Anal. Chem. 1994,66, 2115-2182
Nickel-Titanium Alloy Electrode as a Sensitive and Stable LCEC Detector for Carbohydrates Peffang F. Luo and Theodore Kuwana’ Center for Bioanalytical Research and Department of Chemistv, University of Kansas, La wrence, Kansas 66047-2535
A highly sensitive and stable amperometric detector for the liquid chromatographic (LC) analysis of carbohydrates has been developed on the basis of the use of a nickel-titanium (Ni-Ti) alloy electrode. Between an applied potential of +0.40 and +0.60 V vs Ag/AgCl, the Ni is electrochemicallyoxidized to the Ni(II1) state as the nickel oxyhydroxide. This Ni(II1) is believed to be the active form that oxidizes the carbohydrate in an “ec” catalytic sequence. This catalysis is similar to that proposed for a pure nickel electrode. Cyclic voltammetric studies with a pure titanium electrode indicated that the titanium did not participate in the carbohydrate oxidation. However, its presence greatly improves the reproducibilityand lengthens the useful lifetime of the Ni-Ti electrode in comparison to that of pure Ni. In particular, the Ni-Ti alloy with a 5545 (w/w) composition, which gives an intermetallic compound with a 1:l atomic ratio, was effective as an LC detector for carbohydrates in strongly alkaline media. For example, a 100 pmol sample of glucose injected repetitively over a 17 h period showed a relativestandard deviationof less than 1.5%. Linear responses covering the concentration range of 10-3-10-6M wereobtained for both reducing and nonreducing sugars. Glucose was detected at concentration levels as low as 5 X lo4 M (0.5 pmol). At an applied potential of +0.50 V vs a Ag/AgCl reference, the Ni-Ti electrode exhibited stability for over 40 days of use as an LC detector for the analysisof various carbohydrateswithout the need for any treatments to reactivate the electrode. The effect of experimental conditionssuch as the applied potential, mobile phase composition, and flow rate to the sensitivity and stability is discussed for this Ni-Ti electrode. Advances in understanding the biological function of carbohydrates have stimulated the need for highly precise and accurate analysis of carbohydrates. The development of high-performance liquid chromatography (HPLC) has provided an effective method for separating carbohydrates, thereby making it possible to assay complex biological samples. However, since carbohydrates exhibit only weak optical absorbance in the UV-visible range and refractive index methods are also rather limited in sensitivity, more sensitive methods of detection are desired. Detections based on electrochemical oxidation of carbohydrates have recently received considerable attention. For example, the amperometric detection with either Pt or Au electrodes has been studied.*.2 Additionally, instrumentation capable of implementing the pulse amperometric method using ~~
(1) Johnson, D. C.; Lacourse, W. R. Anal. Chem. 1990,62, 589A-597A. (2) Olechno, J. D.; Carter, S.R.; Edwards, W. T.;Gillin, D. G. Am. Lab. 1987,
Sept./Oct., 38-50, 0003-2700/94/0366-2775$04.50/0 0 1994 American Chemlcal Soclety
a preprogrammed potential waveform with current sampling to determine carbohydrates is commercially available. The method relies on the oxidation of carbohydrate on a clean Pt or Au surface. The Faradaic charge ascribed to the oxidation of the carbohydrate is determined from the difference between the Faradaic component and the capacitive dischargecurrents. Therefore, continuous on-line detection relies on the repetitive application of pulsed-potential waveform to renew the activity of the clean Pt or Au surface. Sensitivity is primarily determined by the precision achieved in discerningthe Faradaic component due to the carbohydrate oxidation from the background charge. LaCourse and Johnson3 have also indicated that the presence of organic modifier in the mobile phase, even for electroinactive organic additives, will suppress the electrode activity due to competitive and often preferential adsorption by organics onto the Pt or Au surface. This latter finding seriously limits the pulse amperometric method in applications involving reversed-phase or ion-pair chromatography. A preferred mode of operation for an electrochemical detector for liquid chromatography (LCEC) applications would be constant potential amperometry because of its instrumental simplicity and inherent sensitivity. The two most often mentioned electrodes for amperometry have been Cu and Ni,4-8 since they can catalyze the oxidation of carbohydrates in alkaline media. The evidence for such catalysis is clearly seen in the cyclic volt ammo gram^^ at Ni and Cu, when glucose, glycine, ethanol, etc. are present in 0.10 M NaOH solution. The anodic current at potentials around +OS0 V vs Ag/AgCl increase substantially above background, while at Pt and Au the anodic current corresponding to oxidation of such analytes is observed only prior to and concurrently with oxide formation, not concurrently and after oxide formation as with Cu and Ni. The above difference in the mode of carbohydrate oxidation could serve as a guide in the selection of metals and alloys for constant potential amperometry. Several studies on LCEC detection of carbohydrates, amino acids, and related compounds under constant potential conditions at Ni and Cu electrodes have been reported.&ll However, a slow decrease in the catalytic activity has been (3) Lacourse, W. R.; Johnson, D. C. Carbohydrate Res. 1991, 215, 159-178. (4) Prabhu, S.V.; Baldwin, R. P. Anal. Chem. 1989,61, 2258-2263. (5) Luo, P. F.; Zhang, F.; Baldwin, R. P. Anal. Chim. Acta 1991,244, 169-178. (6) Zadcii, J. M.; Marioli, J.; Kuwana, T. Anal. Chem. 1991, 63, 649-653. (7) Rcim, R. E.; Van Effen, R. M. Anal. Chem. 1986, 58, 3203-3207. (8) Schick, K. G.; Magcam, V. G.;Huber, C. 0.Clin. Chem. 1978,24,448-450. (9) Buchbergcr, W.; Winsaucr, K.; Breitwieser, Ch. Fresenius Z . Anal. Chem. 1983, 315, 518-520. (10) Goto, M.;Miyahara, H.; Ishii, D. J. Chromatogr. 1990, 515, 213-220.
Analytical Chemistty, Vol. 66,No. 17, September 1, 1994 2775
Table 1. Llsi ot Metals and Alloys Evaluated for Carbohydrate Detectlon
alloys metals
Cu-Ni
Cu-base
Ni-base
stainless steels/low Ni
cu Ni
Cu-Ni (55:45) Cu-Ni-Fe (33:65:2)
Cu-Zn (63:37) Cu-Zn (85:15) Cu-Mn-Ni (85:12:2) CU-W (35:65)
Ni-Cr (8020) Ni-Cr-Fe (72:16:8) Ni-Ti (55:45)
Ni-Cr-Fe (8:18:75) Ni-Cr-Mo-Fe (8:18:3:7 1)
observed with these two metals due to surface corrosion concurrently with the continual buildup of the surface oxide layer. Thus, our interest in the use of alloys of Cu and Ni was predicated by the fact that alloys are quite resistant to corrosion. These alloys, which have been examined to date, with their respective stoichiometric compositions are listed in Table 1. These alloys are Cu-Ni, Ni-Cu-Fe, Cu-Zn, CuMn-Ni, Cu-W, Ni-Ti, Ni-Cr, Ni-Cr-Fe, and Ni-Cr-Me Fe. Unfortunately, all of the Cu-containing alloys exhibited changes in activity, apparently due to the continuous loss of copper under the alkaline conditions required for the oxidation of carbohydrates. For Ni alloys, Ni-Cr and Ni-Ti exhibited the most promising properties of reproducibility in response to carbohydrates. A systematic study of theNi-Cr electrode has been reported recently by Marioli, Luo, and Kuwana.’* This electrode exhibited excellent sensitivity for the detection of sugars with a limit-of-detection (LOD), for example, of 0.5 pmol for glucose. In the search for an improvement to the long-term stability, a fundamental and application-oriented study was undertaken with the Ni-Ti alloy. Like Ni-Cr, Ni-Ti possesses the advantage of being highly resistant to corrosion while it retains the electrochemical characteristics of the base metal (e.g., nickel). The influence of the operational parameters on the analytical performance, such as in the case of Ni-Cr, will be examined with respect to Ni-Ti, so that questions related to the electrode’s reproducibility and long-term stability can be addressed. Additionally, the types of sugars amenable to analysis, the pretreatment protocols for rapid activation, and the use of an organic mobile phase will be examined.
EXPERIMENTAL SECTION Reagents. Carbohydrates and related compounds, purchased from either Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO), wereused as received without further purification. Stock solutions were prepared in NANOpure water (NANOpure System, Barnstead Co., Boston, MA) and brought to the desired concentration and pH by dilution with electrolyte for cyclic voltammetry (CV). For liquid chromatography the stock solutions were diluted with NANOpure water and immediately used. Sodium hydroxide solutions and other modified mobile phases of the desired concentrations were prepared with Aldrich electrolytic grade sodium hydroxide. Mobil phases were degassed and saturated with helium throughout the experiments. Electrodes. The metal and alloy wires were purchased from Goodfellow, Malvern, PA (Ni-Ti: NI205100 (55:45), 0.8mmdiameter. Ni: NI005171(99.98%), 1.Ommdiameter. (1 1) Casella, I. G.; Desimoni, E.; Cataldi, T. R. I. Anal. Chim. Acta 1991, 248, 117-125. (12) Marioli, J.; Luo, P. F.; Kuwana, T.Anal. Chim. Acta 1993, 282, 571-580.
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Ni-Cr: NI055150 (80:20), 1.O mm diameter). The working electrodes used for CV were made by embedding the above wires into Teflon shrinkable tubes and then heating at 300 OC in an oven for 2 min to seal the electrode in the tubing. The electrodes were polished first with emery sand papers in successiveorder of grit sizes 150,240,400,600,and 0000 and then with alumina powders of 1.0, 0.3, and 0.05 pm size on a fiber pad. Surfaces were examined with an optical microscope (45X) to determine the smoothness of the polished surfaces. Finally, the electrodes were washed thoroughly with distilled water, sonicated, and rinsed with NANOpure water before used. The Ni-Ti working electrode was made by embedding a 0.8 mm diameter Ni-Ti wire into a Kel-F block fitted to a Bioanalytical Systems (BAS, West Lafayette, IN) flow cell and polishing similarly to those for CV. Apparatus. CV experimentswere performed with a Cypress Systems (Lawrence, KS) Model CS 1090computer-controlled electroanalytical system, and results were recorded on a Hewlett-Packard ColorPro plotter or a Panasonic KX-P1080i graphics printer. A three-electrode electrochemical cell was used with an Ag/AgC1(3 M NaCl) reference and a platinum wire auxiliary electrode. Flow injection and LC experiments were carried out with a Shimadzu (Kyoto, Japan) chromatographic system, consisting of an SCL-6B system controller, an LC-600 liquid chromatograph pump, an SIL-6B auto injector, a CTO-6A column oven, an L-ECD-6A electrochemical detector, and a C-R4A or CR501 chromatopac data processor (recorder). A Dionex (Sunnyvale,CA) CarboPac PA1 (250 X 4 mm) column or a Wescan (Santa Clara, CA) Anion-R (250 X 4.1 mm) anion-exchange column was used for the LC separations.
RESULTS AND DISCUSSION Cyclic Voltammetry. Earlier studies by Marioli, Luo, and Kuwana12revealed that the alloys of Ni-Cr, Ni-Cr-Fe, and Ni-Cu exhibited improved long-term stability compared to pure Cu or Ni electrodesfor LCEC applications. An important finding was that these alloys apparently retained the electrocatalytic properties of the base metals of Cu and Ni. To take advantage of the stability offered by alloys and to search for the “best” electrodes with long-term high sensitivity and reproducibility, a wide range of Cu and Ni alloys were evaluated by means of CV in 0.1 M NaOH solution. The electrodes examined are listed in Table 1. Generally, the alloys examined can be classified into four groups: Cu-Ni, Ni-based, stainless steels with low Ni content, and Cu-based metals. Among them, the Cu-Ni and Cu-based alloys gave the highest sensitivity toward glucose oxidation, but the reproducibility was poor due to both the dissolution of copper and the buildup of oxide on the surface. The stainless steels exhibited large
1
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h
1
n
I
I
i I1
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-100
-3h
-sbo
-7do
-960
Potential (mVvs Ag/Agc1)
Flgure 1. Traces labeled a-d representingrespectivelythe 1st 2nd, 50th, and 100th cyclic voltammograms obtalned at a NI-Ti electrode In 0.1 M NaOH. Scan rate = 100 mV/s.
background currents, probably due to the anodic dissolution of iron and very little catalysis. Only the Ni-based alloys gave a relatively low background current and stable, catalytic response toward glucoseoxidation. In particular, the Ni-Ti alloy electrode gave a well-defined peak-shaped wave at the redox potential for Ni(I1) to Ni(II1) oxidation and an appreciable catalytic current with glucose. Also, the Ni-Ti alloy possessed mechanical elasticity and "shape-memory" properties that could prove advantageous in both the fabrication and mechanical stability of a LCEC detector. Cyclic voltammograms obtained at Ni-Ti in 0.1 M NaOH at a scan rate of 100 mV/s are shown in Figure 1. With proper electrode polishing and cleaning, an oxidative peak corresponding to initial formation of oxide (Ni(I1) and/or Ti(1V) oxide) at a potential of ca. -0.60 V was observed. As reported earlier for both the Ni and Ni-Cr electrodes,'* such a peak is only observed for the first cycle unless the potential is reversed prior to any additional oxidation of the electrode. The reproducibility of this peak depends on the previous history of the electrode, including any pretreatment protocols. It may appear as a wave with a sharp peak or as a broad peak that extends to higher potentials. When the potential is scanned more anodically beyond +O.O V, a wave due to the oxidation of Ni(I1) to Ni(II1) is observed with a peak potential of +0.47 V. Upon scan reversal at +0.60 V, a cathodic wave is seen with a peak potential of +0.41 V, thedifference between theanodic and cathodic peak potentials being 60 mV. On repetitive scanning, the wave ascribed to Ni(II)/Ni(III) increases in size and becomes sharper. Also, the charge under the cathodic wave approaches that of the anodic wave; the peak shape is that of a surfaceconfined redox reaction. The oxidative wave observed earlier at -0.60 Vis no longer seen after the first scan so that the base current between the potentials of -1.0 to +0.40 V is very low and flat. Depending on the electrode history and scan rate of potential, a smaller cathodic wave may appear as a shoulder to the main wave (see waves c and d, Figure 1). This shoulder is indicative of the presence of more than one oxide form of Ni(II1). It is more pronounced at pure Ni.
5004w300200500~300200500400300200
Potential (mV vs Ag/AgCI) Flgure 2. Cyclic voltammograms of glucose at NI-Ti, NI, and Ni-Cr electrodes in 0.1 M NaOH. Glucose concentration: 0 (fine traces) and 1 mM (bold traces). Scan rate = 50 mV/s.
The general features of the cyclic voltammograms for the Ni-Ti electrode appear similar to those of Ni and the other Ni-base alloys. There are, however, some differences that can be observed by comparing Ni-Ti to pure Ni and the alloy Ni-Cr. For example, the background current in 0.1 M NaOH for Ni-Ti is about half the magnitude observed for the other two electrodes (the diameter of the electrode for Ni-Ti was 0.8 mm and those for Ni and Ni-Cr were 1 mm). This difference is seen in the cyclicvoltammogramsshown in Figure 2 (fine CV traces are for backgrounds). The smaller background current density for Ni-Ti suggests that the amount of oxide formed is less on Ni-Ti than on pure Ni or Ni-Cr alloy. Thus, the thickness of the oxide layer must also be correspondingly less. In the presence of glucose all three electrodes exhibit CV traces (Figure 2, bold traces) that are characteristic of an "ec" catalytic mechanism. That is, the anodic current increases over background due to the catalytic oxidation of glucose by the electrogenerated Ni(II1). The amount of increase is largest, as expected, for pureNi. Further quantitation of the catalysis, such as determining the kinetic rates for glucose oxidation, was not undertaken since the extent of catalysis depended considerably on the past history of the electrodes. Whether or not Ti participates in the catalytic oxidation of the carbohydrates was examined by conducting CV experiments with a pure Ti electrode. The cyclic voltammograms for the Ist, 2nd, and 50th cycles and then in the presence of 1 mM glucose at Ti in 0.1 M NaOH are shown in Figure 3, traces a-d, respectively. Interestingly, a significant oxidative current was observed only in the first scan, starting at a potential of ca. -0.50 V. The current peaked at a potential of ca. -0.30 V and then, after a slight decrease, remained constant at ca. 80%ofthepeakcurrentvalueup to theswitching potential of +0.60 V. On the reverse scan in the cathodic direction, the current decreased rapidly to essentially the baseline. In subsequent scans, the current remained very low without any appearance of the oxidative wave that was seen on the first scan at Ep = -0.30 V. Curve d in Figure 3 is a subsequent CV trace that was run in the presence of 1 mM glucose. There is no evidence that glucose is being catalytically oxidized by Ti in curve d, although titanium oxide must have been formed on the surface during the first scan (Ep = -0.30 Analytical Chemistry, Vol. 66,No. 17, September
I, 1994
2777
-1
I
-3iL-.54 500
100
300
t
I -100
-300
-sal
-700
Potential (mV vs Ag/AgC)
-m
Figure 3, Cyclic voltammograms obtained at a TI electrode in 0.1 M NaOH. Scan rate = 100 mV/s. Traces labeled a-c represent the lst, 2nd, and 50th (plotted twice) cyclic voltammograms. Trace d Is the cyclic voltammogram with 1 mM glucose added and run immediately after the 50th cycle.
V). Curve c (without glucose), when replotted from the initial potential onto curve d (with glucose), superimposes exactly throughout the entire potential range scanned. Clearly, Ti does not catalytically oxidize glucose (nor any other carbohydrates tested). However, Ti appears to play an important role in keeping the nickel oxide surface from deteriorating in its catalytic activity, as we shall subsequently see.
Figure 4 shows cyclic voltammograms for several representative mono- and disaccharides (solid curves) in 0.1 M NaOH at a Ni-Ti electrode in comparison to the CV scans in only NaOH, taken prior to the addition of the analytes (dashed curves, changed slightly with the number of cycles scanned). The principal feature exhibited in the CV waves for these sugars is their similarity in terms of the current increase due to the "ec" catalytic mechanism of the Ni(II1) oxidizing the carbohydrates. The decrease in the magnitude of the current for the reverse cathodic wave, due to the reduction of Ni(II1) to the Ni(I1) oxide, is also consistent with such a mechanism. Some general conclusions can be drawn from a detailed comparison between the magnitude of the catalytic current and the molecular structure of the carbohydrates. First, the simple sugars exhibit higher currents than the larger oligomeric sugars. Second, when the degree of oligomerization is similar, the responses do not distinguish aldoses from ketoses nor hexoses from pentoses. The catalytic currents for glucose (hexaaldose), fructose (hexaketose), and ribose (pentaaldose) arequalitatively similar. Third, the extent of catalytic oxidation applies to both reducing (maltose and lactose) and nonreducing sugars (sucrose). Fourth, the deoxygenated and alkylated sugars exhibit smaller responses than their original sugars (not shown) due to the decrease in the number of available hydroxyl groups for forming metalsugar complexes. In summary, all the sugars appear to be electrocatalytically oxidized at the Ni-Ti alloy electrode in
PI-'.
/
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AH-
T 1
maltose
fructose
1UA
lactose
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1 200
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I
1
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Potential (mV vs A d A g C 1 ) Figure 4. Cycllc voltammograms of glucose, fructose, ribose, sucrose, maltose, and lactose (as marked) obtained at the NI-TI electrode in 0.1 M NaOH. Analyte concentration: 0 (dashed line) and 1 mM (solM line). Scan rate = 20 mV/s. 2778
Ana!bMa/Chemistry, Vol. 66, No. 17, September 1, 1994
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0 560 .600 600
a3 r-450 450 --
3-
.-
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300 v
P
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0
Time @ O W
Figure 8. LCEC response of glucose at the NI-TI electrode at the potentials of +0.46 V (I/), +0.48 V ('), +0.50 V (A),and +0.52 V ([XI), respectively,during the Initialp l o d beglnnhgwith freshly polished surfaces. Mobile phase = 0.1 M NaOH; flow rate = 0.5 mL/mln during experiments and 0.01 mL/min during overnight off perlods. T = 35 OC. Sample glucose injected contained 100 pmoi(l0 pL of 10 pM).
150 +j
63 0
360
380
400
4 5 0 420 440 460 480 500 520 540 Potential (mV vs Ag/AgCI) peakcurrent -+ background noise
-
5100 560
Figure 5. Hydrodynamic voltammograms of glucose (0)and background wlthout glucose (+). The current scale for the backgroundand the noise level (*) is given on the right-hand Y-axis of the two graphs. The upper graph shows results when the potential was lncremented by 20 mV steps from 0.36 to 0.56 V, whereas the lower graph shows results from incrementhg the potential by 20 mV from 0.56 to 0.36 V. Mobile phase was 0.1 M NaOH. Flow rate = 0.5 mL/mln; T = 35 OC. Each glucose sample Injected Contained 100 pmol(l0 pL of 10 pM glucose).
the strongly alkaline solution. The only difference is the variations in the magnitude of the catalytic current. Chromatographic Detection. As has been mentioned earlier, our goal is to develop a highly stable, sensitive, and reproducible LCEC detector for carbohydrates and possibly other oxidizable bioanalytes. To characterize and optimize the Ni-Ti electrode as an LCEC detector, we have focused our attention on examining the effect of the applied potential, electrode pretreatment protocol, short- and long-term reproducibility, and the LC parameters on the LCEC performance. The latter includes the effect of different mobile phase compositionson the LC separation and the Ni-Ti performance as an LC detector. The first step in this process has been to examine the hydrodynamicvoltammetry with glucose as the test compound using a Wescan Anion-R column. The separation of the sugar mixtures was conducted with a Dionex CarboPac PA1 column. Figure 5A shows the hydrodynamic peak current responses as a function of the applied potential, as it is increased incrementally in 20 mV steps from 0.36 to 0.56 V, for the repetitive injection of 100 pmol of glucose (10 uL of 10 uM glucose) in 0.1 M NaOH as the mobile phase. Also shown are the background currents (right-hand current scale in nA) and the noise levels (right-hand current scale in PA) over the same potential range for the mobile phase alone. Figure 5B is the same hydrodynamic experiment except with the potential decreasing incrementally from +0.56 to +0.36 V. After each
20 mV change in potential, 30 min of stabilization time was allowed prior to the injection of the glucose sample. Each point in the hydrodynamic voltammetric curve for glucose represents an average of five separate injections. Although there is little difference in the background currents and noise levels between Figure 5A and B, there is a noticeable difference between the response curves for glucose. That is, the peak currents rise rapidly in the 40 mV interval between 0.44 and 0.48 V and reach a maximumvalue at ca. 0.50 V. In the case of the potential being incrementally decreased, the peak currents are smaller in magnitude and more drawn out without any substantial decrease until a potential of +0.39 V is reached. Similar types of hysteresis have been observed in the cyclic voltammetric curves when the scan rates were less than 10 mV/s. This phenomenon reflects the potential dependence of the catalytic effectiveness of the Ni-Ti surface, which is related to the nature and amount of the Ni(II1) oxyhydroxide. Additional surface studies are underway in an attempt to further understand this phenomenon. The next step in the optimization study was to determine the value of the potential at which the electrode response would be independent of time and number of samples analyzed. Because the current responses in the hydrodynamic experiments depend on the direction of the potential change, which value of potential may be optimal was not obvious. The results are shown in Figure 6 in which the peak currents versus time are plotted for the applied potentials of 0.46,0.48, 0.50, and 0.52 V. For each potential setting, a freshly polished electrode was used. When the potential is initially applied, a large anodic background current is observed. This current decreases exponentially so that, within a few minutes, the current would decay from a value of 1 uA to about 100 nA. Within another 2 h, the current would decrease, for all practical purposes, to a constant value of 8-10 nA at which time the injection of 100 pmol samples of glucose would begin. Although it is quite clear that the peakcurrents for repetitive injections of glucose remained almost constant and independent of time at an applied potential of +OS0 V, another interesting feature emerged with Ni-Ti compared to pure Ni or Cu electrodes. That is, the sensitivity to glucose increases with repetitive Ana&ticalChemistty, Vol. 66, No. 17, September 1, 1994
2779
0
10
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50
60
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Injection Number
Flgurr 7. LCEC response of 100 Injections of glucose solution at a stabilized NI-TI electrode. Injectlons were carried out continuously over a 17 h period, one per 10 mln. Applied potentlal = +0.50 V vs AglAgCI. Mobile phase = 0.1 M NaOH; flow rate = 0.5 mLlm1n. T = 35 OC. Each glucose sample InJectedcontained 100 pmol(10 pL of 10 pM).
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=
Time (day) Peak Current (left)
RSD (right)
Flgure 8. Dally response (current scale on left hand y-axis) of glucose and relative standard deviation (RSD scale on right hand y-axis) at a NI-TI electrode activated at +0.56 V. Detection potential = +0.50 V vs AglAgCI. Mobile phase = 0.1 M NaOH; flow rate = 0.5 mLlmln. T = 35 OC. Sample solution: 10 pL of 10 pM glucose. Average values were calculated on the basis of at least 10 InJectlonseach day, once per hour. The last day the response was obtained after the system was disconnectedfor pump maintenance and reinstalled under the same condltlons without any treatment of the electrode.
injections and time, rather than decreasing, as in the case of pure Ni and Cu. Thus, there is a “conditioning” period in which the sensitivity increases before it levels off, as found when the applied potential was +OS0 or +0.46 V. However, this conditioningperiod is very short when the applied potential is +OS2 V, so that the sensitivity reaches a maximum rapidly and then decreases thereafter. These results in Figure 6 were taken over a period of several days with the LC flow rate set at 0.5 mL/min and then reduced to 0.01 mL/min during the overnight “off” periods. When the flow rate was returned to 0.5 mL/min, the sensitivity would be lower initially and then would gradually increase to the previous day’s level. If the flow rate is maintained at 0.1 mL/min or higher overnight, the time to regain the previous day’s sensitivity is less than 30 min. From the results in Figure 6, the optimal potential for the Ni-Ti electrode appears to be at +OS0 V in the 0.1 M NaOH mobile phase. On the basis of the above results, the stability of the Ni-Ti electrode was tested to the continuous repetitive injection of 2780
Ana&ticalChemistw, Vol. 66, No. 17, September 1. 1994
Ti”in Flgure B, Examples of LCEC separation and detection of sugars at the NI-TI electrode. Stationary phase: DlonexCarboPac PA1column. Mobile phase = 0.1 M NaOH. Flow rate = 0.5 mllmln. Detectlon potential = +0.50 V vs AglAgCI. T = 35 OC. Sample solutlon: 10 pL sugar mixtures containing (1) trehalose, (2) arabinose, (3)glucose, (4) ribose, (5) sucrose, (6) melezltose, (7) cellobiose, (8) stachyose, and (9) maltose. Concentratlon: 10 pM (top) and 0.3 pM (bottom) each.
100 pmol of glucose over a 17 h period. The results for the 100 injections over the 17 h are shown in Figure 7. The LCEC response with the Ni-Ti exhibited a slight increase from 10.67 to 11.OO nA for injection numbers 1 and 97, respectively. The overall relative standard deviation (RSD) was less than 1.5%. This same electrode was used continuously for 44 days under the same conditions except that the flow rate was reduced to 0.1 mL/min during the overnight off periods. The daily average responses and RSD’s are plotted in Figure 8. Ten samples, one per hour, were run each day and averaged. To activate the electrode more quickly each day after the mobile phase flow rate was raised to 0.5 mL/min, a pretreatment potential of +OS6 V was applied for 30 min and then returned to the operating potential of +OS0 V. The average peak current during the first day was 15.5 nA with a RSD of 20%, increasing to 19.5 nA (RSD of 1%) the second day and then decreasing thereafter for the next 15 days by nearly 40%. For the following 30 days, the total variation was less than 10% with an average RSD of 3%. On the 44th day, the LC system was shut down for pump maintenance and then restarted to resume with the lO/day glucose injections. No deactivation was observed for the Ni-Ti electrode. Instead, the average peak current for the 10 injections was 18 nA, which is close
tested. Thedata of retention timeand limit ofdetection (LOD) for these nine sugars are summarized in Table 2. The LOD's range from 0.5 pmol for glucose and arabinose to 10 pmol for maltose. As may be seen from the chromatogram for the higher 10 uM concentration, the peaks are all symmetric in shape except for ribose (peak 4), which has a tailed response. Although not shown, fructose also has a peak shape similar to that for ribose. These two sugars exhibited a positive deviation from linearity at higher concentrations. On the other hand, the larger sugars such as the di- and oligosaccharides showed a negative deviation at higher concentrations. This latter deviation, in comparison to that for the monosugars, may result from the saturation of the anion exchange surface by the larger sized sugars, due to not only exchange but also surface adsorption, as concentration increased. In practice, some sugars may be only partially separated and may require modification of the mobile phase. For example, sucrose and lactose would elute with nearly the same retention time under the conditions for the separations illustrated in Figure 9. By the addition of sodium borate to the mobile phase, the two may be separated due to the formation of a borate-sugar complex,13as illustrated in Figure 10. The resolution for the separation increases as the borate content increases, with sucrose being eluted first; chromatograms B and C in Figure 10 result from the addition of 0.01 and 0.04 M borate, respectively, to the 0.1 M NaOH mobile phase. The peak heights were slightly less as borate was added due to the band broadening with the longer retention times.
Tabk 2. LCEC Rwpome8 of wan at NCTl (55:45)' detection limit
compound
retention time (min)
sensitivity (pA/pmol)
(ng)
(pmol)
trehalose arabinose glucose ribose sucrose melezitose cellobiose stachyose maltose
4.49 6.53 7.70 9.57 11.87 16.53 17.80 19.71 26.95
4.913 18.849 19.619 9.734 5.054 4.020 5.465 5.692 3.139
0.7 0.1 0.1 0.8 0.3 2.5 1.7 1.3 3.4
2 0.5 0.5 5 1
5 5 2 10
a Constant-potential amperometricdetectionat +OS0 V vs Ag/AgCl. StationaryPhasc: CarboPacPAl anion-exchangecolumn. Mobile phase: 0.1 M NaOH. Flow rate: 0.5 mL/min. Calculations were based on the chromatogramsobtained by injections of 10 pL sugar solutions of equal concentration: 0.1,0.3,1, 3, ..., lo00 pM.
to values found for the first few days of working with this electrode. The application of the Ni-Ti electrode as an LCEC detector for other sugars is illustrated in Figure 9. A Dionex CarboPac PA1 anion exchange column with 0.1M NaOH as the mobile phase was used for the separations. The upper and lower LC chromatograms in Figure 9 are the responses for the separation of a mixture containing nine different sugars each at concentrations of 10 uM (upper) and 0.3 uM (lower). Linear plots of peak currents versus concentration were obtained over more than 3 decades of concentration for most of the sugars A
B
C
S
StL
S
T 800
T
PA
800
1
-
0
20
10
0
L
PA
1
1
20
10
0
1
I
10
20
Th/& Flgwo 10. LCEC chromatograms of sucrose (S)and lactose (L) at the Ni-Ti electrode. Statlonary phase: Dlonex CarboPac PA1 column. Mobile phase is (A) 0.1 M NaOH, (B) 0.1 M NaOH 0.01 M borate, and (C) 0.1 M NaOH 0.04 M borate. Flow rate = 0.5 mL/min. Detection potential set at +0.50 V vs AglAgCi. T = 35 OC. Sample solution: 10 pL mixture contalnlng 100 pM each.
+
+
Ana&ticalChemisi?y, Vol. 66, No. 17, September 1, 1994
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I
0
heights of the sugars in these two chromatograms indicates that the Ni-Ti electrodeis unaffected, at least by the presence of acetonitrile. This result suggests that the Ni-Ti electrode can probably be used for separations involving ion-pairing or reverse-phase LC for the detection of a wide range of electroactive analytes. Further studies on the Ni-Ti electrode for LCEC and capillary electrophoretic applications are continuing in our laboratory.
B A
s
4
10
20
3 0 0
10
20
30
mu (d) Flguro 11. LCEC chromatograms of sugars at the NI-TI electrode In the presence of acetonttrlle In the mobile phase. Stationary phase: Dhex C a r W a c PA1 column. Separation mobile phase: 0.1 M NaOH at a flow rate of 0.5 mL/mln. Postcolumn addition reagent: 0.1 M Naotl (A) In H20and (B)In 50% CH&N-HpO at a flow rate of 0.125 mL/mln. Detection potential set at +OS0 V vs Ag/AgCl. T = 35 OC. Sample solution: 10 pL sugar mixtures contalnlng (1) trehalose, (2) arabinose, (3) glucose, (4) ribose, (5) sucrose, (6) melezitose, (7) celloblose, and (8) maltose. Concentration: 10 pM each.
Among LCEC applications, a serious problem is interferences or alterations in the sensitivity of the electrochemical detector arising from the use of organic solvent^.^ Such a situation is encountered often in ion-pair or reverse-phase separations. To test the effect of the addition of an organic solvent to the response of a Ni-Ti electrode, the same sugar mixture was separated by the CarboPac PA1 column with 0.1 M NaOH as the mobile phase except in chromatogram B of Figure 11, where 0.1 M NaOH containing 50% acetonitrile was added postcolumn rather than the same mobile phase of 0.1 M NaOH in chromatogram A. The similarity in the peak (13) Floridi, A. J. Chromatogr. 1971, 59, 61-70.
2782 A n a ~ c a l ~ m i s i r Vol. y , 66, No. 17, September 1, 1994
SUMMARY The ability to conduct long-term constant-potential amperometric detection of various carbohydrates with liquid chromatography has been substantially improved by the use of a Ni-Ti (55:45) alloy electrode. The Ti in the Ni-Ti alloy stabilizes the oxide structure while it allows the electrode to retain the catalytic activity of the Ni. This activity was maintained for the repetitive injection of 100 pmol of glucose in 0.1 M NaOH for a period of 44 days. Reproducibility on a daily basis was the order of 3%. Additionally, the Ni-Ti electrode exhibited a very high sensitivity to carbohydrates with a limit-of-detectionin the low picomole, or in a few limited cases, subpicomole range. Mobile phase modifiers such as borate or acetonitrile also did not alter the response characteristics of the Ni-Ti electrode. The applicability of this electrode to other electroactive analytes separated by LC or capillary electrophoretic methods is under investigation. ACKNOWLEDGMENT This work was supported by a grant from the Shimadzu Corp., Kyoto, Japan, and the Kansas Technology Enterprise Corp. under their Applied Research Matching Grant Programs through the Center for BioAnalytical Research. The authors would like to thank Dr. J. M. Marioli for his assistance and initial contribution to this work. Recehred for revlew March 1, 1994. Accepted May 16, 1994.O Abstract published in Aduancc ACS Abstracts, June 15, 1994.
