Anal. Chem. 1988, 60, 1979-1982
10
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1. Fluoride 1 ppm 2. Carbonate 2 ppm R Chloride 2 DDm
6
Nitrate 4 ppm 7 Phosphate 6 ppm
8 Sulfate 4 ppm 9 Oxalate 4 ppm 10 Chromate 10 ppm 11 Molybdate 10ppm 0
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Flgure 2. Optimization of the separation of 11 anions with the help of an isconductive gradient: (A) isocratic separation using the weaker of the two eluents (A2); (B) isocratlc separation with the strong eluent (82); (C) step gradient from A2 to 8 2 initiated simultaneously with the injection of 100 pL of the standard mixture. For a more detailed description of the eluents A2,B2 and of the ICPak anion column utilized for the above separations, see Experimental Section. I,
4
l I
1. Fluoride 5 ppm 2 . Carbonate 20 ppm 3. Chloride 15 DDm
5. Bromide 2O'ppm 6. Nitrate 20 ppm 7. Phosphate 30 ppm E . Phosphite 20 ppm 9. Sulfate 20 ppm
10. Oxalate 20 ppm 11. Tunastate 25 ppm 12. Chromate 25 ppm Molybdale 25 ppm Thiocyanate 25 ppm
13
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Figure 3. Gradient separation of 14 anions using a high-resolution column (IGPak Anion HR). A step gradient from A3 to 83 was initiated in the moment of injection. At the eighth minute, the flow rate was increased from 1.2 to 1.7 mL/min. Complete description of the column as well as of the eluents is provided in the Experimental Section. Twenty microliters of the standard mixture was injected. help of the stronger of the two eluents are recommended on the basis of the general theory for such mixtures (2). It is also relatively easy to convert most isocratic ion chromatographs to step gradient instruments by an addition of appropriate
1979
solvent select valves. After an evaluation of a delay volume for each particular pump-solvent valve configuration in an experiment similar to that in Figure 1,the weak and strong eluents can be combined to obtain a gradient chromatogram such as the one in Figure 2C. Relative standard deviations of the retention times and of the integrated areas for each of the 11peaks in this separation are given in Table I. Stepwise gradient elution with the borate gluconate anion and the selected countercations proves to be highly reproducible, achieving better than *0.5% precision of retention times for the majority of the analyzed anions. The observed values of percent relative standard deviation for peak areas, 0.8-3.870 for the concentration range of 1-6 ppm and 0.5-1.7% for 10 ppm, make the discussed technique acceptable for many practical applications. Employment of a more efficient anion exchange column based on a smaller diameter of the particles constituting the stationary phase leads to further improvements of the gradient separation. With the additional help of a flow rate gradient, carried out at a time when the stronger eluent determines the elution behavior on the column, 14 different anions can now be separated within a run time of about 14 min, Figure 3. Further work is currently under way involving new eluent compositions for isoconductive gradients and investigations of the influence of various shapes of gradient profiles. LITERATURE C I T E D (1) Engelhardt, H. H@h Performance Liquid Chromatography; Springer: New York, 1979; p 63. (2) Jandera, P.; Churacek, J. Gradient Nution in Column Liquid Chromatography; Eisevier: Amsterdam, 1985; p 243. (3) Sunden. T.; Lindgren, M.; Cedergren, A.; Siemer, D. D. Anal. Chem. 1983, 55, 2-4. (4) Dasgupta, P. K. Anal. Chem. 1984. 56, 769-772. (5) Tarter, J. G. Anal. Chem. 1984, 56, 1264-1268. (6) Shintani, H.; Dasgupta, P. K. Anal. Chem. 1987, 5 9 , 802-808. (7) Berry, V. V.; Waldron, T. Am. Lab. (FairfieM, Conn.) 1988, 18, 57-66. (8) Gjerde, D. T.; Fritz, J. S. Ion Chromatography, 2nd ed.; Alfred Huethig Verlag: New York, 1987; pp 93-128. (9) Schmuckler, G.; Jagoe, A. L.; Girard, J. E.; Buell, P. E. J . Chromatogr. 1986, 356, 413-419.
William R. Jones P e t r Jandik* Allan L. Heckenberg Waters Chromatography Division Millipore Corp. 34 Maple Street Milford, Massachusetts 01757
RECEIVED for review March 18,1988. Accepted May 24,1988.
Flow Injection Analysis of Electroinactive Anions at a Polyaniline Electrode Sir: Recently, Ikariyama and Heineman (I) reported a novel chemically modified electrode (CME) sensor capable of quantitating electroinactive anions in flow injection analysis. The sensor, constructed by electropolymerizing a coating of polypyrrole onto a platinum electrode surface, utilized the repetitive doping and undoping of the polymer by anioncontaining plugs injected into the carrier phase. Because the electrode was poised at a potential sufficiently high to cause oxidation of the polypyrrole to a positively charged form, the presence of anions possessing high enough mobility to penetrate the film facilitated oxidation of the polymer and resulted 0003-2700/88/0360-1979$01.50/0
in the flow of transient, concentration-dependent anodic currents. By this approach, acetate, phcephate, and carbonate could be detected conveniently and reproducibly at concentrations in the 0.01-1 mM range. The most novel aspect of this system is, of course, that at conventional electrodes none of these anions is itself able to be directly oxidized or reduced-and therefore detected-at commonly accessible potentials. In our laboratory, we have observed analogous currents for a variety of electroinactive anions at CMEs where the deposited polymer is formed from aniline monomer units. While 0 1988 American Chemical Soclety
1980
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15. 1988
it seems that the modes of action of both electrode systems are similar, the polyaniline CME appears to have distinct advantages for use in analytical applications. In particular, the polyaniline film is chemically very stable; its oxidation occurs at a lower potential than does that of polypyrrole; and anion incorporation into the polyaniline film is very rapid and reversible. In addition, by varying the background electrolyte composition and concentration, one can significantly alter the anion selectivity. In this communication, we describe the basic characteristics and capabilities of the polyaniline CME for flow injection analysis of anions.
EXPERIMENTAL SECTION Reagents. Aniline was obtained from Fisher Scientific Co. Anion solutions were made from the sodium salts purchased as reagent grade and used as received without further purification. Solutions were prepared with deionized water and were not deoxygenated prior to use. Working Electrodes. The working electrode used in cyclic voltammetry (CV) experiments consisted of a 1.0-cm2platinum plate onto which a film of polyaniline was deposited electrochemically. Prior to film formation, the electrode was polished with I-pm alumina. Subsequently, deposition of the polyaniline film was carried out by immersing the electrode in a stirred 1.0 M H2S04solution containing 0.1 M aniline and scanning three complete cycles between -0.2 V and +1.1 V (vs Ag/AgCl) at a scan rate of 20 mV/s. Other scan rates, numbers of cycles, and H2S04concentrations were examined. However, the above conditions were found to yield CMES possewing optimum sensitivity, stability, and speed of response. Polyaniline coatings were very stable mechanically, chemically, and electrochemically as long as they were not exposed to potentials higher than +0.8 V. The films could be intentionally removed by ultrasonification in concentrated HN03. For flow injection experiments, a Model MF1012 thin-layer platinum-platinum dual electrode (Bioanalytical Systems, West Lafayette, IN) was employed. The polyaniline f h was deposited on one of the two Pt surfaces (area = 0.071 cm2) by following exactly the same procedure as above. Occasionally, the uncoated Pt electrode was also used in flow injection experiments for comparison purposes. Apparatus. CV was performed with a Bioanalytical Systems Model CV-1B potentiostat and a Hewlett-Packard Model 7035B X-Y recorder. All experiments employed a three-electrode cell with a saturated Ag/AgCl reference electrode and a platinum-wire auxiliary electrode. Flow injection experiments were conducted with a Beckman Model llOB pump, an SSI Model LP-21 pulse dampener, a Rheodyne (Berkeley,CA) Model 7125 injector with a 20-pL sample loop, and an IBM Model EC/230 amperometric detector connected to the thin-layer cell. The mobile phase used was either 0.20 M sodium acetate adjusted to pH 5.45 with acetic acid, 0.20 M acetate buffer (pH 5.45) containing 0.10 M sodium perchlorate, or 0.20 M glycine (pH 6.0). Because the conductivity of the latter solution was only 160 pmho/cm, a stable background could be maintained only by adding an external resistance between the reference and auxiliary electrodes. The size of the resistance employed for the glycine experiments here was 301 kQ. The mobile phase flow rate for the flow injection experiments was always 0.5 mL/min. RESULTS AND DISCUSSION Cyclic Voltammetry. The formation of polyaniline films via electropolymerization of aniline has been the subject of several recent investigations (2-8). Ordinarily, the polymerization process is carried out simply by immersing a bare electrode into an acidified aniline solution and stepping or cycling the potential to a value positive enough to initiate oxidation. The resulting polymer coating can be reversibly oxidized and reduced a t low positive potentials by a pH-dependent le-/N process. Interestingly, both the fully oxidized and the fully reduced forms of the film are insulators; but partially oxidized films are excellent electronic conductors (6,
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.
.
.
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Flgure 1. Cyclic voltammograms obtained at polyaniline-film-coated Pt electrode in (A) 0.20 M pH 5.45 acetate buffer, (B) 0.20 M acetate buffer containing 0.10 M CIO,, and (C) 0.20 M glycine.
8). In this work, the polyaniline CMEs were generated by slow potential cycling in 1.0 M HzS04because this procedure was found to produce electrodes exhibiting the greatest sensitivity to solution electrolyte conditions. The qualitative behavior was similar for CMEs prepared by slightly different procedures, but quantitative characteristics of the electrodes were quite sensitive to the specific conditions of the aniline electropolymerization. The CVs shown in Figure 1 illustrate the electrochemical behavior of a polyaniline CME immersed in three different electrolytes: 0.10 M NaC104 in 0.20 M acetic acid/sodium acetate buffer, 0.20 M acetic acid/sodium acetate by itself, and 0.20 M glycine. The same CME was employed for all three CVs, and the results for each could be repeated regardless of whether (or how often) the electrode was exposed to the other electrolytes during intervening CV scans. The nearest-to-ideal behavior was obtained with the perchloratecontaining medium and consisted of a pair of redox waves at +0.34 V and +0.03 V vs Ag/AgCl. This behavior was typical of that observed not only for C104- but for a group of anions including chloride, bromide, nitrate, and nitrate as well. In all these cases, the polyaniline oxidation wave was much broader than the reduction; but the areas under both waves were the same, roughly 5 x C. This corresponded to a surface coverage of 55 (&LO) nmol/cm2 for the aniline monomer. As expected for a surface-bound redox couple, the peak currents for both waves increased directly with increases in potential scan rate. Somewhat less ideal CVs were obtained with acetate alone (and also for phosphate, sulfate, and iodate by themselves), where the currents were smaller and the anodic process was broader and shifted to higher potential than with C10,present. Finally, with the zwitterion glycine serving as sole background electrolyte, no redox waves at all were observed for the CME over the entire potential range examined. In fact, because of the low conductivity of the glycine solution,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
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Flgure 3. Hydrodynamic voltammogram obtained at polyaniline CME for injection of 0.010 M NO3- into 0.20 M acetate buffer.
Figure 2. Signals obtained at poiyaniline CME for 20-pL injections of 0.010 M NO3-, 0.010 M CT, and 0.010 M SO,” when the mobile phase consisted of (A) 0.20 M acetate buffer containing 0.10 M C104-, (B) 0.20 M acetate buffer by itself, and (C) 0.20 M glycine. For (A) and (B),E = +0.20 V vs Ag/AgCI; for (C), E = +0.50 V.
the background current observed in this medium was only a fraction of that in the other electrolytes. In most respects, the CV behavior seen in Figure 1with the C101-containing electrolyte resembles that previously reported for polyaniline CMEs (2-8). The major difference is that, whereas several of the earlier studies ( 3 , 4 , 8 )indicated that the CMEs showed little or no current for pH values greater than 3-4, facile oxidation and reduction of the modifier was seen to occur here at pH values well above this limit as long as the proper choice of electrolyte anion was made. There are several possible reasons for this discrepancy. First, it is not clear what electrolytes were employed for the previous higher pH experiments in which the polyaniline coatings were reported to be nonconductive. In addition, differences in polymer film thickness and morphology might also help to account for the behavior seen here. Our own experience with low-pH media confirmed that the polyaniline electrochemistry was always well-behaved for pH values in the 1-2 range and that the addition of C10, or other activating anion had little or no effect on the CVs obtained under these conditions. The most obvious explanation for the effects reported in Figure 1 for different anions is that, as with polypyrrole (I), the presence of anions capable of diffusing into the polymer is required to offset the buildup of the positive charge generated by oxidation of the fiim. Of course, this is only one possibility, as the electronic and chemical processes occurring upon polyaniline oxidation are complex and as yet not fully understood. Flow Injection Analysis. In view of the CV enhancements generated at the polyaniline CME by selected anions, it seemed worthwhile to see if similar effects could be carried over into a flow environment. This was accomplished by placing the CME in a conventional thin-layer electrochemical flow cell, continuously passing over it a mobile phase con-
taining the desired electrolyte composition, and then periodically injecting a small volume of the anion of interest into the mobile-phase stream. The results obtained for the injection of several anions are shown in Figure 2. During this set of experiments, the CME was maintained constantly at +0.20 V vs Ag/AgCl for the acetate and ClO;/acetate systems; note that this potential corresponds to a setting near the half-wave potential for the polyaniline oxidation under these conditions. For glycine, maximum anion currents required a more positive potential-in this case, +0.50 V. Just as in CV, drastically different results were obtained, depending on the electrolyte composition and concentration of the mobile phase employed. In general, three different types of behavior were identified. First, when C10; or another anion capable of strongly activating the polyaniline film was present in the mobile phase, a high but steady background current occurred and practically no response was observed upon injection of any of the anions examined. When an electrolyte of intermediate activity (e.g., acetate alone) was placed in the mobile phase, large anodic flow injection peaks occurred for C1-, Br-, NO2-, NO3-, and C104-. Finally, for a mobile phase containing glycine, responses were seen not only for this group of ions but also for acetate, S042-, C032-,and Pod3-. In addition, the peaks were substantially larger in magnitude than those seen in the acetate case. Flow injection experiments performed exactly as those in Figure 2, except that a bare platinum electrode was substituted for the polyaniline CME, produced very small current transients, less than 3% by comparison, for all of the a n a l e s and mobile-phase conditions examined. This is important because it clearly indicates that sample-related changes in solutionphase properties were not responsible for the signals seen at the CME. In particular, conductivity increases associated with the passage of the anion plugs through the thin-layer cell could clearly be dismissed as a major factor in the CME signals, even for the poorly conductive glycine mobile phase. Further confirmation that the flow injection responses were due to the oxidation of the polyaniline film was provided by the potential dependence of the CME signals. As exemplified by the hydrodynamic voltammogram shown in Figure 3 for NO3- injections into plain acetate buffer, the response for the anions
1982
Anal. Chem. 1988, 60, 1982-1984
commenced a t low positive potentials near the onset of the oxidation of the polyaniline modifier. Maximum response, however, required the application of only +0.15 V vs Ag/AgCl, a value well below the CV peak potential seen in Figure 1for this electrolyte. In all cases, the CMEs themselves were extremely durable. Ordinarily, the same CME surface could be used for CV and flow injection studies for periods of 2 weeks and longer with no evidence of chemical or mechanical deterioration. The CV response of the CME to various anions was also maintained at the same level over the entire electrode lifetime. However, for the analytical response to remain constant for an extended series of flow injection experiments, some provision had to be made for discharge of the anions accumulated over the course of repeated exposures. For example, for a series of 20-pL injections of 1.0 X M NO3- at +0.40 V vs Ag/AgCl, the peak oxidation currents seen in acetate solution declined to such an extent that, after 10 such exposures, only 50% of the initial response was obtained and, after 20 injections, only 30% remained. Naturally, the decrease in response was much less severe when lower anion concentrations were employed. (It is interesting to note that, although integration of the CV response of the above CME indicated the presence of about 4 nmol of electroactive aniline monomer, the amount of charge passed in the flow experiments corresponded to the uptake of no more than 1 pmol of anion per injection. In view of the CME’s rather rapid loss of response under these conditions, it would appear that only a fraction of the polyaniline coating is accessible in the flow experiments.) Two procedures were available for reversing the anion uptake and thereby maintaining a stable analytical response. First, as has been shown earlier for polypyrrole electrodes ( I ) , the CME could be briefly exposed to a reducing potential (on the order of -0.20 V) between successive anion exposures. In this way, the polyaniline film was cycled through its fully reduced state with the accompanying discharge of any anions that had been incorporated as a result of polymer oxidation. The second, usually preferable approach involved making all anion measurements at a lower potential (e.g., +0.20 V for acetate electrolyte) where the polyaniline was neither fully oxidized nor fully reduced. Under these conditions, any accumulated anions were apparently able to leave the polymer film as soon as the anion-containing sample plug had passed through the detector cell. This was evidenced by the occurrence of a shallow cathodic through, which followed the anodic peaks associated with anion uptake. This effect, which was most noticeable a t high anion concentrations and can be seen for NO3- and C1- in Figure 2, part B, was sufficient to maintain the CME response indefinitely without any intervening change in applied potential. Thus, at +0.20 V, 20 injections of 0.010 M NO3- over the course of 2 h exhibited a relative standard
deviation of only 2%, with the last injection producing 97% as much current as the first. Even a t this early stage, the analytical capabilities of the polyaniline CME appear extremely promising. In the most favorable cases (e.g., NO3- or Clod- injected into the glycine mobile phase), detection limits (signal/noise = 3) were 0.10 ppm or lower; the linear range (correlation coefficient 3 0.99) extended to concentrations at least 3 orders of magnitude higher. Even the nonideal anions such as SO:- gave signals detectable below the parts per million level. In the acetate buffer, anion signals were somewhat smaller and detection limits correspondingly higher than what was seen with glycine. NO3-, for example, could be detected only down to the 1-ppm level when the acetate mobile phase was employed. Perhaps the most interesting aspect of the CME response was that is selectivity was tunable to a large degree by manipulation of the composition of the electrolyte solution in which the sample was presented. Thus, when the background solution was glycine, all anions examined gave large, analytically useful signals. But, as seen in Figure 2, a simple change to an acetate-based electrolyte served to deactivate the CME toward all but a group of small, singly charged anions including C1-, Br-, ClO,-, NO3-, and NOz-. This capability presents a new route for selectivity control that merits further study and application. Registry No. C1-, 16887-00-6;Br-, 24959-67-9; NOz-, 1479765-0; NOS-, 14797-558;ClO;, 14797-73-0;SO-,: 1480879-8;COSz-, 3812-32-6; PO4%,14265-44-2;acetate, 71-50-1;platinum, 7440-06-4; polyaniline, 25233-30-1. LITERATURE CITED (1) Ikariyama, Y.; Heineman, W. R. Anal. Chem. 1986, 58, 1803-1806. (2) Mohilner, D. M.; Adams. R. N.;Argersinger, W. J., Jr. J . Am. Chem. SOC.1962, 84,3618-3622. (3) Diaz, A. F.; Logan, J. A. J. Electroanal. Chem. Interfacial Electrochem. 1980, 1 1 1 , 111-114. (4) Kobayashi, T.; Yoneyama, H.; Tamura, H. J. Electroanal. Chem. Interfacial Electrochem. 1984, 177, 281-291. ( 5 ) Kobayashi, T.; Yoneyama, H.; Tamura, H. J . Nectroanal. Chem. I n terfacial Electrochem. 1984, 177, 293-297. (6) Paul, E. W.; Ricco. A. J.; Wrighton, M. S . J. Phys. Chem. 1985, 89, 1441-1 447. (7) Sasaki, K.; Kaya, M.; Yano, J.; Kunai, A. J . Electroanal. Chem. Interfacial Electrochem. 1986, 215, 401-407. (8) Giarum, S . H.; Marshall, J. H. J. Electrochem. Soc. 1987, 134, 142- 147.
Jiannong Ye Richard P. Baldwin* Department of Chemistry University of Louisville Louisville, Kentucky 40292 RECEIVEDfor review February 2,1988. Accepted May 2,1988. This work was supported by the National Science Foundation through EPSCoR Grant 86-10671-01.
Development of a Solid-Substrate Room-Temperature Luminescence Immunoassay Sir: Luminescence immunoassays most commonly employ a fluorescent compound for labeling antibodies and antigens. While fluorescent labels have obvious advantages over the traditional radioactive labels, the background fluorescence of biological samples can be a problem. Time-resolved fluoro-
metry avoids the short-lived background fluorescence, and immunoassays using long-lifetime lanthanide chelates as labels are among the most sensitive ( I ) . Phosphorescent and delayed fluorescent compounds are alternative labels with attractive features. Their lifetimes are
0003-2700/88/0360-1982$01.50/00 1988 American Chemical Society
