Articles Anal. Chem. 1995, 67, 13-18
Membrane-Based On-Column Mixer for Capillary Electrophoresis/Electrochemistry Jianxun Zhou and Susan M. Lunte*
Center for Bioanalytical Research, 2095 Constant Avenue, University of Kansas, Lawrence, Kansas 66047
One of the drawbacks of chemically modified electrodes for electrochemical detection in conjunctionwith capillary electrophoresis (CE) is that the optimal run buffer for the CE separation is often not compatible with the electrolyte conditions necessary for maximum detector response. This paper reports the development of a membrane-based on-column mixer for CE which makes it possible to perform the separation and detection using ditrerent buffer conditions. The mixer consists of a cellulose acetate tube ca. 2-mmlength and 50-mmi.d. The pH and/ or ionic strength of the CE run buffer can be altered at the detector end by placing the mixer in a cathodic buffer reservoir containing a m o m solution of either HCl or NaOH. Ions from the modifying solution diffuse through the membrane into the detector end of the capillary and cause the desired change in pH. The use of this system was demonstrated for the detection of several biologically important compounds by capillary electrophoresiselectrochemistry (CEEC) using chemically modified electrodes. This included the use of a CoPC electrode for the detection of ribonucleosides, a a amino acid oxidase electrode for the detection of amino acids, and a mixedvalent RuCN electrode for the detection of disulfides. Detection limitsand detector stabilitywere improved over those reported previously for unoptimized systems. Since its introduction in 1988 by Wallingford and Ewing,' electrochemicaldetection (EC) has been established as a useful method of detection for capillary electrophoresis (CE) and has been the subject of several Most initial studies of capillary electrophoresis with electrochemicaldetection (CEEC) were focused on the detection of catecholamines using carbon fiber working electrodes.6-8 More recently, other electrode materials as well as chemically modified electrodes (CMEs) have been exploited in CEEC for the determination of biological compounds. These include the detection of thiols using a Hg/ (1) Wallingford, R A; Ewing, A. G. Anal. Chem. 1987,59,1792. (2) Cum, P. D., Jr.; Engstrom-Siverman, C. E.; Ewing, A. G. Electroanalysis 1991,3,587. (3) Yik, Y. F.; Li,S. F.Y . Trends Anal. Chem. 1992,1 1 , 325. (4) Lunte, S. M.; O'Shea, T. J. Electrophoresis 1994,15, 79. (5) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994,66, 527A. (6) Ewing, A. G. J. Neurosci. Methods 1993,48, 215. (7)Sloss, S.; Ewing, A. G. Anal. Chem. 1993,65, 577. (8) Olefirowicz, T. M.; Ewing. A. G. J. Chromatogr. 1990,499,713. 0003-2700/95/0367-0013$9.00/0 0 1994 American Chemical Society
Au amalgam microelectrodeg or cobalt phthalocyanine- (CoPC) containing carbon paste microe1ectrodes.l0 Carbohydrates have been determined using pulsed amperometric detection (PAD)at Au microe1ectrodes1lJ2 and constant potential amperometric detection at copper microelectrode^.'^ These new electrode materials and CMEs facilitate the detection of substances that normally exhibit slow electron transfer kinetics at bare carbon fiber electrodes. The implementation of micro-CMEs for CEEC has significantlyexpanded the applicability of this technique in pharmaceutical and biomedical analysis. However, there is still much work to be done in the development of the CME-based CEEC systems, if one considers the wide variety of CMEs that have been employed for liquid chromatography with electrochemical detection (LCEC) .14 One drawback of many of the CME-based detectors is that there is often a large discrepancy between the buffer composition needed for the optimal CE separation and the best conditions for EC dete~ti0n.l~ This problem could be overcome if a method for changing the buffer composition at the end of the capillary were developed. The composition of the run buffer could then be easily manipulated postcolumn as required by the particular CME-based EC detection method. Only a few devices that could be used as the interface for postcolumn pH manipulation have been reported. Rose and Jorgenson16 devised a coaxial capillary reactor for postcapillary derivatization for fluorescence detection. Albin et al." used a gap junction reactor for postcapillary derivatization. In both of these cases, the reactor is placed in the high potential end of the capillary. Avdalovic et a1.l8 and Dasgupta and Baolg have described a suppressor for conductivity detection of ions following CE separation. The suppressorwas employed mainly to minimize the conductivity of the final eluent arriving the detection cell; however, it can also be used for postcolumn alteration of the pH (9) O'Shea, T. J.; Lunte, S. M. Anal. Chem. 1993,65, 247. (10) O'Shea, T. J.; Lunte, S. M. Anal. Chem. 1994,66, 307. (11) O'Shea, T. J.; Lunte, S. M.; LaCourse, W. R Anal. Chem. 1993,65, 948. (12) Lu, W.; Cassidy, R M. Anal. Chem. 1993,65, 2878. (13) Colon, L A; Dadoo, R; Zare, R N.Anal. Chem. 1993,65, 476. (14) Baldwin, R P.; Thomsen, K N. Talanta 1991,38, 1. (15) Zhou, J.; OShea, T. J.; Lunte, S. M. J. Chromatogr., 1994,680, 271. 117. (16) Rose, D.; Jorgenson, J. W. J Chromatogr. 1988,447, (17) Albin, M.; Weinberger, R; Sapp, E.; Moring, S.Anal. Chem. 1991,63,417. (18) Avdalovic, N.; Pohl, C. A; Rocklin, R D.; Stillian, J. R Anal. Chem. 1993, 65, 1470. (19) Dasgupta, P. K; Bao, L. Anal. Chem. 1993,65, 1003.
Analytical Chemistry, Vol. 67, No. 1, January 1, 7995 13
of the run buffer. Since the suppressors are composed of ionexchange membranes, they are not generally applicable to all analytes. Problems can arise due to electrostatic interactions between charged analytes and the suppressor membrane. In this work we report the construction of a membrane-based mixer and its application in CEEC. The mixer, which has an inner diameter very close to that of the separation capillary, consists of a 2-mm length of cellulose acetate (CA) tubing between the separation and detection capillary. It is supported on a glass slide and immersed in the cathode buffer reservoir. By virtue of selective permeability, external buffer ions from the cathode reservoir diffuse through the membrane and mix with the run buffer, while analytes of much larger molecular size are prevented from “escaping” through the CA tubing. In this manner, the composition of run buffer can easily be modified postcolumn, as required by the particular CME-based electrode process. This mixer is demonstrated for the CEEC detection of several classes of biological compounds, including detection of nucleosides using a CoPC-modified microelectrode, determination of amino acids using a amino acid oxidasebased microbiosensor, and monitoring of disullides using a mixed-valent ruthenium cyanide filmdeposited carbon fiber electrode. EXPERIMENTAL SECTION
Chemicals and Solutions. All chemicals were obtained from Sigma (St. Louis, MO) and used as received, except for CoPC, which was purchased from Kodak (Rochester, NY), and cellulose acetate (39.8%acetyl content), which was acquired from Aldrich (Milwaukee, WT). Stock solutions of all analytes were prepared daily with NANOpure water (Sybron-Barnstead,Boston, MA) and were passed through a 0.2-pm pore size membrane filter prior to CE. Apparatus. The construction of the home-built CE system has been described in detail elsewhere-ll However, in this case, separation of the high voltage from the detector cell was accomplished using the CA membrane mixer as described in the text rather than an on-column Nafion joint. Samples were injected using a laboratory-built pressure injection system. Mixer Construction. The CA mixer was made according to a procedure similar to that reported for the improved NAon joint20 Fused silica capillaries of 75-pm id., 360-pm 0.d. (Polymicro Technologies, Phoenix, AZ) were used throughout this work. A l k m piece of Ni/Cr (80/20) alloy wire of 50-pm-diameter (Goodfellow Corp., Malvern, PA) was carefully inserted into the bore of the terminus end of a length of the capillary with ca. 5 cm protruding from the capillary end. A 2cm segment of the capillary was cut using a capillary cutter and moved along the axis of the Ni/Cr guide wire, with a gap of 2-2.5 mm left between the detection capillary and the longer separation capillary. These two segments were carefully aligned with the aid of the guide wire and a low-power microscope and were fixed onto a supporting glass slide using a two-component epoxy glue. M e r the epoxy was cured, a drop (ca. 5 pL) of 12%(w/v) CA solution (in acetone) was carefully placed around the guide wire and around a small portion (1-2 mm) of the end of each capillary. By applying a gentle stream of air, a cellulose acetate “tube” of approximately 50;um id. was produced. The CA tubing was allowed to air-dry for ca. 1h. The mixer assembly was then glued into a 5mL plastic buffer reservoir, leaving ca. 1.5 cm of the detection capillary (20) Park, S.; Lunte, S. M.; Lunte, C.E. Cum. Sep, 1993,12, 120.
14 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
t I
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Flgure 1, Schematic diagram of CA mixer (A) and CA joint (5) assembly.
sticking out of the vial. The mixer (CA tubing) assembly was placed in a 100-mL glass beaker filled with 0.15 M NaOH and was hydrolyzed overnight (12 h). The guide wire was then removed by holding the portion (ca. 0.5 cm) protruding from the terminus end of the capillary and gently pulling the wire out of the 2-cm detection segment. A schematic diagram of the mixer is shown in Figure 1. Construction of Micro-CMEs. The construction of CoPCcontaining carbon paste microelectrodes has been described in detail elsewhere.1° Briefly, the electrode was made by packing a portion of CoPC/graphite mixture into one end of a fused silica capillary (150-pm i.d., 360pm 0.d.) to a depth of -5 mm. In this work, the content of the catalyst (CoPC) was 2% (w/w) rather than 5% as used previously. The amino acid oxidase-modified electrode was made according to a procedure similar to that reported previously for glucose oxidase.1° In this case, the electrode was made by incorporating 5%w/w of D-amino acid oxidase into the carbon paste. The mixed valent rutheniumcyanide (mv-RuCN) carbon fiber electrode was prepared as described previously,15with the exception that a 33pm carbon fiber was used as the electrode substrate instead of a carbon fiber array microelectrode. Evaluation of Mixer. Capillary electrophoresis was performed with a 7 k m separation capillary at 20 kV using the run buffers listed in Table 1. A strong acidic or basic solution (herein HCl or NaOH, respectively), also listed in Table 1,was placed in the cathode buffer reservoir, which served as both the cathode buffer and the external modifying solution. The pH of the final eluent was measured with short-range pH paper (Fisher Scientific, Fair Lawn,NJ) after a 15-min equilibration under CE separation conditions. RESULTS AND DISCUSSION It was found that prehydrolysis of the cellulose acetate was necessary for normal function of the mixer. If the mixer was employed without prehydrolysis, two problems were immediately apparent. First, it took more than 5 h to establish the equilibrium between the run buffer and the modifying solution in the cathode
Table I. Use of the CA Mixer as a Postcolumn pH Adapter
final pH run buffer
original pH
10 mM NaHzPOs, 10 mM HCl
2.5 5.0 7.0 10.0 12.0
20 mM sodium acetate 20 mM TES* 10 mM CAPSc 10 mM NaOH, 10 mM NazC03
concn of NaOH‘ (mM) 100 20 5
100
9.5 12.0 12.5 11.0 12.5
1.5 1.5 2.0 4.5 7.5
7.8 9.2 9.5 10.5 12.0
6.5 6.5 8.0 10.0 12.0
concn of HCP (mM) 20 2.0 3.5 3.5 7.0 9.0
5 2.5 4.5 4.5 8.5 10.5
Modifying solutions. N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonicacid. C 5 (Cyclohexy1amino)-1-propanesulfonicacid.
buffer reservoir. Second, the membrane swelled within 30 min of exposure to the cathode buffer solution, blocking the flow passage and resulting in a dramatic decrease in the flow rate of effluent from the detection end. This led to long migration times, accompanied by a significant loss of column efficiency. Both of these problems were overcome by the alkaline prehydrolysis procedure described in the Experimental Section. Using the prehydrolysis procedure, the equilibration time was reduced to ca. 15 min, and swelling of the CA membrane was minimized. Further hydrolysis of the CA membrane by an alkaline modifying solution (as described below) was also minimized when the prehydrolysis procedure was employed. Similar hydrolysis procedures have been reported for the preparation of CA-coated electrodes.21~22 In our case, a more concentrated alkaline solution and longer hydrolysis time were employed due to the large membrane thicknesses involved. The CA membrane in these experiments is ca. 120-pm thick, compared with several micrometers for the CA coating on an electrode.zz The use of the CA membrane-based mixer as a postcolumn pH adapter was evaluated. A strongly acidic or basic solution (i.e., HCl or NaOH, respectively) was placed in the cathode buffer reservoir, which served as both the ground for the CE system and the source of the external moddying medium. Analyte eluting from the capillary merged with the modifying solution that had entered the membrane from the cathodic reservoir. The two solutions are mixed inside the 2-cm detection capillary segment and pushed toward the detection end by the remaining EOF. In this way, the pH of the final effluent was effectively moditied relative to that of the run buffer. The pH adapting capacity of the mixer is shown in Table 1. The system was evaluated for a wide range of buffer concentrations (10-100 mM), pH values (2.5-12.0), and ionic strengths and included both lowconductivity zwitterionic buffers and strong electrolytes. The mixer exhibited a broad capacity for postcolumn pH modification. The stability of the modulated pH of the final eluent was also examined over an 80 h period. The pH remained constant over a 3l/~dayperiod, and a stable baseline was also observed. Mixers were found to last 3-4 days before clogging. The analyticalpotential of the mixer was evaluated using three different electrochemical detection systems where the pH required for optimal EC detection was quite different from those necessary for the corresponding CE separation. CEEC of Ribonucleosides at the CoPC-ContainingMicroelectrode. Ribonucleosidesare essential components in RNA and related molecules of biochemical importance. Electrochemical (21) Wang, J.; Hutchins, L. D.Anal. Chem. 1985,57, 1536. (22) Hutchins, L. D.;Wang, J.; Peng, T. A d . Chem. 1986,58, 1019.
detection using the CoPCcontaining CME following separation by LC has been applied to the determination of this group of compounds. The employment of the CoPC CME reduces the overpotential for the oxidation of these analytes and therefore significantly improves the sensitivityand selectivity of the detector compared to those of the bare carbon electrode^.^^ One limitation of the CoPC CME-based detection scheme is that the current response for the analytes is a function of concentration of hydroxide ion, and a concentration greater than 50 mM was needed for optimal sensitivity. For CE separations, the use of strong alkaline buffer has several obvious disadvantages. The long-term stability of the fused silica capillary is problematic under these extremely high pH ~onditions.2~ More importantly,the high ionic strength leads to high separation currents, subsequent Joule heating, and loss of efficiency. Micellar electrokinetic chromatography (MEKC) at pH 8.5 as been successfullyapplied to the separation of ribonucleosides with good resolution and high column effi~iency.2~ However, CMEs do not work well under these separation conditions due to an inadequate concentration of OH-. Figure 2A shows the detection of adenosine and uridine at pH 8.5 using the CoPC-moditied electrodes. There is no detectable response. The discrepancy between the optimal run buffer pH and that needed for detection was overcome through use of the membrane mixer. To obtain optimal detection sensitivity and adequate resolution, a cathodic buffer of 0.5 M NaOH was employed, while the run buffer was pH 8.5 phosphate. Under these conditions, the pH of the final eluent was increased from 8.5 to > 13, and catalytic oxidation of the solutes was accomplished. The responses for adenosine and uridine using this system are shown in Figure 2B. Some “tailing” was observed, which is believed to be due to slow kinetics at the electrode surface. Other workers have also observed this phenomenon under FIA condition^.^^ Detection limits for both compounds were approximately 0.4 pM (S/N = 3). CEEC Determination of Amino Acids Using a amino Acid Oxidase-Modified Electrode. Amino acids constitute another group of compounds of biochemical significance. The lack of chromophores or electroactive functional groups in the molecular structure of most amino acids makes their separation and detection an analyticalchallenge. So far, relatively few efforts have been made toward the CE separation and detection of native amino a ~ i d s .Amino ~ ~ , ~acid ~ oxidase (AAO) has proven to be an (23) Tolbert, A M.; Baldwin, R P.;Santos, L. M. Anal. Left. 1989,22, 683. (24) Bushey, M. M.; Jorgenson, J. W. J. Chromntogr. 1989,480,301. (25) Cohen, A S.; Terabe, S.; Smith, J. A; Karger, B. L. Anal. Chem. 1987,59, 1021. (26) Kuhr, W. G.; Yeung, E. S.Anal. Chem. 1988,60, 1832.
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
15
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Time (min) 15
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Time (min) Figure 2. Electropherogramsof 100 pM adenosine and uridine at the CoPC CME with the CA mixer-based CEEC. Run buffer, 25 mM borax, 15 mM phosphate, 50 mM SDS, pH 8.5; voltage, 10 kV; capillary length, 64 cm; detection potential, 400 mV vs Ag/AgCI. Cathode buffer (A) run buffer; (B) 0.5 M NaOH.
efficient biocatalyst for the determination of amino acids%and has been incorporated in a postcolumn solid-phase reactoP and in amperometric b i o s e n s ~ r sfor ~ ~the ~ ~detection ~ of amino acids in LC and FIA systems. The use of AAO-containing amperometric sensors in the CEEC system should be a reasonable and interesting option for sensing the native form of the amino acids. Again, there is a discrepancy in the pH necessary for AAO CMEbased EC detection versus that required for the CE separation. Since the PI values of many amino acids are -6-7, most amino acids cannot be resolved using a separation buffer of pH 7.0, although the sensitivity of the enzyme-based electrode is highest at this pH.30,31The separation of six amino acids is illustrated in Figure 3 4 only one of these is resolved from the remaining five under these conditions. However, at a pH greater than 9, the majority of the amino acids bear a net negative charge and are therefore more efficiently separated by CE.2‘j Unfortunately, the use of a high-pH run buffer significantly decreases the sensitivity and stability of the AAO-based microelectrode as a result of the depressed enzymatic activity of the electrode, and no useful current response was obtained. The CA membrane-based mixer was used to modify the eluent to make it compatible with the AAO-based microelectrode. In (27) Engstrom-Silver”, C.E.; Ewing, A G.J. Microcolumn Sep. 1991,3,141. (28) Devlin, T.M., Ed. Textbook ofBiochemisby, 3rd ed.; Wiley-Liss: New York,
1992 p 480. (29) Kiba, N.; Kaneko, M. J. Chromatogr. 1984,303,396. (30) Ianniello, R M.; Yacynych,A M.Anal. Chem. 1981,53,2090. (31) Guilbault, G.; Lubrano, G. J. Anal. Chim. Acta 1973,64,439.
16 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
Figure 3. Electropherogramsof D-amino acids at AAO-containing amperometric sensor. Run buffer, (A) 20 mM phosphate, pH 7.0; voltage, 20 kV; catholyte, 20 mM phosphate, pH 7.0; (B) 20 mM sodium carbonate, pH 9.7; voltage, 27 kV; catholyte, 50 mM HCI. Capillary length, 84 cm. Detection potential, 900 mV vs Ag/AgCI. Samples: (1) 40 pM proline, (2) 60 pM alanine, (3) 200 yM glycine, (4) 800yM leucine, (5) 100pM phenylalanine, and (6)200yM serine.
this case, 50 mM HC1 was placed in the cathode reservoir, and amino acids were separated at pH 9.7. This resulted in an eluent pH of 7.2, which is ideal for detection using the AAO-based microelectrode. Figure 3B shows the CEEC electropherograms of six amino acids separated under these conditions. The concentration detection limits (S/N = 3) for the amino acids tested were 0.35 pM for proline, 1.1pM for aniline, 5.6 pM for glycine, 10pM for leucine, 2.3 pM for phenylalanine, and 4.4pM for serine. This method is comparable in sensitivity to other CE-based methods using indirect fluorescence detection26 or amperometric detection with a copper microelectr~de.~~ CEEC of DisuEdes at a Mixed-ValentRuthenium Cyanide Fi-Deposited Carbon Fiber Electrode. Thiols and disulfides play important roles in drug metabolism and protein synthesis, as well as being used as radioprotective agents and as antibiotics. The determination of disuliides is a particularly challenging analytical problem because most possess no distinguishing chromophores, and the thiol group is no longer available for derivatization. In addition, the disulfide bonds are oxidatively electroinactive within a useful potential range at bare carbon substrates. In 1989, Cox et al.32introduced a method for the detection of disulfides involving the use of a mv-RuCN-modified electrode. This electrode has been employed for the detection of cystine and insulin during flow injection a n a l y s i ~ . ~ ~ ” ~ Recently, we applied the mv-RuCN-basedmicroelectrode to the detection of disulfides by CEEC.15 However, the sensitivity and (32) Cox,J. A; Gray, T.J. Anal. Chem. 1989,61, 2462. (33) Cox,J. A; Javorleski, R K; Kulesza, P.Electroanalysis 1992,3, 869.
cssc I CSSC
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0.2 0.4
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0
1. 7
14
Time (min)
Figure 4. Electropherograms of 5 x M cystine (CSSC) and oxidized glutathione (GSSG)at the mv-RuCN film-deposited carbon fiber electrode. Run buffer, 20 mM MES, pH 7.0; voltage, 25 kV; capillary length, 85 cm; cathode buffer, 50 mM HCV0.5 M NaCI. Detection potential, 850 mV vs Ag/AgCI.
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Time (mh)
stability of the electrode response were not optimal due to the discrepancy in the buffer composition needed for detection and that necessary for the CE separation. The mv-RuCN-containing electrode requires a buffer of low pH (around 2.0) and a high cation concentration for long-term stability of the electrode and for the catalytic oxidation of dis~lfides.’~,~ Unfortunately, the use of low pH for the CE separation leads to low separation efficiencies and long analysis times. In addition, high cation concentrations in the run buffer cause Joule heating. Using the CA membrane-based mixer, the detection of disulfides by the mv-RuCN CMEbased CEEC is greatly improved. In this case, a zwitterionic buffer, 20 mM 2-[N-morpholinolethanesulfonic acid (MES), pH 7.0, was used for the separation. The higher pH run buffer decreases the analysis time and substantially improves column efficiency over the previous ~eparati0n.l~ More importantly, operation at relatively high pH ensures adequate EOF so that sample loss at the mixer is m i n i i e d (see below). Lastly, the use of low-conductivity zwitterionic buffers decreases the detector noise level at the EC detector, leading to an improvement in S/N for the whole CEEC system. In order to detect the disulfides under optimal conditions, a solution of 50 mM HC1500 mM NaCl was placed in the cathodic buffer. Using the mixer, the pH of the eluent was decreased to ca. 2.0, and an adequate amount of cation (Na+) was added. The detection of cystine (CSSC) and oxidized glutathione (GSSG) using the mv-RuCN CME electrode with the CA mixer is shown in Figure 4. As a result of the separate optimization of the buffer composition for CE separation and EC detection, the detector stability and sensitivity for disulfides was significantly improved in comparison to those obtained earlier.15 Detection limits were approximately 0.2 pM (S/N = 3), almost an order of magnitude lower than that reported previously. The stability of the detector response was also improved so that 85% (rather than the prior 40%) of the initial response remained after 8 h of successive operation. Sample Loss and Band Broadening. Sample loss and band broadening are not unexpected in a CE system that involves the use of a joint1335336or f r a ~ t u r e ~for ~ ,sample ~ * introduction, continu(34) Cox, J. A; Gray, T. J. Anal. Chem. 1990,62, 2742. (35) O’Shea, T. J.; Greenhagen, R D.; Lunte, S.M.; Lunte, C. E.; Smyth,M. R; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593, 305. (36) Whang, C:W.; Chen, 1.-C. Anal. Chem. 1992,64,2461. (37) Linhares, M. C.; Kissinger, P. T. Anal. Chem. 1991,63,2076. (38) Chen, 1.-C.; Whang, C.-W. 1.Chromatogr. 1993,644, 208.
Figure 5. Electropherograms of catecholamines at carbon fiber electrode with CA joint (a) and CA mixer (b) CEEC. Run buffer, 20 mM glycine, pH 11.O; voltage, 20 kV; capillary length, 66 cm; detection potential, 800 mV vs Ag/AgCI. Samples: M dopamine ( l ) , norepinephrine (2),and catechol (3).
ous sample collection, or offcolumn electrochemical detection. Therefore, for the proper evaluation of this system, it was important to determine the sample loss and band broadening. A separate CEEC system using an oncolumn CA joint was constructed using the same procedure as that reported by Whang and C h e r ~ .In~ ~this case, the joint was used only to isolate the EC detector from the high separation voltage. Sample loss and band broadening were determined by comparison of the peak area and half-peak width of the test compounds obtained using the CA mixer-based CEEC system with those from the CA joint-based CEEC system. There are two reasons for using the CA joint (rather than a joint- (or decoupler-) free CEEC system) as the “reference”point. First, the performance of CEEC without any joint is impractical for capillaries of greater than 25pm i.de7 Second, sample loss and band broadening have already been demonstrated to be minimal in a CE system using the on-column CA For the proper measurement of sample loss and band broadening, two CE systems of identical dimensions and the same separation conditions were used. The only difference was that the “reference”system employed the on-column CA joint as the decoupler, while the “test” system used the CA membrane mixer. A cylindrical carbon fiber electrode of 33-pm diameter was used as the sensing electrode in both experiments. The outer side of the electrode (total length, ca. 500 pm) was largely (ca. 350 pm) covered by a thin layer of UV glue (UVEXS, Sunnyvale, CA), and the unglued portion was completely inserted into the end of the detection capillary. This ensured the unity of the effective working electrode area between the two systems. Figure 5A,B depicts CEEC electropherograms of catecholamines obtained with the CA joint-based reference system and the CA mixer-based test system, respectively. The average loss of peak area was 6.3%,with a range from 4.2%for catechol to 8.8% for norepinephrine. Dopamine and norepinephrine were intentionally barely resolved (R= 0.55) under the reference conditions, in order to demonstratethe effect of the mixer on their resolution. Following the use of the mixer, the R value was 0.35. Band Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
17
I
Table 2. Sample Loss and Band Broadening of Catecholamines.
a
peak area plate (nC) sample number bqd voltage loss (%) -broademng (%) Orv) compd Aob AC (Ao -A)/Ao Nob Ne (No -N)IN 20 DA 3.36 3.16 6.0 0.72 0.66 8.3 NE 10.75 9.80 8.8 0.57 0.50 12.3 CA 9.41 9.01 4.2 1.27 1.15 9.4 15 DA 3.29 2.90 11.8 0.75 0.67 10.6 NE 14.6 10.25 8.75 0.60 0.50 16.7 CA 9.11 7.96 12.6 1.29 1.11 13.9 10 DA 3.21 2.65 17.4 0.68 0.57 16.2 NE 9.90 7.92 20.0 0.53 0.42 20.7 CA 8.86 7.14 19.4 1.08 0.88 18.5
T I -
2 nA
All the operation parameters are the same as in Figure 5 4 except the high voltage. Dopamine @A) and norepinephrine WE)are run separately. Plate number is expressed as lo4 N . * Obtained with the (I
CA joint-based CEEC (reference) system. Obtained with the CA mixer-based CEEC (test) system.
broadening, measured by the relative loss of plate number (N), was ca. 10%(see Table 2). The effect of EOF on sample loss and efficiency was also evaluated by changing the applied voltage and is shown in Table 2. The EOF had a more profound effect on peak area than on separation efficiency (e.g., for catechol, the loss of peak area increased from 4.2%at 20 kV to 19.4%at 10 kV, while the loss of plate number increased from 9.4%to 18.5%). This is not unexpected considering that a lower EOF resulted in a longer exposure time of the sample zone to the CA membrane and, consequently, a greater possibility of sample loss due to diffusion through the membrane. The separation efficiency was affected to a lesser degree by low EOF since the inner diameter of the CA mixer is very close to that of the capillary, and, in this case, there should be no dilution by the cathodic buffer solution since its composition was the same as that of the run buffer. Another CEEC system was tested in which the EOF was intentionally depressed by lowering the pH of the run buffer, and the sample loss and band broadening were reevaluated. In this case, leucine enkephalin and methionine enkephalin were employed as test analytes. Figure 6A,B shows the electropherograms of enkephalins using a separation buffer of pH 2.5 with the reference and test CEEC system, respectively. Under these conditions, there was considerable sample loss (as high as 70% and 73% for leucine enkephalin and methionine enkephalin, respectively), and band broadening was around 20%. Based on these observations,the operation of the mixer at very low pH is not recommended and should be avoided if possible. In addition to significant sample loss, the use of low pH run buffers can result in irreproducible migration times due to the presence of a discontinuous buffer system. This has been reported to be a problem under low EOF conditions.39 On the other hand, we found that the use of discontinuous buffer systems did not perturb the CE separation if the pH was greater than 7.0. At pH values (39) Weinberger, S.R; Schlabach, T. Abstracts of the 1991 Conference on H g h Performance Capillary Electrophoresis (HPCE '91), San Diego, CA, 1991.
18 Analytical Chemistty, Vol. 67, No. 1, January 7, 7995
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10
Time (min) Figure 6. Electropherograms of enkephalins at carbon fiber electrode with CA joint (a) and CA mixer (b) CEEC. Run buffer, 30 mM phosphate, pH 2.5;voltage, 27 kV; capillary length, 60 cm; detection potential, 1000 mV vs Ag/AgCI. Samples: M Leuenkephalin (1) and Met-enkephalin (2).
above 7, migration times for all test compounds were quite reproducible, with relative standard deviation values less than 5% under normal operation. In conclusion, a membranebased mixer has been developed which is capable of adjusting the pH and/or ionic strength postcapillary without a significant loss in efficiency. The mixer is especially useful for CEEC since it makes possible the independent optimization of the separation and detection buffers. Therefore, the electrochemical detection can be accomplished under optimal conditions, yielding the best limits of detection. Further work will concentrate on increasing the durability of the mixer and applying it to other electrochemical detection systems. ACKNOWLEWMENT The authors acknowledge the Kansas Technology Enterprise Corp. and the Center for Bioanalytical Research at the University of Kansas for financial suppport of this work and Bioanalytical Systems for the contribution of a potentiostat. They also thank Sangryoul Park for helpful discussions and Nancy Harmony for assistance in the preparation of the manuscript. Received for review June 27, 1994. Accepted October 17, 1994.@ AC9406423 .a Abstract
published in Advance ACS Abstmcts, November 15, 1994.
