Effect of ultramicroelectrode array structure and analyte properties on the detector response in flow-injection analysis

Analytica Chimica Acta, 246 (1991) 315-324 Elsevier Science Publishers B.V., Amsterdam

315

Effect of ultrarnicroelectrode array structure and analyte properties on the detector response in flow-injection analysis Ewa Dabek-Zlotorzynska, Deportment

of Chemistry,

Kalsom

Ahmad

and Anna

Brajter-Toth

*

University of Florrda, Gainesville, FL 3261 I-2046 (U.S.A.) (Received

30th July 1990)

Abstract

A tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) microarray electrode was tested as a surface for amperometric electrochemical detection of simple biomolecules in a flow cell. The behavior of this electrode was compared with that of other arrays, glassy carbon polished with alumina and electrochemically pretreated glassy carbon. Hydroquinone, dopamine and ascorbic acid were selected as test probes. The results show that the fraction of active electrode area and the kinetics of the analytes determine the analytical sensitivity. Under flow-injection conditions the dependence of analyte response on flow-rate does not follow theoretical predictions. In the presence of a liquid chromatographic column a different (and very small) dependence on flow-rate is observed, which is consistent with the predictions for microarray detectors. Keywords: Cyclic

voltammetry;

Amperometry;

Flow system;

0 1991

Elsevier Science Publishers

carbon

electrodes;

Microelectrode

arrays

tions controlled by mass transport and those controlled by the rate of charge transfer or other surface phenomena. The microelectrode arrays that have been used in flowing streams have included random arrays obtained by dispersing carbon particles in a polymeric matrix [ll-131 or by using a reticulated vitreous carbon (RVC) with an insulator to fill the pores [lo]; arrays based on disks produced by embedding carbon fibers in a suitable insulating polymer [8,9] and an ordered array of gold in a membrane [14] have also been reported. Other workers have reported lithographic techniques to obtain linear arrays of noble metal, usually gold, which is vapor-deposited on a glass support [15,16]. The microarray electrodes with linear geometry with the lines oriented perpendicular to the direction of flow are the most efficient in flow cells [6,17]. Recently, Magee and Osteryoung [6] described a method for fabricating glassy carbon

Recently, microelectrode arrays, which have been shown to have favorable properties in flow systems, especially from the point of view of signal-to-noise ratio (S/N ), have been the subject of many investigations [l]. Electrochemical detectors employing microelectrode arrays are expected to be more sensitive than electrodes whose entire surface is active owing to partial replenishment of the diffusion layer with an analyte during the passage of the solution between microelectrodes [2-61. Since detector noise is proportional to the active area of the electrode [7], S/N enhancement is also expected. Another advantage of arrays as detectors in flowing streams is the fact that at low flow-rates ( < 1 .O ml min ~ ’ ), the dependence of signal on flow-rate is suppressed [g-lo]. Also, better detection limits at high applied potentials have been observed with microelectrode arrays [4] owing to the ability to discriminate between electrode reac0003-2670/91/$03.50

Glassy

B.V.

316

linear array electrodes. A novel method for the preparation of a microelectrode ensemble using micropores of aluminium anodic film as template was proposed by Uosaki et al. [18]. This paper examines the behavior of a random microelectrode array formed by the one-dimensional organic conductor tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) as a surface for amperometric electrochemical detection in a flow cell. The behavior of this electrode is compared with that of widely used glassy carbon (GC) electrodes. The glassy carbon electrodes tested were of two types, so-called inactive GC, polished with alumina, and active GC formed by electrochemical activation (ECGC). It has recently been shown that these GC surfaces have array-like structures [19,20]. The main difference among these surfaces is the fraction of active to inactive area. The electrodes were tested for amperometric detectton in flow-inJection measurements of several simple biomolecules so that the effect of analyte activity on the response of an array could also be evaluated. Hydroquinone, dopamine and ascorbic acid were selected as test species as they represent small biomolecules of considerable analytical interest and different activity. In addition, the stability of TTF-TCNQ electrodes was investigated. The organic conductor TTF-TCNQ is of interest as an electrode material owing to its interesting electrochemical properties such as the apparent catalytic response [21-241 and improved stability towards passivation [22,25]. These properties are of special interest in the determination of biological compounds, which, because of slow kinetics on GC, have low sensitivity and which, together with redox reaction products, foul the electrode surface

P61EXPERIMENTAL

Materials Unless noted otherwise, the chemicals were of ACS reagent grade and were used without further purification. Potassium hexacyanoferrate(II1) was obtained from Mallinckrodt, hydroquinone from Chem Service, ascorbic acid, dopamine, tetrathiafulvalene (TTF) and poly(viny1 chloride) (PVC)

E DABEK-ZLOTORZYNSKA

ET AL

from Sigma and 7,7,8,8-tetracyanoquinodimethane (TCNQ) from Aldrich. Solutions of probe molecules were prepared immediately before use by dissolving the compounds in 0.05 M phosphate buffer (pH 7.0). Phosphate buffer was made up fresh each day from buffer stock solution (1 M). Prior to use, the buffer was filtered through a 0.45~pm membrane filter (Alltech). Doubly distilled water was used and solutions were deaerated with nitrogen for 10 min. Cyclrc voltammetry Cyclic voltammetry, chronocoulometry and glassy carbon electrode pretreatment procedures were effected with a Bioanalytical Systems BAS100 electrochemical analyzer. The data were stored and processed with an IBM PS/2 50 computer, equipped with a Hewlett-Packard LJ II printer. Cyclic voltammograms were background subtracted. The TTF-TCNQ polymer paste (pp) electrodes used in cyclic voltammetric experiments were constructed using the procedure described by McKenna et al. [22]. Polymer paste was made by mixing a 13 : 1 (w/w) ratio of ‘ITF-TCNQ [22] to PVC in THF. The suspension was stirred until a thick slurry formed and was then spread into the platinum-fitted Teflon holders with a recessed depth of ca. 1.0 mm. The electrodes were cured overnight and were used without further treatment. Glassy carbon electrodes used for cyclic voltammetry were prepared by sealing a ca. 3 mm diameter rod of glassy carbon (Electrosynthesis) in a glass tube with epoxy resin (1C White, Dexter Corp., Hysol Division). Before each experiment, the electrodes were polished with l-, 0.3- and 0.05~pm alumina slurries (Mark V Laboratory) on a microcloth (Buehler), using a polishing wheel, for 1 min each. Next, the electrodes were rinsed and ultrasonicated for 10 min in doubly distilled water to remove alumma particles. The electrodes were then rinsed under a stream of distilled water. The electrochemical pretreatment procedure for GC was sm-nlar to that reported by Engstrom [27]. First, the electrodes were polished with a l-pm alumma slurry for 1 min and rinsed thoroughly

EFFECTS

ON DETECTOR

RESPONSE

IN FIA

with deionized water. After polishing, the electrodes were sonicated in deionized water for 10 min. Next, electrochemical pretreatment of glassy carbon was effected in deaerated phosphate buffer (0.1 M, pH 7.0) by poising the potential at 1.75 V vs. SCE. Then the electrodes were poised at - 1.2 V vs. SCE for another 10 s, followed by potential cycling between 1.75 and - 1.2 V at a scan rate of 10 mV s-’ until a stable background was achieved [28,29]. The geometric electrode areas were determined by chronocoulometry with a pulse width of 250 ms using potassium hexacyanoferrate(II1) in 0.1 M KC1 and D, = 7.63 x lop6 cm* s-t [30]. The geometric areas for GC and ECGC electrodes used in cyclic voltammetry were 0.069 and 0.23 cm’ for TTF-TCNQ. The estimated electroactive area fraction of TTF-TCNQ polymer paste electrode was obtained from [24], whereas the electroactive area fractions of polished GC and ECGC were calculated using the particle density of copper deposits of 4.1 x 10’ and 36 X 10’ sites cm-*, respectively, estimated from scanning electron micrographs (SEM) on GC and ECGC [19]. The microscopic areas obtained by measuring the average size of copper deposits in the micrographs for polished GC and ECGC were 7.4 X lop9 and 3.7 X 10e9 cm*, respectively. An active area fraction was obtained by multiplying particle density by microscopic area. Estimated active area fractions of 0.3 and ca. 1.0 were obtained for GC and ECGC, respectively. As a result of more scratches and cracks at ECGC [19,31,32], large copper deposits were obtained at each active site and the estimated area fraction was larger than 1.0. In cyclic voltammetric experiments, platinum wire served as the auxiliary electrode and silver/silver chloride (3 M NaCl) as the reference electrode. Flow-rnjection system The hquid chromatograplnc system, which consisted of a Rainin-HP pump, a Rheodyne Model 7125 inJector (20 ~1) and a Bioanalytical Model LC-4 amperometrtc detector connected to a stripchart recorder (Fisher Recordall 5000) was used as a flow-injection-electrochemtcal detection sys-

317

tem. In most experiments the analyte solution was introduced through a 50 or 35 cm x ca. 0.25 mm i.d. length of tubing to the detector. A B&J 0C5 C,, column (15 cm X 0.46 cm i.d.) was used in flow-rate dependence experiments. The amperometric detector was equipped with a Model TL-4 thin-layer glassy carbon electrode with a geometric area of 0.07 cm*. The electrode was alumina polished by the same procedure as applied to the GC electrodes used in cyclic voltammetric experiments. The TTF-TCNQ polymer-paste (pp) detector was prepared as follows. The lower block of the thin-layer cell was machined from a Teflon block and a hole of ca. 2 mm depth and ca. 3 mm diameter was drilled in the center of the block. A platinum disk, to which electrical contact with a copper wire was made with nickel paint (GC Electronics, Rockford, IL), was fitted into the hole. Next the TTF-TCNQ polymer paste prepared as described earlier was spackled (packed with paste) mto the hole. Electrodes were allowed to cure for at least 1 day in the dark at room temperature. In a flow cell electrodes were first poised at 0.35 V vs. Ag/AgCl to check the value of the baseline (background current). Electrodes with baseline currents below 100 nA were used in the experiments. A potential region of electrode stability from 0.5 to -0.2 V vs. SCE with several supportmg electrolytes has been reported [24]. The electrodes had a geometric area of ca. 0.07 cm*. The cell was completed by combimng with a commercial upper stainless-steel block (TL-4, Bioanalytical Systems). The thin-layer ECGC electrodes were prepared using a machined Teflon block where a ca. 3 mm diameter rod of glassy carbon (Electrosynthesis) was sealed in the hole drilled in the block using epoxy. The hole was fitted with platinum. Nickel paint was used to attach a copper wire for electrical contact. The geometric area of this electrode was 0.059 cm*. The electrochemical treatment of GC was described under Cyclic voltammetry. Teflon spacers of a thickness of 0.0125 and 0.0050 cm were used to separate the component blocks of the electrochemical cell. The flow channel was 0.48 cm wide and 1.7 cm long, resulting in a total cell volume of 10 or 4 ~1. The Ag/AgCl (3

E DABEK-ZLOTORZYNSKA

318

M NaCl) reference and platinum auxiliary electrodes were placed in a Kel-F holder (Bioanalytical Systems) downstream from the electrode in a standard design. Unless noted otherwise, all potentials are reported versus Ag/AgCl reference electrode. Hydrodynamic voltammetric (HDV) experiments were done by makmg repeated mlections of a standard solution and stepping the potential of the detector to the desired value between the mlections after the baseline current had stabilized. Each pomt in an HDV experiment was the average of three inlections. Response time was determined by measuring the peak width at half-height for inlections of a 20-~1 sample containing 25 PM of hydroquinone at a flow-rate of 1.0 ml min-‘.

RESULTS

AND

DISCUSSION

Electrode structure and electrochemical behavior of probe molecules Table 1 shows the capacitive current densities (I~,~) measured at 0.10 V at the three electrodes used. The estimated electroactive area fractions given in Table 1 were determined as described under Experimental. The ITF-TCNQ pp electrode has the lowest estimated active area fraction, of ca. 0.1, owing to the separation of the conducTABLE

TABLE

1

Electrode

characterlzatlon

Surface

Electroactwe area fraction

Compound

(PA/cm’) TTF-TCNQ GC ECGC

pp

0 1b 0.3 c ca 1.0 ‘

13 03 387

a Current normahzed over geometnc area, measured at 0 10 V vs. Ag/AgCl m 0 05 M phosphate buffer (pH 7.0). scan rate, 50 mV s- ’ b Results obtamed from [24] ’ Estimated from data m [19]

tor by the insulating PVC [20,24]. The results in Table 1 show that polished GC, which has a ca. three times higher estimated active area fraction than TTF-TCNQ pp, has a 24-fold lower capacitive current density. The large value of the capacitive current density of TTF-TCNQ pp is probably due to the faradaic contribution to the background current at TTF-TCNQ [25]. The large increase in background current of ECGC is a result of the increase in the microscopic surface area and the increase m the faradaic contribution to the background current caused by the redox reactions of the surface functional groups [33-361. Cyclic voltammetric results for hydroquinone (HQ), dopamine (DA), ascorbic acid (AA) and hexacyanoferrate(II1) are summarized m Table 2.

of cychc

voltammetry

results

A EP

Surface

Sensitivity, (nA 1 pmol~‘cm-*)

09 mF-TCNQ GC ECGC

pp

DA

TTF-TCNQ GC ECGC

AA

TTF-TCNQ

0 35 + 0.02 0 27 + 0 02 0.13 f 0.02

_ 0 32 0.14

pp

025*002 0.23 f 0 02 0.20 f 0 05

0.16 f 003 0.13 f 003 0095*0003

416 320 796

pp

0.32 + 0.02 030*0003 0 12 * 0.005

_ _

304 174 323

0 21 024*0006

0 18 0 15

GC ECGC Fe(CN)i-

Capacltwe current, I=,~ a

2

Summary

HQ

ET AL

TTF-TCNQ GC

pp

f 0.03 f 0 003

_

f 0.01

343 302 541

40 23

04 mM, Fe(CN)iconcentration, * Hydroqumone (HQ), dopamme (DA) and ascorbic acid (AA) concentration, electrolyte, 0 05 M phosphate buffer (pH 7 0), scan rate, 50 mV s-‘, potentials measured vs Ag/AgCl (3 M NaCI).

4.4 mM,

EFFECTS

ON DETECTOR

RESPONSE

IN FIA

319

Consistent with previous results [37,38], oxidations of HQ and DA on GC electrodes are quasi-reversible. Peak separations for hydroquinone and dopamme are 0.32 and 0.13 V, respectively, indicating that HQ oxidation is more irreversible. Oxidation of both HQ and DA is controlled by diffusion on polished GC, as determmed by a linear relationshtp between peak current (I~) and the square root of scan rate (0); the slopes of log lp vs. log u plots were 0.52 and 0.40 for HQ and DA, respectively. The slope lower than 0.5 observed for dopamme is probably due to the sensitivity of this probe towards partial activity of the surface [24]. On the TTF-TCNQ pp ultrarmcroelectrode under the experimental conditions (scan rate 50 mV s-l), a limiting steady state IS approached, where radial diffusion layers form at individual electrodes. The steady state is approached at scan rates higher than 2 mV s-‘. The electrochemical kinetics of dopamine and hydroquinone on the TTF-TCNQ pp electrode are similar to those on pohshed GC (Table 2) which IS consistent with a similar fraction of active area of the two surfaces. Table 2 shows that at TTF-TCNQ pp ascorbic acid oxidizes at the same potential as on polished GC. Oxidation of hexacyanoferrate(II1) at both TTF-TCNQ and polished GC surfaces occurs at similar potentials (Table 2). The large values of the peak separation indicate that the electrochemical kmetics of hexacyanoferrate(II1) are slow. The

larger peak separation on TTF-TCNQ pp compared with that reported previously [20] is due to the difference in the method of electrode preparation. Electrochemical activation of GC leads to the expected improvement in the electrochemrcal kinetics [31,33,39]. As shown m Table 2, the oxidatton peak of hydroqumone after pretreatment shifts to ca. 0.13 V vs. Ag/AgCl, close to the 2e- formal potential (E” = 0.045 V vs. SCE at pH 7.0) [40], and AE, decreases. Following pretreatment, the oxidation peak of dopamme IS shifted only slightly m the cathodic direction from 0.23 to 0.20 V vs. Ag/AgCl, nearly to its thermodynamic potential (E” = 0.13 V vs. SCE at pH 7.0) [41]. The peak separation of DA on ECGC also decreases. After electrochemical pretreatment of GC, the peak of AA is shifted cathodically from 0.30 to 0.12 V (Table 2). Thts 1s the largest shift seen for all the probe molecules used in this study. Others [20,29,37] reported that oxidation of AA at active electrodes occurs at 0.0 V vs. SCE, close to the 2e- formal potential (E” = - 0.061 V vs. SCE at pH 7.0) [35]. Table 2 also shows the sensitivity for the probe molecules, measured in the linear region of the cahbratron graph and normalized over the geometric area. The sensitivity of all probes is highest on ECGC, followed by TTF-TCNQ pp and polished GC. The sensitivities of hydroquinone and ascorbic acid on ECGC increased ca. 1.8-fold after electro-

TABLE 3 Summary of hydrodynamic Compound

HQ

DA

AA

voltammetrlc

Surface

data Slope ’

E plateau*

E platea” b

E l/2 =

(V)

(V)

(V) _

_

0 70 045

045 0.14

0.011 0.013

TTF-TCNQ

0 91

GC ECGC

0 66 049

TTF-TCNQ

0 60

_

_

_

GC ECGC

0.55 0.38

0.65 042

0 46 0 20

0012 0.026

TTF-TCNQ

0 80

_

_

_

GC ECGC

0 74 0.60

0 70 045

044 0 15

0 010 0016

a PredIcted from cychc voltammetnc data 142) b Measured by HDV at a flow-rate 10 ml/rmn-’ obtalned from the plot of In(r/r, d - I) vs E

’ The E,,,

and slope values were

E DABEK-ZLOTORZYNSKA

320

chemical pretreatment, less than that of dopamine, which increased ca. 2.5-fold, consistent with fastest cyclic voltammetric results. Despite the similar slow kinetics of probes on TTF-TCNQ pp and GC surfaces, the sensitivity is higher on TTF-TCNQ than on pohshed GC. Surface interactions at TTF-TCNQ may be responsible for this behavior [24]. Hydrodynamic tlon The potentials

voltammograms

and flow detec-

at whrch the plateau of HDV should appear ( Eplateau ) were estimated using the method of Anderson et al. [42]. Table 3 summarizes the results. The dtfference between the experimental and estimated values of Eplateau at GC and ECGC electrodes is less than 10%. The estimated Eplateau at the TTF-TCNQ pp electrode is at potentials where decomposition of TTFTCNQ occurs [43]. Figure 1 shows a comparison of HDVs for hydroquinone and dopamme at the three tested surfaces under the same experimental conditions. At GC, the optimum response in the plateau region occurs at ca. 0.70 V for hydroquinone, ascorbic acid and dopamine. The results are summarized m Table 3. At TTF-TCNQ pp electrodes, the plateau cannot be obtained because the electrode material is unstable at positive potentials. Under the experi-

ET AL

mental conditions used, the stable potential window is between 0.40 and -0.05 V vs. SCE [22]. The optimum detector potential for the TTF-TCNQ pp electrode is 0.35 V vs. Ag/AgCl. Hydrodynamic voltammograms of HQ at TTF-TCNQ pp and at polished GC are similar. For DA and AA the hydrodynamic voltammograms shift to less positive potentials on TTFTCNQ in comparison with polished GC. This is consistent with the cyclic voltammetric results at the two surfaces (Table 2). Consistent with the cyclic voltammetric results, at ECGC the half-wave potentials (E,,,) decrease compared with those obtained on polished GC (Fig. 1 and Table 3); for AA, HQ and DA, the values are more positive than the thermodynamic formal potentials. The hydrodynannc background current response at the three tested surfaces is shown in Fig. 2. The large background current observed at TTF-TCNQ at potentials higher than 0.4 V is due to the decomposition of the organic salt. Flow-rate dependence The dependence of peak current on the carrier flow-rate measured at 0.35 V at all three electrodes without a column and at polished GC with a column is shown in Fig. 3. Hydroquinone at a concentration of 25 PM was used as a test probe. The flow-rates ranged from 0.25 to 2.0 ml mm-‘.

Dopomine

Hydroqufnone

Y : a

ooow_ 0 50

0 00

E(V)

vs.

oooJL

5000

i

100

Ag/AgCI

1 50

000

0 40

E(V)

vs.

0 80

1 20

Ag/AgCI

Fig. 1 Hydrodynaouc voltammograms of hydroqumone and dopamine at (A) TTF-TCNQ pp. (0) GC and (0) ECGC electrodes mJectlon volume, 20 pl; concentration of probes, 25 pM. 0.05 M phosphate buffer (pH 7 0) Flow-rate, 1.0 ml mn-‘;

in

EFFECTS

ON DETECTOR

RESPONSE

,,,u 000

321

IN FIA

020

040

060

E(V)

vs.

Ag/AgCI

100

080

Fig. 2 Background currents at (A) TTF-TCNQ pp, (0) GC and (0) ECGC electrodes Condltlons as In Fig. 1.

With the column, the influence of flow-rate (F) on current (I) is small; a slope of log z vs. log F of - 0.17 was obtained in comparison with a slope of -0.48 m the absence of the column. At the three tested surfaces, the log i vs. log F gave similar least-squares slopes of -0.59, -0.48 and - 0.50 for TTF-TCNQ pp, GC and ECGC electrodes, respectively, when the lo-$ cell volume was used.

t 250

Y 0

00

10000

: 50 00

x000

.I

”

,,

~,

IIIIIIIII,III/I’I”II(I”III(I”“‘II(’I’II’(’II’I 0 00 0 50 1 00

Flow

Rate,

x

.,

1 50

ml

2_op

2 50

min

Fig 3 Dependence of peak current on flow-rate at (A) TTFTCNQ pp. (0) GC and (0) ECGC electrodes without a C,s column, and (x) at the GC electrode with a C,s column Apphed potential, 0 35 V; other condltlons as m Fig 1.

A similar flow-rate dependence was obtamed on a GC electrode at the plateau potential region of HDV; the relationship between log z and log F had a slope of - 0.47 at 0.8 V. The dependence on flow-rate which is observed does not correspond to any particular flow model described m the literature [3,15,44,45]. This may be due to dispersion of an analyte carried by pump strokes winch can occur under conditions of laminar and/or non-laminar flow. For example, Meschi and Johnson [46], using the classical treatment of Taylor [47], have shown that for a tubular electrode the predicted peak current depends on the 1/3rd root of flow-rate for low dispersion and the -1/6th root of flow-rate for high dispersion. High dispersion was obtained when a large retention volume, compared with the injection volume, was used [46]. Ruzicka and Hansen [48] have shown that the dispersion in a fluid stream increases with the tube length between the inJector and cell, tube diameter, and flow-rate, and is inversely proportional to the volume of sample inJected. When the cell volume was decreased from 10 to 4 ~1 by reducing the spacer thickness from 0.0125 cm to 0.005 cm, the relationsl-np between measured current and flow-rate did not change; the slope of log z vs. log F of - 0.43 was obtained at GC in a 4+1 cell in comparison with -0.48 when a lo-$ cell was used. Also, when the connectmg tube length was decreased from 50 to 35 cm no change in the dependence of current on flow-rate was observed. It has been reported [49] that the effect of the connecting tube on the analyte zone dispersion is negligible if the length of the capillaries (0.15-0.5 mm in diameter) does not exceed ca. 5 cm. The baseline noise was measured at TTFTCNQ pp and polished GC electrode. The baseline fluctuations that were observed at both electrodes can be attributed to flow irregularities. In contrast to theory, the baseline fluctuations observed at a non-polished TTF-TCNQ pp microarray were larger than those at polished GC. This can be attributed to the fact that an unpolished TTF-TCNQ pp is not microscopically smooth. A similar phenomenon has been observed with other composite electrodes [2,10]. For example, for RVC electrodes, Sleszynslu et al. [50] observed such

E DABEK-ZLOTORZYNSKA

322

deviations, which were confirmed cal analysis.

TABLE

by topographi-

TABLE

4

Reproduclbthty

Electrode stab&y To charactenze the reproducibility of current measurements at TTF-TCNQ pp detector electrodes, a series of 50 consecutive injections of 10 PM HQ were made. A mean peak current of 52 nA, with a range of 50-56 nA and a relative standard deviation of 3.8%, was obtained. For the last ten injecttons, the average of the current was 50.2 k 0.3 nA. Over a 60-min period, the background current (baseline current) did not change significantly, from 57 nA to 52 nA at 0.35 V. Although the reproducibility is good, the TTF-TCNQ electrodes were found to be less stable m the flowing stream than under static conditions [25]. So far, the TTF-TCNQ electrode has worked continuously under flow-injection conditions without a significant loss of sensitivity for only 2-3 days (ca. 50 injecttons per day). To characterize the stability of the TTF-TCNQ pp detector response with time and to evaluate the lifetime of the electrodes, the same electrode was employed to prepare calibration graphs on a few consecutive days with standard solutrons of hydroquinone in the concentratron range 2 x 10P63 x lop5 M. The sensitivity of the TTF-TCNQ pp detector, as indicated by the slope (5.0, 4.8 and 1.8 nA 1 pmol-’ for the first, second and third days, respectively), was found to decrease by ca. 60% with time. However, the electrode response vs. concentration was linear up to the third day; a correlation coeffictent of 0.9987 was obtained on

Electrode 1 2 3 4 5

ET AL

of TTF-TCNQ

Correlation coefflctent

0.9986 0.9936 0.9988 0 9991 0 9999

pp electrode

Slope (nA I j.imol-‘)

Intercept

5.0 4.0 4.8 30 48

3.5 6.5 6.0 18 2.2

preparatton R s.d. (W)

(W 22 65 2.7 14 06

a Condltlons for obtalmng workmg data same as m Fig. 1, except that the apphed potential was 0 35 V Seven hydroqumone standards m the range l-30 PM were used for each data set

the third day. Mechanical instability is the major reason for the decrease in the TTF-TCNQ pp electrode response with time. Table 4 summarizes calibration results obtained for hydroquinone with five TTF-TCNQ pp electrodes. These data suggest that varratrons in the electrode surface preparation can alter the sensitivity of the TTF-TCNQ pp electrode by as much as 20%. The average of the set of five values of the slope was 4.3 + 0.8 nA 1 pmol-‘. Lmearlty of response The response time of the electrode is an tmportant parameter m flowing streams. With a lo-p1 detector cell, the half-widths of the peaks were 12 s at both TT’F-TCNQ pp and GC electrodes and 18 s at ECGC. Broadening of peaks may be due to dispersion. For example, when the cell volume was decreased from 10 to 4 ~1, sharper flow-mjection

5

Summary

of cahbratlon

Surface

data a

Compound

Lmear range (PM)

TTF-TCNQ

GC

ECGC

* Condltlons

Correlation coefflclent

Slope (n.4 1 pmol-‘)

Intercept

R s.d

(nA)

(S)

BQ

l- 30

DA AA

lI-

30 30

0 9988 0 9990 0 9975

48 51 45

60 48 53

2.7 32 50

HQ

l- 60 l-100

0.9980 0.9995

33 27

29 49

3.0 15

HQ

l- 90

DA

l-

0.9990 0 9990

49 5Sb

9.6 11.4

20 20

DA

as m Table 4 Seven standards

90

a

were used for each data set b Normahzed

for 0 070-cm’

geometric

area.

EFFECTS

ON DETECTOR

RESPONSE

IN FIA

peaks were obtained; the half-widths of the peaks were 6 s at GC. The larger peak widths at ECGC than at TTF-TCNQ or GC electrodes are probably a result of the porosity of ECGC [34]. This is supported by the slope of 0.57 (0.52 at GC) of the log(peak current) vs. log(scan rate) measured m cychc voltammetry for hydroquinone at ECGC, which indicates an adsorption contribution to amperometric response. The lower sensitivity of probes on ECGC than on GC at plateau potentials (Fig. 1) is a result of larger peak widths and ca. 20% lower geometric area of ECGC (0.059 cm2) than GC. Calibration graphs for flow mlection obtained at 0.35 V and a flow-rate of 1.0 ml min-’ show lmearity (typical correlation coefficient 0.999) in the concentration range 1 x lo-‘-3 x 10e5 M (Table 5). This concentration range corresponds to concentrations of injected analyte. Dispersion (0) in the system estimated as D = 1.8 at a GC electrode makes the concentrations seen by the detector lower. Sensttivities determmed from the slope of the calibration graphs at the TTF-TCNQ pp electrode are 5.1, 4.8 and 4.5 nA 1 pmol-’ for DA, HQ and AA, respectively. The same order of probe sensitivtties at the TTF-TCNQ electrode was observed in cyclic voltammetric experiments (Table 2). The surface with the highest activity and highest estimated active area gives the highest lmear dynamic range, as shown in Table 5. The TTF-TCNQ pp electrode shows a better sensitivity than GC, consistent with cyclic voltammetric results, indicatmg that the low surface activity may be an underestimation. Conchons The results obtained show that the analytical sensitivity at microarray surfaces increases with the fraction of active area of the array, m agreement with previous findings of Wetsshaar et al. [ll]. Sensitivity is also affected by the kinetics of the analytes. Differences m sensittvtty at microarray electrodes for processes controlled by mass transfer and heterogeneous kmetics have been reported [4]. Apparent heterogeneous kinetics of dopamme under the experimental conditions in thts study are faster than those of hydroquinone but are not suffictently fast for the response to be

323

mass transfer limited. Because of the reasonably fast kinetics, dopamine shows a higher sensitivity to the relatively small differences in the fraction of the active area of the investigated surfaces than the kinetically slower hydroqumone and ascorbic acid, and the sensitivtty of detectton of dopamme is higher. In agreement with theoretical predictions [51], the increase m the fraction of the active area of the ECGC microarray compared with the GC array leads to faster apparent heterogeneous kinetics for all the analytes and a resulting stgmficant improvement in sensitivity. The significantly better sensitivity for TTF-TCNQ compared with GC cannot be explained solely on the basis of microarray structure and may be due to the chemistry of the electrode surface [24]. The relative sensitivity m flow-mlection amperometric detection increases with the fraction of active area for kinetically faster probes as described above, can be predicted from cyclic voltammetric experiments and from the more time consuming HDV. Kmetically slow hydroquinone was used to determine the effect of flow-rate on amperometrtc response. There is a clear decrease in the dependence of the response on flow-rate at GC when the column, which is expected to minimize dispersion, is introduced into the flow system. The resulting independence of signal on flow-rate is consistent with previous results [8]. The same changes in spacer thickness as those m [8] produced no sigmftcant changes in flow-rate dependence. In the absence of the column, a similar significant dependence of response on flow-rate at low flowrates has been observed [53,54]. It has been proposed [53,54] that the observed decrease in response with increase m flow-rate is a result of slow heterogeneous kinetics at the electrode surface which become dominant at htgher flow-rates. This is not consistent with the significant decrease m the dependence of signal on flow-rate when the column is present. Relative effects of kinetics and mass transport have to be evaluated quantitatively to determine when each process becomes limiting. This research was supported in part by NIH grant BMT GM35451-03 A2 and the Division of Sponsored Research at the University of Florida.

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