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Anal. Chern. 1987, 5 9 , 1762-1766
Capillary Zone Electrophoresis with Electrochemical Detection Ross A. Wallingford and Andrew G. Ewing* Department of Chemistry, T h e Pennsylvania State University, University Park, Pennsylvania 16802
A system for interfacing electrochemical detection as well as other “off-column” detectors wlth capillary zone electrophoresis Is demonstrated. This system couples two pleces of column together with a section of porous glass caplllary thus forming a jolnt that Is electrlcaliy conductive. The jolnt Is kmnersed in a M e r reservoir along with the ground electrode in order to allow the separatlon potential to be applied across only the first section of capillary. The strong electroosmotic flow generated in the flrst section of capillary serves to force solvent and analyte zones past the jolnt and through the second section of capllary to the detector. This configuration effectively separates the detector from the high applied voltage via the resistance of the detection capllary. We have used this system to successfully perform electrochemlcal detection wlth capillary zone electrophoresis and have obtained separation efficiencies on the order of 180 000 theoretical plates for the separation of catechol from catecholamines. Preliminary data indicate that a back pressure created by solvent within the second capillary is a significant contrlbutor to zone broadening. However, this effect is minlmized when a short second capillary segment Is employed.
Capillary zone electrophoresis (CZE) has been introduced by Mikkers et al. ( 1 ) and Jorgenson et al. (2-4) as a highly efficient separation technique for ionized solutes. Indeed, for proteins having small diffusion coefficients, efficiencies approaching 1million theoretical plates have been obtained (5). Separations of relatively small molecules have yielded efficiencies on the order of several hundred thousand theoretical plates ( 4 , 6 ,7). Capillary electrophoresis has also been applied to neutral molecules through solvophobic association with tetraalkylammonium ion (8) and by micellar solubilization (9-12). These methods have extended the realm of compounds that can be separated with capillary electrophoresis techniques. Although CZE has advanced rapidly since its inception in 1979, some notable limitations still exist, the major of which is the lack of detection modes available. Due to the small column dimensions and the extremely small zone widths encountered (7), on-column detection is preferred in order to preserve the high efficiency of CZE. Detection is further hindered by the need to keep both ends of the column immersed in buffer reservoirs. For this reason, “spectroscopybased” detectors capable of on-column detection before the cathodic reservoir have been used exclusively. The CZE work reported to date has employed arc-lamp fluorescence (2-4,13, 14), laser fluorescence (12,15, 16), and UV absorbance ( 5 , 6 , 8-1 1,17-19) detection. Fluorescence detection is the most sensitive mode presently available for CZE, giving subfemtomole detection limits in the laser excited configuration. One drawback of fluorescence detection is the need to derivatize most samples of interest. UV detection, although slightly more versatile, has poorer sensitivity with detection limits generally reported in the picomole range. One area of chemistry that should benefit from advances in CZE is that of ultrasmall sample analysis, especially those samples taken from biological microenvironments (Le. single
cells). This area has been largely unexplored to date. CZE is amenable to ultrasmall sample separations mainly due to the extremely small column volumes encountered. Our interests are in neurochemical systems, where sample acquisition and separation from regions such as discrete brain regions and, ultimately, single neurons are extremely exciting. Such separations require a microinjector (ZO),capable of efficiently removing samples from microenvironments, and extremely sensitive detection. Since many neurochemicals of interest are easily oxidized, electrochemical detection could be most useful with CZE. In addition, electrochemical detectors for microcolumn techniques have been shown to be extremely sensitive (21-23). Laser-excited fluorescence may provide the needed sensitivity for these separations; however, difficult microderivatization would be necessary, thereby limiting the usefulness of this technique for the separations of interest. Thus far electrochemical detection has not been applied to CZE. This is primarily due to the requirement of keeping the detection end of the column in a buffer reservoir and also the problem of performing electrochemistry in the presence of a high-voltage electric field. Recently, a method has been reported that couples postcolumn amperometric detection to capillary isotacophoresis (24)with a “T”piece and an auxiliary flow. While this system is successful in isolating the detector from the driving current, this configuration exhibits considerable zone dispersion. This paper deals with a CZE system that eliminates both of the aforementioned detection problems. The system described is based on a porous glass joint created in the column near the cathodic end. The porous joint rather than the end of the capillary is submersed in a buffer reservoir along with the cathode (Figure 1). The applied potential is dropped across the capillary prior to the porous joint and the resulting electroosmotic flow acts as a “pump” to push buffer and solute bands through the short section of capillary after the joint. Detection is performed at the end of the second segment of capillary and is consequently removed from the effects of the high-voltage electric field. In addition to the electrochemical detection described herein, the ability to collect effluent from CZE should permit interfacing with a wide range of detectors that must receive eluent from the end of the column, such as flame ionization detectors and mass spectrometers.
EXPERIMENTAL SECTION Apparatus. The apparatus used for capillary zone electrophoresis is pictured in Figure 1A. This system is similar to that described by Jorgenson and Lukacs (2),with the exception of the porous glass joint submersed in a buffer reservoir. Fused silica capillaries with an inside diameter of 7 5 p m were obtained from Scientific Glass Engineering. After forming the porous glass joint (described below), capillaries were filled with 0.1 M, pH 10 CAPS buffer (3-(cyclohexylamino)-l-propanesulfonicacid) (Sigma Chemical) with a helium-pressurized solvent reservoir. A Spellman high-voltage de power supply provided the electric field for electrophoresis. The high-voltage end of the system was housed in the Plexiglas box equipped with an interlock for operator safety. Platinum wires were used as the electrodes. Injections were made by inserting the anodic end of the column into a sample reservoir and applying a potential for 1-3 s. The end of the column was then placed into the buffer reservoir and the separation potential applied.
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F@IW 1. (A) Sdvmatic of coupled CZE system: A. buffer rese4vocS; E. Separation capillary; C. detection capillary; D. eluent. (E) Detailed schematic d porous phl: A, microscnpe slide: E. fused silica capillary; C. porous glass capillary; D. joint: E, epoxy: F. polymer coating
Detection of fluorescent solutes was performed with a locally constructed on-column fluorescence detector as descrihed previously (20).The on-columndetection cell was created by burning off a small sertion of the polymer coating on the capillary. Cell lengths ranged from 0.&1.3 mm,which corresponds to cell volumPs in the range of 4 4 nL for 75 wn i.d. capillaries. Porous Glass Joint. A detailed schematic of the porous glass joint is shown in Figure 16. A small section (1 cm) of polyimide mating was removed from the capillary approximately 5 cm from the end. The exposed section of fused silira was placed over a small section of mirroscope nlide (3 cm X 2.5 cm) and glued in place with a small amount of DUCO rement (Devcon Corp.) at each end of the exposed region. The surface of the unccated silica was lightly srored near the center with a diamond-tipped glass cutter. Gentle pressure applied to the scored region caused the column to break rleanly leaving a joint that was easily re-formed. Under an optical micrmpe, a small section (2-5 mm) of Corning Thirsty Glass” capillary (no. 7930.27ft305 pm i.d.) was placed fully onto the end of one section of capillary. The two sections of column were then positioned to form a clean joint and the porous glaqs capillary was placed into position over the fracture. Epoxy (Elmer’s Super-fast) was placed on each end of the porol~s glass w e n t and along the entire length of the capillary in contact with the microwope slide. This step sealed the porous glass jnint and affued the a$rembly to the minoscope slide for rigidity. After being allowed to cure at room temperature. the assembly was placed into a plastir rontainer having 1-2 m m slots 180’ apart. The slide was glued into place with epoxy and the slots were sealed so that the plastic container was leak-free. After filling the rouplrd capillary with buffer, the anode end was placed into a buffer reservoir as per normal electrophoresis. The plaqtic container holding the porous glass joint was also filled with buffer. Platinum wires were placed into earh reservoir so that a potential could be applied across the longer segment of capillary. A potential of 30 k V was used for all separations. Electmchemical Detector. The electrochemical detector used (Figure 2) is similar to that described by Knecht et al. (21). A single carbon fiber (10.,m diameter) was aspirated into a glass capillary (A-M Systems, standard capillary glass. 6270). The capillary was pullcd around the fiher with a vertical microeledrode puller ( H a r v d Bioscience). Under an optical mirrwope, a drop of epoxy was applied tn the area where the fiber entered the glass capillary. After cnring, the fiher was cut with a scalpel tu an exposed length of 0.1-1 mm. The open end of the lass capillary waa filled with mercury, a segment of nichrome wire placed into it, and sealed with a drop of DUCO cement. The carbon fiber detector was cemented onto a microscope slide so that the end rontaining the expmed fiber protruded from the edge of the slide. The entire detprtor asxemhly was then placed onto a mirronianipulator (Oriel Corp.). The detection segment
of the electrophoresis capillary was positioned so that the end was just above (0.34.6mm) a stainless steel plate which served as the auxiliary electrode. A drop of operating buffer was placed onto the plate so that it covered the end of the column. While being observed under a microscope, the exposed carbon fiber was manipulated inside the capillary bore. A buffer-filled plastic pipet tip plugged with cotton contained a saturated calomel reference electrode (SCE). The tip of the reference electrode assembly was placed into the pool at the column and to complete the threeelectrode electrochemical cell. Electrochemical detection was performed in the amperometric mode with a locally constructed low-noise potentiostat. Electrode pretreatment can be performed to enhance sensitivity. This was accomplished by oxidation of the carbon fiber at a potential of +1.3 V w. SCE for 90 s while simultaneously flowing operating buffer past the detector. This was done prior to each run in order to maintain constant sensitivity. Chemicals. CAPS buffer (3-(cyclohexylamino)-I-propanesulfonic acid) and TES buffer (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonicacid) were obtained from Sigma Chemical Co. Buffer solutions were prepared with doubly distilled water and were adjusted to the proper pH with solid sodium hydroxide. Amino acids were obtained from Sigma (nL-glutamic acid, DL-histidine, L-alanine, L-arginine, histamine) and from Aldrich Chemical Co. (glycine). Fluorescamine was obtained from Sigma and was used as an acetone solution. Dopamine, (*)-epinephrine, and catechol were also obtained from Sigma. (R)(-)-norepinephrine hydrochloride was obtained from Aldrich Chemical Co. Solutions of catechol and its derivatives were prepared in 0.05 M TES at pH 7.42. Riboflavin was obtained from Sigma end was used as a 9 X M solution prepared in the operating buffer. All chemicals were used as received.
RESULTS AND DISCUSSION Characteristics of the Porous Glass Coupler. The CZE system presented herein consists of two segments of capillary: a separation capillary and a detection capillary. The heart of this system is the porou%glassjoint created a t the junction of the two capillary segments. T h e porous-glass capillaries used to form this joint are created by heat-treating and leaching a special alkali-borosilicate glass (25-28). This p d u m causes the dissolution of a boric oxide/alkali phase, leaving a porous high-silica structure (28). The composition of porous glass is reported to be 96% SiO,, 3% B203,0.4% h03+ RO, (predominately A1,03 and ZrO,), and traces of Na,O and Asa03(27). The average pore diameter is 50 A and the pore size distribution is very narrow, with 96% of the pores being within 0.3 b, from the average radius (28). In the system described, the porous glass joint serves as an “electrical connector”, which permits the application of a potential gradient over one segment of capillary. The pores within the porous-glass joint are large enough to allow permeation of small electrolyte ions, thereby allowing current to flow upon application of a potential to the system. Larger analyte and solvent molecules are excluded by size from permeating through the pores. The currents measured with
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15,
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this system are identical with those currents observed with a normal CZE system when comparing equal column and separation capillary lengths, respectively, for the two systems. Applying a potential gradient across the separation capillary generates a strong electroosmotic flow (1, 2, 29, 30) in the separation capillary, which forces buffer and analyte zones past the porous joint and through the detection capillary. This process is readily observed since a drop of buffer forms at the end of the detection capillary when a potential is applied to the system. Thus, this system provides a type of “electroosmotic pump”, which eliminates the need for placing the end of the column in a buffer reservoir. Also, the detection capillary is effectively isolated from the applied potential field. Finally, the system described is very reliable and durable. Most columns prepared in the prescribed manner have withstood more than 50 electrophoretic separations without deterioration as long as the coupler assembly was kept immersed in buffer to avoid drying out. The porous glass capillaries are extremely fragile. Immobilization of the coupler assembly on a microscope slide was found to be necessary to allow subsequent handling and manipulation. CZE with Electrochemical Detection. Figure 3 shows an electropherogram of an equimolar M) mixture of catechol and various catecholamines obtained with the coupled CZE system described and electrochemical detection. For this separation, detection was performed with a 10-pm-diameter carbon fiber electrode inserted into the end of the detection capillary. Each peak corresponds to approximately 5 pmol of material injected onto the column. The excellent separation efficiency obtained with this system is evident in the catechol peak which exhibits approximately 180000 theoretical plates based on the half width. Given the small sizes and correspondingly large diffusion coefficients of these molecules, this represents excellent efficiency. This separation was performed with a detection capillary having a length of 5.7 cm, which was a convenient length to use with the electrochemical de-
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Figure 4. Electropherograms of fluorescamine-labeled amino acids: (A) fluorescence detector 8.75 cm before porous joint; (B) fluorescence
detector 7.37 cm after porous joint. tector. The rather pronounced tailing encountered in the first three peaks is thought to be due to electrostatic interactions similar to those observed in separations of proteins with CZE (5,13,31). No attempt was made to optimize this separation. The use of electrochemical detection combined with CZE is possible because electroosmotic flow originating in the separation capillary forces the solvent and solute zones through the detection capillary. High electrical resistance through the solution in the small bore capillary serves to effectively isolate the electrochemical cell from the high-voltage power supply. However, we have observed increased detector noise in systems that produce high electrophoretic currents. Although the end of the detection capillary is not totally isolated from the applied potential field, the use of relatively high-resistance buffers minimizes adverse effects from this interaction. This separation and detection scheme demonstrates that “offcolumn” effluent-receiving detectors can be used with CZE. Band-Broadening Considerations. To evaluate the solute band broadening resulting from use of the porous glass coupler, detection must be performed prior to as well as after the coupler. This evaluation scheme is not possible with electrochemical detection. For these comparisons, on-column fluorescence detection was used at points located before and after the coupler. Figure 4 shows separations of six fluores-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
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flow velocity and the detection capillary length (correlation coefficient, 0.998;slope, -0.0026 s?). These data complement the separation efficiency vs. detection capillary length data and support the back pressure explanation. The nature of flow occurring in the detection capillary is necessarily laminar, which gives rise to laminar flow spreading of zones after they have passed the joint. The contribution of laminar flow spreading to the total zone broadening will be proportional to the amount of time spent in the detection capillary. This can be represented as the factor Ldc/(Ldc + LSc)where Ldc and L,, are the lengths of the detection and separation capillaries, respectively. This contribution to the total zone broadening can be minimized simply by employing short detection capillaries. The interface of the two capillary sections must also be considered in a discussion of zone-broadening contributions. Attempts to couple two independent sections of capillary resulted in poor efficiency. A poor connection between the capillary segments creates a dead volume, which acts as a stagnant area for solutes to diffuse into. The use of a single length of capillary, which is immobilized and carefully fractured in a controlled manner, results in relatively tight joints and excellent efficiency as was shown in Figure 3. Since the porous joint is immersed in a buffer reservoir held at ground potential vs. the anodic reservoir, the flow of anions, cations, and neutral species past the joint must be considered. One would expect that ions with a negative charge would be repelled from the negative electrode and, hence, would be swept through the joint and the detection capillary by the strong electroosmotic flow. This is indeed observed experimentally with ascorbate anion (data not shown). Conversely, cations, being attracted towards the negative electrode might be expected to be held up or adsorbed at the coupler. Experimentally this does not happen. Dopamine at pH 6.86 (cation) migrates fairly rapidly with a migration velocity of about 0.22 cm/s and produces peak shapes that are not indicative of any delay or adsorption at the coupler. At pH 4.13, dopamine is also detectable with symmetrical peak shapes and a linear velocity of about 0.14 cm/s. The large difference in migration velocities at the two pHs is expected since electroosmotic flow is pH dependent (30). Apparently, the electroosmotic flow force produced in each case is significantly greater than the electrophoretic attraction of cations for the negative electrode through the porous coupler.
CONCLUSION This paper demonstrates the feasibility of performing “off-column” detection with capillary zone electrophoresis without compromising the high efficiency inherent in this method. Exploitation of the phenomenon of electroosmotic flow allows the previous detection limitations of CZE to be overcome. This system permits successful application of electrochemical detection with excellent efficiency. When careful construction procedures are employed and the detection capillary length is minimized, zone broadening resulting from column coupling can be decreased to a point where it becomes negligible. The prospects for using this system to interface CZE with such detection systems as flame ionization, photoionization, thermionic emmission, and mass spectrometry are indeed exciting. The ability to use “offcolumn” detection should dramatically increase the versatility and expand the usefulness of capillary zone electrophoresis.
ACKNOWLEDGMENT The authors are grateful to Thomas Elmer of Corning for providing technical information concerning porous glass capillaries. Registry No. Dopamine, 51-61-6; epinephrine, 51-43-4; norepinephrine, 51-41-2; catechol, 120-80-9.
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LITERATURE CITED (1) Mlkkers. F. E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M. J. Chromatogr. 1979, 769, 11-20, (2) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218,209-216. (3) Jorgenson, J. W.; Lukacs, K. D. Clln. Chem. (Winston-Salem,N . C . ) 1981, 27. 1551-1553. (4) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (5) Lauer, H. H: McManigill, D. Anal. Chem. 1986, 58, 165-170. (6) Tsuda. A.; Kazuhiro. N.; Nakagawa. G. J. Chromatogr. 1983, 264, 385-392. (7) Green, J. S.;Jorgenson, J. W. J. Chromatogr. 1986, 352, 337-343. (8) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1988, 5 8 , 479-481. (9) Terabe, S.;Otsuka, K.; Ichikawa, K.; Tsuchlya, A; Ando, T. Anal. Chem. 1984, 5 6 , 113-116. (IO) Otsuka, K.; Terabe, S.;Ando, T. J. Chromatogr. 1985, 348, 39-47. (11) Terabe, S.;Otsuka, K.; Ando T. Anal. Chem. 1985, 5 7 , 834-841. (12) Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J. Chromatogr. Sci. 1986, 24, 347-351. (13) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222. 266-272. (14) Green, J. S.;Jorgenson, J. W. HRC CC. J . High Resolut. Chromatogr. Chromatogr. Common. 1984, 7, 529-531. (15) Gassman, E.; Kuo, J. E.; Zare, R. N. Science 1985, 230. 813-814. (16) Gozel, P.; Gassmann, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987. 59,44-49. (17) Tsuda, S.;Nakagawa, G.; Sato, M.; Yagi, K. J . Appl. Biochem. 1983, 5 , 530-336. (18) Walbroel, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 375, 135-143. (19) Fujiwara, S.;Honda, S.Anal. Chem. 1986, 58, 1811-1814.
(20) Waliingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 678-681. (21) Knecht, L. A.: Guthrie, E. J.; Jorgenson, J. W. Anal. Chern. 1964, 56, 479-482. (22) St. Claire, R. L., 111; Jorgenson, J. W. J. Chromatogr. Sci. 1985, 23, 186-191. (23) White, J. G.; st. Claire, R. L., 111; Jorgenson, J. W. Anal. Chem. 1986, 58,293-298. (24) Kaniansky, D.;Havasi, J.; Marak. J.; Sokoiik, R. J. Chromatogr. 1988, 366, 153-160. (25) Hood, H. P.; Nordberg, M. E. US. Patent 2 106744, Feb. 1, 1938. (26) Nordberg, M. E. J. Am. Ceram. SOC. 1944, 27(10), 299-305. (27) Elmer, T. H.; Nordberg, M. E.; Carrier, G. B.; Korda, E. J. J. Am. Ceram. SOC.1970, 53(4), 171-175. (28) Elmer, T. H. J. Am. Ceram. SOC. 1983, 62(4), 513-516. (29) Pretorius, V.; Hopkins, B. J.; Shieke. J. D.J. Chromatogr. 1974, 132. 23-30. (30) Lukacs. K. D.; Jorgenson, J. W. HRC CC, J . High Resolut. Chromatogr . Chromatogr Commun . 1985, 8 , 407-4 11. (31) Lauer, H. H.; McManigill, D. TrAC, Trends Anal. Chem. (Pers. Ed.) 1986, 5 , 11-15.
.
RECEIVED for review January 20,1987. Accepted April 1,1987. This material is based upon work supported by the National Science Foundation under Grant No. BNS-8504292 and the National Institutes of Health under Grant No. 1 R 0 1 GM37621-01.
Liquid Chromatography/Electrochemical Detection of Carbohydrates at a Cobalt Phthalocyanine Containing Chemically Modified Electrode Leone1 M. Santos and Richard P. Baldwin* Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
Numerous carbohydrates can be oxldlzed at low positive POtentlals at chemically modified carbon paste electrodes containing added cobalt phthaiocyanlne (CoPC). Although no response is observed at plain carbon paste electrodes, a diverse group of carbohydrates including mono- and disaccharides, pyranose and furanose rings, and reduclng and nonreducing sugars are readily oxldlzed at the modlfled electrode surface. I n 0.15 M NaOH, the oxldatlons exhlblt a cyclic voltammetric peak potential of 4-0.40 V vs. Ag/AgCI, the waves decreasing In magnltude and shiftlng to more posltlve potentials at less baslc pH. The CoPC electrodes can be used for electrochemical detectlon of the carbohydrates In liquid chromatography as long as the applled potentlal Is regularly pulsed to -0.3 V or lower. Detection Umlts obtained in this manner range from 100 pmol injected for glucose and maltose to 500 pmoi Injected for fructose and sucrose.
In recent years, several electrochemical approaches have been proposed for use in the flow injection or high-performance liquid chromatographic (HPLC) analysis of carbohydrates (1-15). These approaches are of particular interest because carbohydrates do not exhibit significant absorption a t wavelengths above 210 nm and thus are not well suited for the absorption and fluorescence detection methods most commonly employed in HPLC. As a consequence, monitoring of sugars has ordinarily been performed either by refractive index detection of the intact carbohydrates or by chemical derivatization with strongly absorbing or fluorescing groups. Many carbohydrates-most notably, the reducing sugars-are known for the ease with which their chemical 0003-2700/87/0359-1766$01.50/0
oxidation can be made to take place (16). Thus, it might be expected that electrochemical detection following liquid chromatography (LCEC) should provide a relatively straightforward monitoring approach. Unfortunately, utilization of such an approach has been stymied by the fact that carbohydrates, including the reducing sugars, have a large overpotential toward electrooxidation at the glassy carbon or carbon paste electrodes most commonly used in LCEC. As a consequence, inordinately high detector potentials are required for the redox processes to occur to an appreciable extent. Thus, direct electrochemical detection is not a viable option for these compounds when carried out at conventional electrodes in the ordinary manner. Alternatively, several new electrochemical detection schemes have been developed for carbohydrates. These schemes have been of two varieties. In the first, metallic sensing electrodes such as platinum (1-3))gold (4-61,and nickel (7-9) have been used in place of the usual glassy carbon or carbon paste. Although the mechanism involved in carbohydrate oxidation appears to be somewhat different a t each of these surfaces, each permits the oxidation to occur at modest potentials (-0.2 to -0.8 V vs. Ag/AgCl for Pt, +0.15 V for Au, and +0.45 V for Ni) and thereby provides very sensitive LCEC detection of these compounds. However, with Pt and Au, where the electrocatalysis proceeds with adsorption of the starting carbohydrate (Pt) or of resulting oxidation products (Pt and Au), stable response is obtained only if appropriate cycles of oxidative cleaning of adsorbed material and reductive removal of the resulting oxide layer are applied between detection intervals. Thus, the use of dual- or triple-pulse potential waveforms is generally required for operation of these electrode materials to be practical (3, 6). With Ni, where the 0 1987 American Chemical Society
