Anal. Chem. 1003, 65,948-951
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Detection of Carbohydrates by capillary Electrophoresis with Pulsed Amperometric Detection Thomas J. O'Shea and Susan M. Lunte' Center for Bioanalytical Research, University of Kansas,2095 Constant Avenue, Lawrence, Kansas 66047 William R. LaCourse Department of Chemistry, University of Maryland, Baltimore County, 5401 Wilkens Avenue, Baltimore, Maryland 21228
INTRODUCTION Carbohydrates are important in a number of biological processes. They are a significant energysource for both plants and animals and play a substantial role in biological recognition of proteins. The analysis of carbohydrates in protein samples is becoming increasingly important with the emergence of a number of biotechnology-derivedpharmaceuticals into the marketplace. Capillary electrophoresis (CE) is a powerful tool for the separation of a wide variety of biological compounds. The distinguishing characteristics of CE are its ability to analyze extremely small volumes and the high separation efficiencies that can be obtained. The analysis of carbohydrates is a particularly challenging analytical problem since these compounds possess no unique chromophore or fluorophore necessary for direct detection. Carbohydrates have been detected using CE with indirect fluorescence detection.' However, this method suffers from high concentration detection limits, lacks specificity, and requires isolation of the sugars from other charged species prior to analysis of any biological samples. Two groups have employed precolumn derivatization with UV or fluorescence detection for carbohydrate analysis. Honda and co-workers separated 12 monosaccharidesby CEUV following precolumn derivatization with 2-aminoppidine.2 Although the separation of the monosaccharides was quite impressive, detection was limited to millimolar concentrations of the carbohydrates. A more sensitive method was reported by Liu et al., who were able to successfullydetect carbohydrates at the nanomolar level using a two-step reaction including reductive amination and precolumn derivatization by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde.3The disadvantage of this process is that it takes several hours to accomplish, including 1-2 h for the reduction step and an additional hour for the derivatization reaction. In addition, other amines present in the sample can interfere with the analysis, Electrochemical detection is an ideal method of detection for microcolumn-basedseparation systems because detection is based on a reaction at an electrode surface.P6 Therefore, in contrast to optical methods where the response of the detector is dependent on path length, cell volumes can be made very small with no decrease in sensitivity. This is a particular advantage in CE, where the path length is typically less than 100fim. Capillary electrophoresis-electrochemistry has been used extensively for the analysis of catecholamines (1)Garner, T. W.; Yeung, E. S. J. Chromatog. 1990, 515, 639-644. (2) Honda, S.; Iwase, S.; Makino, A.; Fujiwara, S. Anal. Biochem. 1989,
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(3) Liu, J.; Shirato, 0.;Wiesler,D.; Novotny, M. Proc. Natl. Acad. Sci. V.S.A. 1991,88, 2302-2306. (4) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987,59,1762-1766. (5) Curry, P. D.; Enastron-Silverman, C. E.; Ewina, A. G. Electroanalysis 1991, 3, 587-596.(6) O'Shea, T. J.; Greenhagen,R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatog. 1992,593, 305-312. 0003-2700/93/0365-0948$04.00/0
in single cells and for the detection of analytes obtained during microdialysateperfuei~n.~-l~ Detection limits for electroactive compounds usually are in the nanomolar range. Pulsed amperometric detection (PAD) at Au electrodes followinghigh-performance anion-exchangechromatography (HPAEC) has become the method of choice for the determination of carbohydrates in a variety of samples, including foods and beverages,biotechnologicallyderived products, and physiological fluids."-13 PAD at noble metal electrodes exploits surface-catalyzed oxidations of various functional groups (e.g., aldehyde, alcohol, amine, and sulfur-containing moieties). The high electrocatalytic activity at Au or Pt electrodes for a constant applied potential is often accompanied by fouling of the electrode surface.14 LaCourse and Johnson have used pulsed voltammetry (PV) to evaluate and optimize carbohydrate response a t a gold electrode.15 PAD overcomesfouling of electrodes by combining amperometric detection with alternated anodic and cathodic polarizations to clean and reactivate the electrode surface. Hence, PAD utilizes multistep potential-time wave forms (Et)to maintain uniform and reproducibly high electrode activity. Although there are numerous studies employing PAD at macroelectrodes, little has been done with PAD at microelectrodes. Recently, Ewing et al. employed PV to obtain voltammetric information in static biological microenvironments at Pt microelectrodes.16 In this paper, the feasibility of PAD as a detection method for capillary electrophoresis is explored. This is the first report involving PAD at a microelectrode in a flowing stream. A system has been developed using off-column detection and a gold wire microelectrode. The separation and detection by CE-PAD of several charged carbohydrates of biological interest will be presented. In addition, the use of this detector for the determination of glucose in blood will be demonstrated.
EXPERIMENTAL SECTION Reagents. All carbohydrates were obtained from Sigma Chemical Co. (St. Louis, MO) and used as received. Semiconductor-gradesodium hydroxide pellets, used in the preparation (7) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990,62,1872-1876. (8) O'Shea, T. J.; Telting-Diaz, M.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R. Electroanalysis 1992,4, 463-468. (9) O'Shea, T. J.; Weber, P. L.; Bammell, B. P.; Lunte, C. E.; Lunte, S. M. J. Chromatogr. 1992, 608, 189-195. (10) Chien, T. B.; Wallingford, R. A.; Ewing, A. G. J. Neurochem. 1990, 54, 633-638. (11)Johnson, D. C.; Lacourse, W. R. Anal. Chem. 1990, 62, 589A597A. (12) LaCourse, W. R.; Johnson, D. C. Carbohydr. Res. 1991,215,159178. (13) Johnson, D. C.; LaCourse, W. R. Electroanalysis 1992, 4, 367380. (14) Gilman, S. InElectroanalyticalChem~t~;Bard,A. J.,Ed.;Marcel Dekker: New York, 1967; Vol. 2, pp 111-192. (15)LaCourse, W. R.; Johnson, D. C. Anal. Chem. 1993,65, 50-55. (16) Chen,T. K.;Lau,Y. Y.; Wong, D. K. Y.;Ewing,A. G.Anal. Chem. 1992,64, 1264-1268.
0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 7,APRIL 1, 1993 048 of the operating buffer,were also obtained from Sigma. All other chemicals were analytical reagent grade. All solutions were prepared in NANOpure water (Sybron-Barnstead, Boston, MA) and passed through a membrane filter (0.2-pm pore size) before use. Stock solutions of carbohydrates were prepared daily. Apparatus. Electrophoresis in the capillary was driven by a high-voltagepower supply (Spellman Electronics Corp., Plainview, NY). Polyimide-coated fused-silica capillary columns of 360-pm 0.d. and 75-pm i.d. were obtained from Polymicro Technologies (Phoenix, AZ). Sample introduction was accomplished usinga pressure injectionsystem,and the volume injected was calculated in the continuous-fill mode by recording the time required for the sample to reach the detector. The microelectrode was constructed using a 50-pm-diameter gold wire (Johnson Matthey Electronics, Hertfordshire, UK) which was bonded to a length of copper wire using silver epoxy (Ted Pella Inc., Redding, CAI. Capillary tubes were pulled with a Liste-Medical (Greenvale,NY) Model 3A vertical pipet puller to a narrow tip. The gold wire was inserted through the capillary until it protruded approximately 0.5 cm from the tip. Silicon adhesivewas then applied to the tip at the junction of the capillary and the gold wire. Once cured, the gold wire was cut to the desired length, 300-350 bm, using surgical scissors. The gold electrode was then washed with ethanol followed by copious amounts of deionized water. The construction of the complete CE system with electrochemical detection has been described in detail elsewhere.6 The exception is the preparation of the Nafion joint, which is used to isolate the detection end of the column from the effects of the applied electrical field. Due to the large currents (ca. 40 pA) generated using the hydroxide electrolyte in this study, a modification of the designwas used to further lower the resistance across the Ndion joint. Thus, the final section of capillary was epoxied to a glass slide and a fracture was made at a previously prepared score mark. The fracture was then covered with Nafion solution (5 wt % solution in a mixture of lower molecular weight alcohols and 10%water) which was purchased from Sigma.This was allowed to dry fully. This joint was then submerged in the cathodic buffer reservoir and the system completed as described previously.6 A three-electrode configuration was used, and the electrode connectionswere made to an EG&G Princeton Applied Research (Princeton, NJ) Model 400 electrochemical detector, which provided the potential wave form and current output. A Ag/AgCl reference and a platinum auxiliary electrode were employed in the CE-EC experiments. A Faraday cage was used to shield the electrochemical cell from external noise sources. Pulsed Voltammetry (PV) Experiments. Pulsed voltammetric data were obtained at a 50-pM Au electrode with a computer-controlled potentiostat (Model RDE-4; Pine Instrument Co., Grove City, PA) using a DAS-20 high-speed AID-D/A expansion board (MetraByte Corp., Taunton, MA) in an IBMAT-compatible computer (Gateway Co., Sioux Falls, SD). The reference electrode employed was a SCE. Pulse voltammetry wave formswere generated with ASYST Scientificsoftware (Asyst Software Technologies, Inc., Rochester, NY). Separation Conditions. For the CE separations, a capillary length of 95 cm with an applied voltage of 25 kV was employed. The run buffer was 10 mM NaOH containing 8 mM sodium carbonate, pH 12. For detection of carbohydrates, the following PAD wave form was employed El (detection) +325 mV, tl199 ms; El (oxidativecleaning) +800 mV, t 2 166ms; E3 (reactivation) -600 mV, t 3 249 ms applied. All potentials are vs Ag/AgCl. Sample Preparation. For the determination of glucose levels in blood, 20 pL of human blood was diluted in 1mL of operating buffer,filtered, and centrifugedin a GelmanSciences(AnnArbor, MI) two-spin filtration unit. The filtered sample was injected directly.
RESULTS AND DISCUSSION
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Flguro 1. Pulse voltammetric response of glucose at a Au mlcrcb electrode In 10 mM NaOH-6 mM Na2C03. Condltlons: partially deaerated with N2. Wave form: = -800 to +QOO mV, a,= 500 ms, 0,= 240 ms, 4, = 200 ms; Eoxd= 800 mV, I&,, = I66 ms; = -600 mV, & = 249 ms. Summation of nlne PAD cycles per polnt. Solutions: (-) residual; (- -) 10 pM glucose; (- -) 50 pM glucose; (-) 100 pM glucose.
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because this response is relevant to the PAD wave forms (in which the anodic response is measured following a positive step from the cathodic potential pulse (Ed)to the detection potential (E&), which is much greater than E d ) . The cathodic and anodic signals for the residual correspond to reduction of dissolved 02 (wave A; ca. -600 to -100 mV), the formation of surface oxide (wave B; greater than ca. +300 mV), and 02evolution from water anodization (waveC, greater than ca. +700 mV). The "background-corrected" PV responses for glucose at 10,50, and 100 pM are also shown in Figure 1. Glucose produces a two-step anodic response during the positive scan. The first anodic step corresponds to the oxidation of the aldehyde group to the carboxylate anion in this alkaline media (wave D; ca. -600 to ca. -100 mV). The second step has the form of the peak (wave E; ca. -100 to +450 mV) and corresponds to the combined oxidations of the aldehyde and alcohol groups of glucose. The anodic signal during the positive scan is inhibited (greater than ca. +300 mV) corresponding to the onset of oxide formation (wave B). This is as expected for detections which are catalyzed at oxidefree s u r f a c e ~ . ~b~y JE~d e t v d u e in the range of 0 to +300 mV vs SCE will be acceptable for detection of glucose by PAD under these conditions. Application of this potential in PAD will enable the universal and direct detection of virtually all carbohydrates. By judicious selection of the detection potential, the applicability of this system can be extended to simple alcohols11 and aliphatic amines and amino acids,17JS as well as numerous sulfur compounds.19~20 Determination of Glucose. Glucose was the analyte of choice for the initial investigation of the performance of the detector with CE because of ita general importance in a number of biological systems and ita proven record with PAD detection. The column appeared to be quite stable at the high pH with migration times not exceeding 3% RSD within 1 day. A column used continuously over a 3-week period showed variations in migration times of not more than 10% RSD. Eight successive injections of 1 X 1V M glucose exhibited RSDs for migration time and peak height of 1.1% and 3.3 76, respectively. (17) Polta,J. A.;Johnson, D. C. J. Liq. Chrornatogr. 1983, 6, 17271743. ~. (18)Welch, L. E.; Lacourse, W. R.; Mead, D. A.; Johnson, D. C. Anal. Chern. 1989,61, 555-559. (19) Polta,T.2.;Johnson, D. C. J.Electroanal. Chern. 1986,209,159~
Pulsed Voltammetry of Glucose. Figure 1 shows the current-potential (i-E)response during the positive scans for glucose at a Au microelectrode in 10 mM NaOH-8 mM Na2C03. Only the data for the positive scans are shown
169. (20) Ngoviwatchai, A.; Johnson, D. C. Anal. 1-12.
Chim.Acta 1988, 215,
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 7, APRIL 1, 1993
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Flguro 4. Electropherogram of human blood. Peak corresponds to 85 pM glucose. Separation conditions as outlined In Flgure 2.
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time (mins.) M glucose using a 75-pm capillary column: operatlng buffer, 10 mM NaOH-8 mM Na2C03; separatlon voltage, 25 kV; PAD wave form as described in the Experimental Sectlon.
Flgure 2. Electropherogram of 1 X
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Flguro 3. Separatlon of 1 X lo-‘ M (1)glucosamine, (2) glucosaminic acid, (3)glucosamine &sulfate, and (4) glucosamine 6-phosphate. Separatlon conditlons as outlined in Figure 2. The PAD response for glucose was linear over the range 1 10-5 to 1 X 10-3 M with a correlation coefficient of 0.9997 (n = 7). Calibration curves prepared over this range yielded slopes of 299 nAImM. A detection limit of 9 X M based on a SIN = 3 waa estimated from the electropherogram obtained from an injection of 1 X M glucose shown in Figure 2. Based on an injection volume of 25 nL, this corresponds to a mass detection limit of 22.5 fmol. The mass detection limits are 5-fold higher than those reported using indirect detection;’ however, in the latter method 5-pm capillaries were employed, which leads to increased mass sensitivity. Therefore, although mass detection limits are comparable, because the injection volume is smaller in 5-pm columns, the concentration detection limit is poorer with indirect detection. In this study, 75-pm4.d. capillaries were used. The mass detection limit could be substantially improved by the use of columns of smaller inner diameter. Separation of Charged Carbohydrates. CE-PAD was employed for the separation and detection of several biologically important charged carbohydrates, including glucosamine, glucosaminic acid, glucosamine 6-sulfate, and glucosamine 6-phosphate. Figure 3 shows the separation achieved for these four compounds, with glucosaminic acid X
and glucosamine 6-sulfate nearly baseline-resolved. Garner and Yeung achieved a separation of the “neutral” sugars sucrose, glucose, and fructose in less than 10 min at a similar pH using 20-pm capillaries.’ However, many other sugars exhibited the same migration time as these three. Under the conditions reported here, we were not able to achieve the high efficiencies necessary for this separation of these three neutral sugars. This could be due to the fact that a larger amount of current is generated by the hydroxide electrolyte in a 75-pm capillary than in a 20-pm capillary, leading to Joule heating. An additional factor is the time constant of the detector, which is designed for LC applications. Future research will involve the evaluation of new buffer systems, complexing agents, and other additives to better facilitate the separation of uncharged carbohydrates. Determination of Glucose i n Blood. To investigate the selectivity of the detector for the analysis of carbohydrates in real samples, the determination of glucose in blood was examined. Illustrated in Figure 4 is the electropherogram of a 1:50 dilution of human blood;the only sample pretreatments were centrifugation and filtration. The glucose peak represents an injection of 85 pM glucose, which corresponds to a concentration in the blood of 4.25 f 0.13 mM (n = 3). This agrees well with that reported in the literature.21 Only a few other small peaks are present in the electropherogram, demonstrating the high degree of selectivity this technique has toward carbohydrates. There are several potential advantages of this method over other methods for carbohydrate analysis. In CE-PAD, unlike LC-PAD, buffers of high pH can be employed for the separation without column degradation. Capillaries are also much less expensivethan the ion-exchangecolumns presently used for LC separations. The small volumes required for analysis by CE make it possible to apply this method in those cases where one is limited by sample size. In addition, this method permits direct detection, eliminating problems inherent in derivatization procedures currently employed for CE analysis (dilution of sample, incomplete reaction, side products, long reaction times). The high degree of selectivity has been demonstrated by the electropherogram of glucose in blood. One drawback of this technique is the high pH necessary for electrochemical detection of carbohydrates, which limits the pH range that can be employed for the separation. However, this problem can be overcome either by using Pt electrodes, which may allow one to work at lower pH values, or through postcolumn addition of base using one of the previously described methods for postcolumn derivatiza(21)The Merck Manual, 15th ed.; Berkow, R., Ed.; Merck Sharp & Dohme Research Laboratories: Rahway, NJ, 1987; p 2413.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 7, APRIL 1, 1993
tion.22p23Oxygen can be a problem when Pt electrodes are employed; however, deoxygenation has been shown to be relatively simple to accomplish with CE because of the small volume of buffer involved.24 The emphasis of future studies will be on the separation and detection of carbohydrates present on glycoproteins.The use of this method for the detection of other PAD-active (22) Rose, D. J.; Jorgenson, J. W. J. Chromatogr. 1988,447,117-131. (23) Pentoney, S., Jr.; Huang,X.; Burgi, D.; Zare, R. N. Anal. Chem. 1988,60, 2625-2629. (24) O’Shea, T. J.; Lunte, S. M. Anal. Chem., 1993, 65, 247-250.
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functional groups, such as amines and alcohols, will also be investigated.
ACKNOWLEDGMENT The authors thank Nancy Harmony for help in the preparation of the manuscript. This work was supported by the Center for Bioanalytical Research and the Kansas Technology Enterprise Corp. RECEIVED for review October 5, 1992. Accepted December 30, 1992.
