Anal. Chem. 1998, 70, 1496-1501
Improved Long-Term Reproducibility for Pulsed Amperometric Detection of Carbohydrates via a New Quadruple-Potential Waveform Roy D. Rocklin,* Alan P. Clarke, and Michael Weitzhandler
Dionex Corporation, 1228 Titan Way, Sunnyvale, California 94088-3603
A new quadruple-potential waveform is introduced for detection of carbohydrates using pulsed amperometry. The new waveform cleans the electrode by application of a potential more negative than the potential limit. In contrast to a commonly used triple-potential waveform, negative cleaning allows the time during which gold oxide is formed to be minimized, thus minimizing the dissolution and resulting recession of the gold working electrode as a result of gold oxide formation/reduction cycles. Preventing gold electrode recession is shown to improve long-term reproducibility. Waveform parameters were chosen to maximize signal-to-noise ratio and freedom from electrode fouling caused by matrix components in the sample. Compared to the triple-potential waveform, the quadruple-potential waveform shows similar minimum detection limits but greatly improved long-term reproducibility. Pulsed amperometric detection is commonly combined with anion-exchange chromatography to provide a sensitive and selective means of determining carbohydrates and other oxidizable species such as amines and sulfur compounds.1,2 Since introduction of this technique in the early 1980s, it has become a popular method for determining carbohydrates in samples ranging from foods and beverages to the carbohydrate portion of glycoproteins. Pulsed amperometric detection works by applying a repeating potential vs time waveform to the working electrode in a flowthrough detector cell. The waveform period is typically from 0.5 to 2 s. Analyte molecules are oxidized at the working electrode, and current from this oxidation is measured during each period. A waveform3 in common use for the detection of carbohydrates at a gold working electrode is shown in Figure 1A (this triplepotential waveform will be referred to here as the standard waveform). The first portion of the waveform is at potential E1 and is the potential at which analyte oxidation current is measured. As shown in the cyclic voltammetry in Figure 2, E1 is chosen such that carbohydrates can be detected at a potential with low
Figure 1. Standard (A) and quadruple-potential (B) waveforms for pulsed amperometric detection of carbohydrates. Detector response is the charge (in coulombs) from integration of the carbohydrate oxidation current between 0.2 and 0.4 s.
* To whom correspondence should be addressed. E-mail: Roy_Rocklin@ Dionex.com. Fax: 408-732-2007. (1) Johnson, D. C.; Dobberpuhl, D.; Roberts, R.; Vandeberg, P. J. Chromatogr. 1993, 640, 79-96. (2) LaCourse, W. R. Pulsed Electrochemical Detection in High-Performance Liquid Chromatography; Wiley: New York, 1997. (3) Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using Dionex Pulsed Electrochemical and Amperometric Detectors; Technical Note 21; Dionex Corp.: Sunnyvale, CA, 1996.
background current. Following a delay during which charging current resulting from the step from the final potential back to the initial potential decays, analyte oxidation current is measured by integration for a fixed duration, in this case 200 ms. The detection portion is followed by steps to potentials more positive (E2) and more negative (E3) than the detection potential. The purpose of these steps is to clean the electrode and maintain a catalytically active surface. Pulsed amperometric detection does not measure an intrinsic quantity of the analyte, as, for example, in UV absorbance detection. Instead, the measured charge (in coulombs) resulting from integration of analyte oxidation current is proportional to the rate of the oxidation reaction. Therefore, reproducible response is obtained by maintaining constant the variables that govern the oxidation reaction rate. One of these variables is the rate of analyte transport to the surface of the working electrode, for which one of the controlling factors is the dimensions of the
1496 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
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Figure 2. Rotated disk cyclic voltammetry of 0.5 mM glucose at a gold working electrode in degassed 100 mM sodium hydroxide.
electrochemical detector cell. Studies have shown that current in a thin-layer type amperometric cell is inversely proportional to thin-layer channel thickness raised to the power of two-thirds.4,5 An increase in thin-layer channel thickness will both lower the linear velocity of flow over the surface of the working electrode and broaden the parabolic laminar flow profile. Both effects will cause a decrease in cell current. In this publication, we show that gold from the surface of the working electrode is slowly lost as a result of the use of the 200ms positive cleaning potential (E2) in the waveform shown in Figure 1A. As gold is removed from the surface of the electrode, the electrode becomes recessed below its original position (Figure 3). Over time, this causes an increase in thin-layer channel thickness, a decrease in the velocity of fluid flow over the electrode surface, and a decrease in detector response to a given concentration of analyte. Dissolution of gold from the electrode and the resulting decrease in detector response can be greatly minimized by avoiding the application of high positive potentials for long times. However, it is this removal of the electrode surface that is responsible for maintaining a clean electrode. In previous studies in which modifications to the triple-potential waveform were presented, electrode cleaning at positive potentials was used.6-8 Recently, Jensen and Johnson9 showed that products from the oxidation of glucose could be cleaned off of the electrode at high negative potentials rather than at high positive potentials. We used this concept to develop a new four-potential waveform for carbohydrate detection (Figure 1B) that maintains a constant response by greatly minimizing dissolution and recession of the electrode, thereby improving the long-term reproducibility of carbohydrate peak area measurements. Potentials and their durations in the waveform were chosen to maximize signal-tonoise ratio and freedom from electrode fouling caused by matrix components in the sample. (4) Elbicki, J. M.; Morgan, D. M.; Weber, S. G. Anal. Chem. 1984, 56, 978985. (5) Rocklin, R. D.; Tullsen, T. R.; Marucco, M. G. J. Chromatogr. A 1994, 671, 109-114. (6) Andrews, R. W.; King, R. M. Anal. Chem. 1990, 62, 2130-2134. (7) LaCourse, W. R.; Johnson, D. C. Carbohydr. Res. 1991, 215, 159-178. (8) LaCourse, W. R.; Johnson, D. C. Anal. Chem. 1993, 65, 50-55. (9) Jensen, M. B.; Johnson, D. C. Anal. Chem. 1997, 69, 1776-1781.
Figure 3. ED40 thin-layer channel schematic diagram. Recession of the working electrode increases the volume between the working electrode and the counter electrode, decreasing the linear velocity of flow over the surface of the working electrode. This causes decreased transport of analyte to the electrode surface and decreased detector response.
EXPERIMENTAL SECTION All experiments were performed using Dionex DX-500 liquid chromatographs. The systems consisted of a GP40 gradient pump with on-line degas, an LC30 chromatography oven with the temperature set to 30 °C, and an ED40 electrochemical detector. The ED40 detector uses a thin-layer type amperometric cell.5 The working electrode is made from a 1-mm-diameter gold rod, forcefit into a Kel-F plastic block, and polished to a mirror finish with 1-µm alumina. Working electrodes with significant recession were sanded flat prior to polishing with No. 600 silicon carbide sandpaper. The counter electrode is the titanium cell body across the 25-µm thin-layer channel from the working electrode. An Ag/ AgCl reference electrode is downstream from the thin-layer channel. Working electrode recession was measured by viewing the electrode with a microscope and comparing the depth of recession to the thickness of wires of known diameter. For some experiments, a 5-Hz activation waveform was used for 1 h to cause the electrode to reach stable response more rapidly after polishing. This waveform used potentials and times of +0.1 V, 100 ms; +0.75 V, 30 ms; and -0.15 V, 70 ms. Chromatography of glucose and sucrose was performed on a CarboPac PA1 column (4 mm × 250 mm) with 100 mM sodium hydroxide at a flow rate of 1 mL/min. Monosaccharides were separated at 1.5 mL/min using a CarboPac PA10 column (4 mm × 250 mm) and an AminoTrap column (4 mm × 50 mm) to minimize electrode fouling from amino acids.10 For monosaccharide chromatography,11 separation using 18 mM sodium hydroxide was followed by a rinse for 7 min with 200 mM sodium hydroxide, followed by a 21 min equilibration with 18 mM sodium hydroxide. Total run time was 40 min. For sialic acid (10) Weitzhandler, M.; Pohl, C.; Rohrer, J.; Narayanan, L.; Slingsby, R.; Avdalovic, N. Anal. Biochem. 1996, 241, 128-134. (11) Glycoprotein Monosaccharide Analysis Using High-Performance AnionExchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD); Technical Note 40; Dionex Corp.: Sunnyvale, CA, 1997.
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Table 1. Percent Change in Peak Area over 14-Day Periodsa monosaccharides wave form
Fuc
GalN
Gal
Man
NANA
NGNA
quadruple potential standard
-10 -29
+2 -33
-11 -28
-8 -27
+2.4 -22
-6.6 -32
a
Figure 4. Peak area from repeat injections of 100 pmol of glucose using the quadruple-potential and standard waveforms. Injections were made every hour for the standard waveform and every 2 h for the quadruple-potential waveform. The quadruple-potential waveform used was as shown in Figure 1, except that the negative cleaning potential (E2) was -1.5 V.
chromatography,12 constant 100 mM sodium hydroxide was used with a gradient from 70 to 300 mM sodium acetate over 10 min, followed by reequilibration at 70 mM for 16 min. A CarboPac PA10 column was used at 1 mL/min. New columns as well as those which had not been used for a week or more were rinsed with mobile phase overnight prior to use. Mobile phases were prepared from 18-MΩ deionized water and 50% sodium hydroxide (Fisher) and were maintained under helium to prevent contamination from atmospheric carbon dioxide. Carbohydrate samples were stabilized with 20 ppm sodium azide to prevent bacterial growth. All chromatography data acquisition and instrument control were performed using Dionex PeakNet software. Detector noise was measured peak-to-peak over a 2-min period of flat baseline. Noise is dependent on many factors, such as the age of the working electrode and the stability of pump flow. It is typical for noise measurements to vary by a factor of 2 for different, properly working systems. Comparison of noise measurements between two sets of conditions can only be accomplished if there is little time between experiments. Rotated ring-disk voltammetry was performed using a Pine Instruments model AFCBP1 bipotentiostat to control the ring and disk electrodes. Electrode rotation (900 rpm) was controlled using a Pine Instruments analytical rotator, model AFMSRX. The disk electrode has a diameter of 0.457 cm. The detection waveform to the ring electrode was generated using LabVIEW software. An AT-MIO-16X I/O board (National Instruments) in a PC was used for data acquisition. RESULTS AND DISCUSSION Electrode Recession. Peak area trends for glucose measured during repeat injections over 2-week periods using the standard waveform and the new quadruple-potential waveform are shown in Figure 4. After an initial increase in response over about 8 h, peak area using the standard waveform decreased approximately exponentially over 14 days, while response with the quadruplepotential waveform remained relatively constant. (The initial response increase was avoided with the quadruple-potential waveform experiment by applying an activation waveform after polishing the electrode; see Experimental Section.) At the conclusion of the 2-week test periods, the working electrode was (12) Rohrer, J.; Thayer, J.; Weitzhandler, M.; Ardalovic, N. Glycobiology 1998, 8, 35-43.
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sialic acids
See Experimental Section for method details.
recessed approximately 45 µm when the standard waveform was used but remained flush with the surface of the surrounding plastic block when the quadruple-potential waveform was used. With the standard waveform, the addition of 45 µm to the initial thin-layer channel thickness of 25 µm increased the thin-layer channel thickness to 70 µm. This caused a 45% decrease in measured peak area for glucose from the initial value (excluding the rise at the beginning by extrapolating to zero time). The magnitude of response decrease is in agreement with the predicted decrease based on the cell current being inversely proportional to thin-layer channel thickness to the two-thirds power, which would predict a decrease of about 50%. Two-week runs comparing the quadruple-potential waveform to the standard waveform were also performed using conditions developed for the separation of glycoprotein monosaccharides11 and for the sialic acids N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA).12 The results (summarized in Table 1) are similar to those observed for glucose, with response using the standard waveform declining much more than that observed using the quadruple-potential waveform. After 2 weeks, during which peak areas for four monosaccharides were measured, there was no electrode recession when the quadruplepotential waveform was used, but the electrode was recessed approximately 35 µm when the standard waveform was used. Although there was some decline in response for the neutral sugars with the quadruple-potential waveform, most of the change was within the first 5 days. The change in response during the next 9 days was about 1%. During the 151 injections over the 9-day period, peak area RSDs were 0.9% for fucose, 0.5% for galactosamine, 0.8% for galactose, and 1.0% for mannose. These results show that, although there is still some peak area change following electrode polishing, detector response reaches a steady state, during which there is little or no electrode recession and virtually constant detector response. Following completion of 2-week runs of sialic acids, the working electrode was recessed approximately 45 µm using the standard waveform but was flat and unrecessed when the quadruple-potential waveform was used. Peak areas changed little during the quadruple-potential waveform runs but declined 22% (NANA) and 32% (NGNA) when the standard waveform was used. The cause of electrode recession is dissolution of gold from the electrode surface. Rotated ring-disk voltammetry was used to determine the conditions which cause this dissolution. Reaction products formed at the center disk electrode are swept to the surrounding ring electrode and are detected. Our experimental setup is similar to that outlined by Jensen and Johnson.9 A triangle wave is applied to the gold disk electrode, while a pulsed amperometric detection waveform (similar to that in Figure 1A)
Figure 5. Rotated ring-disk voltammetry at 900 rpm in 100 mM sodium hydroxide at gold electrodes. Both disk and ring currents are plotted as a function of disk potential. Gold oxide is formed at the disk at region a. This releases a small amount of reducible gold species which is reduced at the ring in region b. Reduction of gold oxide at the disk in region c produces a much higher concentration of reducible gold, which is reduced at the ring in region d. A pulsed amperometry waveform similar to the standard waveform was used to detect gold at the ring. Potentials used were E1 ) 0 V, E2 ) +0.8 V, and E3 ) -1.2 V.
is applied to the gold ring electrode. Both current at the disk and detector response measured at the ring are plotted as a function of potential applied to the disk. By a suitable choice of E1 in the detection waveform applied to the ring, species released from the disk during oxide formation and reduction can be detected at the ring. In this way, the potential dependence of gold dissolution at the disk electrode was assessed. Cyclic voltammetry for the disk electrode in 100 mM sodium hydroxide and detector response at the ring, both measured as a function of the disk electrode potential, are plotted in Figure 5. The value for E1 at the ring electrode was 0 V. During the positive-going potential scan, gold oxide formation at the disk commences at approximately +0.20 V and continues until the potential scan is reversed at +0.60 V (trace a in Figure 5). While this oxide is being formed, a small cathodic current is measured at the ring (b). This indicates that, while gold oxide is formed at the disk, a small amount of a reducible gold species is dissolved from the disk and can be detected at the ring. During a similar experiment, in which the detection potential at the ring was set to +0.3 V, no appreciable anodic current was measured at the ring (data not shown). Therefore, the gold species being dissolved at the disk should be mostly Au(III) rather than a form of gold that can be further oxidized. During the negative-going sweep at the disk, a large cathodic current is observed at the ring with a peak at approximately +0.10 V (Figure 5d). This corresponds to reduction of the gold oxide layer on the disk back to elemental gold (c). This is indicative of a large amount of gold being dissolved from the disk electrode into solution, which is then detected by reduction at the ring. When E1 at the ring electrode is set to +0.30 V, an anodic current is measured at the ring electrode during the disk oxide reduction (data not shown). This indicates that both Au(I) and Au(III) species are being dissolved off the disk. From the relative magnitudes of the peak currents at the ring electrode, it is clear that most of the gold dissolution at the disk electrode occurs during reduction of the gold oxide layer. To avoid excessive
electrode dissolution and subsequent diminution of signal in the pulsed amperometric detection of carbohydrates, the extent of gold oxide formation should be minimized. This is achieved by the quadruple-potential waveform. Waveform Optimization. Detection Potential (E1). Each of the four potentials shown in Figure 1B performs a different function. The first potential (E1) is the detection potential at which the current from carbohydrate oxidation is integrated. As can be seen in the cyclic voltammetry shown in Figure 2, the peak current for glucose is at 0.23 V. Using pulsed amperometric detection, graphs of peak height as a function of detection potential show that the maximum response for most carbohydrates is from about 0.1 to 0.2 V. A value of 0.1 V was selected for E1. An advantage to detection at this potential is that amines contained in samples are detected at potentials above 0.15 V and are prevented from interfering. Also, monosaccharide peaks begin to tail as the potential is raised to values which form surface oxide. The tail is barely noticeable at 0.15 V and becomes more prominent as the potential is increased. The cause of the tail is not known. There is no one optimum value for the duration during which carbohydrate oxidation current is integrated. Detector response is directly proportional to the integration period, while noise was found to be independent of the integration period. Therefore, increasing the integration period produces a proportional increase in signal-to-noise ratio. However, a total waveform period that is too long and the resulting decrease in the data acquisition rate will degrade reproducibility for high-efficiency peaks. The recommended duration of 200 ms is a compromise between obtaining a good signal-to-noise ratio and maintaining a short (0.5 s) total waveform period. The same consideration holds for the delay time prior to the integration period. A long delay time decreases noise and has only a minor effect on signal. Using 18 mM sodium hydroxide, noise was measured at 30 pC with a 50-ms delay time and 18 pC with a 200-ms delay time; 200 ms is recommended for columns of normal efficiency. As these noise measurements show, a shorter delay time and higher data acquisition rate can be used for high-efficiency separations with only a modest increase in noise. Cleaning Potential (E2). With the standard three-potential waveform, the working electrode is cleaned by removal of gold from the electrode following each repetition of the positive potential. The cleaning function is accomplished in the quadruplepotential waveform at high negative instead of high positive potential. A plot of baseline noise and detector response for monosaccharides as a function of the negative cleaning potential is shown in Figure 6. The results show that noise increases as the potential is made more negative and that detector response plateaus at approximately -1.5 V. To obtain the maximum signal, it is necessary to exceed the negative potential limit (see Figure 2). Similar results were obtained for glucose and sucrose using 100 mM sodium hydroxide mobile phase (results not shown). It would appear that the optimum potential is -1.5 V, because this potential is effective at removing adsorbed sugar oxidation products. Previous studies have shown that amines from samples can cause electrode fouling.10 Amino acids and small peptides can elute from the column and adsorb on the working electrode. This causes a reduction in carbohydrate response until the amines Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
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Figure 6. Peak areas for four monosaccharides and baseline noise plotted as a function of the negative cleaning potential, E2. Five injections were made at each potential.
Figure 7. Effect of matrix components on electrode poisoning during detection of monosaccharides from 2.18 µg of hydrolyzed fetuin using the quadruple-potential waveform with E2 set to -2.0 (A) and -1.5 V (B). Peak area from five consecutive injections is plotted as the percent of peak area from the first injection. The more negative potential is more effective at removing adsorbed matrix components and preventing them from fouling the electrode.
are removed by the potential pulses. The AminoTrap column causes most peptides and all amino acids except arginine to elute after the monosaccharides, so electrode poisoning is the result of amino acids being adsorbed and not completely desorbed from the working electrode during previous runs. Repeated injections of a fetuin hydrolysate showed that -1.5 V was not as effective at removing matrix components from the electrode as was -2 V (compare parts A and B of Figure 7). A similar study using the standard waveform showed that it is also effective at maintaining constant detector response for successive injections of the fetuin hydrolysate. The mechanism of cleaning at negative potential could be displacement of adsorbed molecules on the electrode surface by hydrogen atoms produced from the reduction of water. Oxidation of these adsorbed hydrogen atoms at the detection potential is 1500 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
the likely cause of noise observed as the negative cleaning time is increased or the potential is made more negative. Adding an extra 10 ms to the negative cleaning potential duration increased baseline noise by approximately 50%. The recommended 10-ms hold at -2 V is selected to accomplish effective cleaning without excessive noise. For clean samples that do not contain components which foul the working electrode, a potential of -1.5 V produces near maximum response with less noise. Conversely, samples containing high ratios of amines to carbohydrates may require considerable electrode cleaning. Improved electrode cleaning can be accomplished by increasing the time during which the negative cleaning potential is applied, but at the cost of increasing noise. Activation Potential (E3). The purpose of the third potential at +0.6 V is to maintain an active electrode. Catalytic sites on the gold electrode are thought to be low-coordination gold atoms such as those at grain boundaries, those at steps in the gold crystal plain, or adatoms.13,14 It is believed that these catalytic sites are created by forming and then reducing surface oxide.13 We have found that omission of this transient oxide formation step results in rapidly decreasing detector response. A possible explanation for this is the elimination of active sites during exhaustive reduction of the gold surface via a reconstruction type mechanism.15 Measurement of response as a function of E3 showed response to be largely independent of potential from +0.4 to +0.9 V. The effect of using different potentials showed up in longterm studies in which repeat injections of glucose and sucrose were made for a period of 2 weeks. When +0.4 V was used, response was constant for 10 days and then fell off rapidly, although there was no electrode recession. Presumably, the rate of formation of catalytic sites was not sufficient to maintain electrode activity. When +0.6 V was used, response was constant over at least 14 days, as shown in Figure 4. In a 2-week experiment in which +0.75 V was used, response was also constant, but the electrode showed slight recession at the edges. To avoid electrode recession, this positive activation potential is applied for the shortest possible time. As shown in Figure 1B, potential is ramped up to +0.6 V and then down to -0.1 V, without holding at +0.6 V. When the potential was, instead, held at +0.6 V for 10 ms, the electrode was recessed up to 20 µm (mostly around the edges) after 2 weeks, and there was a noticeable decrease in response of glucose and sucrose after 6 days. Ramping up to +0.6 V and then down to -0.1 V without holding at +0.6 V maintained a catalytically active electrode and prevented recession. Oxide Reduction Potential (E4). The purpose of the fourth potential at -0.1 V is to reduce oxide formed at the positive activation potential. It is this formation and then reduction of gold oxide which is responsible for the creation of catalytic sites on the electrode surface. This potential was chosen to be negative of the gold oxide reduction wave, but not so negative as to reduce dissolved oxygen. The reduction of oxygen from the sample at E4 is responsible for the baseline dip observed at approximately 14 min (CarboPac PA1, 1 mL/min). Avoidance of an excessively (13) Johnson, D. C.; LaCourse, W. R. In Carbohydrate Analysis: High Performance Liquid Chromatography and Capillary Electrophoresis; El Rassi, Z., Ed.; Elesevier Science: Amsterdam, 1994; Chapter 10. (14) Burke, L. D.; Buckley, D. T.; Morrissey, J. A. Analyst 1994, 119, 841-845. (15) Hamelin, A. Electroanal. Chem. 1995, 386, 1-10.
Table 2. Performance Comparison for Monosaccharides:a Quadruple-Potential and Standard Waveforms
response ratios at 1 nmol, QP/std detection limit,c pmol, QP WF detection limit,c pmol, std WF
Fuc
2-dGlcb
GalN
GlcN
Gal
Glc
Man
1.34 0.25 0.25
1.57 0.31 0.38
1.08 0.14 0.11
1.04 0.19 0.15
1.30 0.24 0.23
1.21 0.24 0.22
1.62 0.33 0.40
a Method for determination of monosaccharides as described in the Experimental Section using 18 and 200 mM sodium hydroxide. b Used as an internal standard.11 c Three times noise level: 10.3 pC with the quadruple-potential waveform and 7.6 pC with the standard waveform.
Figure 8. Effect of sodium hydroxide mobile-phase concentration on detector response for 100 pmol of glucose and sucrose. Waveform potentials E1 and E4 were adjusted -0.06 V per increase of 1 pH unit from the quadruple-potential waveform shown in Figure 1B, which was used with 100 mM sodium hydroxide.
negative potential for E4 minimizes this dip and also improves baseline stability, since changes in mobile-phase oxygen concentration will have less effect on the baseline level. Reduction of gold oxide is relatively fast, so the time spent at this potential is not critical. The duration used was chosen to round out the waveform to 0.5 s. The potentials chosen for the quadruple-potential waveform were selected from experiments performed using 18 and 100 mM sodium hydroxide. However, the optimum potentials display approximately Nernstian behavior with changing pH as a result of the pH-dependent shift of gold oxide formation and reduction potentials.14,16 Therefore, the waveform potentials should be shifted -0.06 V for each increase of 1 pH unit that the mobile phase pH is changed from pH 13, and they should be shifted +0.06 V per pH unit decrease. The detection potential E1 and the oxide reduction potential E4 are the only potentials which need to be shifted, and only if the mobile phase is shifted by more than 1 pH unit. The cleaning potential E2 and the oxide formation potential E3 show little potential dependence, so there is no need to shift these potentials as pH is changed. A plot of detector response for glucose and sucrose as a function of mobile-phase pH using a -0.06 V per pH unit shift is shown in Figure 8. The maximum response is at a sodium hydroxide concentration of about 50 mM. Performance. A comparison of sensitivity and detection limits for monosaccharides using the two waveforms is shown in Table 2. The baseline noise level using the quadruple-potential waveform is not as low as that measured using the standard waveform. However, sensitivity is better for all monosaccharides. Minimum detection limits (3× noise level) are similar and are in the low (16) LaCourse, W. R.; Mead, D. A.; Johnson, D. C. Anal. Chem. 1990, 62, 220224.
Figure 9. Monosaccharides linearity, plotted as response factor (peak area divided by picomoles injected) vs picomoles injected. A horizontal line is a linear calibration. The linear range is 3 orders of magnitude from the minimum detection limits up to several hundred picomoles.
hundreds of femtomoles. Monosaccharide linearity, plotted as response factor vs the log of the quantity injected, is shown in Figure 9. The same measurements of linear range were performed using the standard waveform, and with similar results. A linear calibration produces a horizontal line on this type of plot. The linear range extends approximately 3 orders of magnitude from the minimum detection limit up to several hundred picomoles injected. A similar curve was obtained for glucose and sucrose using 100 mM sodium hydroxide mobile phase, but with the linear range extending up to 1 nmol for glucose. Quantities above 1 nmol should be determined using a multilevel calibration with a quadratic fit. Better yet, samples should be diluted to place the analyte concentrations within the linear range. CONCLUSION Compared to the standard waveform, use of the quadruplepotential waveform greatly improves long-term reproducibility for pulsed amperometric detection of carbohydrates. In addition to the advantage of improved reproducibility on a single chromatograph, minimizing electrode recession also minimizes the response differences between working electrodes. The quadruplepotential waveform should, therefore, be useful in quality control and in other situations where results on one chromatograph must be compared to results on another, or where changes in peak area over time must be monitored. ACKNOWLEDGMENT The authors thank Prof. Dennis Johnson for his helpful discussions and his insight into the mechanisms of electrode cleaning. Received for review August 19, 1997. Accepted January 29, 1998. AC970906W Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
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