Anal. Chem. 1093, 85, 2878-2881
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Pulsed Amperometric Detection of Carbohydrates Separated by Capillary Electrophoresis Wenzhe Lu and Richard M. Cassidy’ Chemistry Department, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 0 WO.
Working conditions for operation of a 10-pm Au electrode have been evaluated for the detection of eight carbohydrates separated by capillary electrophoresis. The voltage pulse sequence consisted of 300 mV (vs SCE)for 165 ms, 1200 mV for 55 ms, and -800 mV for 165 ms; carbohydrate oxidation was monitored in the first pulse. Samplingtimes of 55 ms were used during the 300-mV pulse, and maximum sensitivity was observed for the second 55-ms sampling period. Detection limits were in the low-femtomole range for separation in 10-pm capillaries with a 0.1 mol/L sodium hydroxide electrolyte. A linear working range was observed from 104 to 10-4 mol/L, and peak height reproducibility for inositol was 2%. Separation efficiencies were in the range of 100000-200000 theoretical plates, but some degradation of capillary performance was observed with time.
INTRODUCTION Constant-voltageamperometry with ultramicroelectrodes has been successfully used for detection of many organic, inorganic, and biological molecules separated by capillary electrophoresis (CE).1-4 However, there have been very few evaluations of pulsed techniques reported for CE detection. Pulsed amperometric detection (PAD), particularly at Au electrodes, has been successfully used to monitor carbohydrates separated by liquid chromatography (LC), and the application of PAD in LC was reviewed recently.5 The reaction mechanism of the detection process has been studied in some detail,”12 and those studies indicated that the rate of carbohydrate oxidation a t Au electrodes was accelerated through the interaction of the carbohydrates with hydrous oxide formed on Au (or Pt) electrode surfaces in basic solutions. Passivation of the electrode activity due to accumulation of the products of the carbohydrate oxidation can be minimized by applying a series of voltage pulses to clean and recondition the electrode surface. Such cleaning strategies have also been applied to the electrochemical
detection of other organic and inorganic substances.4~~J0J3 Recently, pulse voltammetry, a combination of cyclic voltammetry with pulse amperometry, has been used for the detection of ferrocyanide,glucose,catechol,and other organics a t platinum microelectrodes in static solutions.14 In another recent study, a two-step pulsed amperometric technique was used with ultramicroelectrodes to detect metal ions separated in 25-pm-i.d. capillaries; the metal ions were first reduced at a negative potential and then oxidized with a positivepotential step, which also served to clean the electrode surface prior to the next sampling stage.4 It can be expected that PAD techniques may prove useful for detection in CE in general, and in particular for carbohydrate analytes. Carbohydrates are of interest in many scientific and industrial areas, and many approaches to their analysis have been investigated. For additional background information, the reader is referred to a recent publication that provides an excellent summary of current approaches to carbohydrate analy~e5.l~ Recently two papers have described amperometric CE methods for carbohydrate detection.16J6 One of these reported the use of 25 pm X 400 pm copper filaments and 50-pm4.d. capillaries; values of height equivalent to theoretical plate (HETP), 13.8 pm, were good, but separation voltages had to be a t 1 1 O O O O V.16 Pulsed amperometric detection has also been reported recently for 50 pm X 350 pm gold filaments with 75-pm-i.d. capillaries and a grounded detection zone;l6 values of HETP were quite large ( 50 pm) and detection limits were 10-8 mol/L. The work presented here describes the results of an investigation of the application of PAD with 10-pm disk gold electrodes for carbohydrates separated in 10-pm-i.d. capillaries. Eight common carbohydrates were chosen to evaluate the effect of electrolyte composition on the separation, and the effect of the choice of voltage pulse and data sampling periods on the detector performance was also examined.
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EXPERIMENTAL SECTION Apparatus. Fused-silica capillaries, 25 pm i.d. X 365 pm 0.d. and 10 pm i.d. x 150 pm o.d., were obtained from Polymicro Technology Inc. (Phoenix,AZ). Before use, the new capillaries were washed with water and operating electrolyte (NaOH solutions);this was done by aspirating these solutions through the capillaries using a syringe at a linear flow rate of -0.5 cm/ min. The capillarieswere stored overnight by placing both ends of the capillary in operating electrolyte with a height differential (1) Olefirowicz,T. M.; Ewing, A. G. Anal. Chem. 1990,62,1872-1876. (2) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, to maintain electrolyte flow through the capillary. The high63,189-192. voltage power supply,with reversible polarity (0 to i 3 0 kV),was (3) OShea,T.J.;Greenhagen,R.D.;Lunte,S.M.;Lunte,C.E.;Smyth, obtained from Spellman (Model RHR30PN30, Plainview, NY). M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593,305-312. The high-voltage input was placed in a Plexiglas box with an (4) Lu, W.; Caesidy, R. M., Anal. Chem. 1993,65, 1649-1653. interlock on the access door for protection. The detection cell (5) Johnson, D. C.; Lacourse, W. R. Anal. Chem. 1990,62,589A-597A. (6) Larew, L. A.; Johnson,D. C. J.Electroanal. Chem. 1989,262,167and detector were housed in a faradaic cage to minimize the 182. interference from external noise. PAD was performed with a
(7) Lacourse, W. R.; Johnson, D. C. Anal. Chem. 1993,65,50-55. (8)Hughes, S.; Johnson, D. C. Anal. Chim.Acta 1983,149, 1-10. (13) Stulik, W.; Hora, V. J. J . Electroanal. Chem. 1976, 70, 253. (9) Edwards, P.; Haak, K. K . Am. Lab. 1983, (April), 78-87. (10) Johnson,D.C.;Polta,J.A.;Polta,T.Z.;Neuburger,G.G.;Johnson, (14) Chen, T. K.; Leu, Y. Y.; Wong, D. K. Y.; Ewing, A. G. 1992,64, 1264-1268. J. L. J. Chem. SOC.,Faraday Trans. 1 1986,82,1081-1098. (15) Colon, L. A.; Dadoo R.; Zare, R. N. Anal. Chem. 1993,65,47€-481. (11) Rocklh,R. D.;Pohl,C. A. J.Liq. Chromatogr.1983,6,1577-1590. (16) OShea, T. J.; Lunte, S. M.; Lacourse, W. R. Anal. Chem. 1993, (12) Marincic, L.; Soeldner, J. S.; Colton, C. K.; Giner, J.; Morris, S. J. Electrochem. SOC.1979,126, 43-49. 65, 948-951. 0003-2700/93/0365-2S78$04.00/0
0 1993 American Chemical Society
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D in the order of 106 cm2 s-l, the first and second terms become equal a t 49 s, but for a 10-pm electrode, this occurs after 4.9 ms. The rapid response of 10-pm disk electrodes has been confirmed experimentally.20 Since considerable differencesare expected between the response of conventional electrodes and ultramicroelectrodes,the behavior of the gold ultramicroelectrode was examined briefly prior for its application to the detection of carbohydrates. Cyclic voltammetry was used to initially evaluate the electrochemical behavior of Au ultramicroelectrodes with and without carbohydrates present. Studieswere made a t various concentrations of NaOH (0.001-0.5 mol/L) and different sweep rates (0.005-0.5 V/s) for the carbohydrates glucose, inositol, and fructose. The cyclic voltammograms observed were similar to those reported by Johnson and Lacourses for large Au electrodes used for LC applications and thus are not reported here. Consequently, a three-step waveform similar to that used in LC5 was chosen for evaluation of the electrode operating conditions. Modified potential waveforms were briefly evaluated in terms of the potential value and the time period for each applied pulse. The voltage ranges examined, which covered the range of values used in pulsed amperometric studies,"llJ6 were from 0 to 440 mV (vs SCE) for detection, 800 to 1300 mV for the oxidative cleaning, and -400 to -1100 mV for reductive conditioning of the Au electrode surfaces. The ranges of time sequences examined were from 55 to 440 ms for the detection pulse, 55 to 220 ms for the oxidative cleaning pulse, and 55 to 440 ms for the reductive conditioning pulse. The range of voltage waveforms that gave the best (&lo%)signal-to-noise ratio (S/N) in this system were as follows: 200-350 mV and 11G220 ms for detection step (see discussion of detection below), 900-1200 mV and 55-110 ms for oxidation, and -500 to -lo00 mV with 110-330 ms for reduction. It was found that values in the upper range of RESULTS AND DISCUSSION reduction and oxidation potential were required to condition the electrodes when shorter conditioning periods were used. Optimization of Detection Parameters. In LC appliThe time period chosen for measurement of the analyte cations of PAD for carbohydrates, the current is often faradaic current will also affect detection limits because the integrated for a relatively long time (up to 200 ms)7 during S/N will depend on the ratio of residual and faradaic currents the last part of the sampling voltage pulse (typical total a t different sampling time periods and the precision and duration of this voltage pulse is -400 ms'9. A delay of stability of these currents. Consequently, the effect of the 50-100 ms is used to minimize residual currents from the choice of the time period for data collection on detection was double-layer charging current.5 The time constant, t,, for examined briefly. Figure 1shows how peak height changed changes in charging current can be calculated from the as the time for data collection was varied across the voltage following equation: pulse used for detection of glucose on a 10-pm gold electrode after separation by CE. Each peak in Figure 1was plotted t, = RCd = ?V2Ch(?TQ/r)= kr (1) from data collected during one CE experiment. When this where R is the resistance, c d and Ch are the double-layer experiment was repeated either in static or in flowing solution, capacitance and the specific double-layer capacitance (-20 some variation in the response ratios of the first two 55-ms pF/cm2), respectively, q is the specific resistance of the periods was observed, but the response from the first 55-ms solution, and r is the radius of the electrode. For a period was always less than that for second sampling period. conventional 1-mmdisk electrode, t, is -0.1 ms in a 0.1 mol/L Similar results were obtained for the constant-potential NaOH solution; for a 10-pm electrode, t, is -0.1 ps. oxidation of catechol at 10-pm gold electrodes, which is an The choice of delay time used before the current is measured electrochemically reversible reaction. If the electrode process will also depend on the response curve for the faradaic current. was controlled solely by diffusion, maximum current is The decrease in a diffusion-controlled current at a disk expected in the first 55 ms. The response observed for the electrode can be approximated by the following e q ~ a t i o n : ' ~ J ~ application of a potential to a resistor was essentially constant for all 55-ms sampling periods, and thus the time constant i = nFAD'/2C/(?rt)'/2 4rnFDC (2) for the voltage waveform generator was not a factor in the where n is the number of electrons transferred, F is the lower initial response for carbohydrates. Studies in static Faraday constant, D is the diffusion coefficient, C is the solutionsshowed that the currents for background electrolyte concentration, and r is the electrode radius. A steady-state and 10-4mol/L glucose or catechol solutions were essentially current will be obtained when the second term becomes a t steady-state conditions from the second 55-ms point on. dominant. For a 1-mm disk electrode, assuming a value of At this time it is not clear whether the low initial response is caused by change in signal vs time due to the complex (17)Lu,W.; Caseidy, R. M.; Baranski, A.S.,J. Chromatogr. 1993,640, reactions taking place a t the gold oxide surface? by structural 433-440. faults in the electrode seal, or by instrumental problems. Since (18)Wight", R. M.Anal. Chem. 1981,53,1125A-1134A. (19)Ewing, A.G.;Dayton,M.A,;Wightman,R. M.Anal. Chem. 1981,
threeelectrode system; the construction, circuit diagram, and performance of this detection system has been described elsewhere17 (the capacitorsshown previously in the circuit diagram1? were removed for pulse studies). Detection was controlled with a 386DX/40 MHz IBM personal computer equipped with PCL818high-performance data acquisition card (B & C Microsystem Inc., Sunnyvale, CA). "In-house" computer programs were developed to generate different voltage pulse trains, to collect data at specific time periods, and to display the data. The construction of the detection cell assembly has been described previously.'' An Auultramicrodik (10pm in diameter) was used as the working electrode;the fabrication of this electrode was described previously.' Before use the electrode was polished with carborundum abrasive paper (Diamond waterproof paper, No. 600) and 0.3-pm alumina powder. The position between the capillary, which was mounted on a micropositioner, and the electrode was measured with an optical scale under a microscope (to within 1-3 pm) for both top view and side view directions. The electrode was placed directly in front of the capillary exit at a distance of 3-5 pm. The reference electrodewas a saturated calomel electrode (SCE) (Miniature model, Fisher Co., Ottawa, ON, Canada), and the auxiliary electrode was platinum foil with an exposed surface area of 0.5 cm2. Chemicals. All solutions were prepared from double distilled and deionized water (Coming, Mega-Pure system, MP-6A & D2, Corning, NY). The background electrolyte for the separation and detection of the carbohydrates was 0.025-0.1 mol/L NaOH [97%, British Drug Houses (BDH), Toronto, ON, Canada]. Sample solutionsof glucose (BDH),inositol, sorbitol,rhamnose, and xylose (Aldrich, Milwaukee, WI), and D-fructose, maltose, and arabinose (Fisher, Fair Lawn, NJ) were prepared as 0.01 mol/L stock solutions in water. Samples were diluted to the desired concentrations with operating electrolytes prior to use. All solutionswere filtered through a 0.2-pm Nylon-66membrane syringe filter (Cole-Parmer Instrument, Chicago, IL) prior to use.
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53,1842-1847.
(20) Scharifker, B.; Hills, G. J . Electroclnal. Chem. 1981,130,81-97.
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Flgure 1. Electropherogram of glucose peaks obtalned wlth PAD at dlfferent data sampllng tlmes over the detectton period. Except for the first peak, each subsequent peak has been displaced by 25 s on the X-axls. Experimental conditlons: separation voltage, 30 kV over 10 pm X 60 cm caplllary; electrode, 10 pm (In diameter) Au dlsk; electrode potentlel, 300 mV (vs SCE) for 275 ms (detection perlod), 1200 mV for 110 ms, and -900 mV for 220 ms; electrolyte, 0.025 mol/L NaOH; glucose concentration, 1 O4 mol/L; electromlgratton Injection, 30 kV for 5 8, Peak Mentlflcation: (1)sampllng at 0-55 ms, (2)sampling at 56-1 10 ms, (3)sampling at 1 1 1-165 ms, (4)sampllng at 166-220 ms, and (5)sampling at 221-275 ms.
it was not possible to change the applied potential timing to periods less than 55 ms without extensive changes to the computer software, the behavior of the currents in the first 55 me could not be examined in more detail. Although fast pulse waveforms and sampling periods of less than 55 ms may be required for some CE applications where peaks are very narrow, 55-ms samplingperiods proved to be sufficiently fast for this evaluation. The pulse sequenceused for detection of carbohydrates in this study was as follows: 300 mV (vs SCE) for 165 ms with data collection over the second or third 55-ms period, 1200 mV for 55 ms to clean the electrode, and -1OOO mV for 165 ms to condition the electrode. CE Separation of Carbohydrates. It has been shown that the CE separation of carbohydrates can be achieved with the use of borate as a complexingreagent in the electrolyte.21~ Consequently, a borate buffer (pH 10-13) was evaluated in these studies. Unfortunately for both cyclic voltammetric studies and CE detection in a pulsed mode, very small electrode response was observed for carbohydrates in borate electrolytes. Carbohydrates, which are weakly acidic,2s are negatively charged in basic solutions, and this property can be used for the CE separation of carbohydrates. It was found that higher concentrations of base improved resolution; for example, resolution for glucose and maltose changed from 0.5 in 0.025 mol/L NaOH to -5 in 0.1 mol/L NaOH, and this is in agreementwith a recently published report.16 However, Joule heating was a problem at higher concentrations of the electrolyte. Dissipation of the heat from the large currents ( 50 p A ) in 25 pm X 60 cm capillaries was improved through the use of smaller capillaries (-7 p A in 10 pm X 60 cm capillaries). The effect of the addition of organic solvents to decreasethe conductivityof the electrolyte was also examined
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(21)Honda, S.;Iwase, S.; Makino, A.; Fujiwara, S. Anal. Biochem. 1989,176,72-77. (22) Hoffstatter-Kuhn, S.; Paulus, A.; Gassmann, E.; Widmer, H. M. Anal. Chem. 1991,63,1541-1547. (23)Rendleman, J. A. In Training of Literature Chemists;Gould, R. F., Ed.; Advances in Chemistry Series 17;American Chemical Society: Waehington, DC, 1973; p 51.
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Electropherogram of carbohydrateswlth PAD. Experimental condltlons: separatlon voltage, 30 kV over 10 pm X 60 cm caplllery; electrode, 10 pm (in dlameter) Au disk; electrode potentlal, 300 mV (vs SCE) for 165 ms (sampling at 1 1 1-165 ms), 1200 mV for 55 ms, and -1000 mV for 165 ms; electrolyte, 0.1m d R NaW electromlgratbn injection, 30 kV for 3 s; sample concentrations, 1 X lo4 mol/L for Inositol and 2 X lo4 mol/L for others. Peak Identification: (1)Inositol, (2)sorbitol, (3) unknown, (4) maltose, (5) glucose, (6) rhamnose, (7) arablnose, (8) fructose, and (9) xylose. Flgurq2.
briefly; the solvents examined were dimethylformamide, methanol, and acetonitrile and the percent volume (HzO organic) ratios examined ranged from 9:l to 41. The addition of these solvents gave a moderate decrease( -20 % ) in current, but decreased detection sensitivity. Consequently,0.1 mol/L NaOH electrolyte and 10-pmcapillaries were chosen for the CE separation of carbohydrates. Figure 2 shows the electropherogram obtained for the detection and separation of the test carbohydratesin 0.1 mol/L NaOH (injection at 30 kV for 3 8). The S/N was -100 for inositol, 90 for sorbitol, 70 for glucose, 50 for fructose, 50 for xylose, 45 for arabinose, 35 for maltose, and 30 for rhamnose. Detection limits (based on 2X peak-to-peak noise) were determined by injection of lower sample concentrations (10-6 mol/L for inositol and 2 x 10-6 moVL for the other carbohydrates) at 30 kV for 5 s. The detection limits obtained were -0.28 fmol for inositol, 0.60 fmol for sorbitol, 0.61 fmol for glucose, 0.75 fmol for xylose, 0.79 fmol for fructose, 0.82 fmol for arabinose, 1.21 fmol for maltose, and 1.21 fmol for rhamnose. Integration of the signal over periods larger than 55 ms could improve detection limits, but this is of limited use since any significant increase in time would result in a serious reduction in the number of sample points per peak. Separationefficiencies, which were calculated from the peak width at half peak heights, were 200 OOO theoretical plates for xylose, 180 OOO for fructose, 160 OOO for arabinose, 150 OOO formaltme, 130 OOOforrhamnoee,120 OOOforglucose,100 OOO for inositol, and 70 OOO for sorbitol. During several studies, it was observed that the best separation efficiency was obtained with new capillaries. Separation efficiencies decreased after the capillaries were used for few days, and the peak tailing became more obvious. This may be due to a change in the properties of the internal wall of the capillaries, resulting in interaction between the carbohydrate and the wall. In an attempt to minimizethe changes in the properties of the capillary wall, three Surfactants were evaluated in an attempt to coat and stabilize the properties of the capillary wall. The test surfactants included Waters OFM, Carbowax 20M, and Triton-X 100. With OFM (0.5 mmol/L) and Triton-X 100 (0.2 mmol/L) the carbohydrates could not be detected, perhaps due to interference from the adsorption of surfactants onto the electrode surface. With higher concentration of Carbowax 20M (>1%), detection sensitivity also
ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993 2881
decreased. At lower concentrations (0.1% ) of Carbowax 20M, the detection sensitivity did not change appreciably, but no improvement in separation efficiencywas observed. Further detailed studies are needed to develop experimental conditions that will minimize changes in separation efficiency as a function of age of the capillary. Except for arabinose, the test carbohydrates exhibited reproducible migration times, either day to day or within 1 day. With arabinose, a positive or negative drift of 5 s was observed within any 1day, but no significant changes were observed in the peak shape. The maximum variation in the migration time over a 5-day period was also -*5 s. The exact cause for this variation is unclear, but it may be related to temperature changes, which could have an effect on the distribution of different conformationsfor this carbohydrate. The potential of this separation system for the analysis of carbohydrates was briefly examined using inositol as a test solute. A relative standard deviation of 2 % in peak height (24) Cassidy, R. M.; Janoski, M. LC-CC 1992,10, 092-696.
was observed for seven samples introduced by electromigration a t 10 kV for 10s. Calibration plots gave good correlation coefficients (0.9961, and detector linearity, over the concentration range from 10-6 to 10-4 moUL, was evaluated further by plotting sensitivity, corrected for nonzero intercept, vs concentration (five concentration points) as described elsewhereSu The maximum difference between the values of sensitivity observed in this concentration range was 22 % . This is considered to be quite reasonable considering that these data were obtained with 5-s injections at 30 kV.
ACKNOWLEDGMENT We acknowledge the financial support of the Natural Science and Engineering Council of Canada and Waters/ Millipore.
RECEIVED for review February 2, 1993. Accepted July 14, 1993.' 0
Abstract published in Advance ACS Abstracts, September 1,1993.
