Chapter 26 Electrochemical detection of carbohydrates at constant potential after HPLC and CE separations

Ziad El Rassi (Editor) Carbohydrate Analysis by Modern Chromatography and Electrophoresi s Journal of Chromatography Library, Vol . 66 © 2002 Elsevier Science B .V. All rights reserved

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Electrochemical Detection of Carbohydrates a t Constant Potential after HPLC an d CE Separation s RICHARD P. BALDWI N Department of Chemistry, University of Louisville, Louisville, KY 40292, USA

26 .1 . INTRODUCTION Without question, the determination of carbohydrate compounds in complex sample s represents both an extremely important and an extremely challenging task for th e modern analytical chemist . First, there is the difficulty of separating the carbohydrate s of interest from each other and from other components of the sample matri x usually carried out by high performance liquid chromatography (HPLC) or capillar y electrophoresis (CE) . In addition, there is the difficulty of detecting a set of compound s that are poorly suited by nature to the optically based detection approaches normall y employed with these separation techniques . In this chapter, I focus on the latte r detection issue for carbohydrates and, in particular, consider the progress that ha s been made in developing alternative electrochemical (EC) detection strategies for thes e compounds . Certainly, the driving force for the interest in EC detection of carbohydrates ha s been the fact that these compounds lack a native chromophoric group that can b e accessed at convenient UV-visible wavelengths . Therefore, without prior conversio n of the carbohydrate to a suitable derivative, conventional absorbance and fluorescenc e methods provide far less than optimum sensitivity and selectivity for HPLC and C E applications . However, most carbohydrate species including not only simple sugar s but also alditols, sugar acids, aminosugars, oligo- and polysaccharides, and many suga r conjugates can undergo facile chemical oxidation and therefore make attractiv e candidates for direct EC detection with no need for derivatization . Thus, EC detection , if successful, offers the possibility of simpler, faster, and more efficient HPLC an d CE assay procedures for carbohydrate compounds . In addition, the amperometric E C systems involved are generally among the most economical and most flexible o f detection techniques available . For example, the instrumentation required to carry ou t References pp . 958—959



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potential control and current measurement, as required for EC detection, is relativel y unsophisticated and inexpensive . Further, the electrodes themselves can be easily varied in composition, size, and shape and, most important, can usually be miniaturized fo r applications where electrode size may be critical, e .g ., for in vivo sensing, CE detection, or incorporation onto lab-on-a-chip devices . For EC detection applications, the choice of electrode material is nearly alway s a critical one . This is certainly the case for the electro-oxidation of carbohydrat e compounds for which several common electrode materials are not well suited . Fo r example, mercury is easily oxidized itself and cannot withstand the higher oxidatio n potentials required by carbohydrates ; and carbohydrate oxidation at carbon electrodes i s kinetically unfavored and requires potentials too high for practical use . Consequently , the electrodes that have been successfully employed for such detection have consiste d of either a noble metal or a transition metal . The use of noble metal electrodes (e .g ., Pt or Au) for HPLC applications, firs t reported by Hughes and Johnson in 1981 [1], has typically been carried out with a pulsed potential format . This approach, usually termed pulsed amperometric detectio n (PAD) or, more recently, pulsed electrochemical detection (PED), is necessary becaus e adsorption of free radical intermediates produced during carbohydrate oxidation leads t o the fouling and rapid deactivation of the Pt or Au surface when the electrode is simpl y held at a constant oxidizing potential . PAD/PED addresses this problem by applicatio n of a controlled sequence of potential steps chosen to desorb the adsorbed specie s anodically and then regenerate the active state of the electrode surface cathodically. The resulting applications of PED/PAD over the past 20 years have been extensive an d leave no question concerning the broad utility of this technique ]2-5] . In fact, a separat e chapter devoted specifically to PAD/PED theory and applications has been included i n this volume (Chapter 25) . Therefore, no further discussion of noble metal electrodes an d the pulsed detection approach needs to be provided here . In addition, carbohydrate oxidation and detection has also been carried out a t electrodes made from several different transition metals (e .g ., Cu, Ni, Co, and Ru ) ]6] . Accordingly, the focus of this chapter will be the operation and performance o f such transition metal electrodes for carbohydrate analysis . In general, the applicatio n of these electrode materials has not been as extensive or as systematically developed as has that of Pt and Au . However, compared to Pt and Au, the transition metals offe r some unique detection features . Most important, electrodes from such materials al l exhibit stable activity for carbohydrate oxidation without the need for the continuou s application of surface cleaning and reactivation potentials . As a result, although thei r analytical performance is roughly comparable to that of pulsed detection at Pt and Au , the transition metals do not require specialized pulse instrumentation and thus are full y compatible with the simple potentiostatic control instrumentation commonly used for EC detection in existing HPLC and CE systems . In the discussion below, I will begi n with an overview of these electrode materials and the principles of their operation i n both HPLC and CE . Subsequently, I will review their most important applications t o date for carbohydrates, highlighting some of the most exciting new developments in th e field .



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26 .2. OVERVIE W 26.2.1 . Electrode materia l The transition metals that have been employed most extensively for EC detectio n of carbohydrates in HPLC and CE have been Cu [7–10], Ni [11,12], and Co [13,14] . For this reason, the discussion here will focus on these materials . In addition, severa l other metals, including Ru [15] and Ag [16,17], have been shown to be suitable fo r carbohydrate detection but have not in practice seen very much use for this purpose . Of course, as carbohydrate detection at these electrodes is nearly always performed i n strongly basic solution and at oxidizing potentials, the active electrode surfaces in al l these cases should more accurately be described as the metal oxide or hydroxide . The most usual form for the electrode to take in practice is simply a wire or dis k of the pure metal . Such electrodes can be obtained commercially in a wide range of shapes and sizes as needed, and incorporation into a cell suitable for flow analysis mus t be done in-house but is normally straightforward . Furthermore, films of these metals , especially useful for on-capillary CE detection or lab-on-a-chip applications, can b e readily generated by routine electroplating, sputtering, or vapor deposition procedures . Of course, the thickness selected for such films should keep in mind that, with use , there may be a gradual loss of metal via dissolution at the metal–metal oxide/solution interface and the presence of underlying bulk metal insures that such loss should no t adversely affect electrode functioning . In addition to the pure metal, a variety of other electrode structures, includin g alloys and composite materials, have been investigated as well . A variety of Cu and N i alloys has been investigated by Kuwana's group for the purpose of improving electrod e performance, especially with respect to long-term stability of response 118–201 . Ni–T i and Ni–Cr mixtures were reported to exhibit the best results here, and Morita et al . have recently reported the further optimization of the Ni–Ti alloy composition to permi t extremely sensitive glucose detection in microbore HPLC [21 I . Alternatively, electrode s have been prepared for several metals by incorporating particles of the insoluble meta l oxides into carbon paste or polymer films 115,22–291 . Finally, chemically modifie d electrodes containing one of the active transition metals in a specific molecular form , e .g ., Co as cobalt phthalocyanine [30,311 or Ni as nickel tetramethyl-dibenzo-tetraaz a [15] annulene 132], have also been employed as well . Apart from the applied potential required for optimum detection, the differen t electrode materials and formats tend to exhibit more similarities than differences i n overall operation and performance . One of the few systematic studies comparing th e performance of different electrode materials was that by Luo and Baldwin [331 wh o showed that the sensitivity and stability afforded by Cu and Ni for flow detection o f carbohydrates was superior to those found for most of the alternative materials . For most of the electrode systems, the carbohydrate oxidations are thought to be electrocatalyti c in nature, initiated by the electrochemical generation of a high oxidation state of th e metal substrate (such as Ni(III) or Co(III)) . Beyond this, the specific electrode reaction s involved are generally not well established ; and most of the time reaction product s have not been well characterized . Although it has often been postulated that the initia l References pp . 958—959



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oxidative event occurs at the carbonyl group of the carbohydrate, the fact that mos t of the electrode materials oxidize alditols in a similar fashion suggests that this may not be the case . Of the different metals, the one whose electrode reactions have bee n most thoroughly investigated is Cu where carbohydrate oxidations have been show n by exhaustive electrolysis to consist of many electron processes (n = 14 for glucose ) and to yield single carbon units such as formate and carbonate as final products [33] . Fortunately, lack of knowledge of electrolysis mechanisms and products does no t preclude useful analysis applications to be developed and put into practice .

26.2 .2. Separation considerations One detection requirement shared by all of the metal-based electrode system s (including Pt and Au) is that they exhibit maximum response for carbohydrate oxidatio n only under high pH conditions typically, pH 13 and seldom below pH 12 . Becaus e the pK a values of simple sugars are 12–13 [34], this means that the carbohydrate specie s will normally have some anionic character under these conditions and must be separated as such unless post-separation pH adjustment of the mobile phase or CE buffer is carrie d out . Accordingly, for HPLC applications, anion-exchange chromatography has normall y been the separation method of choice when EC detection is employed . Of course, thi s precludes the use of conventional silica-based stationary phases . Fortunately, specialized polymeric ion-exchange columns suitable for these separations are now commerciall y available . For CE applications, the high pH requirement has actually proven to be quit e convenient as the accompanying deprotonation serves to provide the carbohydrate analyte with the charge needed for effective electrophoretic migration and separation . Otherwise, an additional procedure involving formation of a charged derivative o r reliance on a more complex form of CE such as capillary electrochromatograph y would be necessary . In fact, manipulation of the pH of the CE buffer has been show n to provide an effective means for optimizing carbohydrate separation . For example , e .g ., glucose , Ye and Baldwin [35] showed that related carbohydrate compounds glucitol, and gluconic, glucaric, and glucuronic acids are ionized to different degree s in and easily separated by CE in 0 .10 M NaOH . Furthermore, many traditionall y difficult problems, such as the separation of different alditols from one another or th e resolution of complex polysaccharide mixtures, can be overcome by measures as simpl e as optimizing the pH of the CE buffer [35] or adjusting the electroosmotic flow b y surfactant addition [36] .

26 .2.3 . Instrumentatio n One issue that must be acknowledged with respect to CE/EC of carbohydrates i s that, at the time of this writing, the instrumentation required is not yet commerciall y available in a pre-packaged or `black-box' format with which the non-electrochemist



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might feel most comfortable . Rather, CE/EC devices must be assembled in-hous e from the readily available components . At the minimum, a commercial potentiosta t identical to that used for EC detection in HPLC must be acquired and then interface d to an existing CE instrument. This requires the solution of two instrumental problem s unique to EC detection in CE namely, the electrical isolation of the CE and EC electronics and the physical alignment of the EC electrode with the capillary outlet . As both of these problems, though important, are common to all CE/EC systems and no t just to those intended for carbohydrate detection, they will be described only briefl y here . The need to isolate, or `decouple', the CE and EC systems arises from the fact tha t CE separation voltages are typically 5–30 kV and generate 11A-level electrophoresi s currents as a background . This is in contrast to typical EC detection potentials of 1 V or less and currents in the nano- to pico-amp range . Thus, for practically useful CE/E C operation, it is imperative that electrical overlap of the two systems is minimized . Suc h electrical decoupling can be carried out by either of two general approaches . In th e first, decoupling is accomplished by creating a fracture in the capillary wall 1 cm o r so before the outlet end [37] . The fracture is typically covered with a porous coatin g such as Nafion in order to inhibit solution flow and stabilize the capillary and is the n immersed in an electrolyte solution that contains the high voltage CE electrode . The physical end of the capillary is immersed in a separate solution that contains the E C detection electrodes . With this arrangement (which is termed `off-column' detection) , the CE voltage and current are dropped across the capillary only up to the point of th e fracture and should have little or no interaction with the downstream EC system . In th e alternative decoupling approach (which is referred to as `end-column' detection), th e EC electrodes are simply placed at the physical end of the capillary in the same buffe r reservoir as the CE electrodes . No special isolation procedures are employed except to insure that only relatively narrow capillaries, 25 p,m or less in diameter, are used . In these cases, the ohmic resistance of the capillary is sufficiently high that the CE curren t is low in magnitude and nearly all of the CE voltage is dropped across the capillary itsel f [38,39] . Both the off-column and end-column CE/EC configurations are illustrated i n Fig . 26 .1 . The need to insure accurate capillary/electrode alignment arises from the fact tha t both the CE capillaries and the EC electrodes typically used have diameters from 5 to 100 Rm . Optimum sensitivity requires that the electrode is positioned very clos e to the capillary outlet from the outset of the analysis, and optimum reproducibilit y requires these positions are maintained throughout the entire analysis process . In th e earliest CE/EC designs, proper electrode placement was carried out by attaching th e EC microelectrode, in the form of a wire or fiber, to an x,y,z-micropositioner and the n moving it as close as possible to, and perhaps inside, the capillary tip . This is referred to as an `in-capillary' detector configuration [37,40] . Alternatively, the EC electrode can take a flat, disk-shaped form and then simply be pushed up against the capillary tip s o that exiting solution flows onto and then radially out across its surface . Because such a `wall jet' electrode is physically much larger and can be seen and worked with fairl y easily, optimum placement is far easier to establish initially and subsequently maintai n over an extended series of CE/EC experiments [41] . This advantage can be enhanced References pp. 958—959



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Fig . 26.1 . Off-column (a) and end-column (b) CEEC configurations . W, R, and A represent working , reference, and auxiliary electrodes, respectively.

further by fixing both the capillary and the wall-jet electrode in a mechanical support that serves to hold both in place more rigidly [42] . Finally, `on-capillary' electrode s in which the capillary and the EC electrode are incorporated into a single integrate d unit have also been described . This can be accomplished by gluing a wire electrode onto the capillary tip [43] or by sputter-coating the capillary tip with a thin layer of the desired electrode material [44] . As a result, the position of capillary and electrod e is permanently fixed ; and alignment is no longer an issue . Rather, the integrate d capillary/electrode unit is simply immersed in the terminating CE buffer reservoir at th e start of the CE/EC experiment . As a result of these instrumental developments, both the convenience and th e performance of CE/EC have been enhanced considerably . For example, both the wall jet and the on-capillary configurations have shown peak height variations of less tha n 2% for repeated injections with the same capillary—electrode arrangement and of les s than 5% for different electrodes or capillary—electrode alignments . Typically, the same electrodes can be used for continuous day-long experiments and for discontinuou s experiments spanning several weeks . Although this may not always be the case, on capillary electrodes have sometimes proven to be more durable than the capillar y itself.



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26 .3. APPLICATION S To date, nearly every variety of carbohydrate compound has been detected unde r constant potential conditions at one or more of the transition metal electrodes describe d above . A representative listing of these applications is given in Table 26 .1 . No attempt was made here to provide a comprehensive compilation, but rather the listing highlights those reports that seem either to possess a historical significance to the field or t o address particularly unusual or important carbohydrate analytes . Because the focu s here is especially on the EC detection step rather than the separation process, example s involving flow-injection analysis (i .e ., with no formal separation process) are included i n addition to those where the EC detection is coupled to HPLC or CE . In the former cases , the detection could clearly be coupled to either or both of these separation systems, i f desired . Among the carbohydrate compounds successfully detected and quantitated at constant potential electrodes have been not only simple mono- and oligosaccharides but als o the following related species : alditols, aldonic, aldaric, and uronic acids, aminosugars , nucleosides, aminoglycosides, and cyclodextrins . Fig . 26 .2 illustrates a typical separation of several such compounds from the glucose and galactose families via HPL C on a commercially available high pH anion-exchange column and with detection at a Cu-based electrode [511 . The samples included all the alditol and acidic sugar derivatives for each family, and the elution order observed was exactly that expected on th e basis of the compounds' pK a values and their resulting ionic character in the 0 .13 M NaOH/0.02 M Na 2 SO 4 mobile phase employed . This particular system was intended t o showcase the separation of analytes with very different charges, ranging here from th e nearly uncharged glucitol and galactitol to the di-anionic glucaric and galactaric acids . In fact, a more challenging problem is presented by the separation of a group of mor e closely related carbohydrate compounds e .g ., a series of alditols . Such a separation , achieved here by CE with EC detection again at a Cu electrode, is illustrated in Fig . 26 . 3 [35] . The electropherograms shown here were obtained for a series of eight differen t alditols at four different NaOH concentrations . In all cases, the pH was sufficiently hig h for the operation of the Cu electrode ; but only under the most alkaline conditions were the alditols (pKa = 13—44) sufficiently deprotonated to permit their separation by CE . An obvious point of interest is the question of whether detectability is limited t o relatively low molecular weight species, which certainly represent the compounds tha t have been the most frequently studied, or can be extended to larger polysaccharides a s well . On this issue, there have been two specific studies reported to date . Zhou an d Baldwin used CE with EC detection at Cu electrodes to investigate some commerciall y available polysaccharide samples [36] . These included enzymatically hydrolyzed starc h with polysaccharide fragments containing 10—15 glucose units and dextran sample s with nominal molecular mass as high as 18,300 Da (or containing approximately 10 0 monosaccharide units) . Also, Lewinski et al . reported the detection of heparin, a highl y sulfated glucosamine/glucuronic acid polysaccharide with a molecular mass in th e 15,000-Da range, via flow-injection analysis at both Ru oxide and Cu oxide electrode s [29] . Because the separation difficulties encountered with samples as complex as thes e and the lack of available standards, it was not possible in either of these studies t o References pp. 958—959

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Fig . 26 .2 . Chromatograms of glucose and galactose families . Stationary phase, Dionex Carbopac PA 1 column ; mobile phase, 0 .020 M Na2 SO4 /0 .134 M NaOH ; working electrode, Cu CME at +0 .50 V vs . Ag/AgCI . Labeled peaks correspond to glucitol (A), glucose (B), gluconic acid (C, 8 x 10 -6 M), glucuroni c acid (D), glucaric acid (E), galactitol (F), galactose (G), galactonic acid (H, 2 .5 x 10 -5 M), galacturonic aci d (I), and galactaric acid (J) ; all concentrations were 4 x 10 M unless indicated otherwise .

evaluate the electrodes' response for specific polysaccharides . However, it is clear that EC at transition metal electrodes offers a viable detection approach for carbohydrate s well above the oligosaccharide level .

26 .4. CONCLUSIONS In assessing the present state and future possibilities of constant potential E C detection of carbohydrates, it seems beneficial to take two different points of reference . First, one can try to compare the strengths and weaknesses of the approach vs . its mos t References pp. 958—959



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Electrochemical Detection of Carbohydrates at Constant Potential

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immediate alternative, pulsed EC detection at Au or Pt . Alternatively, one might try to evaluate EC detection in general vs . other usually spectroscopy-based carbohydrate detection strategies . In what follows below, I will try to address both of these questions directly.

26 .4 .1 . Constant potential vs . pulsed detection EC detection of carbohydrates using a pulsed potential format at Au and Pt electrodes represents a firmly established analysis approach . Thanks largely to the persistent effort s of Dennis Johnson's group at Iowa State University, the electrode processes involve d in PED are well characterized ; the instrumentation needed for PED, at least for HPL C applications, can be purchased commercially ; and PED's ability to provide sensitive an d relatively stable analytical performance has been amply demonstrated . By comparison , constant potential detection at transition metal electrodes offers possible advantages i n two respects . First, the instrumentation required for constant potential detection is b y nature simpler than that required for PED . Second, the constant potential detection mod e should, in principle, provide better detectability and lower detection limits because, al l other things being equal, noise (especially in the form of charging current) should b e minimized when the applied potential is invariant . In practice, however, these advantages have not proven to be decisive factors to users of the EC technique . Even a cursory scan of the relevant literature shows that there have been over the years, and continue to be today, many more reported applications of PE D for carbohydrate detection than of the constant potential approach . This preference fo r PED is especially apparent for HPLC applications where pre-packaged PED instrumentation is already available and many separation and detection protocols have alread y been documented . This preference, based largely on convenience, holds much les s for CE-based applications where EC instrumentation of both types must normally b e assembled in-house and there are very few longstanding and well-established analytica l procedures . Interestingly, Casella et al . have recently compared the quantitative detectio n capabilities of PED and constant potential detection at Au/Cu [49] and Au/Ni [501 composite electrodes that are capable of supporting both detection modes and have foun d that the latter gives consistently lower detection limits and wider linear ranges in HPLC , usually by a factor of 5-10 . Nevertheless, this modest gain in detectability with constan t potential detection has not been sufficiently important to remove PED from its dominan t position in HPLC studies . It may be expected, however, that the constant potential ap proach may find itself better able to compete with PED in CE applications and espe cially those involving new lab-on-a-chip instrumentation where issues of instrumen t simplicity and ruggedness may turn out to be more important than they are in HPLC .

26 .4 .2. EC detection in general It must be generally admitted that EC-based detection methods have usually bee n slow to be adopted fully by the non-electrochemical community when more familia r References pp . 958—959



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Chapter 2 6

optical detection methods are available as an alternative . To a large extent, this has been true for carbohydrates for which analyses involving complicated and time-consumin g derivatization with strongly absorbing or fluorescing functional groups continue to b e widely used despite the fact that EC techniques normally allow direct detection withou t the need to carry out such operations . In the future, two factors may act in the favo r of the EC approach . Certainly, the accessibility of EC methods has increased to th e extent that a highly specialized background in electrochemistry is no longer required fo r their implementation . Furthermore, the ease of miniaturization of EC electrodes and th e simplicity and affordability of the associated EC instrumentation make this approac h ever more attractive for CE applications than for HPLC . For the development of full y microfabricated instrumentation, these aspects of EC detection should be even mor e appealing . Thus, for these newer and still emerging separation methodologies, it shoul d be expected that interest in EC detection in both the pulsed and the constant potentia l formats will increase and intensify in the years to come .

26.5. ACKNOWLEDGEMENTS This work was supported by a grant from the U .S . National Science Foundatio n XYZ-on-a-Chip Program .

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95 9

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