Amperometric detection of catecholamines with liquid chromatography at a novelly constructed prussian blue chemically modified electrode

Tahmra, Vol. 39,No. 3, pp. 235-242,1992 FYintcd in GreatBritain.All fightamcrvcd

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AMPEROMETRIC DETECTION OF CATECHOLAMINES WITH LIQUID CHROMATOGRAPHY AT A NOVELLY CONSTRUCTED PRUSSIAN BLUE CHEMICALLY MODIFIED ELECTRODE JIANXUN ZHOU and ERKANG WANG*

Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of sciences, Changchun, Jilin 130022, People’s Republic of China (Received 20 February 1991. Revtied 8 July 1991. Accepted 10 July 1991) Bummary-A novel Prussian blue chemically modified electrode (CME) was constructed and characterixed for liquid chromatography electrochemical detection (LCEC) of catecholamines. Both anodic and cathodic peaks could be obtained by monitoring at constant applied potential at ancdic and slightly cathodic potential ranges (0.3-0.7 and -0.241 V vs. SCE), mpectively. When arranged in a series configuration, using the modified electrodes as generating and collecting detectors, extremely high effective collection elliciencies of 0.91 (for norepinephrine) and 0.58 (for dihydroxyphenylacetic acid) were achieved in dual-electrode LCEC for catecholamines; and a linear response range over 3 orders of magnitude and a detection limit of 10 pg were obtained with a downstream CME as the indicating detector.

Liquid chromatography with electrochemical detection (LCEC) continues to grow in popularity for the sensitive determination of trace compounds in complex samples,’ and is most highly publicised for its applications in neurochemistry. Up to 1986, over 800 papers had been published dealing with LCEC to solve neurochemical problems which would otherwise have been more time-consuming or even impossible to attack.2 Among electrochemical flow detectors, amperometric detection at constant potentials has proved to be the most popular tool for solving a wide variety of practical analytical problems in neurochemical research by virtue of the facility in operation, sensitivity and excellent selectivity;3 and catecholamines have been the primary target compounds among the major neurotransmitter groups penetrated by LC amperometric detection procedures. Singleelectrode detectors at a constant applied potential have been widely employed for the determination of catecholamines in biological fluids,“O including catecholamines in plasma&’ and brain tissue homogenate.7-‘0 Amperometric dual-electrode detectors have recently become popular in LCEC monitoring of catecholaminerelated species, which have three configurations termed parallel-adjacent, series, and parallelopposed dual-electrode detectors,” according to *Author for correspondence.

the orientation of the electrodes with respect to the flow axis. Parallel-opposed dual-electrode detection has been explored to improve detectability of catecholamines in blood plasma” and serun~‘~ Parallel-adjacent detectors have been employed to aid in identification of catecholamines in brain tissue.‘4,‘5 Series dualelectrode configurations have been applied mainly to substantially enhance the selectivity for the determination of catecholamines,‘G’O because the system which is routinely operated in oxidative+reductive mode discriminates against chemically irreversible reactions upstream. For series dual-electrode detectors, the fraction of upstream products that are converted at the downstream detector, termed collection efficiency (N), is the governing parameter for analytical applications of the downstream signal. The N value is highly dependent on the electrodes’ size, shape and the configuration with respect to one another,‘*‘* as well as the cell designs. Although as high as 60% collection efficiency would be obtained theoretically with horseshoe cell configuration,2 series dualelectrode detection with N values less than 40% is the typical occasion encountered in LCEC procedures of catecholamines and other redox ~pecies.‘*~ High performance collection of catecholamines (N of 0.546) with LC amperometric detection has recently been achieved with an interdigitated microarray electrode (IDAE).*’ 235

236

JUNXUNZHOUand ERKANGWANG

This paper reports the attempts on LC amperometric monitoring of catecholamines with a chemically modified electrode (CME). A number of unique features were obtained compared with conventional LCEC procedures of catecholamines. These include extremely high collection efficiencies that could be readily obtained with series dual-electrode configuration which are highly recommended for the CMEs analytical applications in LCEC of catecholamines and perhaps other neurotransmitters. EXPERIMENTAL

Reagents

Analytical reagent potassium ferricyanide was from Beijing Chem. Co. Epinephrine (E) was from Serva. Norepinephrine (NE), dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were purchased from Fluka A.G. All these chemicals were used as received. Other chemicals were of analytical grade. Doubly distilled water was used for the preparation of all solutions. Catecholamines stock solutions (1 mg/ml) were prepared with O.lM perchloric acid, stored at 4” in the dark. The mobile phase was always O.lM phosphate buffer (pH 5.0) containing ImM EDTA delivered at 1 ml/min, unless stated otherwise. Apparatus

The instrumentation used was the same as in the previous report. **The electrochemical detector was a TL-SA thin-layer cell (BAS, U.S.A.) incorporating two glassy carbon electrodes of the same size. The auxiliary electrode was positioned across the thin-layer channel from the working electrodes to minimize ohmic losses for adequate potential control,” when the series dual-electrode arrangement was utilized. All potentials were measured and reported against a saturated calomel electrode (SCE), unless stated otherwise.

ferricyanide solution in 0.5M potassium chloride. Electrochemically pretreated GC electrode (EPGC), The polished and washed GC electrode

was subjected to potential cycling as in the above procedure in 0.5M potassium chloride containing no ferricyanide. RESULTS

AND DISCUSSION

CME electrochemistry

On removal of the electrode from the ferricyanide solution after the modification was completed, no obvious changes of the electrode surface was observed. However, cyclic voltamperograms (CVs) similar to the ferro/ ferricyanide redox couple were obtained (Fig. 1) when the electrode was washed and examined in 0.5M potassium chloride devoid of the redox species. Sharp and narrow peaks for oxidation and reduction exhibited characteristics of a surface reaction, with the peak currents, both anodic (h) and cathodic (i,), increasing linearly with scan-rates (5-100 mV/sec). This indicated that such a modified electrode did incorporate a stably bound redox group characteristic of the adsorption type. The nature and mode of the modification of the ferro/ferricyanide redox couple is not immediately clear yet. On extending the anodic sweep limit to + 1.3 V vs. Ag/AgCl during the potential cycling with the CME in 0.5M potassium chloride, a second redox couple with E,, of ca. + 1.0 V was repeatedly observed (not

Working electrodes Prussian blue chemically modjied electrode (PB-CME). A glassy carbon (GC) electrode

was modified with the procedure similar to that described earlier. 23 Prior to modification, the GC electrode was polished with a OS-pm alumina suspension on a smooth cloth, thoroughly ultrasonicated in a water bath and rinsed with water. The GC electrode was then subjected to potential cycling between - 0.4 and 1.5 V vs. SCE at 1 V/set for 30 min in 1mM

I

08

I 04

Wmt101,

I 0

I -04

V, vs Ag/AgCC

Fig. 1. Cyclic voltamperograms (CVs) with the CME in OS4 KC1 at a scan-rate of 5, 25, 50, 75 and 100 mV/sec.

Amperometric detection of catecholamines

shown), which is suggested to correspond to the Prussian blue/Berlin green redox couple on the electrode surface. 23 Based on evidence of the occurrence of this second redox couple, it was established that the incorporated species corresponded to Prussian blue (PB)F3 Figure 2B shows the effect on the cyclic voltamperograms (CVs) obtained at the CME in OSM potassium chloride upon addition of dopamine to the blank supporting electrolyte. In the presence of dopamine, no changes of the background peaks at the CME was observed, but another pair of redox waves (with Ep and Epc of 0.40 and 0.34 V vs. Ag/AgCl at 100 mV/sec, respectively) appeared, corresponding to the dopamine redox group itself. Compared with CVs of dopamine at the bare GC electrode (Fig. 2A), the peak potential separation AEp was significantly reduced (from 600 mV at the bare GC to 60 mV at the CME at 100 mV/sec), and the peak currents increased, indicating a greatly improved reversibility of the dopamine electrode process. In view of the fact that the CME modification procedure has in effect involved an

(B)

0.9

025

-0.4

Fbtential, V, vs.Ag/AgCl

Fig. 2. Cyclic voltamperograms of dopamine at the bare GC (A), CME (B) and EPGC (C) electrodes. Scan-rate, 100 mV/sec. Dopamine concentration is 60 ppm for (A) and for (C); 0 ppm for curve 1 in (B), 10 ppm for curve 2 in (B) and 60 ppm for curve 3 in (B).

231

electrochemical treatment process by alternative anodic and cathodic polarization at high speed, the effect of such electrochemical pretreatment on CVs of dopamine at the bare GC was determined with an EPGC electrode and is shown in Fig. 2C. Interestingly it was found that the CV behaviour of dopamine at the EPGC electrode was nearly the same as that at the CME, which might suggest that the redox couple (Prussian blue) sustained on the CME had little effect on the electrode process of dopamine. The small and smooth redox couple with Ep of ca. 0.20 V vs. Ag/AgCl observed on CVs at the EPGC corresponded to quinone groups bound at the GC surface.2k26 Flow-through amperometric detection Single electrode detection. Figure 3(A) shows the hydrodynamic voltamperograms (HDVs) for catecholamines at the single PB modified CME, with O.lM phosphate buffer (pH 5.0) containing 1mM EDTA as mobile phase. In the anodic potential region, a plateau response level was achieved at +0.45 V vs. SCE and beyond. Cathodic currents, however, arose in the cathodic potential range (more negative than 0.20 V), reaching maximal levels at around +0.05 V. The absence of ImM EDTA in the mobile phase had little effect on the HDV behaviour of the analytes in the anodic response region; however, the cathodic currents decreased to a certain degree (20% at the maximum response level) in this EDTA free solution. The flow-rate dependence on the anodic peak current of LC amperometric detection of catecholamines was studied and is shown in Fig. 4. It is clear that the current response always decreased with increasing flow rate, contrasting sharply with theoretical predictions,2728 with practical considerations for conventional LCEC of catecholamines with unmodified electrodes.29 This unique flow-rate dependence is of advantage for the compatibility with microbore HPLC, high sensitivity being attained at rather low flow-rate of the mobile phase. All these observations indicated that the detector response is not controlled by the process of direct electron transfer between the analytes and the GC surface. The specification of actual mechanism for the appearance of both anodic and cathodic peaks with LCEC of catecholamines, using the single CME working electrode remains difficult. There would be a possibility that the detector response was governed by the complexation of the solutes with iron ions

238

01 0

01 02 03 04 05 06

-01 0

01 02 03 04 05 0.6

Fot6nti01, V ,yS .SCE

Fig. 3. Hydrodynamic vohamperograms of 1 ppm each of catecholamines at single CME (A) and downstream CME in series configuration (B) with the upstream CME monitored at 0.60 V us. SCE. Column, Nucleosil C,, (7 pm) 200 x 4 mm i.d. Mobile phase, O.lOMphosphate buffer @H 5.0) containing 1mM EDTA at a flow-rate of 1 ml/min. Injection volume, 10 pl.

[Fe(II) or Fe(III), determined by operating po tential] present in the PB molecule.m The current response at the CME might result from the differences in solubility and electroactivity of complexes between the components in the mobile phase (EDTA, etc.) and the ligands (ana-

0104’

I

I

I

08

12

16

I 20

Fig. 4. Effect of mobile phase flow-rate on current response in LCEC of catecholamines at the CME. Potential, 0.45 V IX SCE. Chromatographic conditions as in Fig. 3.

lytes) injected with Fe(I1) or Fe(II1) sustained on the CME surface. Eluted catecholamines may form more or less favoured soluble and electroactive iron complexes than the mobile phase components with Fe(II1) or Fe(II), respectively, while monitoring the detector at either anodic or cathodic potential regions, and thereby resulting in an increased or a decreased current for iron ion redox reactions, and hence in amperometrically positive or negative peaks. Considering the EDTA effect on the HDV behavior of the analytes, these complexation kinetics would more likely occur in the cathodic response region. Figure 5 shows the dual-electrode chromatograms of catecholamines with paralleladjacent configuration, using a bare GC electrode and a CME as indicator detectors, respectively. Distinct cathodic peaks of adequate sensitivity were observed with the CME at the applied potential of 0.0 V 0~. SCE which exhibited a moderate amount of tailing compared with those obtained at the bare GC electrode.

Amperometric dete&on of catecholamines

239

40

35

DA

30

NE F

.

25

I DA

I

P

4nl I!

I( + 1

5

IO

15

Retentiontime, min t

Fig. 5. Parellel-adjacent dual-electrode chromatograms of catecholamines at a bare GC (WI) and the CME (W2). Potential, 0.60 V for Wl and 0 V for W2. Flow parameters as in Fig. 3.

The effects of the mobile phase pH on peak current with the LCEC of catecholamines at the bare GC and the CME are shown in Figs. 6 and 7, respectively. With the bare GC electrode, the current response of epinephrine at higher pH ( > 6) was double that at lower pH (2.5). Increasing pH had less effect on current response for norepinephrine and negligible change for dopamine. This is due to the dramatic effect of pH on the electrode mechanism involving ECE reactions.’ Higher pH favoured the indoline form of the epinephrine molecule which has the known high rate of nucleophilic addition, resulting in a net four electron transfer and thereby the detector response may double. All these observations were consistent with previous work.’ DOPAC exhibited maximum current response at pH 5.0. Lowering the pH is disadvantageous for DOPAC oxidation due to a more positive half-wave potential,’ whereas higher pH is also unfavourable for DOPAC oxidation probably due to the lower heterogeneous electron transfer rate constant at higher PH. With the CME, however, the case is quite different. As shown in Fig. 7, catecholamines

34567

8

Mobib phase pH

Fig. 6. E&t of mobile phase pH on the peak current in LCEC of catecholamines at the bare GC electrode. Potential, 0.60 V t)s. SCE. Analyte concentration: 1 ppm for NE and E, 1.5 ppm for DA and 2 ppm for DOPAC. Other conditions as in Fig. 3.

(except for NE) exhibit maximal current response at pH 5.0. This unique pH dependence was ascribed to the altered electrode mechanism involved at the CME compared with that on the bare GC electrode. It should be noted that the response sensitivity was greatly improved with the CME in the LCEC of catecholamines. Series dual-electrode configuration. Figure 3(B) shows the hydrodynamic voltamperograms of catecholamines at the downstream CME in a series dual-electrode configuration, monitoring the upstream CME at constant potential of 0.60 V us. SCE. It is clearly seen that the anodic plateau response levels were lower than those at the upstream CME [Fig. 3(A)], indicating that the latter depleted a significant portion of the analyte in the diffusion layer before the mobile phase arrived at the downstream detector. However, the cathodic current response levels dramatically increased at the downstream CME in a series arrangement, reaching maximal response levels at an identical potential of 0 V. The enhancement of current responses should

JUNXUN THOU

240 log

r

k/=-J

and

/-‘*

‘-6 hkblle phase pH

Fig. 7. Effect of mobile phase pH on LCEC of 1 ppm each of catecholamines at the CME. Potential, 0.50 V us. SCE. Flow conditions as in Fig. 3.

be ascribed to the reduction of catecholamine oxidation products upstream. Figure 8 shows dual-electrode chromatograms of catecholamines with generation (oxidation)collection (reduction) mode, using bare GC electrodes, PB modified electrodes and EPGCs as working electrodes, respectively. The current measurements at the CMEs and EPGCs, both anodic and cathodic, were much higher than those obtained at bare GC electrodes. The effective collection efficiency (Ne) (determined from the ratio of the current at the downstream electrode to the current at the upstream electrode measured under given experimental conditions) for catecholamines was no more than 30% with bare GC electrodes (see Table 1). Ne values, however, were greatly increased at EPGCs due to the improved reversibility of catecholamines in the electrode process, as mentioned above. With CMEs, extremely high collection efficiencies were readily obtained, with the maximal value of 91% for NE, resulting from the total contributions of the complexation reaction between the analytes and Fe(I1) ion sustained at the CME surface and the reduction of catecholamine oxidation products upstream. Figure 9 shows the series dual-electrode chromatograms of catecholamines with the same oxidativereductive mode, using EPGC (or CME) and CME (or EPGC) as generating and

ERKANG WANG

collecting electrodes, respectively. It was interestingly found that extremely high Ne values were always obtained with the CME as collecting electrode, whether the CME or EPGC was used as the generating electrode, indicating that it was the reduction of upstream oxidation products of catecholamines that contributed a significant portion to the high collection efficiencies, and not the upstream complexation reaction between the analytes and Fe(II1) ion sustained on the CME surface. Table 1 summarizes the effective collection efficiencies of catecholamines obtained with the series oxidative-reductive operation mode, using various electrodes in the upstreamdownstream arrangements. It is essential that the CME be used as the collecting electrode in order to obtain highly enhanced effective collections. Using the PB modified electrodes as generating and indicating detector in a series configuration and the oxidative-reductive mode (at constant applied potentials of 0.60 and 0.0 V 11s. SCE), LCEC of catecholamines gave linear response range over 3 orders of magnitude, with a correlation coefficient greater than 0.99, and detection limits (S/N = 3) of 10 pg (for NE) and 50 pg (for DOPAC), readily obtained, which are much lower than those obtained at the interdigitated microarray electrode.21 In the past few years, a number of investigations have appeared3S36 concerning the preparation, electrochemical properties and analytical applications of chemically modified electrodes based on the mixed-valence hexacyanides (Prussian blue and its analogues). Li and Dong3’ and Net?” prepared thin adherent films of PB on platinum substrate by controlled current electrolysis and studied the electrochromism of the resultant PB CMEs. Cox and co-workers33*” and Kulesza et aL3$designed PB analogue CMEs for the electrocatalysis and determination of inorganic ions and organic compounds, including ruthenocyanide CMES”~~~and a nickel ferrocyanide CME.3S All these PB CMEs and analogues were reported to possess good stability under pH less than cu. 5.5; higher pH conditions would destroy the films sustained on the electrode surfaces.3’ Recently, Hou and Wang6 applied the simple adsorption procedure to the glassy carbon electrode to construct a PB/GC CME and used it in flow amperometry by electrocatalytic oxidation for hydrazine. It was found that such a PB/GC electrode was not stable in flow system at high

241

Amperometric detection of catecholamines NE

I

(6)

(A)

(Cl

NE

NE 20nA

I

0

I

I I

I

I

I

I

I

7

14 0

7

14

0

7

14

Retentmn tame.min

Fig. 8. Series dual-electrode chromatograms of 1 ppm each of catecholamines with bare GC electrodes (A), CMEs (B) and EPGCs (C) as working electrodes. Potential, 0.60 V for upstream WI, 0.0 V (B and C) and -0.20 V (A) for downstream W2. Other conditions as in Fig. 3.

positive applied potentials (0.9 V and beyond). In view of the fact that electrocatalysis is the fundamental point of all these previous works,33-36 and not the case in our research, from an analytical point of view the stability of the PB CME designed should be our primary concern. It should be noted that the PB/GC CME made in this work possesses favoured stability over those mentioned above.W36 This is evidenced by three factors: (a) the CME permits hundreds of potential cycles with the anodic scan limit up to + 1.5 V us. Ag/AgCl, without any obvious changes in the peak potentials and peak currents; when used in a flowing stream, the cathodic and anodic peak currents of catecholamine responses retained more than 90%

of their initial levels after two days of continuous service, even with high positive applied potentials (1 .OV and beyond); (b) the CME can be normally operated over a wide pH range (typically 1.540), examined by batch (CV) and flow (FIA) experiments. This makes the CME completely compatible with conventional reversed-phase liquid chromatography systems. Finally, the CME appears to possess excellent chemical and mechanical stability, as immersing the electrode in concentrated nitric acid for several minutes or submitting it to an ultrasonic water bath and polishing for a long period of time (typically more than 10 min) could not completely destroy the CME examined by cyclic voltammetry in a blank supporting electrolyte.

Table 1. Effective collection efficiencies for catecholamines with series dual-electrode LCEC’ Upstream Wl Downstream W2 Ne (ic/ia)

NE E DA DCPAC

Bare GC Bare GCt

CME CME

EPGC EPGC

EPGC CME

CME EPGC

0.27 0.26 0.30 0.12

0.91 0.82 0.80 0.58

0.47 0.44 0.51 0.34

0.88 0.79 0.82 0.59

0.35 0.35 0.50 0.16

*Potential: 0.60 V for Wl and OV for W2 us. SCE. tPote.ntial: -0.20 V us. SCE.

JIANXUN ZHOUand ERKANG WANG

242 NE

E ,

NE

5

WI=CME

NE

W2’CME I

0

NE

WI=EFGc

E

I

IO

I

I5

I

0

Retention time,

w2 = EFGC I 5

I

I 15

IO

min

Fig. 9. Series dual-electrode chromatograms of 1 ppm each of catecholamines with EPGC and CME as generating and collecting electrodes (A) and vice versa (B). Potential: 0.60 V for upstream Wl and 0.0 V for downstream W2. Flow conditions as in Fig. 3.

This confirms that modification readily occurred in depth at the GC surface. When a fresh electrode surface is needed, the old CME surface could be effectively depleted by performing several potential cycles between -0.40 and + 1.5 V us. Ag/AgCl in O.lM potassium hydroxide. Acknowledgements-The support of the National Natural Science Foundation of China is gratefully appreciated.

13. M. Goto, G. Zou and D. Ishii, J. Chromarogr., 1983, 268, 157.

14. G. S. Mayer and R. E. Shoup, ibid., 1983, 255, 533. 15. D. A. Roston, R. E. Shoup and P. T. Kissinger, Anal. Chem., 1982, 54, 1417A. 16. C. L. Blank, J. Chromatogr., 1976, 117, 35. 17. L. C. Lunte, P. T. Kissinger and R. E. Shoup, Anal. Chem., 1986, 57, 1541. 18. M. Goto, T. Nakamura and D. Ishii, J. Chronturogr., 1981, 226, 33.

19. Idem, ibid., 1982, 238, 357. 20. Ji Huamin and Wang Erkang, Chinese J. Chromalogr., 1988, 6, 137.

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