent an improvement by a factor of 3-4 over the results obtained on a 75-pm-i.d. column under similar conditions (data not shown). Improved separation efficiency is expected with smaller bore capillaries on the basis of more efficient heat dissipation (1, 2, 32). However, one might expect capillary bore alignment to be much more difficult as the bore size is decreased. The dramatic increase in efficiency observed with the smaller capillaries implies that the column/detector interface is not the limiting factor determining separation efficiency in these experiments. Electrochemical detection also affords excellent detection limits with small-bore capillaries. The total amounts of each compound injected onto the column in CZE can be determined with the following equation
Q=
(F + 1osm)VACti
L
(1)
where Q is the quantity injected, V is the injection voltage, A is the cross-sectional area of the capillary, C is the concentration of the sample, ti is the injection time, L is the length of the column over which the potential is applied, and (u + u-) is a term expressing the effective electrophoretic mobility of the component under the influence of electroosmotic flow (4). The amounts injected in Figure 2 were determined to be approximately 37 fmol for dopamine, 36 fmol each for norepinephrine and epinephrine, and 47 fmol for catechol. Detection limits ( S I N = 3) on the order of 0.2-0.4 fmol have been extrapolated for each solute. The volume of the sample plug injected was approximately 0.23 nL, which is sufficiently small to preclude any overloading effects which can be detrimental to efficiency (6,32). One reason for the excellent sensitivity
JALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988
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3rnM SDS
8rnM SDS
80rnM SDS
0 I
L
-a2
02
0.6
0.02
0.04
0.06
0.08 0
SDS CONCENTRATION (M)
ID
E ( V V S SSCE)
M Flgure 4. Oxidation current vs SDS concentration for 5 X catechol (O),dopamlne (+I, noreplnephrine ( O ) ,and 4methylcatechol
Flgure 3. Oxklatlon of 5 X M catechol In pH 7 phosphate buffer wlth varied SDS concentrations at a cyllndrlcal carbon-flber electrode. Scan rate, 100 mVls.
(A)at cylindrical carbon-flber electrodes. Currents normalized to geometric electrode area for comparison. Scan rate, 100 mV/s; pH 7 phosphate buffer.
obtained a t smaller bore capillaries with carbon-fiber electrochemical detection is the increased coulometric efficiency obtained as the annular flow region around the electrode decreases (26, 27). Electrochemical Considerations for Detection i n MECC. In order to evaluate the critical parameters for amperometric detection in MECC, cyclic voltammetric experiments with cylindrical carbon-fiber microelectrodes were performed in solutions of several catechols at various concentrations of SDS. Significant effects were observed concerning the half-wave potential (Elp)and the limiting current. Although electrochemical detection will only be affected marginally by shifts in voltammetric potential, the ability to quantitate eluting peaks is critically dependent upon the oxidation efficiency, which changes in micellar solutions. It is obviously important to understand and characterize these parameters before using electrochemical detection with MECC. Figure 3 shows typical voltammograms of catechol obtained at carbon-microcylinder electrodes at various concentrations of SDS. The voltammograms are sigmoidal, indicating near-steady-state behavior as has been observed experimentally for microcylinder electrodes with small radii (26,33). It may be noted from Figure 3 that an increase in the concentration of SDS results in a shift in Ellz to more negative values. This suggests that the oxidation product, orthoquinone, is more soluble in the SDS micelles than is the catechol. Increasing the concentration of SDS above the cmc (8-10 mM for SDS) causes the formation of a larger number of micelles, resulting in a shift in Nernst potential to more negative values. The average shift in of catechol measured at three electrodes was 85 mV over the range of 3-80 mM SDS. The largest change in Ellz occurred in the pre-cmc region of 3-8 mM SDS, where an average shift in Ellz of 33 mV was observed. This effect may result from interaction between the relatively hydrophobic catechol species and a layer of surfactant adsorbed on the electrode surface. The practical implications of a shift in Ellzat high surfactant concentration is that lower electrode potentials may be employed for the oxidation of neutral electroactive species. Lower electrode potentials generally translate to lower background currents with electrochemical detection. The voltammetry of both neutral and cationic catechols displays a fairly dramatic dependence of limiting current on SDS concentration (Figure 4). The data shown in Figure 4 were normalized with respect to the response of 5 X M solutions of each compound at an electrode of known surface area and are reported as current densities. All data reported
are averages for at least two electrodes. An increase in the limiting current is observed for constant concentrations of the nonionic catechols (catechol, 4-methylcatechol) following small additions of surfactant. Under the same conditions, a decrease in the limiting current is observed for the cationic catechols (dopamine, norepinephrine). The limiting current drops off as a function of surfactant concentration at levels above the cmc; however, the decrease in signal is much more dramatic for the cationic species than for the nonionic species. It appears that several competing equilibria must occur to explain the voltammetric behavior observed. The enhanced limiting currents for the neutral species at low surfactant concentration are puzzling, since these data would appear to indicate either an increase in electrode surface area or a decreased solution viscosity and, hence, increased diffusion coefficient. The first possibility is not likely given the observation that the behavior is reversible. The second possibility is contrary to viscosity effects observed with surfactant solutions (34). Attenuated limiting currents for dopamine and norepinephrine at low surfactant concentration might result from interaction with small surfactant aggregates, thereby reducing the effective concentration of solute. Alternatively, norepinephrine or dopamine might interact wth the anionic surfactant resulting in an ion pair with a reduced diffusion coefficient. At surfactant concentrations above the cmc, nonionic species will partition into the micelles while cations can either associate with the anionic micelles or ion pair with free monomer during or before micellar solubilization. Electron transfer apparently cannot take place between electrode and solute if the solute is located inside the micelle core (35). Thus, as the micelle concentration is increased, a decrease in the effective solute concentration is observed resulting in a decreased limiting current for all solutes. As a practical consideration, the limiting current dependence on SDS concentration will affect the sensitivity and calibration of detection in MECC. These effects are much more dramatic for the cationic solutes examined where calibration can be affected by 60440% with varied SDS concentration. Since solute retention in MECC is dependent on the SDS concentration, trade-offs in sensitivity may be necessary in order to achieve the desired resolution of components. MECC with Electrochemical Detection. Electrochemical detection of three nonionic species (catechol, 4-methylcatechol, and 4-ethylcatechol) electrophoretically separated from a cation (norepinephrine) in phosphate buffer at pH 6.98 is shown in Figure 5. This separation was obtained on a 52-pm-i.d. fused-silica column with a 65.1-cm separation capillary and a 2.4-cm detection capillary. As expected, only
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988 B
I
T
1 nA 11
0
/I
A
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(min.)
8
Flgure 5. Capillary (52-pm i.d.) electropherogram of catechols with electrochemical detection: 0.01 M phosphate buffer (pH 6.98); injection, 3 s at 20 kV; separation potential, 20 k V (10 pA); electrode potential, 0.7 V vs SSCE; A, norepinephrine; B, catechol, 4-methylcatechol, 4-ethylcatechol. A
B
croelectrode was employed for comparative experiments and was pretreated prior to initiating experiments with each mobile phase by scanning from -0.2 to 1.8 V vs SSCE at 200 mV/s in phosphate buffer. Such anodic pretreatments occasionally affect the sensitivity of the detector; however, detector noise changes proportionately. Linearity was verified in both SDS solutions by digital integration of the catechol peaks (correlation coefficient, 0.997 for 0.01 M SDS; 0.999 for 0.025 M SDS). The minimum quantities of catechol detected were on the order of 19 fmol in 0.01 M SDS ( S I N = 3.6) and 17 fmol in 0.025 M SDS (SIN = 2.4). Lower operating voltages serve to lower noise levels and increase coulometric efficiency thereby lowering detection limits further; however, this occurs at the expense of analysis time. Detection limits can also be improved by employing smaller diameter columns where the coulometric efficiency of the detector is increased (26, 27). The elution order observed in Figure 6 illustrates an interesting phenomenon. The nonionic catechols elute in order of their relative hydrophobicities as expected in MECC. However, norepinephrine, a cation at pH 7, elutes much later than expected for a cation undergoing only electrophoretic effects. The norepinephrine cation may be solubilized by two slightly different mechanisms. The first mechanism concerns electrostatic interaction of the cation with the negatively charged Stern layer of the SDS micelle (36). The second mechanism involves ion pairing of the cation with free SDS monomer and solubilization of the ion pair by the micelle. At surfactant concentrations above the cmc, a constant concentration of surfactant monomer equal to the cmc always exists (9, 10). The free SDS monomer (DS-) can ion pair with norepinephrine ion to form a neutral species as indicated by the following equilibrium: OH I
0
4
12
8
TIME
Imin.)
16
20
Figure 6. Capillary (52-pm i.d.) electrokinetic separation of catechols with electrochemical detection: 0.01 M phosphate buffer (pH 6.98) with 0.01 M SDS; Injection, 2 s at 13 kV; separation potential, 13 kV (8 PA); electrode potential, 0.7 V vs SSCE; A, catechol, B, 4methylcatechol; C, 4-ethylcatechol; D, norepinephrine. two peaks are observed. Consistent with CZE theory, the cationic norepinephrine elutes first, followed by the three neutral catechols which elute with a velocity corresponding to the electroosmotic flow velocity. The calculated amounts injected are approximately 3.5 pmol for norepinephrine and 1.0 pmol each for the three catechols. Separation selectivity for neutral species can be enhanced dramatically by adding a surfactant to the buffer at a concentration above the cmc (7,8,14-16,21,23).Figure 6 shows a chromatogram obtained on the same column used in Figure 5 employing the same phosphate buffer with 0.01 M SDS added. In this system the neutral catechols now appear to partition into the hydrophobic interior of the micelles and are retained based on their relative hydrophobicities. Total amounts of solutes injected are approximately 190 fmol for norepinephrine, 170 fmol for catechol, 150 fmol for 4methylcatechol, and 280 fmol for 4-ethylcatechol. The differences in voltammetric limiting current as a function of SDS concentration warrant a careful characterization of electrochemical detection in MECC. Detection limits and linearity were determined for injections of catechol in 0.01 M and 0.025 M SDS solutions. A 52-pm-i.d.column consisting of a 64.3-cm separation capillary and a 1.7-cm detection capillary was used for this study. The same carbon-fiber mi-
OH I
ion pair
The neutral ion pair is very hydrophobic and can be solubilized by the micelles, thus constituting a possible mechanism for increased retention of ionic species in MECC. Ion pairing in this system is analogous to ion pairing in HPLC where surfactants (ion-pairing reagents) are added at concentrations below the cmc in order to form ion pairs, which are partitioned between the stationary and mobile phases (37). Reactions of ions with surfactant monomer in micellar solutions have been previously proposed by several research groups investigating electrochemistry in micelle-containing solutions (34,38-40). Several reports (36,38)postulate subsequent solubilization of the neutral ion pair into the micelle interior. It is not clear at this time which solubilization mechanism is in effect in this system. High separation efficiencies and an ability to control retention selectivity of neutral and ionic solutes coupled with the low sample volume capabilities of MECC suggest a very powerful separation technique, especially for biological separations. The literature contains several reports of separations of a wide variety of compounds with efficiencies of greater than 100000 theoretical plates for neutral species being common (7, 14-16,21,23). The first three peaks in Figure 6 exhibit separation efficiencies of 74 000, 124 000, and 137 000 theoretical plates, respectively, based on peak half-widths. The last peak corresponding to norepinephrine exhibits only about 10 OOO plates and a high degree of tailing. This result is not unexpected. The low efficiency of the norepinephrine peak can be attributed to the secondary and tertiary equilibria involved in the retention of the norepinephrine cation. When secondary equilibria are employed in HPLC to enhance se-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988
lectivity, efficiencies are seldom as great as those obtained with normal HPLC separations (42). In addition to the solubilization equilibria postulated, adsorption of cationic solutes to the surface of the fused-silica capillary is also potentially problematic. Tailing of the norepinephrine peak is indicative of interactions with the capillary walls, a fact that is supported by the tailing also observed for the norepinephrine peak in Figure 5 when no SDS was present. However, since tailing is greater in the presence of SDS micelles, interactions between the cationic norepinephrine and the micelles appear to be the dominate factor involved. Work is currently being carried out to improve the efficiency for cations in MECC since this technique has the potential to greatly enhance selectivity over conventional C Z E The ultimate goal, however, is to obtain optimal resolution of solutes. Sacrificing separation efficiency for selectivity is satisfactory, provided detection limits are not severely compromised. The system described herein provides an opportunity to develop low-volume high-resolution separations with extremely sensitive detection of electroactive solutes. Registry No. SDS, 151-21-3; epinephrine, 51-43-4; catechol, 120-80-9; dopamine, 51-61-6; norepinephrine, 51-41-2; 4methylcatechol, 452-86-8; 4-ethylcatechol, 1124-39-6.
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RECEIVED for review August 17, 1987. Accepted October 15, 1987. This material is based upon work supported by the National Institutes of Health under Grant No. 1 R 0 1 GM37621-01. A.G.E. is the recipient of a Presidential Young Investigator Award (CHE-8657193).
