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TECHNICAL NOTES On-Detector Injector for Flow Injection Amperometrlc Measurements. The Wail-Jet/Thin-Layer Cell Joseph Wang* and Liang Chen' Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
INTRODUCTION Because of its attractive features, flow injection analysis (FIA) has received considerable attention for high-speed analysis of discrete samples (I, 2). The technique involves the injection of small sample volumes into a carrier stream that transports the sample zone toward a detector. The detector is normally placed at the end of the system (Figure 1A). However, with the growing trend toward miniaturization and the development of active detectors, several groups have recently demonstrated the advantages of placing the detector within the injection valve. Hence, on-valve optical monitoring of extraction processes (3) or amperometric biosensing of glucose (4) have been successfully accomplished. Other efforts have focused on the coupling of the separation and detection functions in FIA systems (5). Reported here is a novel approach based on placing the sample injector directly within a common electrochemical detector. Electrochemical detectors account for one-third of the detectors used in FIA (5). Several cells, originally developed for liquid chromatography/electrochemistry(LC/EC), have been employed in FIA, with the thin-layer (6) and wall-jet (7)configurations representing the most popular ones. however, considering the different requirements and conditions of FIA and LC systems and the growing popularity of FIA, it is desirable to design new detectors that address the specific needs of FIA systems. The injector/detector described in the present work separates the sample and carrier introduction functions within the detector body (Figure 1B). This is accomplished with the carrier stream flowing parallel to the working electrode while the sample striking its surface perpendicularly. For this purpose, a conventional (commercial) thin-layer cell is modified for accommodating the sample introduction opposite the working electrode (Figure 2). Samples are reproducibly injected, with a microsyringe, through a rubber septum placed in a cavity above a nozzle inlet. (Such gas chromatographic like injection is facilitated by the low pressures of FIA systems.) The resulting hybrid wall-jet/thin-layer injection/ detector unit exhibits a very attractive FIA performance, the characteristics of which are explored and illustrated in the following sections. EXPERIMENTAL SECTION Apparatus. The wall-jet/thin-layer cell design is shown in Figure 2. A "homemade" upper block was made of Plexiglas to combine with a commercial lower block of a thin-layer detector (Model TL-5, Bioanalytical Systems (BAS)),containing the glassy carbon working electrode and the carrier solution inlet and outlet. A hole (3 mm narrowed to 0.5 mm) was drilled through the center of the Plexiglas body to accommodate the rubber-diaphragm injector and to serve as inlet nozzle through which samples impinge perpendicularly onto the working electrode. Teflon spacers maintained the channel width at 175 pm. The reference and Permanent address: Anhui University, Hefei, People's Republic
of China.
0003-2700/91/0383-1499$02.50/0
auxiliary electordes were located in a downstream compartment (Model RC-BA, BAS). All potentials were reported versus the Ag/AgCl reference electrode (Model RE-1, BAS). Samples (usually 10&) were injected with a Hamilton mimyringe (Model 705). The carrier reservoir (a 400-mL Nalgene beaker) was connected to the cell through Teflon tubing. Flow of the carrier solution was maintained by gravity. The FIA system and thinlayer cell, used in comparison studies, consisted of carrier and sample reservoirs, interconnecting tubing, a Rheodyne Model 5020 injection valve, and a Model TL-5 (BAS) detector. The detector was placed 6 cm from the valve. Electrochemical detection was performed in the amperometric mode with an EG&G PAR Model 173 potentiostat/galvanostat, the output of which was displayed on a Houston Omniscribe strip-chart recorder. Reagents. All solutions were prepared with double-distilled water. The chemicals used were the following: caffeic acid, glucose (Sigma), and potassium ferrocyanide (Baker). Glucose oxidase (Type X-S,EC 1.1.3.4) was also purchased from Sigma. The poly(eatel-sulfonic acid) polymer (Eastman AQ-55D) was obtained from Eastman Kodak Co. Supporting electrolytes were 0.05 M potassium nitrate and 0.05 M phosphate buffer (pH 6.5). Procedure. In most experiments, the polished glassy carbon electrode was potentiostated at +0.5 V and transient currents were allowed to decay. Glucose measurements were carried out at +0.95 V. Theae measurements were performed after covering the surface with a 10-pL drop of the mixed polymer/glucose oxidase solution (1.4% polymer/l mg/mL enzyme). The coatings were then dried for 15 min with a heat gun, held 40 cm above the surface.
RESULTS AND DISCUSSION The attractive features of the hybrid wall-jet/thin-layer injector/detector are best illustrated when comparing its FIA response with that of a conventional thin-layer cell. Such comparison is shown in Figure 3, from the amperometric peaks for 20 p L ferrocyanide solutions; both operations employed the same working electrode under otherwise identical conditions. The negligible dispersion, associated with the short residence time of the sample in the wall-jet/thin-layer detector, results in a very fast response (b). The baseline peak widths are 6 and 4 s (at 0.6 (A) and 1.1 (B) mL/min, respectively), as compared to 21 and 14 s at the thin-layer cell (a). Even much shorter response times can be obtained by coupling smaller sample volumes with faster carrier flow rates. For example, with 5 pL and 1.9 mL/min, the baseline peak width is only 0.7 s (c). Such significantly short response times of the integrated unit correspond to extremely high injection rates of more than 3600 samples/h. The fast response indicates that injected samples are rapidly replenished (and "pushed") from the surface by the flow-by carrier solution. Note atso that the sensitivity of the wall-jet/thin-layer detector compares favorable with that of the thin-layer one (ca. 45% higher). The latter is attributed to the convection "pulse" (associated with the injection action), which leads to further reduction of the diffusion-layer thickness. The high speed and sensitivity of the wall-jet/thin-layer detector are coupled with excellent reproducibility and absence of carryover effects. These are illustrated from the series of 0 1941 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 14,JULY 15, 1991 P
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Figure 1. Configurations of different FIA systems. (A) Conventional F I A (B) FIA with ondetector injector; (C) FIA wtth ondetector reagent introduction; (D) reversed FIA wRh ondetector carrier introduction.
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Flgure 4. Repetitive injections with the walCjetlthtn-Iayerdetector. (A) Carryover between sequential samples of 1 X lo4 M (L) and 2 X lo4 M (H) caffeic acid. (B) Precision for replicate injections of a 3 X lo4 M ferrocyanide solution. Sample volume, 10 LLL;other conditions, as in Figure 38. FLOW RATE (cm3min-’]
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Figure 2. Wall-jet/thin-layer detector. (A) Carrier solution inlet; solution outlet; (C) sample introduction; (D) working electrode; reference electrode; (F) auxiliary electrode.
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TIME Figure 3. Flow injection current-time profiles for 3 X lo4 M ferrocyanide at the thin-layer (A) and wall-jetlthin-layer (B) detectors: sample volume, 20 (a, b) and 5 (c) pL; operating potential, +0.5 V; flow rate, 0.6 (a), 1.1 (b), and 1.9 (c) mL/min; carrier and electrolyte, 0.05 M KNOS.
sequential injections of caffeic acid solutions differing in concentration and from 54 repetitive measurements of a 3 x lo4 M ferrocyanide solution (Figure 4, A and B, respectively). The former exhibits no observable carryover, while the later yields a relative standard deviation of 1.7%. Note (again) the extremely sharp response peaks. The high precision indicates that the microsyringe offers reproducible sample introduction. The reproducibility, and overall performance, may be further improved through automation. Response characteristics resulting from the unique coupling of flow-by (carrier) and flow-onto (sample) have been examined. Figure 5 shows the effect of several experimental variables upon the response of the wall-jet/thin-layer cell. The
Figure 6. Flow injection peaks for 3 X lo4 M ferrocyanide at carrier flow rates of 0 (A), 0.7 (B), and 1.5 (C) mL/mln. Other condtions as in Figure 3B.
flow injection peaks are independent of the carrier flow rate (A), indicating that it is the jet emerging from the nozzle (Le., the injection action) that governs the analyte flux a t the surface. Such flow rate independence can be attractive to various FIA applications, as it minimizes the carrier consumption (without compromising the sensitivity) and offers high reproducibility (even when the flow rate fluctuates). The peak height increases slowly with increasing sample volume
Anal. Chem. 1881, 63,1501-1504
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TIME Figure 7. Flow injection peaks for glucose at the glucose oxidase walCjet/thin-layer detector: (A) 6 X lo4 M; (B) increasing concentrations (2-6) X lo4 M (a-c). Sample volume, 10 wL; operating potential, +0.95 V; fbw rate, 0.9 mLlmin; carrier and electrolyte, 0.05 M phosphate buffer (pH 6.5).
(between 5 and 20 rL), after which it starts to level off (B). In addition, the peak decreases slowly as the inlet-electrode distance increases between 250 and 500 Wm (C). A unique situation occurs for a zero carrier flow rate (Figure 6). In accordance with the flow rate independence (reported in Figure 5),such a flow rate yields a peak current similar to that observed at higher flow rates (A versus B and C). However, in the absence of the carrier pushing action, the fast rise in the current is followed by a very slow decay (in a manner analogous to stopped-flow FIA experiments). Repetitive injections of ferrocyanide solutions of increasing concentrations ((1- 6) X 10" M) were used to estimate the linearity and detectability. The response was linear over the entire concentration range (slope, 2.61 pAlmM; intercept, 14 nA; correlation coefficient, 0.999 (not shown)). The signalto-noise characteristics ( S I N = 3 ) of the 1 X M ferrocyanide peak indicated a detection limit of 7 X lo-' M. Analogous measurements of caffeic acid (e.g., Figure 4A(L)) yielded a detection limit of 3 X lo-' M. The placement of the sample injector onto the detector is particularly attractive in connection with active detector surfaces. Highly specific enzyme electrodes, resulting from the judicious immobilization of biocatalysts onto the glassy carbon surface, represent such an opportunity. Figure 7 illustrates the performance of a glucose oxidase based walljet/thin-layer detector. The enzyme electrode responds rapidly and reproducibly to the dynamic changes in the glucose concentration. For the 24 repetitive injections (shown in B) the relative standard deviation is 1.2%. At least 240 glucose measurements can be made every hour at physiolog-
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ically relevant concentrations. The enzyme immobilization was accompplished by incorporation within a Kodak-AQ poly(ester-sulfonic acid) film. Because of the additional (protection and germselective) functions offered by AQ polymers (8),such incorporation is extremely attractive for integrated injectorlreactorldetector systems for FIA (which lack solution handling capability). In conclusion, the on-detector injector has been demonstrated to be a very practical device, which addresses the trends toward integration and miniaturization of FIA systems. In addition to its ultrafast (flow-rate independent) response and high sensitivity, such a wall-jet/thin-layer configuration holds promise for performing other FIA procedures. In particular, the versatility and flexibility obtained by introducing the carrier and sample through different inlets (on the detector body) allows additional schemes. These may include on-detector reagent introduction (with minimum dispersion of the product, Figure 1C)or reversed FIA (with on-detector injection of the carrier into the flowing sample solution, Figure 1D). Such schemes should further minimize reagent consumption and enhance the sensitivity, in comparison to analogous FIA systems with separated injector and detector components (1,9). Stopped-flow, flow-reversal, and titrimetric procedures, performed a t the surface, can also be envisioned. The separate control of the sample and carrier introduction should facilitate such schemes and may offer new FIA procedures. As such, the on-detector injection is much more versatile than on-valve detection. In addition, such modification of commercial flow cells offers greater convenience, simplicity, and reliability than the on-valve microelectrode detector ( 4 ) . The concept of on-detector injector should be suitable for other (nonelectrochemical)detection schemes. A direct injection into a large optical (absorption) flow cell has already been reported (10). Registry No. Eastman AQ-55D, 116326-12-6; glucose, 50-99-7.
LITERATURE CITED (1) Ruzicka. J.; Hansen, H. E. Flow In/ection Analysis; Wiiey: New York, 1981. (2) Christian, G.: Krull, I.; Tyson, J. Anal. Chem. 1990, 62, 455A. (3) Canete. F.; Rios. A.; Luque de Castro, M. D.; Valcarcei, M. Anal. Chem. 1988. 60. 2354. (4) Wang, J.; Li, R. Anal. Chem. 1990, 62, 2414. (5) Valcarcel, M.; Luque de Castro, M. D. Anal. Roc. 1989, 26, 313. (6) Kissinger. P. R. J. Chem. Ed. lB83, 60, 308. (7) Fleet, 0.; Little, C. J. J . Chromatogr. Sci. 1974, 12, 747. (8) Wang, J.; Golden, T. Anal. Chem. 1989, 61, 1397. (9) Johnson, K. S.; Petty, R. L. Ami. Chem. 1982, 54, 1185. (10) Eswara Dutt. V. V. S.; Mottola, H. A. Anal. Chem. 1975, 47, 357.
RECEIVED for review December 18,1990. Accepted April 8, 1991.
Application of Scanning Electrochemical Microscopy to GenerationKollection Experiments wlth High Collection Efficiency Chongmok Lee, Juhyoun Kwak, and Fred C. Anson* Arthur Amos Noyes Laboratories, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 The technique of scanning electrochemical microscopy (SECM) was introduced by Bard and co-workers (1)who have utilized i t in a variety of experiments (2). The use of a microelectrode placed close to a substrate electrode to detect
* To whom correspondenceshould be addressed. 0003-2700/9 110383-150 1$02.50/0
electroactive products generated at the latter, as described by Engstrom and ceworkers (3,4),is one attractive application of the SECM technique (I). In previous studies, this type of generationldetection experiment has usually been carried out with the microelectrode used as the detector electrode (1-5). In the present study, a larger (100 pm diameter) substrate 0 1991 American Chemical Society
