Anal. chem. 1003, 65, 1893-1896
1803
On-Line Monitoring of Hydrophobic Compounds at Self-Assembled Monolayer Modified Amperometric Flow Detectors Joseph Wang,' Hui Wu, and Lucio Angnest Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Substantial improvements in the selectivity of amperometric monitoring of flowing streams are obtained by using detectors coated with neutral n-alkanethiol monolayers. Permselective transport properties, based on solute polarity, are obtained and add a new dimension of information to amperometric detection. An elegant way of varying the transport properties of hydrophobic drugs and the exclusion of hydrophilic compounds is achieved by varying the chain length (Le., hydrophobicity)of the n-alkanethiolmodifier.The dynamic behavior of the detector is evaluatedwith respect to the thiol chain length or concentration, flow rate, solute concentration, and other variables. The surface coating, and hence the discriminative properties, are highly stable under the vigorous hydrodynamic conditions existing in the flow cell. Highly selective flow injection measurements of chlorpromazine in an untreated urine sample are illustrated.
INTRODUCTION Flow-through amperometric detectors have proven themselves as highly powerful means for on-line monitoring of electroactive species.14 For example, liquid chromatography with electrochemical detection (LCEC) has become one of the most widely used approaches for trace organic analysis, while flow injection amperometric procedures have found useful applications for industrial processing and quality control testing. While offering excellent sensitivity and high specificity (toward electroactive species), amperometric detectors lack the ability of distinguish between solutes possessing similar redox properties. Further improvements in their selectivity is thus desired to meet new challenges posed by increasingly complex sample matrices. One promising avenue to impart higher selectivity to amperometric detection is to cover the surface with an appropriate permselective coating."' Such surface barriers effectively exclude from the detector undesired interfering species while allowing transport of the analyte. Such a rapid separation step, performed in situ a t the detector surface, often alleviates the need for time-consuming sample pretreatments. A large variety of discriminative coatings have
* To whom correspondence should be addressed.
Permanent address: Instituto de Quimicada USP,Sa0 Paulo, Brazil. (1)Stulik, K.; ParBkova, V. Electroanalytical Measurements in Flowing Liquids; Ellis Horwood: Chichester, 1987. (2)Kissinger, P. R.Electroanalysis 1992,4,359. (3)Johneon, D.C.; Lacourse, W. R. Anal. Chem. l990,62,589A. (4)Stankovic, R.;Bond, A.M.; Butler, E.Electroanalysis 1992,4,453. (5)Wang, J. Anal. Chzm. Acta 1990,234,41. (6)Baldwin, R. P.; Thomeen, K. N. Talanta 1991,38,1. (7)Wang, E.;Ji, H.; Hou, W. Electroanalysis 1991,3,1. t
0003-2700/93/0365-1893$04.00/0
been suggested for this purpose, including size-selective cellulose acetateas@or poly(1,2-diaminobenzene),10 chargeselective Ndion,llJ2 Eastman AQ,13 or poly(vinylpyridine)14 and hydrophobic lipid15 layers. In the present paper we describe the behavior, utility, and advantages of an amperometric flow detector coated with self-assembled monolayers (SAMs) of unsubstituted n-alkanethiols. The formation and structure of SAMs are of considerable recent interest.l&Z4 This monolayer self-assembly technique is expected to yield a very simple, and yet highly versatile, controllable and stable approach for tailoring electrode surfaces. The well-defined SAM films have already proven to be extremely useful for studying ion binding,1'-21 for incorporating redox couples into electrochemical interfaces,19920 for studying protein adsorption,18922 or for blocking electron transfer between redox species and electrode surfaces.16Js.24 In the latter case (published upon completion of this work) a carboxylic acid terminal group was used to exert permselectivity based on charge. Yet, despite the growing attention given to electrodes coated with SAMs, and despite their great analytical prospects, such surfaces have not been used for enhancing the capabilities of amperometric flow detectors. In the followingsections we illustrate that coverage of walljet electrochemical detectors with neutral SAM layers can impart high selectivity toward hydrophobic compounds (through discrimination against coexisting polar interferences). In particular, the flow injection response is elegantly manipulated via a judicious choice of the chain length of the bound n-alkanethiol molecule. Long, and hence hydrophobic, alkyl chains result in the permeation of hydrophobic drugs into the SAM film. The finely controlled access toward the detector surface is coupled with a fast and sensitive response (toward target analytes) and good mechanical stability, as desired for on-line monitoring of flowing streams. (8)Sittampalam, G.; Wilson, G. S. Anal. Chem. 1983,55,1608. (9)W a g , J.; Hutchins, L. D. Anal. Chem. 1985,57,1536. (10)Sasso, S.;Pierce, R.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990,62,1111. (11)Ji, H.;Wang, E. J. Chromatogr. 1987,410,111. (12)Shimazu, K.; Kuwana, T. J. Electrochem. SOC.1988,135,1602. (13)Bremle, G.; Persson, B.; Gorton, L. Electroanalysis 1991,3,77. (14)Wang, J.; Golden, T.; Tuzhi, P. Anal. Chem. 1987,59,740. (15)Wang, J.; Lu, Z.Anal. Chem. 1990, 62,826. (16)Porter, M.; Bright, T.; Allara, D. Chidsey, C. J. Am. Chem. Soc. 1987,109,3559. (17)Steinberg, S.;Tor, Y.; Sabatani, E.; Rubinstein, I. J. Am. Chem. SOC.1991,113,5176. (18)Prime, K.; Whitesides, G. Science 1991,252,1164. (19)Chidsey, C.; Bertozzi, C.; Putvinski, T.; Mujsce, A. J . Am. Chem. SOC.1990,112;4301. (20)Tsutsumi. H.;Furumoto, S.; Morita, M.; Matauda, Y. J. Electrochem. SOC.1992,139,1522. (21)Sun,L.;Johnson, B.; Wade, T.; Crook, R. J. Phys. Chem. 1990, 94,8869. (22)Collinson, M.; Bowden, E.; Tarlov, M. Langmuir 1992,8,1247. (23)Chen, Q.;Brajter-Toth, A. Anal. Chem. 1992,64,1998. (24)Malem, F.;Mandler, D. Anal. Chem. 1993,65,37. 0 1993 Amerlcan Chemlcal Society
1894
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
EXPERIMENTAL SECTION Apparatus. The “home-made”flow injection systemconsisted of a 400-mL carrier reservoir, a Rainin Model 5041 sample injection valve (20-rLloop), interconnecting tubing, and a large volume wall-jet detector. Details of the detector configuration were given earlier.25 The nozzle was kept 1 mm away from the center of the working electrode. Flow of the carrier solution was maintained by gravity. Experiments were performed using an EG&G Model 264 voltammetric analyzer connected to a Houston Omniscribe strip-chart recorder. All potentials were measured againstthe Ag/AgC1(3M NaC1) reference electrode (Bioanalytical Systems (BAS),Model RE-1). Cyclic voltammetry was carried out in a 10-mL cell (Model V-2, BAS). Electrode Preparation. Before its modification, the gold disk electrode (1.6-mm diameter, Model MF-2014, BAS) was polishedwith a 0.05-rm alumina slurry,rinsed with distilled water, sonicated in a water bath for 5 min, and dried. Modification of the gold electrode was accomplished by soaking it in a quiescent 50150% (viv) ethanol/octane solution containing 25 mM of the desired n-alkylthiol. After a 1-min equilibration, the electrode was removed from the solution, rinsed in ethanol, and allowed to dry. Reagents. All solutions were prepared with double-distilled water. Ascorbic acid, potassium ferrocyanide (Baker), hexanethiol (Kodak), ethanethiol, dodecanethiol, octadecyl mercaptan, n-octane (Aldrich), dopamine, promethazine, chlorpromazine, hydrogen peroxide, and ethanol (Sigma) were used without further purification. The supporting electrolyte1carrier solution was a 0.05 M phosphate buffer (pH 7.4). The urine sample was obtained from a healthy volunteer and diluted with the supporting electrolyte solution. Procedure. Flow injection measurements were made by applying the desired working potential (usually +0.80 V) and allowing the transient current to decay. All experiments were performed at room temperature.
A 1
4
~
~
a /
-
b
b
,
C ____
d e I
-’
e
de--
O G 0.2 - 0 2 0.G 0.2 -0.: 0.G 0.2 -0.2 06 0 2 -02
POT ENTI A L /V Figure 1. Cyclic voltammograms for 1 X M promethazine (A), chlorpromazine(B), potassium ferrocyanide(C), and hydrogen peroxide (D) at the bare (a)and CT (b), CE- (c),C12- (d), and CIE-SAMcoated (e)electrodes. Scan rate, 50 mV s-l;electrolyte, 0.05 M phosphate buffer (pH 7.4).
RESULTS AND DISCUSSION Transport Characteristics a n d Dynamic Behavior. Batch cyclic voltammetric experiments were used first to evaluate and demonstrate the ability to control the transport toward the gold surface via the use of neutral n-alkanethiol (CnH2,+1SH) molecules of different chain lengths. The adsorption of these compounds a t gold electrodes is known to yield monolayers of thiolates.26 Figure 1 displays cyclic voltammograms for promethazine (A), chlorpromazine (B), potassium ferrocyanide (C),and hydrogen peroxide (D) utilizing SAM films with chain length varied from n = 2 to n = 18 (b-e). Also shown is the corresponding response at the bare gold surface (a). All four compounds are readily transported through the coating made of the shortest (n=2) alkanethiol. In contrast,the hexanethiol- and dodecanethiol(n=6 and 12) coated electrodes effectively exclude the ferrocyanide ion and greatly diminish the hydrogen peroxide contribution, while exhibiting a large response toward the hydrophobic drugs. The coating made of the longest (n=18) alkanethiol offers a nearly complete exclusion of all compounds. Also notice the gradual anodic shift (of up to 60-70 mV) in the peak potential upon increasing the alkyl chain length. These changes and the increased peak separation for ferrocyanide (a vs b) indicate change in the electron-transfer rates. The data of Figure 1 clearly illustrate that different permeabilities are obtainable via judicious choice of the chain length and that polarity plays a major role in the transport mechanism. In particular,the change in the barrier properties (with the chain length) differs from solute to solute in accordance to its polarity. (25) Wang, J.; Freiha, B. Anal. Chem. 1985, 57, 1776. (26) Widrig, C. A.; Chung, C.; Porter, M. D. J . Electroanal. Chem. 1991,310, 335.
0.2
1 aa
0.0
1 2 3 4 5 0 5 15 25 COMPOUND CONCENTRATION/mM
Flgure 2. (A) Permeability of the C,Ji2+lSH-coated flow detectors, using different chain lengths: n=6 (a),n= 12 (b),and n= 18(c). Solutes used were ferrocyanide (l),ascorbic acid (2), hydrogen peroxMe (3), chlorpromazine (4), and dopamine (5). (B) Effect of the CIOHBISH concentration upon the permeability of ascorbic acid (a) and chiorpromazine (b). Flow injection conditions: operating potential, +0.80 V; flow rate, 1 mL min-I; carrier, 0.05 M phosphate buffer; analyte concentration, 1 X lo-‘ M. Subsequent work was thus focused at exploiting the controllablepermeability of SAM coatings for improving the selectivity of amperometric detection for flowing streams. Figure 2A shows changes in the permeability of three such films for five different analytes. The ratio between the flow injection peak current a t the SAM-coated electrode over that a t the bare detector surface (idit,)is used as a measure of the permeability. Distinct permeability profiles, and an increase in the transport are observed upon changing from the polar to the nonpolar species. The resulting trend in permeability is the following: dopamine > chlorpromazine > hydrogen peroxide > ascorbic acid > ferrocyanide. Additional factors, such as the solute charge and size also appear to affect the response (although to a much lesser extent). The access to the detector surface is strongly influenced by the SAM chain length. For example, while the octadecyl-mercaptan (CIS) coating blocks nearly complete the transport of ferrocyanide and ascorbic acid species, the hexanethiol (CB) and dodecanethiol (C12) films offer only ca. 50-60% attenuation of
ANALYTICAL CHEMISTRY, VOL. 85, NO. 14, JULY 15, 1993
I
0.0 0.3
0.9
1.5
1895
2.1
FLOW RATE (cm3min-1) Flgure 4. Effect of the flow rate upon the flow Injection response to 1 X lo4 M dopamine, at the bare surface (A), and Clr (e), and Clr (C) SAM coated detectors. Condltlons as In Flgure 2.
I B
A
TIME FJouro 3. Flow Injection peaks for 1 X 10-4 M dopamlne (a) and chlorpromazine (b) are the bare and Cl&oated electrodes(dotted and solld lines, respecthrely). Other conditions as In Figure 2.
their peaks. The permeabilityof hydrogen peroxide decreases from 0.52 to 0.07 upon increasing the chain length from n=6 to n=18. The more hydrophobic chlorpromazine and dopamine exhibit high permeability at the different films (with iPd ip,b values between 0.31 and 0.97). These data indicate that the packed assembly forms an effective barrier for the transport of polar compounds. Hydrophobic compounds, in contrast, are readily partitioned and permeate through the monolayer. The practical detection utility of such fine tuning of the response will be discussed later on in this paper. Comparison of the permeability data of Figures 1 and 2A indicate a similar trend upon changing the chain length. Yet, the exact permeability values differ, as expected in the change from quiescent to flowing solutions. Also shown in Figure 2 is the effect of the n-alkanethiol concentration (in the “modification”solution) on the film permeability (B).The permeability decreases upon increasing the concentration between 5 X 10-8M and 2.5 X 10-2M. The latter provides a 99% attenuation of the ascorbic acid, while retaining 32% of the chlorpromazineresponse. A modifier concentration of 2.5 X 10-2 M was thus used for all subsequent work. The response time of the coated electrode is an important parameter when monitoring dynamic flowing streams. Because transport through the film is a major contributor to the total diffusional resistance, slower response characteristics are expected at the coated electrode. Figure 3 displays characteristic flow injection current-time profiles for dopamine (a) and chlorpromazine (b) at the bare and C18-coated electrodes (dotted and solid lines, respectively). The coated electrode exhibits rapid increase and decrease of the current, indicating a facile transport of these analytes. The peak widths (at 0.6 i,) are 8 s, as compared to 6-7 at the uncoated electrode. Some peak tailing is also observed versus the bare surface (widths at 0.1 i, of 50 s vs 35 s), indicating slower ‘wash out” characteristics. This slight broadening is not a matter of major concern as high injection rates (of ca. 40 samples/h) are maintained. Figure 4 shows the dependence of the peak current upon the flow rate at the bare (A), and Clz- (B)and CIS-(C)coated electrodes. At the uncoated detector, the current rises sharply between 0.3 and 1.5 mL/min, and then it starts to level off. In contrast, both coated detectors exhibit reduced flow rate
1,,t
, 12 min
TIME Flgure 5. Flow lnjectlon response of the ClrSAM coated (A) and bare (e)detectors to (a) 1 X lo4 M dopamlne; (b) 1 X lo4 M ferrocyanlde; (c and d) as (b) but after additions of 1 X 1O4 M dopamine. Condltions as In Flgure 2.
dependence, with a slightly increasing response between 0.3 and 0.9 mL/min, followed by a return to the original value at 1.5 mL/min, and no further change at higher flow rates. Such profiles reflect conditions of a mixed film/solution control, with the current approaching film control at higher flow rates.9~~’The decreased response above 0.9 mL/min can be attributed to the faster passage of the sample plug over the film. As expected, the trend in sensitivity is bare > Clzcoating > Cia-coating. The slight loss in sensitivity toward the target analytes is not of major concern, considering the inherent (ne) detectability of amperometric detection. Analytical Utility. As desired for routine detection applications, the present procedure relies on the use of conventional gold electrodes and a short self-assemblytime. The fine control of the permselectivity of SAM coatings, via a judicious choice of their chain length (in accordance with the profiles of Figure 2), providesan elegantway for enhancing the selectivity of amperometric flow detection. Particularly attractive is the successful exclusion of small and neutral solutes (e.g., hydrogen peroxide) which represents a major challenge to most common permselectivefilms which rely on size or chargepermselectivity. The selectivity advantage that accrues from the permselective transport of the SAM-coated detedor is illustrated in Figure 5. Due to its additive response, it is not possible to use the bare electrode for selective detection (27) Gough, D.A.; Leypoldt, J. Anal. Chem. 1979,51,439.
1896
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
A
E
E2
i)
u
I l l
3 min
2
4 TIME
Flow injection peaks of the bare (A) and Cl8-SAMcoated (6)detectorsto 1 X lo4 M ferrocyanide (a),dopamine (b), and ascorbic acid (c). Numbers indicate the time interval (in hours) between these series of injections. Conditions as in Figure 2. of dopamine in the presence of ferrocyanide (B). In contrast, the coated electrode(A) effectively excludesferrocyanidefrom reaching the surface (b), and dopamine can be conveniently detected in the mixture (c and d). Note also the similar size of the dopamine peaks in the absence (a) and presence (c) of ferrocyanide. Similar improvements were observed for selective flow injection measurements of dopamine (0.1-0.3 mM) in the presence of 0.1 mM ascorbic acid (not shown). The ability to selectively measure dopamine in the presence of ascorbic acid was illustrated recently in connection with cyclic voltammetric experiments at charged-exclusion SAM layers.24 The concentration dependence of the SAM- (Cle-) coated detector was evaluated for flow injection measurements of increasing concentration of chlorpromazine and dopamine (eight successiveinjections over the 0.5-4.0 mM range). The peak response increased linearly with the concentration (not shown); the slope of the resulting calibration plots corresponded to a sensitivity of 23.2 and 19.9pA/mM, respectively (correlation coefficients, 0.999). A series of 20 repetitive injections of a 1 X 10-4 M dopamine solution was used to evaluate the precision of the response [conditions as in Figure 2A (c)]. Highly reproducible results were obtained, with a relative standard deviation of 1.2% (mean, 1.84 FA; range, 1.78-1.88 PA). Such precision compares favorably with that common at bare-electrode detectors. An attractive property of the SAM-coated detector is the inherent stability of the film under the vigorous hydrodynamic conditionsof amperometric flow cells. The long-termstability was studied from repetitive injections of ferrocyanide, dopamine, and ascorbic solutions (at a flow rate of 1.0 mL/ min) over a 6-h period, Typical results (at 2-h intervals) are shown in Figure 6B. Also shown, for comparison, are the corresponding peaks at the uncoated detector (A). These data indicate that the permselective response is maintained throughout this prolonged period, with an effective rejection of the polar ascorbic acid and ferrocyanide species and no apparent change in the dopamine response. Only a slight increase in the permeability is observed (e.g., from 0.043 to 0.055 for ascorbic acid). Even more dramatic and significant are the resulb obtained with complex physiological fluids, such as urine (Figure 7B).
b
c
d
e
TIME
6
Figure 6.
b
B
a 0
a
Flgure 7. Flow injection peaks of the bare (A) and C18-SAMcoated (B) detectors to a diluted (1:20) urine sample (a, c, d, and e) and to the diluted urine containlng 1 X lo4 M Chlorpromazine (b). Peaks c, d, and e were recorded in 2-h intervals (after a and b). Conditions as in Figure 2.
The SAM film effectively restricts all electroactive constituents of the urine from reaching the surface (a, c, d, and e), while allowing a highly selective and sensitive detection of the spiked (1 X 1V M) chlorpromazine drug (b). The exclusion of endogenous components is retained over the 6-h period of operation (compare a and e). The bare detector, in contrast (A), exhibits a large contribution of the urine constituents (a) which does not permit selective detection of the spiked drug (b). Overall, the data of Figures 6 and 7 indicate that the integrity (organization and packing) of the SAM layer remain intact over long periods. In conclusion, the experiments described above confirm the expectation placed in the utility of SAM-coatedelectrodes for amperometric monitoringof flowing streams. Such neutral monolayers add a new dimension of information, based primarily on polarity, to amperometric flow cells. Such discriminative properties are usually difficult to achieve utilizing common (size- or charge-) permselective polymeric films. Particularly attractive is the ability to fine tune the permselective behavior via the use of different chain lengths. Such rational approach for designing interfaces should have important implications on flow analysis. The effective separation step, performed in situ at the detector surface, has been useful for "fishing out" a single drug from a complex biological matrix. While the concept has been illustrated in connection with flow injection systems, it should greatly benefit other analytical flow systems, such as liquid chromatography. For example, amperometric chromatograms of complex matrices at flow-through gold detectors should be greatly simplified by the isolation of hydrophobic compounds from coelutinginterferences. Multifunctional coatings, which couple the permselective transport of SAM layers with electrocatalytic, preconcentration, or biocatalytic functions should offer additional advantages. Mixed or multilayers should also be considered for this task. Applications to other electrochemical devices, including amperometric biosensors, are also anticipated. Finally, flow injection systems can serve as useful tools for elucidatingthe transport properties of SAMcoated electrodes.
RECEIVED for review January 27, 1993. Accepted April 9, 1993.
