Anal. Chem. 1996, 68, 2432-2436
Improvements in the Selectivity of Electrochemical Detectors for Liquid Chromatography and Flow Injection Analysis Using the Self-Assembled n-Alkanethiol Monolayer-Modified Au Electrode Zhiming Liu, Jinghong Li, Shaojun Dong,* and Erkang Wang*
Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
4-Aminophenol (4-AP), paracetamol (PRCT), norepinephrine(NE), and dopamine (DA) (all somewhat hydrophobic compounds) were HPLC electrochemically detected while the signals from uric acid (UA) and ascorbic acid (AA) (both hydrophilic compounds at the pH studied) were minimized, taking advantage of the permselectivity of the self-assembled n-alkanethiol monolayer (C10-SAM)-modified Au electrodes based on solute polarity. The effects of various factors, such as the chain length of the nalkanethiol modifier, modifying time, and pH value, on the permeability of C10-SAM coatings were examined. The calibration curves, linear response ranges, detection limits, and reproducibilities of the EC detector for 4-AP, PRCT, NE, and DA were obtained. The result shows that the EC detector can be applied in the chromatographic detection of 4-AP, PRCT, NE, and DA in urine, effectively removing the influence of UA and AA in high concentrations existing in biological samples. As a result, a great improvement in the selectivity of EC detectors has been achieved by using Au electrodes coated with neutral n-alkanethiol monolayer. Electrochemical (EC) detection in a flow system such as liquid chromatography or flow injection analysis has become a powerful tool for chemical, clinical, pharmaceutical, and environmental sciences, owing to its excellent sensitivity and moderate selectivity toward electroactive compounds. However, further improvements in the selectivity and stability of electrochemical detection are needed to extend the power and scope of this technique, because the electrode is usually subject to a gradual loss of activity in the presence of many electroactive species for complex sample analysis. Several detection schemes have been proposed for this purpose, such as multielectrodes,1 potential scanning detection,2,3 pulsed amperometric detection,4 indirect EC detection,5 post- and precolumn derivatization,6 and chemically modified electrodes (CMEs).7-10 (1) Roots, D. A.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982, 54 , 1417A. (2) O’Dea, J.; Osteryoung, J. Anal. Chem. 1980, 52, 2215. (3) Reardon, P. A.; O’Brien, G. E.; Sturrock, P. E. Anal. Chim. Acta 1984, 162, 175. (4) Austin-IIarrison, D. S.; Johnson, D. C. Electroanalysis 1989, 1, 189. (5) (a) Yeung, E. S. Acc. Chem. Res. 1989, 22, 125. (b) Ye, J.; Baldwin, R. P.; Ravichandran, K. Anal. Chem. 1986, 58, 2337.
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CMEs exhibit better selectivity for some species in complex matrixes based on the properties of modifying substance, e.g., electron-transfer mediation; acceleration or inhibition of electrode reactions; chemical, electrostatic, or steric selectivity; photosensitivity; and so on. So far, a series of discriminative coatings, including size-selective cellulose acetate,7,8 poly(1,2-diaminobenzene),11 charge-selective Nafion,12,13 Eastman AQ,14 or poly(vinylpyridine),10 and hydrophobic lipid15 layers, have been suggested. Joseph Wang et al.16 have described recently that a Au electrode coated with self-assembled monolayers (SAMs) of unsubstituted n-alkanethiols has high selectivity toward hydrophobic compounds chlorpromazine and dopamine (through discrimination against coexisting polar interferences such as ferrocyanide, hydrogen peroxide, and ascorbic acid). They fitted the Cn-SAM-coated Au electrode into an amperometric flow detector to improve its selectivity. Paracetamol (PRCT) and 4-aminophenol (4-AP) are two commonly used analgetic drugs. Norepinephrine (NE) and dopamine (DA) are two important neurotransmitters. For the determination of these compounds in biological fluids (plasma, serum, and urine), it is necessary to understand the clinical pharmacokinetics and neurochemistry. Liquid chromatography (LC) with electrochemical detection17,18 has been applied to the determination of these compounds where large amounts of uric acid (UA) and ascorbic acid (AA) from biological samples and physiological fluids affect greatly the determination. In the paper, a well-jet electrochemical detector coated with neutral SAM layers, which have high selectivity toward hydrophobic compounds, has been used to prevent the interference of UA and other electroactive species in the liquid chromatographic determination of PRCT, 4-AP, NE, and DA in urine. The voltammetry and hydrodynamic voltam(6) Krull, I. S.; Selavka, C. M.; Duda, C.; Jacobs, W. J. Liq. Chromatogr. 1985, 8, 2845. (7) Sittampalam, G.; Wilson, G. S. Anal. Chem. 1983, 55, 1608. (8) Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 57, 1536. (9) Hutchins-Kumar, L. D.; Wang, J.; Peng, T. Anal. Chem. 1986, 58, 1019. (10) Wang, J.; Golden, T.; Peng, T. Anal. Chem. 1987, 59, 740. (11) Sasso, S.; Pierce, R.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990, 62, 1111. (12) Ji, H.; Wang, E. J. Chromatogr. 1987, 410, 111. (13) Shimazu, K.; Kuwana, T. J. Electrochem. Soc. 1988, 135, 1602. (14) Bremle, G.; Persson, B.; Gorton, L. Electroanalysis 1988, 3, 77. (15) Wang, J.; Lu, Z. Anal. Chem. 1990, 62, 826. (16) Wang, J.; Wu, H.; Angnes, L. Anal. Chem. 1993, 65, 1893. (17) Pang, K. S.; Taburet, A. M.; Hinson, J. A.; Gillette, J. R. J. Chromatogr. 1979, 174, 165. (18) Zhou, J.; Wang, E. Anal. Chim. Acta 1990, 236, 293. S0003-2700(95)01256-X CCC: $12.00
© 1996 American Chemical Society
metry of UA, AA, PRCT, 4-AP, NE, and DA for the SAM-coated flow detector were described. Its analytical performance as an electrochemical detector and various factors affecting selectivity and stability were also investigated in detail. EXPERIMENTAL SECTION Apparatus. Voltammetric experiments were carried out on a Model 400 EC detector with scan mode (E&G, PAR) in a conventional three-electrode cell, with a 1-mm-diameter gold disk electrode, an Ag/AgCl (saturated KCl) reference electrode, and a platinum wire counter electrode. Flow injection and chromatographic experiments were performed with a Gilson gradient HPLC system (Gilson Medical Electronics, Inc.) consisting of a Model 7125 injection valve (20-µL loop) and a 7-µm particle size Nucleosil C18 column (200 mm × 4.0 mm i.d., Dalian Institute of Chemical Physics, Dalian, Liaoning China) as analytical column. A precolumn (Perisorb RP-18, 30-40µ Pellicular, Upchurch Scientific, Inc.) was placed between the injection valve and the analytical column so that the analytical column could maintain proper pressure and optimal efficiency. The chromatographic operations and data processing were controlled by a Philip’s computer configured as a Gilson 715 HPLC system controller software. A home-made well-jet cell with a nozzle kept 1 mm away from the center of the working electrode and a Model 400 electrochemical detector (EG&G, PAR) were employed. Electrode Preparation. Bulk gold (99.9%) was annealed at 1000 °C in air and etched in freshly prepared Piranha solution (3:1 mixture of concentrated H2SO4 and 30% H2O2 heated to ∼100 °C; Caution! Piranha solution reacts violently with organic material, and it should not be stored in closed containers), and then gold disk electrode (1 mm diameter) was sealed in a soldering glass according to Malem and Mandler.19,20 The prepared electrode was polished first with emery paper followed by alumina slurry (1.0, 0.3, and 0.05 µm), rinsed with distilled water, sonicated in a water bath for 5 min, and then dried. The electrode was repetitively cycled at potential range of 1.5 and -0.3 V vs Ag/AgCl in 1 mol/L H2SO4 until a reproducible voltammogram was obtained. Modification of the gold electrode was accomplished by soaking it in a quiescent ethanol/octane solution containing 20 mmol/L of the desired n-alkanethiol. After 1 min of equilibration, the electrode was removed from the solution, rinsed in ethanol, and allowed to dry. Reagents. Uric acid (>99%, Sigma), ascorbic acid (>99.7%, Beijing, China), n-octanethiol, C8 (>98%, Fluka); 1-decanethiol, C10 (>96%, Aldrich); tetradecanethiol, C14 (>98%, Fluka); and hexadecanethiol, C16 (tech. 92%, Aldrich) were used without further purification. The purity of DA and NE (from Fluka AG) was higher than 98%. PRCT and 4-AP were of pharmaceutical grade and obtained from Harbin Medicine (Harbin, China). All other chemicals were of analytical-reagent grade, and all solutions were prepared with doubly distilled water. The supporting electrolyte/ carrier solution for flow injection analysis was a 50 mmol/L phosphate buffer (pH 7.0). The chromatographic eluent was 90.0% (v/v) phosphate buffer (50 mmol/L, adjusted to the desired pH value with phosphoric acid and NaOH) and 10.0% methanol. The aqueous phosphate buffer was ultrasonically degassed for 5 min under a vacuum of 600 mmHg. (19) Malem, F.; Mandler D. J. Electrochem. Soc. 1992, 139, L65. (20) Malem, F.; Mandler, D. Anal. Chem. 1993, 65, 37.
Figure 1. Voltammograms for 2 mmol/L 4-AP (A), PRCT (B), NE(C), DA(D), AA (E), and UA (F) at the bare Au (curve 1) and C8- , C10- , C14-, and C16-SAM-coated Au electrodes (curves 2-5). Scan rate, 50 mV/s; electrolyte, 50 mmol/L phosphate buffer (pH 7.0).
RESULTS AND DISCUSSION Voltammetry and Hydrodynamic Voltammetry. It is known that the alkanethiol monolayer formed on a gold surface has shown well-organized, densely packed, and electron-insulating characteristics.21,22 The hexanethiol- and dodecanethiol- (n ) 6 and 12) coated electrodes can effectively exclude the Fe(CN)63and hydrogen peroxide contribution while exhibiting a large response toward the hydrophobic drugs.16 A series of voltammetric experiments were carried out using gold electrodes modified with n-alkanethiol (CnH2n+1SH) molecules with different chain lengths. These results were evaluated to determine the optimum alkanethiol chain length required to minimize the transport of hydrophilic species (UA and AA) toward the gold electrode. Figure 1 shows the voltammograms of 2 mmol/L concentrations of 4-AP (A), PRCT (B), NE (C), DA (D), UA (E), and AA (F) in 50 mmol/L phosphate buffer (pH 7.0) at bare Au (curve 1) and Cn-SAM coated Au electrodes (curves 2-5), respectively. The alkanethiol SAMs reduce the anodic peak currents of the six compounds gradually with increasing chain length of the alkanethiols, and the faradaic current was barely measurable at the longest chain lengths of 16. As discussed by Porter et al.23 and by Miller et al.,24,25 these results can be attributed to the formation of densely packed monolayer as(21) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 45. (22) Finklea, H. O.; Robinson, L. R.; Blackburn, A.; Richter, B.; Allara, D.; Bright, T. Langmuir 1986, 2, 239. (23) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. D. E. J. Am. Chem. Soc. 1987, 109, 3559. (24) Miller, C.; Cuendet, P.; Gratzel, M. J. Phys. Chem. 1991, 95, 877. (25) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1992, 96, 2657.
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Figure 2. Hydrodynamic voltammograms of AA (1), UA (2), 4-AP (3), PRCT (4), NE (5), and DA (6) at the C10-SAM-coated Au electrodes. Mobile phase, 50 mmol/L phosphate buffer (pH 7.0); flow rate, 1 mL/min; injection volume, 20 µL; modifying time, 1 min; modifier concentration, 20 mmol/L.
semblies with the thiols having alkyl chain longer than n ) 11 and the hindrance of electron transfer between a metal surface and alkyl chain/electrolyte interface. The gradual anodic shift (of up to 90 mV) in the peak potential upon increasing the alkyl chain length was observed. This also indicates changes in the electron-transfer rates. Moreover, it was observed that a decrease in the anodic peak current caused by the monolayer modification was greater for UA and AA than that for 4-AP, PRCT, NE, and DA. In other words, 4-AP, PRCT, NE, and DA are easier to permeate into the n-alkanethiol SAM formed on a gold electrode than UA and AA. Such differences in the voltammetric response can be attributed to differences in the affinities of these compounds for the hydrophobic alkyl chain assembly of the monolayer. Thus, n-alkanethiol SAM on a gold electrode can significantly decrease the response of AA and UA while maintaining a measurable signal for the other four compounds. Hydrodynamic voltammograms (HDVs) obtained for 1 mmol/L UA, AA, 4-AP, and PRCT and 2 mmol/L NE and DA via flow injection detection with C10-SAM-coated Au electrodes are shown in Figure 2. They were obtained by injecting a fixed concentration of the standard solutions and varying the potential between 0.40 and 1.20 V. The current response almost reached plateau currents above 1.00 V; thus, a potential of 1.00 V was selected for the detection of UA, AA, 4-AP, PRCT, NE, and DA at C10-SAM-coated Au electrodes. Effect of the Alkanethiol Chain Length on the Permselectivity of Cn-SAM Coatings. The voltammograms in Figure 1 show that the current responses of the compounds at Cn-SAMcoated Au electrodes vary with chain lengths of alkanethiols. It is thus possible to improve the selectivity of amperometric detection for flowing streams by exploiting the controllable permeability of the different chain length SAM coatings. We tried to investigate the effect of the chain length on the permeability of Cn-SAM-coated electrodes, where the ratio between the flow injection peak current at the SAM-coated Au electrode and that at the bare Au electrode (im/ib) was used as a measure of the permeability. The results are shown in Figure 3. It was found from Figure 3 that a decrease in the permeability of SAM-coated Au electrodes with increasing the chain length was distinct for 4-AP and AA, but not for UA, PRCT, NE, and DA. To maintain the higher permeability for 4-AP, 2434 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
Figure 3. Effect of the chain lengths on the permeability (im/ib) of Cn-SAM coatings: AA (1), UA (2), 4-AP (3), PRCT (4), NE (5), and DA(6) (2 mmol/L each); Eapp ) 1.0 V vs Ag/AgCl. Other parameters as in Figure 2.
Figure 4. Effect of the modifying time on the im/ib of C10-SAM coatings. The signs and experimental parameters are as in Figure 3.
1-decanethiol (chain length, n, is 10) was chosen as a proper modifier coated on Au electrodes. Effect of Modifying Time on the Permeability of C10-SAMCoated Au Electrode. The effect of modifying time on im/ib is shown in Figure 4. The modifying time is the time the Au electrode remains in the solution containing 20 mmol/L 1decanethiol. Figure 4 clearly shows that, when the modifying time is about 50 s, the permeability of C10-SAMs for UA and AA descends sharply to 10% and changes a little beyond 50 s, but for 4-AP, PRCT, NE, and DA it is still higher between 45 and 60 s. An appropriate modifying time of 1 min was suggested so that the obtained SAM coatings can effectively block UA and AA under the good permeation of 4-AP, PRCT, NE, and DA into SAM coatings. Effect of pH on the Permeability and the Chromatographic Separation. Figure 5 shows the effect of the mobile phase pH on the permeability (im/ib) of C10-SAMs (A) and the logarithm of capacity factors (ln k′) of UA, AA, 4-AP, PRCT, NE, and DA in reversed-phase HPLC. In Figure 5A, 4-AP (curve 3) and PRCT (curve 4) can easily permeate into the SAMs and have higher chromatographic response at pH ) 6.0-6.5, but the permeability
Table 1. Results of Calibration Curves of 4-AP, PRCT, NE, and DAa
compd
equationb
correl coeff
linear range (µmol/L)
detection limitc (µmol/L)
RSDd (%)
4-AP PRCT NE DA
Y ) -0.75 + 4.51X Y ) 2.11 + 0.22X Y ) 7.28 + 0.45X Y ) 11.84 + 0.96X
0.9986 0.9985 0.9981 0.9994
1-1000 5-500 1-1000 1-1000
0.5 1.4 0.2 0.5
4.02 3.53 3.95 9.93
a Conditions as in Figure 6. b In the equation, Y is peak current (nA) and X is concentration (µmol/L). c Signal-to-noise ratio is 3. d Eight parallel determinations.
Figure 5. pH effect on the im/ib of C10-SAM coatings (A) and the logarithm of capacity factors (ln k′, B) of AA, UA, 4-AP, PRCT, NE, and DA in HPLC. Mobile phase, 90% (v/v) 50 mmol/L phosphate buffer + 10% methanol; CUA ) CAA ) 1 mmol/L, C4-AP ) CPRCT ) CNE ) CDA ) 2 mmol/L. Other conditions are as in Figure 3.
quickly goes down at pH > 6.5. NE and DA have good permeability at pH ) 6.5-7.0, which gradually lessens at pH < 6.5. It may result from the deprotonation of phenolic groups in 4-AP and PRCT in neutral and alkaline media and the protonation of amino group in NE and DA in acidic conditions, because the SAMs effectively exclude the polar species. Within the pH range investigated, UA and AA existing in ionic form are excluded away from the Au electrode surfaces by the hydrophobicity of nalkanethiol, so their responses at SAM-coated electrodes are lowered to about 10%. These data suggest that the mobile phase pH range is more favored to be 6.0-6.5 for 4-AP and PRCT, and to be 6.5-7.0 for NE and DA. From Figure 5B, we found that it is difficult to get a proper pH value for mobile phase where the six compounds can be separated satisfactorily in HPLC, so these species were divided into two groups (group A, AA, UA, 4-AP, and PRCT; group B, AA, UA, NE, and DA) in the successive chromatographic studies. The optimal pH values of mobile phase were selected to be 6.0 and 7.0 for group A and group B, respectively. Calibration Curve. Under the optimal conditions chosen above, the calibration curves, linear response ranges, detection limits, and reproducibilities of 4-AP and PRCT at C10-SAM coated Au electrodes, which lower the im/ib of 2 mmol/L AA and 1 mmol/L UA to 10%, were investigated by means of the flow injection detection. The results are shown in Table 1. Obviously, the linear response ranges of the detector for the other compounds except PRCT extend over 3 orders of magnitude of the concentration, with correlation coefficients greater than 0.998. The detection limits of the compounds are between 0.2 and 1.4 µmol/L. The
Figure 6. Stability of C10-SAM coatings in flowing streams. Modifying time, 2 min; mobile phase pH, 6.5; CAA ) 2 mmol/L. The signs and other parameters are as in Figure 3.
above results demonstrate that the C10-SAM-coated electrode can meet the requirements of electrochemical detection in flowing streams. Stability of C10-SAM Coatings in HPLC. The stability of SAM coatings in the continual flowing streams over 13 h is shown in Figure 6. It was found that, after 2 h, the permeabilities of AA, UA, 4-AP, and PRCT at a SAM-coated detector do not have obvious changes for 9 h, and that of NE and DA initially decreases a little and gradually trends to be stable. Chromatographic Selectivity of C10-SAMs for AA, UA, 4-AP , PRCT, NE, and DA. The aim of examining the permselectivity of the C10-SAM-coated Au electrode for these compouds is to use the electrochemical detector with the electrodes to enhance the selectivity of chromatographic detection for practical samples. We examined first the chromatographic behaviors of the urine samples spiked by 0.1 µmol/L 4-AP, 0.2 µmol/L PRCT, and 0.3 µmol/L NE and DA at bare Au electrodes. The urine samples were prepared by diluting the urine taken from a healthy person 10-fold with phosphate buffer solution and rapidly pretreated at a Sep-Pak C18 cartridge (Waters Associates, Millipore). The urine samples were injected directly into a chromatographic system. The chromatograms obtained are shown in Figure 7A and B. It was shown that a large amount of uric acid and other electroactive species can affect the chromatographic detection of low concentrations of 4-AP, NE, and DA in urine. In particular, Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
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restricted the electroactive constituents of the urine reaching to the surface, especially for uric acid, while allowing a highly selective and sensitive detection of the spiked 4-AP, PRCT, NE, and DA. From the chromatograms (not shown) obtained by lengthening the modifying time, it was found that this restrictive rule becomes stronger as the modifying time increases, but the signal of analytes is also reduced, evidently. As a result, we suggest that, when choosing a proper modifying time to improve the chromatographic selectivity and to simplify the chromatograms of complex samples, it is necessary to consider the sensitivity of analytes; that is, on the premise of ensuring considerable detecting sensitivity, the signal of interfering species is restrained as far as possible using C10-SAM coated electrode.
Figure 7. Chromatograms of the urine spiked by 4-AP, PRCT, NE, and DA at bare (A, B) and C10-SAM-coated (C, D) Au electrodes. Modifying time, 2 min. The other parameters are as in Figure 6.
the chromatographic peaks of NE and DA almost overlay with that of the interfering species. Nevertheless, the interferences can be eliminated by modifying C10-SAMs on Au electrodes. Figure 7C and D shows the chromatograms of the spiked urine obtained with electrochemical detectors modified with n-alkanethiol. The results illustrate that the C10-SAM film effectively
2436 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
CONCLUSIONS The experiments described herein confirm the good permselectivity of C10-SAM-coated Au electrodes based on polarity for 4-AP, PRCT, NE, and DA. Particularly attractive is the ability to control the permselective behavior by carefully choosing different chain lengths and modifying times. This advantage is exploited for the amperometric monitoring of flowing streams including flow injection and chromatographic detection. The EC detector with C10-SAM-coated Au electrodes has high selectivity, good stability, and moderate sensitivity. It can eliminate the influence of uric acid at high concentration and simplify the chromatograms for the detection of 4-AP, PRCT, NE, and DA in urine. It is expected that this EC detector will be applied in the chromatographic detection of 4-AP, PRCT, NE, and DA in other biological samples such as serum, plasma, biological fluids, and so on. ACKNOWLEDGMENT The support of the National Natural Science Foundation of China is greatly appreciated. Received for review December 29, 1995. Accepted April 19, 1996.X AC9512564 X
Abstract published in Advance ACS Abstracts, June 1, 1996.
