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Anal. Chem. 1983, 5 5 , 1285-1288
The quantitative applications to analysis demonstrated here suggest clearly that other functions utilizing the discriminating power of sinusoidal hydrodynamic modulation could also make use of the derivative readout technique. A relevant example would be the diagnostic and quantitative functions of modulated currents in selecting and interpreting the voltammetric behavior of redox couple-semiconductor pairings in photoelectrochemical cells (5).
LITERATURE CITED Miller, B.; Bruckensteln, S. Anal. Chem. 1974, 46, 2026. Rosamllla, J. M.; Miller, B. Ant?/. Chem. 1983, 55, 1142. Mlller, 8.; Bruckensteln, S. J . Electrochem. SOC.1974, 721, 1558. Bond, A. M. “Modern Polarographic Methods In Analytical Chemistry”; Marcel Dekker: New York, 1980; Chapter 4. (5) Bruckensteln, s.; hAlller, B. J . Electrochem. SOC. 1982, 729, 2029. (1) (2) (3) (4)
RECEIVED for review ?January21,1983. Accepted April 1,1983.
Selective Voltammetric Detection Based on Adsorptive Preconcentration for Flow Injection Analysis Joseph Wang* and Bassam A. Frelha Department of Chemistty, Mew Mexico State Universi~,Las Cruces, New Mexico 88003
A new electrochemical detection scheme for flow iiijection systems based upon selective adsorptive accumulation of an analyte at the electrode surface followed by Its differential pulse quantitation is described. The remarkable sellectivity of the method (compared with conventional amperometric detection) Is a result of combining the intimate contact between the surface-bound species and the electrode surface with the medium-exchange procedure. The increased seiectivity thus obtained is demonstrated by the determination of chlorpromazine In a 102-foidexcess of nonadsorbabie solution species with similar redox potentials. Enchanced sensitlvity Is obtained as a result of the preconcentratlon step. The response of the system is characterized with resped: to preconcentration period, solution flow rate,,scan rate, concentration, and other variables. The operation of the system was optimized to achieve the desired balance between high sensitivity, rapid assay rate and minimum carry-over effstcts. At a flow rate of 0.3 mL/min, injection rates of 24 samples per hour and detection limits of a few nanograms are obtainable. Reproducible quantitation of chlorpromazine in urine1 is possible with no sample treatment.
Flow injection analysis (FIA) is that type of continuous flow analysis that involves injection of reproduciblesample volume into a continuously flowing unsegmented carrier stream, followed by quantitation of the species of interest at the downstream detection area (1-3). Due to its speed, simplicity, and use of small sample volumes, the technique has proved useful in practical applications in fields as varied as pharmaceutical, clinical, environmental, or agricultural a.nalysis. At present, FIA is very useful to those perform straightforward analyses routinely. Major improvements in the analytical capabilities of FIA systems can be achieved by developing new detection methodologies (in a similar way to the significant contribution of advanced detection modes to liquid chromatography). While photometric detectors are the most widely usred with FIA systems, the electrochemical detectlor is growing in popularity at a fastrate (4).Electrochemical detectors are suitable in flowing liquid streams because of their sensitivity, selectivity (toward electroactive species), economy, and linear response over a wide concentration range. Most electrochemical detectors utilize a constant-potential amperometric detection mode, because it has the advantage of being free of double0003-2700/83/0355-1285$01 SO10
layer charging and surface transient background effects. The combination of FIA with amperometric detection thus results with detection limits down to subnanogram amounts. However, fixed potential measurements do not furnish necessary information on the identity of the compound and, thus, have restricted use in the sense that they will rarely be specific for a particular species. Only when an enzyme is attached to the sensor surface, the selectivity of the enzymatic reaction yields a substrate-selective amperometric sensor (5). In many complex samples the advantages of a selective detector in isolating the species of the interest are apparent, and thus selective electrochemical detection schemes are desirable. In this paper, a new selective electrochemical detection methodology is presented. This approach is based upon selective and controlled preconcentration of the analyte from the flowing solution. Such preconcentration may be accomplished by spontaneous adsorption of the analyte or by its covalent attachment to the surface (via specific functionalities deliberately introduced to the surface). Since the surfacebound species retains its electrochemical characteistics, it can be quantified by using conventional voltammetric procedures. This approach has been employed recently for the batch (beaker) analysis of various analytes (6-10). Both signal enhancement and high degree of selectivity are achieved by the controlled preconcentration step. While the improvement in detectability is significant (detection limits -lo4 M), the main advantage of this methodology, in analyses of complex media, is the high degree of selectivity toward the adsorbed/attached electroactive species. The selectivity is enhanced by employing a medium exchange procedure, i.e., transferring the electrode from the complex sample to a blank electrolyte solution before the measurement step (8, IO). This allows direct determination of drugs in physiological fluids (urine and blood) without any pretreatment of the fluid. The advantages associated with this preconcentration approach have not been exploited yet for selective detection in flowing streams. The characteristics of the new flow injection detection scheme are evaluated by use of chlorpromazine as a model compound. This important antipsychotic drug is known to accumulate onto various carbon electrodes (7,8). EXPERIMENTAL SECTION Apparatus. Carrier and sample reservoirs were 400-mL Nalgene beakers, fitted with Nalgene covers. The sample injection valve was a Rheodyne Model 7010 with a 200-pL sample loop. All interconnections were made with 1.0-mm i.d. Teflon tubing. The length of tubing between the valve and the detector was 2.5 cm. Flow of the electrolyte carrier solution was maintained by 0 1983 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
gravity. Flow rates were calibrated volumetrically. A commercial thin-layer electrochemical detector (Model TL-4, Bioanalytical Systems Inc., West Lafayette, IN) with a carbon paste working electrode was used. The Ag/AgCl reference and Pt auxiliary electrodes were housed in a downstream compartment (Model RC-2, Bioanalytical Systems). The carbon paste was prepared by thoroughly mixing 2.5 g of graphite powder and 1.5 g of Nujol oil. All experiments were performed with a Princeton Applied Research Model 174 polarographicanalyzer, in conjunction with a Houston Omniscribe strip-chart recorder. Reagents. Chemicals and reagents used have been described previously (8). The urine samples were obtained from healthy volunteers and used without any preliminary treatment. All solutions were prepared daily. Procedure. Background and sample determinations were done as follows. The 0.1 M phosphate buffer carrier solution was allowed to flow through the cell. The valve was turned from the load position, in which the loop had been filled by gravity flow of the sample, into the inject position. After 5 s, a sufficient time for the solution to reach the detector, preconcentration was initiated by applying a potential of +0.3 V for a selected time (usually 60 s). At the end of this period, the surface-bound chlorpromazine was measured by applying a differential pulse anodic potential ramp to +0.9 V. A t the end of this scanning period the valve was returned to the load position. Keeping the electrode at +0.9 V for 60 s "cleans" the carbon paste of all chlorpromazine, readying the system for the next injection. Subtractive voltammograms were obtained with the Tektronix 4010 terminal of Prophet, a time-sharing computer system operated by the National Institutes of Health.
FLOW RATE,ml/ min 0
0.25
0.50
I
I
10-
I
a-
--0
a
4
i,, nA 5-
OA
I
I
1
25
50
75
PRECONC. PERIOD,s Flgure 1. Dependence of the FIAIdifferential pulse peak currents on the preconcentration period (a) and volume flow rate (b): 3 X M chlorpromazine in 0.1 M phosphate buffer; scan rate, 20 mVls; amplitude, 25 mV; repetition tlme, 1 s; flow rate (a), 0.2 mL/min; preconcentration period (b), 60 s;preconcentration potential, +0.3 V; sample volume, 200 pL
RESULTS AND DISCUSSION Manifold Procedure. Exploratory experiments were made first to optimize the operation of the system for achieving the desired balance between high sensitivity, rapid sampling rate, and minimum carry-over effects. The unique nature of the proposed methodology, preconcentration followed by potential scanning, dictates the use of lower sampling rate, larger sample volumes, and/or lower flow rates, as compared to those employed in amperometric detection. The resulting manifold procedure thus resembles in part the procedure suggested recently for the adaptation of stripping voltammetry of trace metals as detection mode for flow injection systems (11). In a flow injection system, the preconcentration period can be started as the sample arrives in the cell and terminated before or after the sample plug passed completely through. In the latter case, part of the so-called preconcentration period is not exploited for actual preconcentration due to the arrival of the carrier electrolyte. The manifold procedure has been designed to minimize this part, while still maintaining the advantage of the medium-exchange method, Le., presence of the carrier during the measurement step. Whether or not most of the preconcentration period is exploited for the accumulation depends mainly upon two experimental parameters: solution flow rate and preconcentration period. Figure 1shows the dependence of the differential pulse peak current upon these experimental parameters. As expected (7), the amount of chlorpromazine on the electrode is a function of the preconcentration period. As the length of this period increases, the peak current rises rapidly a t first and then more slowly (curve a). Flow rate of 0.2 mL/min and sample volume of 200 p L were employed in this experiment. Under these conditions, the dispersion of the sample plug is limited (D= 1.6, as was confirmed by amperometric detection of injected K4Fe(CN), solutions), Thus, the sample plug was present throughout the periods employed in this experiment. Due to the slow increase in the response above 60 s, this period was employed throughout the rest of this study as a compromise between sensitivity and speed (sampling rate). The peak current decreases with increasing the solution flow rate (Figure Ib). Increased flow rate results in enhanced transport of chlorpromazine to the surface (while the sample plug is a t the
0.75
4 3V
Figure 2. Differential pulse voltammograms for injections of a 5 X lo4 M chlorpromaztne solution, recorded at scan rates of 5 (a)and 20 (b, c) mV/s: 60 s preconcentration at +0.3 V; flow rate, 0.7 mL/min; electrolyte, amplitude, and repetition tlme, as in Figure 1; conventional mode (a, b), subtractive mode (c).
detector) and the earlier arrival of the carrier to the cell, i.e., shorter residence time of the sample plug in the detector. The net result of these two opoposite effects is a decrease of the response with increased flow rate (continuous steady-state measurements of the sample resulted with increased current at higher flow rates). Because of the reasons discussed above it is obvious that different preconcentration periods and injected volumes would yield different flow rate dependences. Some of the parameters of the differential pulse measurement step may also affect the performance of the system. For example, the potential scan rate may affect the sensitivity and speed of the system. Differential pulse voltammetry is usually performed with slow scan rates (-5 mV d);such rates greatly prolong the duration of the measurement step and thus result in low injection rates. To increase the speed, we examined the feasibility of using faster scan rates than those traditionally used. Figure 2 compares the response for injected 5 X lo4 M chlorpromazine solutions utilizing scan rates of 5 and 20 mV s-l. While a slight degradation in peak shape is observed at the faster scan rate, this rate was used throughout this study because of the significant gain in time (30 s vs. 120 s). The chlorpromazine peak current is accompanied by a background current associated with surface reactions and the oxidation
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8 , JULY 1983 1287
I
i
r“
1
d
JJ e
Figure 3. Effect of solution-phasespecies upon the chlor romazlne peak current. Injected solutions contalning 2.5 X 10-f hd chlorM chlorpromazlne end 5 X lo-‘ M ferropromazine (a), 2.5 X M chlorpromazine and 1 X M cyanide ion (b, c), and 5 X ascorbic acid (d, e). Flow rate was 0.25 (a, b), 0.6 (c, e), and 0.38 (d) mL/min. Electrolyte, preconcentration, and differential pulse ramp were the same as those given in Figure lb.
of water (dotted lines in Figure 2). These background contributions restrict the detectability of measurements a t the submicromalor concentration level. T o compensate the background current we have employed a computerized-subtractive approach in which the blank voltammogram, recorded while passing the carrier stream through the cell, is subtracted from the sample voltammogram. The resulting subtractive response (Figure 2c) shows well-defined peak current for chlorpromazine utilizing 60 s preconcentration. The advantage of this approach is obvious, when the subtractive curve is compared with the corresponding conventional response (curve b). Throughout this study, the measurement step was performed utilizing a flowing solution. Comparison to quiescent solution data, recorded by stopping the flow followiing the preconcentration (as in stripping voltammetry),yielded similar peak response (not shown). The use of continuous flow conditions simplifies the manifold procedure and thus is more suitable for automation. Effective flow analysis of discrete samples requries complete “cleaning” (desorption) of the surface-boundd y t e after each measurement to minimize carry-over effects. The “cleaning” conditions and period may differ from analyte to analyte, depending upon the nature of its interaction to the surface. In the case of chlorpromazine, we found that by holding the electrode at +0.9 V for 60 s, the adsorbed drug completely desorbed from the surface (as indicated from the restored background current and the precision or calibration experiments discussed below). Some analytels (e.g., adriamycin) attach so strongly to the surface (IO) arid may require mechanical renewal of the surface that cannot be achieved under continuous flow conditions. In such cases, on-line chemical or electrochemical surface renewals may also be useful. Overall, the resulting manifold proceduire employed in this work (60 s preconcentration, 30 s measurement, 60 s “cleaning”)allows an injection rate of 24 samples per hour. Analytical Applications. The main advantage of the adsorptive precncentration FIA detection approach is tlhe high degree of selectivity toward the surface-boundanalyte. Figure 3 illustrates the selective measurement of the adsorbable chlorpromazine in samples containing 20-200-fold excess of solution-phasespecies, ferrocyanide ion and ascorbic acid, that oxidize at a similar potential region. With relatively low solution flow rates (0.2-0.35 mL/min), part of the sample plug is still present at the detector during the measurement step. Thus, the chlorpromazine peak is obscurred by the high current associated with the oxidation of ferrocyanide ion and
Figure 4.
(1
Differential pulse voltammograms for injections of diluted
+ 3) human urine of isscendlng chlorpromazine concentrations: (a)
blank; (b-g) successive concentration increments of 1.5 X lo-’ M. Conditions, were the same as those given in Figure 1b except that the flow rate was 0.7 mL/rnin.
ascorbic acid, and its quantitation is not feasible (curves b and d, respectively). At higher flow rates the sample plug is eluted completely and the detector contains only the carrier electrolyte, The resulting differential pulse voltammograms show Well-defined peaks and low base line currents (curves c and e). Thus, the medium-exchange nature of the flow injection system provides discrimination against current8 associated with redox reactions of highly concentrated solution-phase species. f3uch selective measurements cannot b achieved by using conventional amperometricdetection where additive response is obtained; e.g., at a fixed potential of +0.9 V used for the amperometric detection of chlorpromazine, both ascorbic acid and ferrocyanide ion undergo oxidation. The decrease in the analyte peak current (compare curves a and c) is according to its flow rate-dependence discussed earlier (Figure lb). To demonstrate the suitability of the method to analyses of real samples, the concentration dependence and precision studies were performed utilizing urine samples. No sample preparation was used, other than a 1:3 dilution with the supporting electrolyte solution. Figure 4 shows differential pulse stripping voltammograms,obtained after six successive injections of urine samples of ascending chlorpromazine concentration ((1.5-9 0) X lo* M). These data yielded a linear calibration plot (also shown in Figure 4). Least-squares treatment of these data yielded the equation Z(n.4) = (0.48 f 0.03)C (lo* M) 0.31 f 0.18 nA with Syx = 0.19 nA and r = 0.992. At higher concentrations M), a deviation from linearity is expected (8, IO). In such cases, shorter preconcentrationperiods would be expected to allow extension of the linear range, as well as higher sampling rate. On the basis of the signal-to-background characteristics of the response, a limit of detection around 1x lo-’ M chlorpromazine (in diluted urine) is expected by using 60 s preconcentration. This value corresponds to 7 ng in the injection volume used. Even better detectability would be obtainable by using longer preconcentration periods or the subtractive mode (discussed earlier). The former case will result in lower injection rate. None of the urine electroactive constituents was observed to adsorb strongly onto carbon paste under the experimental conditions employed (as indicated from urine blank voltammograms, e.g., curve a). Weak adsorption of an electroactive constituent was reported recently (IO)and attributed to uric acid. Possible adsorption of nonelectroactive urine constituents may affect the number of surface sites available for the analyte adsorption. However, as long as the calibration curve is made by using the particular physiological sample, the eventual quantitatian would not be affected. The precision of the results has an important bearing on the utility of the method. The precision was estimated by eight repeated injections of diluted urine sample spiked with 8 X lo4 M chlorpromazine, over an unbroken 20-min period of operation. The resulting voltammograms are represented
+
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Anal. Chem. 1983, 5 5 , 1288-1291
2
1
4
3
2nA
~
aqueous samples (e.g., compare Figures 2 and 5). In conclusion, we have shown in this paper that voltammetry can be utilized for selective detection of adsorbable analytes in flow injection systems. Solution-phase electroactive species with similar redox potentials do not interfere due to the medium exchange nature of the flow injection systems. At the same time high sensitivity is maintained as a result of the preconcentration step. The simplicity of the method makes it superior to existing techniques for measuring adsorbable analytes. The extensive literature on chemically modified electrodes suggests many possibilities for extensions and elaboration. The characteristic procedure parameterspreconcentration period, flow rate, etc.-must be adjusted to suit the requirements of each particular case (nature of attachment, concentration level, etc.). Work in this laboratory is continuing in this direction. Registry No. Chlorpromazine, 50-53-3.
LITERATURE CITED 5
6
8
7
+
Figure 5. Re etitive injections of a diluted (1 3) urine solution spiked with 8 X I O - M chiorpromazlne. Condtlons were the same as those given in Figure l b except that the flow rate was 0.72 mL/mln.
P
in Figure 5. The mean peak current found was 4.50 nA with a range of 4.25-4.85 nA. The relative standard deviation over the complete series was 4.2%. The precision obtained with the flow injection system compares favorably with the precision reported for analogous batch preconcentration approaches (6,8, IO),and illustrates the absence of carry-over effects. Thus, reproducible data are achievable for a single species, present at the micromolar concentration level in complex samples, such as urine. Chlorpromazine peaks from the diluted urine are only slightly lower than those from
(1) RuiiEka, J.; Hansen, E. H. "Flow InJection Analysis"; Wiley: New York, 1981. (2) Betterldge, D. Anal. Chem. 1978, 50, 832A. (3) RuiiEka, J.; Hansen, E. H. Anal. Chlm. Acta 1978, 9 9 , 37. (4) Pungor, E.;Feher, 2.; Nagy, G.; Toth, K.; Horvai, G.; Gratzl, M. Anal. Chlm. Acta 1979, 109, 1.
Yao, T.; Kobayashl, Y.; Musha, S. Anal. Chim. Acta 1982, 139, 363. Price, J. F.; Baldwln, R. P. Anal. Chem. 1980, 5 2 , 1940. Jarbawi, T. B.; Helneman, W. R. Anal. Chim. Acta 1982, 135, 359. Wang, J.; Frelha, 6 . A. Anal. Chim. Acta, in press. Kaldova, R. Anal. Chlm. Acta 1982, 138, 11. (IO) Chaney, E. N.; Baldwln, R. P. Anal. Chem. 1982, 5 4 , 2556. (1 1) Wang, J.; Dewald, H. D.; Greene, B. Anal. Chlm. Acta 1983, 146, 45. (5) (6) (7) (8) (9)
RECEIVED for review January 28, 1983. Accepted March 21, 1983. This work was supported by a grant from the U.S. Department of the Interior, through the New Mexico Water Resources Research Institute.
Boolean Logic System for Infrared Spectral Retrieval S. R. Lowry" and D. A. Huppler Nicolet Instrument Corporation, 5225- 1 Verona Road, Madison, Wisconsin 5371 I
An lnteractlve retrieval system has been developed for infrared spectral data. Thls system employs Boolean logic operations on sets deflned by the presence or absence of I speclflc peak In a spectrum. The sequentlal appllcation of this procedure can reduce the original llbrary of 3300 spectra down to a few spectra, whlch can be displayed and compared to the unknown spectrum. Options have been Included for both peak locatlon and lntenslty varlatlons. The system has been evaluated by using spectra obtalned from a capillary GCIIR experlment.
The trend toward digital data in most new infrared spectrometers has created a renewed interest in automated spectral identification. This demand and the availability of highquality digital data bases have led to new research into methods for computerized spectral identification. Some of this research has been described in a number of recent papers (1-7). Our laboratory has been extensively involved in this field for several years and recently reported the results of an au-
tomated search system that is part of our GC/FT-IR package (8). This search algorithm utilizes a file of reduced resolution full spectral data as a reference set and performs a point by point similarity measure to determine the best matches. We have also developed a search system that utilizes the Sadtler digital SPEC FINDER data as a reference set (SPEC FINDER is a Trademark of Sadtler Research Laboratory). This data file contains only a reduced set of peak location data and the location of the largest peak for each spectrum. The reference libraries used by these two algorithms approach the opposite ends of the scale of information content for a spectral representation. The "deresolved spectra" used in our GC/IR research still contain most of the peak width and intensity information found in the original spectra. These spectra were created by applying a 17-point polynomial smooth to the original 4-cm-' resolution spectra and saving every fourth point. The spectra were then normalized so that the intensity of the largest absorbance peak could be represented in 10 bits. This compression technique allows an infrared spectrum in the region 4000 cm-' to 450 cm-l to be stored in 230 20-bit computer words with a data point every 8 cm-l and one part in a thousand intensity resolution. Similar
0003-2700/83/0355-1288$01.50/00 1983 American Chemlcal Soclety
