Anal. Chem. 1995, 67,911-918
A Perfluorosulfonated lonomer Joint for Capillary Electrophoresis with On-Column Electrochemical Detection Sangryoul Park, Susan M. Lunte, and Craig E. Lunte*
Department of Chemistry and Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66045
A new fabrication method for a Nafion joint for capillary electrophoresiswith on-column electrochemical detection (CEEC) is described. The Nation joint was cast directly on the capillary column. A tungsten wire was used to define the flow channel during fabrication and removed for operation. This method allowed the construction of N d o n joints over 1 mm in length, which did not significantly contribute to band broadening. The increased length of these joints resulted in a significant decrease in the noise at the electrochemical detector. The relationship between the noise, the magnitude of the electrophoresis current, and the length of the Nafion joint was investigated. The separation efficiency of a CEEC system equipped with this type of Nafion joint was investigated using hydroquinone, phenolic acids, and catecholamines. Using 10 mM phosphate buffer at pH 6.1 for the run buffer, the Nafion joint did not have a significant effect on the separation efficiency of neutral or acidic compounds; however, the cationic compounds exhibited significant band broadening. The interaction of cations with the Nafion was minimized by use of a 100 mM EPPS zwitterion, pH 6.0, running buffer. A detection limit of 5 x 10-loM was achieved for hydroquinone and 2 x M for the phenolic acids using the phosphate running buffer. A slightly higher detection limit of 5 x M was achieved for the catecholamines using the EPPS running buffer. Capillary electrophoresis (CE) has become a powerful analytical method because of the extremely low mass detection limits, small sample volume, and high separation efficiency offered.' A limitation has been that the concentration detection limits that can be achieved are relatively high. Considerable effort has gone into the development of laser-induced f l ~ o r e s c e n c eand ~ ~ ~electro~hemical~-~ detectors for CE in order to lower the concentration detection limits. A major technical difficulty with capillary electrophoresis with electrochemical detection (CEEC) is that the current of the electrophoresis system gives rise to noise at the (1) Wallingford, R A; Olefirowicz, T. M.; Ewing, A G. Anal. Chem. 1989,61,
292A-303A. (2) Gozel, P Gassmann, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987,59, 44-49. (3) Wu, S; Dovichi, N. J.J Chromatogr. 1989,480, 141-155. (4) Wallingford, R. A.; Ewing, A G. Anal. Chem. 1987,59,1762-1766. (5) Wallingford, R A; Ewing, A G. Anal. Chem. 1988,60, 258-263. (6) Wallingford, R. A.; Ewing, A G. Anal. Chem. 1988,60, 1972-1975. (7) Wallingford, R. A; Ewing, A G. Anal. Chem. 1989,61, 98-100. (8) O'Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593,305-312. 0003-2700/95/0367-0911$9.00/0 0 1995 American Chemical Society
electrochemical detector. In general, the noise arising from the CE system has been the limiting factor in the detection limits of CEEC. To overcome this limitation, techniques to minimize the electrophoresis current by using low-conductivity zwitterionic buffersgand/or small inner diameter capillaries ( < l o ym i.d.)'O," have been developed. However, these techniques limit the flexibility of CEEC by placing limitations on the buffers and capillaries that can be used. Another approach to minimize the detector noise from the CE system is to electrically isolate the electrochemical cell from the CE system. This can be achieved by placing an electrically conducting joint in the capillary before the electrochemicalcell. The joint is placed in one of the running buffer reservoirs of the CE system and provides an electrical path to ground for the electrophoresis current. Wallingford and Ewing4-6 developed a porous glass capillary joint and reported M detection limits for several catecholamines. However, the porous glass capillary is not readily available. Huang and ZareI2 modified the design using an on-column frit. This design is also difficult to implement as a laser-drilled hole in the capillary is required to construct the frit. T i e et used a porous graphite tube as the isolating joint. O'Shea et a1.8devised an isolation joint for CEEC using Nafion tubing and reported a 6 x M detection limit for hydroquinone. The Nafion tubing is readily available and the joint can be constructed relatively easily. Finally, a joint made by a simple fracture of the capillary has also been rep01ted.l~ These fracture joints require careful alignment of the separation and detection capillaries to ensure flow across the fracture. Even with careful alignment, leakage from the joint and poor reproducibility limit the usefulness of this design. None of the joints described above provides sufficiently large conduction channels to ground to completely isolate the electrochemical cell, particularly when the electrophoresis current is high. In this report, we describe the construction of a modfied Nafion joint that provides improved detection limits with low dead volume. This joint is constructedby casting the Nafion membrane around a wire defining the flow channel instead of using precast Nation tubing. In this way, joints over 1mm in length and of an inner diameter comparable to that of the CE capillary can readily be fabricated. (9) O'Shea, T. J.; Lunte, S. M. Anal. Chem. 1993,65, 247-250. (10) Olefirowicz, T. M.; Ewing, A G. Anal. Chem. 1990,62, 1872-1876. (11) Huang, X.; Zare, R N.; Sloss, S.; Ewing, A G.Anal. Chem. 1991,63, 189192. (12) Huang, X.; Zare, R N.Anal. Chem. 1990,62, 443-446. (13) Yike, Y. F.; Lee, S. F.; Khoo, S . B.J Chromatogr. 1991,585, 139-144. 99 (14) Linhares, M. C.; Kissinger, P. T. Anal. Chem. 1991,63, 2076-2078.
Analytical Chemistry, Vol. 67, No. 5, March 1, 7995 911
E
F
G
H Figure 1. Top view of the cast Nafion joint assembly (not to scale): (A) anodic capillary end; (B) epoxy board; (C) cathodic buffer reservoir; (D) cast Nafion membrane; (E) septum; (F) carbon fiber working electrode; (G) electrochemical cell; (H) cathode for electrophoresis.
EXPERIMENTAL SECTION
Chemicals. Hydroquinone, catechol, dopamine, norepinephrine, isoproterenol, chlorogenic acid, caffeic acid, protocatechuic acid, gentisic acid, 2-morpholinoethanesulfonicacid (MES) , and N-(2-hydroxyethyl)p~perazineN-?-propanesulfonic acid @PI’S) were obtained from Sigma (St. Louis, MO). All other chemicals were reagent grade or better and used as received. All solutions were prepared in NANOpure water (Sybron-Barnsted, Boston, MA) and filtered through a 0.45 pm pore size Acrodisc syringe filter (Fisher, Fair Lawn, NJ) before use. CE Apparatus. Electrophoresis was driven by a high-voltage dc (0-30 kv) power supply (Glassman High Voltage, Whitehouse Station, NJ). The anodic high-voltage end was isolated in a Plexiglas box fitted with an interlock for operator safety. Experiments were performed at ambient temperature (24 OC). Sample introduction was performed using either pressure or electrokinetic injection as described in the text. The injection volume using pressure injection was calculated in a continuous-fill mode by determining the time necessary for sample to reach the detector. A fused-silicacapillarywas prepared by washing with 0.1 M NaOH for 5 min and then equilibrating in the running buffer for at least 2 h prior to use. The assembled Nafion joint was immersed in the cathodic buffer reservoir, which contained either running buffer or ionic solutions as described in the text. The separation capillary was 65 cm long for the separation of hydroquinone and the phenolic acids or 85 cm long for the separation of the catecholamines. In both cases, the detection capillary was 1.5 cm long. One end of the detection capillary was introduced into the electrochemical cell via a septum. The run buffer for separation of hydroquinone and the phenolic acids was 10 mM potassium phosphate at pH 6.1. The run buffer for separation of the catecholamines was a mixture of 100 mM EPPS and 20 mM MES at pH 6.0. Nation Joint A schematic diagram of the Nation joint assembly is shown in Figure 1. Fused-silica capillaries of 75 pm i.d. and 360 pm 0.d. were obtained from Polymicro Technologies phoenix, AZ). After these capillaries were cut to the desired lengths for separation and detection, one end of each capillary was ground on a 1000 grit finishing paper to a conical shape. This conical shape is to enlarge the area that will contact Nafion with a smooth angle and avoid bubble entrapment. After grinding, slumes in the capillaries were flushed out with water. The capillarieswere then washed with 5%(v/v) Micro cleaning solution NJ) for lo and rinsed with Onternational water for an additional 10 min. An epoxy board for printed circuits 912 Analytical Chemistry, Vol. 67, No. 5, March 7, 1995
was etched and cut to 0.5 cm x 1.5 cm. The separation capillary was placed on the epoxy board and fixed with epoxy. A piece of 85 pm diameter tungsten wire (Hamilton, Reno, NV) was reduced in size to approximately 70 pm in a flame. The final diameter of the tungsten wire was determined by use of a microscope with a vernier eye piece. An approximately 10 cm length of this wire was inserted into the detection capillary, which was then positioned on the epoxy board in line with the separation capillary. The tungsten wire was inserted through the detection capillary and into the separation capillary. The distance between the two capillaries was adjusted under a microscope to the desired length (between 0.01 and 2.0 mm for this work) and the detection capillary then glued to the epoxy board. Nation ion exchange powder (5 wt % solution,Aldrich Chemical Co., Milwaukee, WI) was used to cast the joint between the separation and detection capillaries. Two approaches to casting Nafion membranes were evaluated. In the first method, known as the “recast”method,16Na6on dissolved in low aliphatic alcohols (Le., as received from Aldrich) is used and curing is performed at moderate temperature for a short period. In the second method, known as the “solution-processing” method,17the Nafion is dissolved in less volatile solvents, such as DMF or DMSO, and cured for a longer period at higher temperature. For the solutionprocessing method, the original Nafion solution was diluted 3:l with DMF. For both methods, the Nafion solution was applied dab by dab on the exposed wire between the two capillaries under a 200 W tungsten lamp to accelerate evaporation of solvent. Most of the solvent was allowed to evaporate between application of each drop of Nafion solution. It is critical to slowly build the Nafion film and rapidly evaporate solvent to avoid capillary action of Nafion solution into the capillaries and entrapment of bubbles. When the Nafion layer reached a thickness of approximately 50 pm as determined under a microscope, the joint was dried in an oven at 80 “C for 10 min for the recast method and at 130 “C for 1 h for the solution-processing method. A “dualcasting” procedure was also used in which a 25 pm layer was fabricated by the solution-processing method and then an additional 25 pm layer was added by the recast method. This method was ultimately adopted for the fabrication of Nafion joints. The mechanical strength of the area where the capillary contacts the Nafion was enhanced by application of epoxy glue. The tungsten wire was separated from the Nafion by electrolytic generation of hydrogen. A piece of platinum wire was immersed in the buffer reservoir and served as the anode and the tungsten wire was connected as the cathode. Electrolysis at 0.2-1.0 mAwas performed with visual monitoring under a microscope. Electrolysis is immediately stopped when hydrogen gas formation is observed over the entire length of the tungsten wire, typically 1-2 min of electrolysis. The current required is a function of the length of the gap. After electrolysis, the tungsten wire could easily be removed from the Nafion joint by gently pulling it through the detection capillary. If any Naiion solution penetrated into the capillaries and partially blocked liquid flow, it was removed by injecting a small amount of 50%isopropyl alcohol. Finally, the completed joint was flushed in turn with 5%Micro cleaning solution, 0.1 N NaOH, and water. The capillary was filled with run buffer and equilibrated for 2 h prior to use. (15) Lu.W.; Cassidy, R M. Anal. Chem. 1994,66,200-204. (16)Moore, R B., III; Martin, C. R Anal. Chem. 1986,58, 2570-2571. (17) Moore, R B., 111; Martin, C. R. ?ducromolecuh 1988,21, 1334-1339.
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Electrochemical Detection. The working electrodes for amperometric detection were prepared by inserting a 33 pm 0.d. carbon fiber (Avco Specialty Products, Lowell, MA) into a 5 cm length of fused-silica capillary of the same type as used for the electrophoresis capillary. The carbon fiber extended from both ends of the capillary approximately 1cm. One end of the capillary was inserted into a 2 cm long, 20 gauge stainless steel needle and affixed with epoxy. The needle was filled with carbon powder to make electrical contact to the exposed carbon fiber. This needle was used to connect to the lead for the working electrode
from the potentiostat. The other end of capillary was sealed with silicon adhesive @ow Corning, Midland, MI). After curing at room temperature for 1day and then at 120 "C for 1h, the exposed carbon fiber was cut to an appropriate length (0.2-1.0 mm) with surgical scissors. The fresh carbon fiber was cleaned by sonication in 30% Micro cleaning solution for 2 min and rinsed with water. The working electrode was then inserted into the detection capillary using an X-Y micromanipulator (New Port, Fountain Valley, CQ . . The cell was operated in a threeelectrode configuration with a platinum wire auxiliary electrode and Ag/AgCl reference electrode (Bioanalytical System, West Lafayette, IA). The electrochemical cell was placed in a BAS CC-4 Faraday cage and connected to a BAS PA-1 potentiostat. Data acquisition and control of the system were performed by an IBM-compatible 286 microcomputer via an ADALAB-PC/ADAPT interface board (Interactive Microware, Inc., State College, PA). The potentiostat had a built-in low-pass filter operated at either 0.5 or 5 Hz as described in the text. Data were collected at 3 kHz and signal averaged over 500 ms. The carbon fiber working electrode was Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
913
Table 1. Peak Widths. at Half-Maximum Using Various Types of Electrlcally lsolatlng Joints
fracture joint
Nafion tubingd
compounds
cast Nafion joints 0.5 mm 1.0 mm
10 ym
catechol dopamine norepinephrine isoproterenol chlorogenic acid caffeic acid gentisic acid
2.89 f 0.06 2.87 f 0.06 3.11 f 0.07 3.24 f 0.06 2.95 f 0.07 2.83 f 0.09 3.02 0.10
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4.26 i 0.08 nF nF 4.78 i 0.18 4.40 f 0.10 4.35 f 0.15 4.88 f 0.12
*
2.89 0.04 2.68 f 0.02 2.82 f 0.03 3.00 f 0.01 3.17 i 0.08 3.05 f 0.10 3.22 f 0.08
2.0 mm
2.83 f 0.11 2.58 f 0.05 2.74 f 0.07 2.92 i 0.08 2.79 f0.10 2.70 f 0.05 2.95 k 0.08
2.88 f 0.05 2.79 i 0.01 2.83 f 0.05 3.03 f 0.04 2.85 f 0.09 2.68 f 0.08 2.90 i 0.07
3.18 f 0.07 2.82 f 0.06 3.04 f 0.07 3.19 f 0.07 3.07 f 0.10 3.03 f 0.08 3.12 f 0.10
Peak width in seconds with n = 5 for all determinations. The Nation tubing joint was prepared as described by O'Shea et aL8 except that a 300 pm gap was used between the capillaries. Dopamine and norepinephrine were not sufficiently resolved to determine the peak width using this joint. I
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Figure 6. Electrophoregrams of catechol and catecholamines using cast Nafion joints of various length. Joint lengths: (A) 150, (6)300, (C) 500, (D) 800, (E) 1000, and (F) 2000pm. Peak identities: (1) dopamine, (2) norepinephrine, (3) isoproterenol, and (4) catechol. Electrophoretic conditions: capillary, 75 pm i.d., 85 cm long; run buffer, 20 mM MES and 100 mM EPPS at pH 6.0; separation voltage, 30 kV; injection, electrokinetic for 1 s at 30 kV; electrode, 700 pm long inserted 500 pm; working electrode potential, +0.55 V vs Ag/AgCI.
activated in the running buffer by applying a f 1 . 2 V triangular waveform at 100 Hz for 2 min. Separation Conditions. The run buffer for the separation of hydroquinone and the phenolic acids was 10 mM potassium phosphate buffer, pH 6.1. Stock solutions of 10 mM, except for caffeic acid, which was 1.0 mM, were prepared in 0.1 M HC104 and diluted to the appropriate concentration in run buffer. A separation capillary of 65 cm was used for this separation. The electrophoretic voltage was 30 kV, which resulted in a current of 914 Analytical Chemistry, Vol. 67, No. 5, March 7, 7995
33 PA, Pressure injection at 1 psi for 5 s was used unless otherwise specified. Amperometric detection was performed at +0.80 V vs Ag/AgCl reference. The run buffer for the separation of the catecholamineswas a mixture of 100 mM EPPS and 10 mM MES buffer, pH 6.0. Stock solutions of 10 mM were prepared in 0.1 M HClOd and diluted to the appropriate concentration in run buffer. A separation capillary of 85 cm was used for this separation. The electrophoretic voltage was 30 kV, which resulted in a current of 13 yk Electrokinetic
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Figure 7. Electrophoregrams of catechol and phenolic acids with various solutions in the cathodic reservoir. Cathodic reservoir solutions: (A) 10 mM phosphate at pH 6.1 (run buffer), (B) 1 N KCI, (C) 0.1 N HCI, (D) 0.1 N HCI in 0.9 N KCI, (E) 0.5 N HCI in 0.5 N KCI, and (F) 1.O N HCI. Peak identities: (1) catechol, (2) chlorogenic acid, (3) caffeic acid, (4) protocatechuic acid, and (5) gentisic acid. Electrophoretic conditions: capillary, 75 pm id.,65 cm long capillary with a 1000pm long cast Nafion joint; run buffer, 10 mM potassium phosphate at pH 6.1; separation voltage, 30 kV; injection, pressure at 1 psi for 5 s; electrode, 700 pm long inserted 500 pm; working electrode potential, +0.8 V vs Ag/AgCI.
injection at 30 kV for 2 s was used. hperometric detection was performed at f0.55 V vs a Ag/AgCl reference electrode. RESULTS AND DISCUSSION
Effect of Electrode Position on Detection Parameters. The design of the electrochemical detector cell dramatically affects the observed sensitivitynoise. Therefore, the initial investigation was to determine the optimal configuration of the electrochemical cell. Both the sensitivity and noise of the detector increase as the electrode length increases. The primary parameter in determining the optimal codguration, however, is the depth to which the electrode is inserted into the detection capillary. The detector noise increases dramatically as the electrode is inserted deeper into the detection capillary (Figure 2). This observation has led to the development of end-column detection strategies to minimize detector noise arising from the electrophoretic current.15 However, the sensitivity also increases as the insertion depth increases (Figure 3). End-column detection therefore trades sensitivityfor noise reduction. The use of an electricallyisolating joint between the electrophoretic system and the detection cell allows in-column detection for high sensitivity with a reduction
in detector noise. For the evaluation of this new Nafion joint fabrication procedure, 700 pm long electrodes were inserted to a depth of 500 pm. Comparison of Casting Methods. The two processes for casting Nafion result in membranes of considerably different characteristics.'+-'* "Solution-processed Nafion has superior mechanical properties and much lower solubility in organic solvents relative to recast Nalion. Moore and Martin17explained these differences as a result of a higher crystallinity of the solutionprocessed Nafion. Nafion joints fabricated by the recast method were prone to cracking and partially dissolved in 50% ethanol. Nafion joints fabricated by the solution-processed method do not crack in use and are insoluble in 50% ethanol. However, solutionprocessed Naflon swells considerably in strongly acidic solution. Particularly for longer joints, this swelling led to leaking of the joint. These limitations were overcome by use of the dual-casting method. A solution-processed inner membrane coated with a recast outer membrane resulted in superior mechanical and chemical stability but was more dflcult to fabricate. For most (18) Gebel, G.; Aldebert, P.; Pineri M. Macromolecules 1987, 20,1425-1428.
Analytical Chemistry, Vol. 67, No. 5,March 1, 1995
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2
3
4
5
6
7
Time (minute)
Figure 9. Reproducibility of electrophoregrams of catechol and phenolic acids obtained from consecutive injections.A 1000 pm long Nafion cast joint was used, and the cathodic reservoir was filled with 1.0 N HCI. All other experimental conditions were as described in Figure 7.
experiments, when the joint was less than 1 mm and no acidic solutionswere used, the solution-processedmethod was used for joint fabrication. When a joint longer than 1mm was needed or acidic solutionswere employed, a dual-cast joint was used. Both types of joints have exhibited working lifetimes of several months. Effect of Joint Length on Detector Noise. To evaluate the ability of Nafion joints fabricated by this method to isolate the electrochemical detection cell from the electrophoretic current, the detector noise as a function of joint length and electrophoretic current was determined. These results were compared to a fracture-type joint. For all of the joints, the detector noise is a function of the electrophoretic current. However, longer joints show a far weaker dependence than the shorter joints (Figure 4). For example, the detector noise rises dramatically with electrophoretic currents above 5 p A for a fracture-typejoint and a 10 pm cast Nafion joint while the detector noise with a 1 mm 916 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
cast Nafion joint is independent of electrophoretic current up to 20 p A Figure 5 shows the detector noise as a function of the joint length under the electrophoretic separation conditions of 30 kV. The detector noise exhibits a logarithmic decrease as a function of the gap length. Effect of Joint Length on Peak Shape. An advantage of this fabrication procedure over the use of precast Nafion tubing is that the inner diameter of the isolating joint is roughly that of the inner diameter rather than the outer diameter of the separation capillary. Longer joints can be fabricated to reduce noise without introducing dead volume into the system. Peak widths using a fracture joint,14 a Nafion tubing joint,8 and a cast Nafion joint were compared under identical separation conditions. Table 1lists the peak width at half-maximum determined for catechol, catecholamines, and the phenolic acids at a variety of different Nafion joint lengths. As can be seen, no increase in band broadening is evident, relative to a simple fracture joint, even with cast Nafion joints up to 1mm in length. In contrast, use of a 300 pm Nafion tubing joint resulted in considerable band broadening. The case was not as simple for the catecholamines. If a simple phosphate buffer was used as the electrophoretic run buffer, significant tailing was observed with all systems. This has been reported previously as the result of interactions of the protonated amine groups with negatively charged silanol groups on the wall of the silica tubing? The use of a buffer component with an amine group was reported to mask the silica walls and greatly reduce tailing.6 When these large surface area Nafion joints were used, reduced peak heights were also observed for cationic compounds relative to neutral and anionic compounds. The loss of cations was a function of the length of the Nafion joint. The addition of the zwitterion EPPS to the buffer dramatically reduced the tailing of the catecholamines. Using a EPPS containing buffer, peak width was independent of joint length for the catecholamines (Table 1). While EPPS reduced the loss of cations at the joint, this phenomenon was not totally eliminated with longer joints. As can be seen in Figure 6, while the peak shape is unaffected,
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Time (minute) Figure 10. Electrophoregrams for determining the detection limits for catecholamines: (A) electrophoregram of 10 nM of each catecholamine, and (B) electrophoregram with 100 nM of each catecholamine. Peak identities: (1) dopamine, (2) norepinephrine, and (3) isoproterenol. A 500pm long Nafion cast joint was used. All other experimental conditions were the same as described in Figure 6.
the peak heights for the catecholamines decrease relative to that of the neutral catechol peak height with the joint length. A length of 500 pm provides good noise reduction and little loss of the catecholamines. On-Line pH Adjustment. The optimal separation buffer conditions for the electrophoretic system are often not the optimal buffer conditions for the detection system. For example, it was observed that the detector sensitivity for the phenolic acids was 1order of magnitude lower than for the neutral catechol in a pH 6.1 phosphate buffer. This is thought to be a kinetic phenomenon resulting from charge repulsion between carboxylate functionalities on the carbon fiber and the anionic phenolic acids.Ig The (19) Randin, J.-P. Encyclopedia of Electrochemisty of the Elements, Vol. VII-1: Carbon: Bard, A. J., Ed.; Marcel Dekker: New York, 1976; pp 25-29.
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Time (minute) Figure 11. Electrophoregrams for determining the detection limits for hydroquinone and phenolic acids: (A) electrophoregram with 6 nM hydroquinone and 12 nM each of the phenolic acids, and (B) electrophoregram with 50 nM hydroquinone and 100 nM each of the phenolic acids. Peaks: (1) hydroquinone, (2) chlorogenic acid, (3) caffeic acid, (4) protocatechuic acid, and (5) gentisic acid. A 2000 pm long Nafion cast joint; cathodic reservoir solution, 1 N HCI; (electrokinetic) injection, applying 30 kV for 3 s; electrode, 1300 pm long inserted 1100 pm; working electrode potential, 1.0 V vs AgIAgCI.
strong cation exchange nature of Nafion provides a route to adjusting the buffer pH at the Nafion joint. Using a 1 mm long joint, filling the cathodic buffer reservoir with 1N HC1 resulted in a pH of 1.8 at the detector electrode when the electrophoretic run buffer was 10 mM potassium phosphate, pH 6.1. Even 0.05 N HCl in the cathodic reservoir decreased the pH to 2.5 at the detector. The effect of various solutions in the cathodic reservoir are shown in Figure 7. In order for the pH adjustment to be effective the cast Nafion joint must be of sufficient length. Figure 8 shows electropherograms obtained using various joint lengths and 0.5 N HCl in the cathodic reservoir. By using the cathodic Analytical Chemistry, Vol. 67, No. 5, March 7, 7995
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reservoir and the Nafion joint to adjust the buffer pH after separation but before detection, optimal conditions could be achieved for both. This resulted in between a 5- and l@fold increase in sensitivity for the phenolic acids. To determine whether back diffusion of protons into the separation capillary would change the separation conditions over time, 20 injections of a standard were added over a 4 h period with 1 N HCl in the cathodic reservoir. As seen in Figure 9, peak height, shape, and elution time are very stable over this period. Detection Limits. Using a 1mm joint with 0.1 N HCl in the cathodic reservoir, detection limits of 0.5 nM for hydroquinone, 2.7 nM for chlorogenic acid, 1.4 nM for caffeic acid, 2.7 nM for protocatechuic acid, and 1.4 nM for gentisic acid were determined at a S/N of 3. A representative electropherogram near the detection limits is shown in Figure 10. Using a 500 pm joint, detection limits of 4.8 nM for dopamine, 3.9 nM for norepinephrine, and 3.5 nM for isoproterenol were determined at a S/N of 3 (Figure 11). The actual detection limits should be somewhat lower as degradation of the standards at the low concentrations caused nonlinearity in the calibration curves.
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Analytical Chemistry, Vol. 67, No. 5,March 1, 1995
CONCLUSIONS Fabrication of a Nafion isolating joint for electrochemical detection with capillary electrophoresis by directly casting the Nafion onto the capillaries provides longer joints with low dead volume. The increased length of the joint provides greater surface area and higher conductance, resulting in enhanced shunting of the electrophoretic current to ground. The improved electrical isolation of these joints results in dramatic decreases in the noise at the electrochemical detector during electrophoresis. The low dead volume resulting in this fabrication allows longer joints to be made without loss of separation efficiency. Finally, the joint can also be used to adjust the pH of the electrophoresis separation buffer to one better suited for electrochemical detection. This pH adjustment can result in a l@fold increase in sensitivity. Concentration detection limits as low as 5.0 x 10-lo M were achieved with this Qpe of Nation isolating joint. Received for review September 29, December 19, 1994.@
1994. Accepted
AC940967J @Abstractpublished in Advance ACS Abstracts, February 1, 1995.
