Anal. Chem. 1993, 65, 50-55
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Optimization of Waveforms for Pulsed Amperometric Detection of Carbohydrates Based on Pulsed Voltammetry William R. Lacourset and Dennis C. Johnson’ Department of Chemistry, Iowa State University, Ames, Iowa 50011
Llquld chromatography (LC) wlth pulsed amperometrlc detection (PAD) at Au electrodes has galned promlnence for analysls of complex mixtures of polyalcohols and carbohydrates. The mort commonly used PAD waveform condrts of three potentlals and four tlme parameters, all of whlch can be varkd Independently but the effects of which are not necessarlly mutually excluslve. Choke of potentlal values In PAD waveforms has tradttlonally been based on cycllc voltammetry (CV). However,CV data are uselessfor educlng optlmal values for tlme parameters, and these values are usually approxlmated uslng the trlal-and-error method durlng muttlple Injectlons In LC-PAD sydems. An automated procedure Is described for optlmlzatlon of all potentlal and tlme parameters In PAD waveforms on the bask of pulsed voltammetry (PV) at a rotated dlsk electrode(RDE). General and optlmlzed waveforms are presented for detectlon of carbohydrates,and effectson LC-PAD response are Illustrated.
INTRODUCTION Pulsed amperometric detection (PAD) at Au electrodes has become popular for the sensitive and direct detection of many polar aliphatic organic compounds separated by liquid chromatography (LC).’-3 These compounds typically exhibit little or no chromophoric and/or fluorophoric activity, and furthermore, many do not give persistent amperometric response for a constant (dc) applied potential at noble-metal electrodes. PAD a t Au electrodes in alkaline media exploits surfacecatalyzed oxidations of various functional groups under managment by multistep potential waveforms (E-t) to alternate the processes of amperometric detection with anodic and cathodic polarizations to clean and reactivate the Au surface. Presently, the most prominent application of PAD is for anodic detection of polyalcohols and carbohydrates in flow-through electrochemical cells followingtheir separations by high-performance ion-exchange chromatography (HPIC) using an alkaline mobile phase.1-6 Sensitivity for monosaccharides is comparable for aldoses and ketoses with detection limits at the picomolar level. A typical PAD waveform is described in Table I for carbohydrate detection at Au electrodes in 0.1 M NaOH. The detection potential (Edet)is applied for the time period tdet, and the electrode current is sampled by electronic integration over the time period tint following a delay of tdel to allow charging current to diminish toward zero (i.e., tdet = tdel + + Present address: Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21228. (1)Johnson, D. C.; Lacourse, W. R. Anal. Chem. 1990,62,589A-97A. (2)LaCourse, W. R.; Johnson, D. C. Carbohydr. Res. 1991,215,15978. (3)Johnson, D. C.; Lacourse, W. R. Electroanalysis 1992,4,367-80. (4)Rocklin, R. D.; Pohl, C. A. J. Liq. Chromatogr. 1983,6,1577-90. (5)Olechno, J. D.;Carter, S. R.; Edwards, W. T.; Gillen, D. G. Am. BiotechnoL Lab. 1987,5, 38-50. (6)Paskach, T. J.; Lieker, H. P.; Reilly, P. J.; Thielecke, K. Carbohydr. Res. 1991,215,1-14.
tint). The output voltage signal can be proportional to the integrated charge (q, coulombs) or the average current (iavg = q/tint,coulombs per second). The oxidative desorption of adsorbed detection products and/or solution impurities occurs simultaneously with anodic formation of surface oxide at Eoxd >> Edetapplied during the period toxd. Subsequently,the inert oxide produced at Eoxdis cathodically dissolved atEred +200 mV, reductive dissolution of surface oxide does not occur and carbohydrate response is terminated. Values of E r e d 5 -300 mV are sufficient to minimize oxidation of carbohydrates prior to application of &et, and -300 mV is chosen as the optimum value for Ere+ A secondary consequence of incomplete removal of surface oxide for the chosen values of Erdand trdis that reduction of oxide can persist at E d e t with a significant contribution to the residual response in LC-PAD. This residual response is very sensitive to fluctuations in temperature and pH. Hence, LC-PAD results obtained under conditions of incomplete oxide reduction have exhibited baseline signals which drift for more than 2 h after initiation of the PAD waveform. Conversely, when surface oxide is completely removed in the PAD waveform, baselines are stabilized within ca. 10 min.2 Another consequence of inadequate oxide removal is a decreased analytical signal because the electrode surface is not fully utilized for the detection process. These factors have previously been noted with regard to the determination of amino alcohols by LC-PAD.15 Shown in Figure 11 is the PAD response for glucose (-) and maltose (---I as a function of tr4 for E r e d = -300 mV. The response for glucose reaches a plateau for tr4 > ca. 100 ms, whereas Tred > ca. 300 ms is required to reach a maximum response for maltose. This difference is attributed to the difference in the extent of adsorption of the detection producta for these two compounds. We recommend tred = 360 ms as optimal in the general application of PAD for carbohydrates. Experience indicates this choice of tr4 allows for latitude in the selection of Eoxd within the range +600 to +800 mV.
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(14) Johnson, D.C.; Polta, J. A.;Polta,T. Z.;Neuburger,G.G.; Johnson, J. L. J. Chem. SOC.,Faraday Trans. 1 1986,82,1081-98. (15) Lacourse, W. R.; Jackson, W. A.; Johnson, D. C. Anal. Chem. 1989,61, 2466-71.
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Flgure 11. PV response as a function of r ,for (-) glucose and (- -) maltose at the Au RDE In 0.1 M NaOH. Conditions/solutlons are as In Figure 4. (
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POTENTIAL (mV) Flgure 12. PV response and S/N as a function of for glucose In 0.1 M NaOH. Conditions are as In Flgure 4. Curves: (). aerated supporting electrolyte; (-) aerated 10 pM glucose; (- -) signal-to-
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PV and LC-PAD Response with Optimized Waveforms. Figure 12 shows the PV response for the Au RDE in 0.1 M NaOH with (-) and without (--) the presence of 10pM glucose during the positive scan of Edeb All other waveform parameters correspond to the optimal values given in Table I. The S/N is shown correspondingto the presence of glucose. In these experiments, solutions were not deaerated so to better simulate the inevitable presence of dissolved 02 in LC-PAD. The labels correspond to those shown in Figure 2. For the high sensitivity used, the residual response (-) shows clearly that E d e t = ca. 0 to +300 mV corresponds to very low signals for both oxide formation and 02 reduction. The response for 10 pM glucose is in the form of a broad peak, which is consistent with Figure 2. The corresponding S/N (---) easily = ca. +200 mV (S/N = 190). The identifies the optimal PAD response for glucose at the Au RDE was determined for these conditions to be linear in the range 0-500 pM. The regression line is described by the equation pA = 0.678 (pM) + 1.346 with a correlation coefficient ( r ) of 0.999 996. The above procedure was repeated at the Au electrode of the flowthrough thin-layer cell typically used in LC-PAD. The PV response for a positive E d e t scan during continuous flow of sample was very similar to that in Figure 12for the RDE with a maximum value of S/N for &et = ca. +200 mV. Figure 13 shows the LC-PAD response of a solution containing 10pmol each of sorbitol, glucose,fructose, sucrose, and maltose in 0.1 M NaOH using the optimized PAD waveform described in Table I. The relative standard
ANALYTICAL CHEMISTRY, VOL. 65, NO. 1, JANUARY 1, 1993
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Eord,and Ered are decreased by 20 mV to compensate for the increased alkalinity. For a 0.02 M NaOH mobile phase, commonly applied for assays of monosaccharide mixtures derived from glycoproteins, the PAD potentials should be increased by 40mVto compensate for the decreased alkalinity. Application of a positive pH gradient in HPIC-PAD can be beneficial for separations of mixtures of carbohydrates having a large range of molecular weights with the expected large range of k’ values observed for isocratic elution. However, with an increase in pH comes a negative shift in the potential for the onset of oxide formation. Consequently, a large positive (anodic) shift in the baseline signal can result when Edetis held constant throughout development of the gradient.16 This problem can be alleviated by substitution of a pH-sensitive glass-membrane electrode for the Ag/AgCl reference electrode in the electrochemical cell. The potential response of a glass-membrane electrode is ca. -60 mV pH-1, and hence, the value of Met is automatically adjusted during execution of the pH gradient.16 Typical ionic strengths (0 in HPIC-PAD are in the range 50-100 mM, and variations within this range are of virtually no consequence to the optimization of PAD waveform parameters. However, under conditions of ion-gradient elution (e.g., increasingacetate concentrationin HPIC-PAD), baseline drifts have been observed (data not shown).2 In this case, we recommend postcolumn addition of a solution with high ionic strength to effectively ‘buffer” the ionic strength in the effluent stream passing through the electrochemical detector cell. The presence of organic modifiers to the mobile phases in LC-PAD can have a much greater consequence on the optimized PAD waveform than is caused by changes in Uand pH. This is true especially when the modifiers are adsorbed at the electrode surface and interfere with access by the carbohydrate molecules to specific reaction sites on the electrode with a corresponding attenuation of analytical response. Furthermore, adsorbed organic modifiers can suppress the oxide-formation process with the necessity of increasing Eoxd values. Large temperature fluctuations (i.e., >>1“C) can cause significant changes in the sensitivity of PAD response, especially for compounds whose detection is under mixed or surface control. Whereas the majority of present-day LCPAD applications rely on the thermostatic control normally found in well-designed analytical laboratories, we can foresee applications that can benefit from better control of temperature (i.e.,
