Pulsed electrochemical detection of H2O2 on gold

M. Gerlache, S. Girousi*, G. Quarin and J.-M. Kau�mann{

Institut de Pharmacie, Universite Libre de Bruxelles, Campus Plaine 205/06, Bvd du Triomphe,1050 Brussels, Belgium

(Received 22 September 1997; in revised form 6 November 1997)

AbstractÐThe voltammetric oxidation of hydrogen peroxide at a polycrystalline gold electrode, in neutraland alkaline media, shows two oxidation waves. At pH 7.4, one wave occurs at +490 mV and the secondone at +870 mV vs Ag/AgCl. The latter appears at the expense of the ®rst one. Formation of the ®rstwave is favoured by the presence of an adsorbed layer of hydroxyl-groups on gold but its magnitude ismarkedly a�ected by adsorbed interfering species and gold oxide structures. The quantitative analysis ofhydrogen peroxide, using the ®rst wave, is possible by preferably applying a pulsed electrochemical cleaningwith a detection at +550 mV. In ¯ow injection analysis the response of hydrogen peroxide is linear over awide concentration range i.e., 7 mM±0.1 M with a detection limit of 3 mM. The inhibition of H2O2 oxi-dation by an interfering species is suitable for the indirect quantitative analysis of species which are readilyadsorbed on gold. This is shown in ¯ow injection analysis by recording a stable oxidation current due tohydrogen peroxide in the carrier stream with detection of the inhibition of the signal due to the adsorbedspecies. Application to a surfactant such as Triton X100 at concentrations as low as 0.3 mM is reported.# 1998 Elsevier Science Ltd. All rights reserved

Key words: hydrogen peroxide, gold electrode, pulsed amperometry, Triton X100.

INTRODUCTION

Hydrogen peroxide is a molecule which is often stu-

died in electrochemistry and especially in the devel-opment of electrochemical biosensors. Hydrogenperoxide can be electrochemically detected at elec-

trodes such as gold [1±4], platinum [5±10], metalallows such as Pd/Au, Bi/Au [1, 11±15], and carbonelectrodes modi®ed by metal particles or with the

enzyme horseradish peroxidase [16, 17]. Platinum iscommonly used because of its high reactivitytowards H2O2 oxidation at a relatively low appliedpotential (+600 mV vs Ag/AgCl). The use of plati-

num o�ers also a better reproducibility of the oxi-dation of H2O2 compared to the gold electrode [4].Following a recent investigation, we have been able

to show that the voltammetric oxidation of H2O2

on gold may occur at two di�erent potentials i.e.,at a potential preceding the onset of gold oxide for-

mation (Ep=+ 490 mV) and at a potential in the

gold oxidation domain (Ep=+ 870 mV) in pH 7.4phosphate bu�er depending on the electrode surface

state [4]. The experimental conditions to observethe oxidation wave at +490 mV require a verydrastic control of impurities in the analysed sol-

ution, electrode surface cleanliness and oxidationstate. Actually, the electrode surface must be free ofany adsorbed species which may depreciate or

totally inhibit the oxidation at +490 mV in favorof the more positive one (+870 mV). Yet, it wasobserved that the ®rst wave of H2O2 oxidation wasfavoured by the presence of an adsorbed layer of

hydroxyl ions on the gold surface [4]. The exactstoichiometry of the hydroxide is still not wellde®ned but it may correspond to an incipient oxi-

dation of the gold surface which has been exten-sively described in the literature [18±29]. Thepossibility of using gold to detect the oxidation of

H2O2 is of interest because, at a potential of ap-proximately +500 to +600 mV in pH 7.4 bu�er,the H2O2 wave and the metal oxidation wave are

more clearly separated on gold compared to thesituation on platinum allowing greater sensitivity atthe former. In addition, gold is attracting consider-

Electrochimica Acta, Vol. 43, No. 23, pp. 3467±3473, 1998# 1998 Published by Elsevier Science Ltd. All rights reserved

Printed in Great Britain0013±4686/98 $19.00+0.00PII: S0013-4686(98)00093-0

*On leave of absence from the Aristotle University ofThessaloniki, Greece.{Author to whom correspondence should be addressed.

3467

able interest as electrode substrate for self-assem-bling of molecular assemblies and biosensor

development [30±32].Considering that the amperometric detection of

hydrogen peroxide on platinum and gold at a con-

stant potential is a�ected by accumulation ofadsorbed species and by oxide formation [4, 33, 34],it was of interest to investigate pulsed electrochem-

ical sequences as a means to maintain a reproduci-ble surface state and detect hydrogen peroxide at apotential corresponding to the ®rst oxidation wave.

This work also reports the suitability of the indirectquantitation of readily adsorbed species on gold byinhibition of hydrogen peroxide oxidation in ¯owinjection analysis (FIA).

EXPERIMENTAL

All chemicals were of analytical grade: sodium

laurylsulfate and cetylpyridinium chloride (Acros,Belgium), sodium hydroxide, sulfuric acid, hydro-gen peroxide (30% m/m), boric acid, sodium dihy-drogenophosphate, sodium sulfate, potassium

thiocyanide and sodium chloride (Merck) andcysteine, cystamine, cysteamine, Triton X100 andTween 80 (Sigma). All bu�ers and solutions were

prepared daily with milli-Q water.Cyclic voltammetry was achieved with an electro-

chemical analyzer 100B from BAS using a three-

electrode cell (Ag/AgCl 3 M NaCl as reference,platinum as auxiliary and BAS polycrystalline plati-num or gold disk, 1.8 mm diameter, as working

electrode). Amperometric measurements at a con-stant potential were realized with a CV 27 voltam-

mograph (BAS) using the same cell con®guration.Pulsed amperometric detection (PAD) was per-formed in a ¯ow system setup with an electrochem-

ical unit ED 40 from Dionex using the Dionexthree electrode ¯ow cell con®guration. The carrierstream was assured by a LKB pump and injection

of the samples was performed with a Gilson auto-sampler. All the experiments were made at roomtemperature (22228C) and in 0.1 M sodium sulfate

supporting electrolyte.Smoothing of the disk working electrode was

made using ®rst 1 mm then 0.3 mm alumina powderon a soft tissue for 2 min. Subsequently, the elec-

trode was rinsed with milli-Q water but preferablywithout sonication [4].

RESULTS AND DISCUSSION

As described previously [4], the voltammetric oxi-dation of H2O2 on gold in neutral and alkalinemedia may give only one oxidation wave at

+490 mV (Fig. 1(a)) or at +870 (Fig. 1(b)) or bothwaves may be detected (Fig. 1(c)) depending on thegold surface state. The ®rst wave is very sensitive to

poisoning by readily adsorbed species such as chlor-ide ions, surfactants and by oxide structures ongold. Actually, the second wave occurs at the ex-

pense of the ®rst and it may eventually be the onlyone observed because of complete surface ``fouling''or strong oxide coverage. Such a behaviour can be

Fig. 1. Cyclic voltammograms (CV) of H2O2 at a polycrystalline gold electrode. Phosphate bu�er pH 7.4; (a) gold

polished with alumina, rinsed with water without sonication, (b) polished with a diamond suspension, (c) gold polished

with alumina (CV in phosphate bu�er pH 7.4 + NaCl 10 mM).

M. Gerlache et al.3468

regarded as a result of a competitive adsorptionbetween hydroxyl groups and the interfering mol-

ecules on gold. It is worth mentioning that theseobservations are similar to inhibition phenomenawhich may occur in polarography in the presence of

a surface active compound. For instance, the ad-dition of a surfactant to a cadmium solutionshowed a depression of the cadmium reduction

wave with concomitant formation of a new re-duction wave at more negative potentials [35]. Theformation of the ®rst wave was favoured in alkaline

media i.e., by the presence of adsorbed hydroxylions on gold, and this wave was only poorlydetected in acidic media. Regarding the secondwave, it has been shown that, at an applied poten-

tial of +1000 mV in phosphate bu�er pH 7.4, adirect proportionality between the oxidation currentand H2O2 was established over a concentration

range comprised between 2�10ÿ5 and 6� 10ÿ3 Mwith a detection limit of 4� 10ÿ6 M [4].The determination of H2O2 by amperometry, at

an applied potential corresponding to the ®rst wave(+600 mV), was investigated here (the electrodewas smoothed between each measurement). A line-

arity between H2O2 concentration and the magni-tude of the oxidation current was also observed(I(mA) = 1.44 + (1.3220.06)�CH2O2

(mM) withr = 0.990) in the range 2� 10ÿ4 to 1.4�10ÿ2 M.

Contrary to the amperometric data obtained for thesecond wave [4], quanti®cation at the ®rst waveo�ered less reproducible results (RSD of the

slope = 2.9% for N= 6) because of the necessityto repolish manually the electrode surface betweeneach experiment and risks of impurity adsorption.

Yet, in order to obtain more reproducible data, anelectrochemical multi-scan sequence between nega-tive (i.e. ÿ400 mV) and positive potentials (i.e.+1000 mV) was investigated. This electrochemical

cleaning procedure should allow one to control andclean the surface state of the electrode in a reprodu-cible manner through surface oxidation and re-

duction of the formed oxide. Pulsed electrochemicalcleaning [33] was exploited below as it should allowthe use of the ®rst oxidative wave for quantitative

analysis. The potentials and the time window of thewaveform used have been selected so that, at eachcycle, a new and oxide free electrode surface was

made.

Interferences on the ®rst oxidation wave of H2O2

As mentioned earlier, the presence of surfaceactive compounds can interfere with the ®rst oxi-dation wave of H2O2. Many compounds can be

adsorbed or/and oxidized at a gold electrodesurface [33]. Cyclic voltammetry in phosphate bu�erpH 7.4 suggested that sodium sul®te and sodium

thiosulfate interfere since their oxidation (peak po-tential) occurred at +520 and +220 mV, respect-ively. The presence of sodium chloride in thesolution had a double e�ect on the cyclic voltamme-

try i.e., the intensity of the catalytic wave decreasedand the position of the maximum of the oxidation

peak shifted to more positive potentials e.g.Ep= + 600 mV in the presence of 0.01 M NaCl. Inthe presence of a sulfur derivate, the inhibition of

the H2O2 was more important because of the strongchemical interaction of sul®de groups withgold [33, 36]. This was also observed for potassium

thiocyanide, cysteamine and cystamine. In the pre-sence of 1� 10ÿ2 M cystamine, the linearity of theresponse was only obtained above a concen-

tration of 5�10ÿ3 M H2O2 (I(mA) = 0.29 +(0.01720.002)�CH2O2

(mM) with r= 0.988).Surfactants such as cetylpyridinium chloride orTriton X100 showed physical adsorption e�ects of

the same magnitude as the chemical inhibitione�ects observed for the thiol species.

Pulsed amperometric detection (PAD) of H2O2

The experiments have been carried out in pH 7.4

phosphate bu�er, in the absence of interferents,with a ¯ow through system ®tted with a 20 ml injec-tion loop. The ¯ow rate was adjusted to 1 ml minÿ1.Taking into account the cyclic voltammetric curves(Fig. 1) and the hydrodynamic voltammogram(Fig. 2), the applied potential pulse waveform

selected were: a delay time and integration at+550 mV, oxidative pulse at +1000 mV and reduc-tive pulse at +100 mV (potential corresponding to

gold oxide reduction). After approximately 45 minof stabilisation, this potential sequence allows oneto observe quasi no baseline drift. The H2O2 re-sponse intensity was found to increase with the

applied potential where the current integrationoccurred. However, at a potential higher than+570 mV, the ratio signal/background intensity

decreased quickly because of the gold surface oxi-dation. A measuring potential of +550 mV wasselected as it appeared to be a good compromise. It

was found also that the duration for the reduction(at +100 mV) of the formed gold oxide must belonger compared to the time devoted to the oxi-

Fig. 2. Hydrodynamic voltamogramme of H2O2 at a poly-

crystalline gold electrode in phosphate bu�er pH 7.4.

Electrochemical detection of H2O2 on gold 3469

dation (at +1000 mV) in order to avoid that the

baseline current increased regularly with time. In

order to increase the gold oxide reduction e�ciency,

the value of this potential could also be decreased

but this was not investigated here. If the delay time

before integration of the signal was lowered to

50 ms, the baseline increased very quickly. The opti-

mized waveform sequence selected is displayed in

Fig. 3, i.e., a delay time of 100 ms and a current

sampling duration of 50 ms at +550 mV. Using

this PAD sequence, successive injections of a 10 mMH2O2 solution showed only a slight decrease of the

signal with time (0.3% minÿ1) (Fig. 4). A cali-

bration curve established in such conditions gave a

linear response from 7�10ÿ6 till 0.1 M (Fig. 5).

Note that all the values recorded corresponded to

the integration of the area of the peak given by the

acquisition system. The detection limit was

23� 10ÿ6 M (S/N = 3).

In our previous work, we showed that the ®rst

H2O2 oxidation wave was more readily observed

and less a�ected by interfering species in alkaline

media. Subsequent quantitative attempts were car-

ried out in pH 8.0 and 9.0 phosphate bu�ers. At

pH 8.0 and 9.0, the maximum of the ®rst wave of

H2O2 oxidation shifted by approximatively 40 mV

and 100 mV, respectively, towards less positive po-

tentials compared to the value at pH 7.4 [4]. At pH

8.0, the signal/blank ratio was 1.0320.01 for a

10 mM H2O2 solution and was potential indepen-

dent between 440 and 600 mV. At pH 9.0, the sig-

nal was potential dependent, the value of the signal/

blank ratio decreased by raising the potential of the

measurement likely due to a higher extent of gold

oxide formation than at pH 8.0. However, between

390 and 450 mV, this ratio was more stable

(1.6220.04). The same experiment has been carried

out in borate bu�er pH 9.0 and, here, the ratio

(1.0320.01) showed no potential dependence

between 300 and 500 mV.

The characteristics of the FIA response are sum-

marized in Table 1 for all selected working poten-

tials and di�erent bu�ers. As can be seen in Table 1,

the intensity of the signal decreased by raising the

pH of the phosphate bu�er yet the reproducibility

of the injections was better. At pH 9.0, the intensity

of the signal decreased slightly with time, probably

due to the instability of the hydrogen peroxide at

high pH values. This diminution of the response

with time also appeared in borate bu�er whatever

the applied potential.

From these results, it appeared that the ®rst oxi-

dation wave of H2O2 on gold was suitable for ana-

lytical purposes and that the amperometric pulsed

technique should be preferred to amperometry at

constant potential. Whatever the three pH values

tested (7.4, 8.0, 9.0), the concentration range where

the response was proportional to H2O2 was ap-

proximately the same, i.e. from 7�10ÿ6 to 0.1 M.

Fig. 3. PAD waveform sequence for the analysis of H2O2

on gold. FIA, phosphate bu�er pH 7.4.

Fig. 4. Hydrogen peroxide response in PAD-FIA in

20 mM phosphate bu�er. Flow rate = 1 ml minÿ1,Eapp= + 550 mV.

Fig. 5. Calibration curve of H2O2 in PAD-FIA at the gold

electrode (same conditions as in Fig. 3).

M. Gerlache et al.3470

The response in pH 7.4 phosphate bu�er was higherbut less reproducible, for successive injections of the

same sample, compared to the results at pH 9.0.The baseline and the H2O2 signal decreased by rais-ing the pH, so that the signal to blank ratioincreased with pH. However, if the same calibration

curve was reprocessed twice a day (at the beginningand at the end of the day), the correlation betweenthe two sets of experiments was less good for high

pH values. This may be due to the instability of theH2O2 when the pH is more alkaline or it might beattributed to an evolution of the surface state of the

electrode (oxide buildup).

Application of the PAD to the indirect detection of

surfactants

As shown previously, the ®rst oxidation wave ofH2O2 was highly sensitive to adsorption of interfer-

ing species on gold. If a permanent ¯ow of H2O2

was passed through the amperometric pulsed detec-tor, a constant oxidation current related to H2O2

concentration was observed at +550 mV. In thepresence of an interfering species, when injectedinto the carrier stream, the adsorbed hydroxylgroups on the gold surface may be substituted by

the injected compound and the oxidative current ofH2O2 diminished.In the following series of experiments, the injec-

tion valve was ®tted with a 100 ml loop and the re-duction potential, in the electrochemical cleaningstep, was set at ÿ500 mV in order to obtain a stable

baseline in the presence of a 1�10ÿ2 M H2O2 inthe ¯ow carrier (phosphate bu�er pH 7.4). Usingthese parameters, the baseline was stable over aworking day.

Sodium lauryl sulfate (SLS), an anionic surfac-tant, was investigated as possible interfering species.For the three pH tested, SLS induced a decrease of

the H2O2 baseline only for concentrations higherthan 1�10ÿ4 M. By injection of thiocyanate orchloride ions, which are known to be strongly

adsorbed at the gold surface [24], the perturbationof the base line appeared already at 1�10ÿ6 M ofinterferent. The indirect detection technique was

also tested on a polyethoxylated surfactant which ischaracterized by strong surface active propertiessuch as Triton X100 (TX100). At pH 7.4, a signaldepression by addition of TX100 was detected at

3�10ÿ6 M (Fig. 6). From the peak shaped response

it was inferred that the EC cleaning allowed a rapid

desorption of the surfactant. Yet, further optimiz-

ation would be required since some peak distortion

was observed especially at high surfactant concen-

trations (Fig. 6). Note that here all the measured

values corresponded to the current height given by

the integrator. The trends of the response was not

linear but actually characteristic of an electrode sur-

face saturation behavior (Fig. 7). This saturation

pro®le appeared also for pH 8.0 or 9.0. From a

calibration made at the beginning and at the end of

the day, a shift of the calibration curve was

detected but the sign and the magnitude of this

shift was random from one experiment to the other.

Successive injections of TX100 (3�10ÿ5 M), were

quite reproducible with a RSD of 1.3% (pH 8.0

phosphate bu�er and at a measuring potential at

500 mV). A detection limit of 3�10ÿ7 M TX100

was obtained. The response was relatively fast (less

Table 1.

Characteristics of the hydrogen peroxide response in FIA

Eapplied Phosphate bu�er Borate bu�er

pH 7.4 pH 8.0 pH 9.0 pH 9.0

550 mV 500 mV 450 mV 450 mV 340 mV

Relative signal 100 35 17 80 66

RSD (%) 6.4 3.1 1.2 3.5 4.7

Fig. 6. Indirect detection of Triton X100 by H2O2 inhi-

bition. PAD-FIA, 10 mM H2O2, phosphate bu�er pH 7.4.

Electrochemical detection of H2O2 on gold 3471

than 2 min for a 3� 10ÿ5 M TX100 injected sol-ution) so that 20 injections could be performed in1 h. Some attempts were also carried out with the

non ionic surfactant Tween 80. This polysorbate isextensively used in pharmaceutical formulations asemulsi®er. The system responded also to this mol-

ecule but, the adsorption was so strong that ap-proximately 40 min were necessary to recover thebase line value. Further studies, dedicated to the

optimisation of the pulse potential sequence, areunder investigation for the possible quanti®cationof this surfactant.

CONCLUSIONS

The pulsed amperometric detection of hydrogen

peroxide at a polycrystalline gold electrode com-prised in a ¯ow system e.g. in ¯ow injection analysisor in high performance liquid chromatography

appeared to be a useful method for the quantitativedetermination of H2O2 down to the mM level and ina large linear domain range. The indirect detection

of surfactants by inhibition of the H2O2 oxidationwave on the gold electrode was shown to be suit-able for quantitative purposes. Applications to elec-troinactive compounds but readily adsorbed on

gold may be foreseen, but additional experimentsconsisting of optimization of the pulsed electro-chemical sequence are required for improvement of

the analytical procedure.

ACKNOWLEDGEMENTS

Thanks are expressed to the Ministry of WalloonDistrict in application of the FIRST programme

(M. G.) and to the European Science Foundationfor an ESF-ABI fellowship (S. G.).Acknowledgments are also expressed to the analyti-cal department of the Colgate-Palmolive R and D

centre (Milmort, Belgium) and BAS (Lafayette,U.S.A.) for instrumentation support.

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