1536
Anal. Chem. 1985, 57, 1536-1541
(15) Brainina, Kh. 2. Talanta 1971, 18, 513-539. (16) Nagaosa, Y.; Kabayashi, K. Talanta 1984, 37, 593-596. (17) Stary, J.; Zolotov, Yu. A.; Petruklln, 0. M. "Crltlcal Evaluation of Equibrium Contants Involving 8-Hydroxyquinoline and Its Metal Chelates"; Pergamon press: Oxford, 1979; IUPAC Chemical Data Series 24, p 18.
(18) Milazzo, G.; Caroli, S. "Tables of Standard Electrode Potentials"; Wiley: Chichester, 1978; pp 264-267. (19) Cosovic, B.; Vojvodlc, V. Limnol. Oceanogr. 1982, 27, 361-369.
RECEIVED for review January 25,1985. Accepted April 8,1985.
Thin-Layer Electrochemical Detector with a Glassy Carbon Electrode Coated with a Base-Hydrolyzed Cellulosic Film Joseph Wang* and Lori D. Hutchins Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
The permeability characteristics and analytlcal applications of a thin-layer glassy carbon detector coated with cellulose acetate film are descrlbed. Access to the surface can be manipulated via a controlled base-hydrolysis of the fllm. As a result of excluding macromolecules from the surface, the stablilty and selectivity are greatly Improved. The range of applications can be extended vla a careful choice of the hydrolysis period. By adding a new dimension of selectivity based on molecular slre, hlgh spectficlty toward smaller analytes Is demonstrated In both flow Injection and ilquld chromatographic systems. Electrode polsonlng, due to protein adsorption or accumulation of reactlon products, Is mlnlmlred. Complex chromatograms can be greatly slmpllfled without lowering the operating potential. The coated electrode exhlblts extension of the linear range, In situations where a nonllnear response Is obtalned at the bare electrode. Varlous analytes and lnterferents, representing a wide range of molecular slres, are tested. Appllcablllty to urine samples Is demonstrated.
Electrochemical detection in flowing streams such as liquid chromatography or flow injection has attracted considerable interest in recent years (1,2). Such detection schemes offer excellent sensitivity, selectivity toward electroactive analytes, wide linear range, and low costs. However, improved stability and selectivity are desirable for many practical applications. Most detectors utilize solid electrodes which are subject to a gradual loss of activity. This is primarily due to adsorption of proteins and other surfactants (present in complex clinical or environmental samples) or accumulation of reaction products (as in oxidation of phenolic or aromatic compounds). At present, there is no simple solution to the problem of electrode poisoning. In addition, amperometric detection may lack the desired selectivity when mixtures of electroactive species are concerned. This is usually the case in flow injection analyses of such mixtures, as well as in chromatographic separations with peaks which are overlapped in the time domain. Added selectivity is usually achieved via the redox potential resolution of voltammetry, using multielectrode (3) or potential scanning (4)detection schemes. Nevertheless, it would be desirable to obtain an additional dimension of information. One approach to improve the stability and selectivity is to cover the electrode with an appropriate membrane, thus selecting the components arriving at the surface. The membrane
must be designed to provide rapid diffusion of the desired analyte, as required in most flowing streams. Various bulk membranes have been used in conjunction with different batch and flow electroanalyticalsystems (5-8). In spite of the intense activity to produce polymeric coatings on electrodes (9),little work has been reported on the application of such films for improving the selectivity and stability in flow analysis. Sittampalam and Wilson (IO) coated a platinum detector with a thin film of cellulose acetate to minimize protein fouling during flow measurements of biological matrices. However, their measurements were limited to very small molecules, e.g., HzOz, that can diffuse rapidly through cellulose acetate films. In this paper we describe a surface-modified glassy carbon detector with hydrolyzed cellulose acetate film. Controlled hydrolysis in alkaline medium is known to increase the porosity of cellulose acetate by breaking its chain into small fragments (11). As a result, different permeabilities (molecular weight cutoffs) are obtainable by hydrolyzing the film over different time periods. Such manipulation of access to the surface allows monitoring of a variety of analytesrepresenting different molecular sizes-while achieving significant stability and selectivity improvements. Thus, by adding an in situ separation step (on the electrode surface), based on the size dimension, the scope of applications of coated electrodes is extended. The transport characteristics and analytical applications of a glassy carbon detector coated with hydrolyzed cellulosic films are elucidated below.
EXPERIMENTAL SECTION Apparatus. The flow injection system consisted of the carrier reservoir and two sample reservoirs (400-mL Nalgene beakers), a Rheodyne Model 7010 sample injection valve, interconnecting Teflon tubing, and the electrochemical detector. The use of two sample reservoirs, connected via a three-way stopcock to the injection valve, allowed alternate injections of different samples. Flow of the carrier solution was maintained by gravity. The liquid chromatographic system (Bioanalytical Systems LC-303) was described previously (12). A glassy carbon thin-layer electrochemical detector (Model TL-5, Bioanalytical Systems) was used. The reference and auxiliary electrodes were located in a downstream compartment (Model RC-SA, Bioanalytical Systems). All potentials are reported vs. a Ag/AgCl reference electrode (Model RE-1, Bioanalytical Systems). An EG&G PAR Model 174 POlarographic analyzer was used in the flow injection experiments. The current-time output was plotted on a Houston Omniscribe chart recorder. Coating the Glassy Carbon Electrode with Cellulose Acetate Film. Prior to its coating, the electrode was polished with 1fim a-alumina particles, rinsed with double-distilled water, and allowed to air-dry. The electrode was coated with 10 ILLof
0 1985 American Chemical Society 0003-2700/85/0357-1536$01.50/0
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Figure 2. Dependence of fllm permeability on the analyte molecuiar weight at 30 (a) and 45 (b) rnin hydrolysis times: analytes tested, m-nitrophenol (mol wt 139.1),acetaminophen (mol wt 151.1),dop-
amine (mol wt 189.6), pherphenatine (mol wt 404), potassium ferrocyanide (mol wt 422.4), and NADH (mol wt 709); analyte concentration, M; flow injection conditions, as in Figure 1. 1X
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Flgure 1. Dependence of the peak current on the hydrolysis time for phenol (a), acetaminophen (b), estriol (c), NADH (d), and potassium ferrocyanide(e); flow rate, 0.75 mL/min: sample loop, 20 /ALL; applied potential, +0.8 V (a, b, d, e) and +1.1 V (c); solute concentrations, 6 X lo4 M (a),4 X lo4 M (b-d); electrolyte, 0.05 M phosphate buffer
(pH 7.4).
the 5% cellulose acetate solution (in a 1:l mixture of acetone and cyclohexanone after 12 h of stirring), placed in an eliptical shape, to cover the active disk alid its surrounding. The solvents were allowed to evaporate (for 1h in air), leaving a uniform film over the entire channel, The film was hydrolyzed in a 0.07 M KOH solution. For this purpose, the electrode was immersed in a stirred KOH solution for the desired time. Following this, the electrode was rinsed with double-distilledwater and immersed in a stirred solution of the supporting electrolyte for 10 min to wash out the tesidual solvent. The cell was then assembled by carefully placing the spacer and upper block (according to the manufacturer instructions). The assembled cell was equilibrated with the flowing supporting electrolyte (or mobile phase) solution while applying the working potential. Amperometric measurements were made after the transient current decayed. A film thickness of about 0.01 mm was estimated from measurements with a micrometer, after removing the film from the surface. Reagents. All solutions were prepared with double-distilled water. Stock solutions of acetaminophen, uric acid, mnitrophenol, 2,4-dinitrophenol, perphenazine, NADH (Sigma Chemical Co.), potassium ferrocyanide, ascorbic acid, and phenol (Baker) were made up fresh each day by dissolving the compound in the supporting electrolyte solution. The estriol solution was prepared by dissolving the compound in ethanol and diluting with the supporting electrolyte. Cellulose acetate (39.8% acetyl content) was purchased from Aldrich Chemical Co. Albumin, bovine (98-99%), was purchased from Sigma and dissolved in doubledistilled water. A 0.05 M phosphate buffer (pH 7.4), prepared from K2HP04 and KHzP04was used in the flow injection experiments. The chromatographic mobile phase was a 0.15 M chloroacetic acid solution adjusted to pH 3.5 with perchloric acid; the mobile phase contained 25 mg/L sodium octyl sulfate and 200 mg/L disodium ethylenediaminetetraacetate. The urine samples were obtained from a healthy volunteer, filtered by passing through a glass filter (10-15 pm porosity), and diluted with the mobile phase solution. RESULTS AND DISCUSSION Permeability Studies. Base-hydrolysis is known to increase the porosity of bulk dialysis membranes (13). Similar effects are observed for cellulosic films coated onto the electrode surface. The use of different hydrolysis times results
in different film permeabilities. Figure 1shows the dependence of the flow injection peak current for different analytes, representing different molecular sizes. The nonhydrolyzed film excludes most species (0 min data); only the small phenol molecules (mol wt 94, curve a) diffuse rapidly through its small pores and yield well-defined peak currents. The permeability of the film gradually increases upon increasing the hydrolysis period, as indicated from the gradual increase of the acetaminophen (mol wt 151, curve b), estriol (mol wt 288, curve c), and phenol peaks. Larger species, e.g., NADH or ferrocyanide, diffuse through the film only for hydrolysis times longer than 40 min (curves d and e). Hydrolysis times longer than 50 min result in unstable coating, with the film falling apart from the supporting electrode. Cellulose acetate membranes are permselective primarily based on size (14). Figure 2 represents the dependence of the film permeability on the solute molecular weight for 30 (a) and 45 (b) min hydrolysis times. The ratio between the current at the film coated electrode over that at the bare electrode im/ibis used as a measure of the permeability. With both films, a rapid decrease in the permeability is observed around a molecular weight of 140. Following this, the permeability continues to decrease gradually. For large solutes (mol wt >400), no response is observed a t the 30 min hydrolyzed film. Such discrimination between small and large species provides the basis for the analytical utility of this detector (see below). Bulk cellulosic membranes exhibit similar profiles, with an exponential decrease of the permeability with increasing solute size (15). While molecular size appears to play a major role in the film permeability, other factors such as the net electrical charge, shape, or conformation of the solute may contribute to the mass-transport resistance. The effects of these factors on the permeability of dialysis cellulosic membranes were discussed by Colton et al. (15). Our film coating exhibited lower permeability (than that expected based on size) toward ferrocyanide and ascorbic acid, and higher permeability toward various chlorophenols. While charge repulsion may be responsible for the behavior exhibited toward the former two solutes, thert is something unique about the interaction between cellulose acetate and chlorophenols. This unique factor remains to be elucidated. Figure 3 illustrates characteristic flow injection current-time profiles for acetaminophen using different sample volumes. At both the coated (A) and bare (B) electrodes, peak shapes are similar, with rapid increase and decrease of the current. The peak widths (at 0.6Cm,,) are 5 (20 pL), 19 (200 pL), and 61 (500 pL) s with the coated electrode and 4 (20 pL), 19 (200 pL), and 62 (500 pL) s a t the bare electrode. The response times to reach 90% of maximum signal are also similar at both
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Figure 3. Flow injectlon current-time profiles for 1 X M acetaminophen using 20 (a),200 (b), and 500 (c)pL sample volumes: (A) coated electrode, 30 min hydrolysis; (B) bare electrode; flow rate, 0.64 mL/min; applied potential and buffer, as in Figure 1.
electrodes. The fast response indicates rapid replenishment of the solution from the surface (lack of trapping of the analyte). Such properties are essential for use in dynamic systems such as flow injection or liquid chromatography. Most solutes tested exhibit similar response times at both modified and unmodified electrodes, with no time lags due to diffusion in the film. Only some phenols (e.g., phenol) exhibit longer response times at the coated electrode, indicating some interaction with the film or possible changes in the nature of the electrochemical reaction (5). The film coating exhibits different permeabilities when different volumes are injected. For example, im/ib values of 0.09,0.18, and 0.18 are observed for the 20-, 200-, and 500-pL samples, respectively (note the different current scales employed). The changes in permeability with sample volume are attributed to the different residence times of the sample plug in the detector compartment (i.e,, different time scales to diffuse in and out of the film). Notice the diminished sensitivity associated with the modified electrode, as discussed later in the paper. The value of im/ibremains constant for injections of various concentrations using the same volume (as it reflects the ratio of the slopes of the corresponding calibration plots). For example, for injections of samples of ascending acetaminophen concentration ((2.0-10.0) X M) an im/ib value of 0.34 was obtained (conditions: as in Figure 1, with 40 min hydrolysis time). As will be discussed later, im/ibmay change with the concentration, in situations where the bare electrode yields a nonlinear response. The dependence of the current upon the flow rate was evaluated under steady-state conditions, for flow rates ranging from 0.12 to 1.55 mL/min (conditions: 4 X lov4M acetaminophen, 30 min hydrolysis; applied potential, +0.8 V, 0.05 M phosphate buffer, not shown). A log-log treatment of the resulting data yielded two straight lines with slopes of 0.078 (over the 0.12-0.90 mL/min range) and 0.064 (for the 0.90-1.55 mL/min range). Such dependence reflects combined film and
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solution mass-transport resistances. A theoretical value of 0.33 is expected for bare electrode thin-layer transducers (16). It is well-known (17)that the current becomes less dependent on the flow rate if the electrode is covered by a layer through which solute can diffuse, but which does not allow bulk flow to occur. Under such mixed (film/solution) controlled conditions, the steady-state diffusion current can be described by (5)
where P, and P, are the permeability of the solution or film, respectively, and iL is the current at the bare electrode. At sufficiently low and high flow rates, the current would approach solution and film control, respectively. Analytical Utility. In liquid chromatography of complex sample matrices, the need for detector selectivity is often paramount to detector sensitivity. Even with the most sophisticated chromatographic equipment, unresolved peaks commonly occur when such matrices are concerned. New electrochemical detection schemes, using rapid potential scanning ( 4 ) or multielectrodes (3), have been developed to separate solutes in the potential domain, when the time domain cannot be utilized. Similarly, surface modification procedures have been incorporated with liquid chromatographic detectors to improve the selectivity via preferential catalysis of specific redox reactions (12, 18). By coating the detector with a cellulosic film it is possible to isolate small solutes, from coeluting large ones. This adds a new dimension of information, based on molecular size, to electrochemical detection for liquid chromatography. With this approach in mind, the suitability of the glassy carbon coated detector for analytical determinations in physiological matrices was tested. Figure 4 compared chro-
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Figure 5. Flow injection detection of phenol in the presence of other phenolic compounds, at the film coated (A) and bare (9) electrodes: (a) 2 X lo-’ M phenol; (b) same as (a) but after addition of 2 X lo-’ M m-nitrophenol; (c) same as (b) but after additions of 2 X M 2,4dinitrophenol; applied potentlal, 4-0.9V; flow rate, 0.6 mL/min; nonhydrolyzed film; 0.05 M phosphate buffer (pH 7.4).
Figure 6. Flow inJection peaks for solutions containing increasing acetaminophen concentrations, in the presence of potassium ferrocyanide, at the film coated (A) and bare (B) electrodes: (a) 2 X lo-‘ M acetaminophen and 4 X M potassium ferrocyanide; (b) same M acetaminophen; (c) same as as (a) but after addition of 2 X (b) but after addition of 2 X M acetaminophen; 40 rnin hydrolysis; flow rate, 0.5 mL/min; loop, potential, and buffer, as in Figure 1.
matograms for diluted urine sample obtained at the bare electrode (A) and at the film coated electrode, after 15 (B) and 30 (C) min hydrolyses. More than 15 peaks of variable sizes are observed at the bare electrode. Most of these peaks are eliminated using the 15 min hydrolysis film coated electrode. Only the uric acid yields a well-defined peak (t = 8 min) under these conditions. By increasing the porosity of the film (30 min hydrolysis), five defined peaks-representing small (mol w t
