Electrochemical and FTIR studies of l -phenylalanine adsorption at the Au(111) electrode

Journal of Electroanalytical Chemistry 500 (2001) 299– 310 www.elsevier.nl/locate/jelechem

Electrochemical and FTIR studies of L-phenylalanine adsorption at the Au(111) electrode Hong-Qiang Li a, Aicheng Chen a, Sharon G. Roscoe b, Jacek Lipkowski a,* a

Guelph – Waterloo Center for Graduate Study in Chemistry, Guelph Campus, Uni6ersity of Guelph, Guelph, Ontario, Canada N1G 2W 1 b Department of Chemistry, Acadia Uni6ersity, Wolf6ille, No6a Scotia, Canada B0P 1X0 Received 12 June 2000; received in revised form 21 August 2000; accepted 9 September 2000 Dedicated to Professor R. Parsons on the occasion of his retirement from the position of the Editor in Chief of the Journal of Electroanalytical Chemistry and in recognition of his enormous contribution to the Journal and Electrochemistry

Abstract The adsorption of L-phenylalanine (Phe) at the Au(111) electrode surface has been studied using electrochemical techniques and subtractively normalized interfacial Fourier transform infrared (SNIFTIR) techniques. The electrochemical measurements of cyclic voltammetry, differential capacity and chronocoulometry were used to determine Gibbs energies of adsorption and the reference (E1) and sample (E2) potentials to be used in the spectroscopic measurements. The vibrational spectra have been used to determine: (i) the orientation of the molecule at the surface as a function of potential; (ii) the dependence of the band intensity on the surface coverage; (iii) the character of surface coordination, and (iv) the oxidation of adsorbed Phe molecules at positive potentials. The adsorption of Phe is characterized by DG values ranging from − 18 to − 37 kJ mol − 1 that are characteristic for a weak chemisorption of small aromatic molecules. The electrochemical and SNIFTIR measurements indicated that adsorbed Phe molecules change orientation as a function of applied potential. At the negatively charged surface Phe is predominantly adsorbed in the neutral form of the amino acid. At potentials positive to the pzc, adsorption occurs predominantly in the zwitterionic form with the COO− group directed towards the surface and the ammonium group towards the solution. At more positive potentials electrocatalytic oxidation of Phe occurs and is marked by the appearance of the CO2 asymmetric stretch band in the FTIR spectrum. Thus, relative to pzc, Phe is weakly chemisorbed at negative potentials, changes orientation at potentials close to the pzc and is oxidized at positive potentials. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Adsorption; Infrared spectroscopy; L-Phenyalanine; Amino acid; Gold electrodes

1. Introduction The interest in the interfacial behavior of proteins at solid surfaces originates from the need to understand better the mechanisms of processes associated with their use in advanced technical applications and industrial problems. In order to understand the behavior of proteins, it is important first to understand the adsorption behavior of amino acids. L-Phenylalanine was chosen for this study as it contains the hydrophobic aromatic group and therefore follows a series of small organic compounds (i.e. cyanopyridine [1], benzonitrile [2], and benzoate [3,4], pyridine [5] and 2,2%-bipyridine [6]) that we have been studying using electrochemical * Corresponding author. Fax: +1-519-7661499. E-mail address: [email protected] (J. Lipkowski).

and in situ FTIR to examine their orientation and coordination at the Au(111) electrode surface. The adsorption of phenylalanine (Phe) at metal surfaces has been investigated by a number of techniques. Differential capacitance and radioactive indicators were used to investigate the adsorption of phenylalanine and tyrosine at bismuth and mercury electrodes [7,8]. Other electrochemical studies of amino acids at mercury electrodes were made with glycine [9], glycyl –glycine [10], and methionine [11]. Studies have also been carried out on glycine [12,13], a- and b-alanine [13,14], a-, b- and gaminobutyric acid [13,15], and tyrosine and tryptophan [16] at polycrystalline platinum. A mechanism involving adsorption through the carboxylate group followed by decarboxylation was proposed for the oxidation of the amino acids.

0022-0728/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 0 0 ) 0 0 3 9 1 - 0

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In situ FTIR spectroscopy is a technique that has been used very successfully in the study of electrified interfaces [17 –20]. This technique allows the identification of intermediates and products of an electrode reaction, and it has been widely used to study electrocatalytic oxidation mechanisms of small organic molecules at noble metal electrodes [21 – 23]. Because of the surface selection rules, the technique provides information on the orientation and coordination of molecules adsorbed at metal surfaces. Applications of this technique have been made to the adsorption of CO on platinum group metals [23 – 26], as well as a number of organic molecules on polycrystalline platinum [27,28], mercury [29,30] and gold [31 – 33]. The in situ FTIR studies were concerned either with the identification of species generated at the electrode surface or the determination of the characteristic of their surface coordination in most of these applications. For example, in situ FTIR and electrochemical measurements on the adsorption of glycinate on Pt(111) showed the molecule to be twofold coordinated to the platinum through the carboxylate group and with CO2 formation at the higher potentials [34]. Less work has been done to develop FTIR spectroscopy as a tool for quantitative analysis of adsorbed species that could provide information about the composition of the interfacial region. Only a few papers report quantitative studies [20,35,36]. The objectives of our present work are threefold; (i) to employ the surface selection rules of IR spectroscopy to investigate the potential induced reorientation of the adsorbed molecules; (ii) to use IR spectroscopy to determine the potential range within which the molecules are stable at the electrode surface and to determine the potential for the onset of Phe oxidation, and (iii) to correlate the surface concentrations of Phe molecules determined from electrochemical studies with the intensities of selected IR bands for adsorbed molecules.

2. Experimental

2.1. Reagents L-Phenylalanine is a zwitterionic molecule at the pH of its isoelectric point which corresponds to 5.75 [37]. All experiments described in this work were performed in a neutral unbuffered electrolyte at pH values close to the isoelectric point. In these solutions, Phe exists predominantly in the zwitterionic form. Solutions of 0.01 M L-phenylalanine (\ 99% purity, Sigma Chemical Co.) were prepared from Millipore water (resistivity \ 18 MV cm). The supporting electrolyte was 0.1 M KClO4 (ACS Certified, Fisher Scientific) purified twice according to the procedure described in our previous papers [38,39]. Deuterium oxide, D2O (99.9%, Cam-

bridge Isotope Laboratories) was used in the in situ FTIR measurements without further purification. The electrochemical cell and other glassware were acidwashed and thoroughly rinsed with the Millipore water. The electrolyte solution was de-aerated by purging with argon for about 20 min before starting the measurements, and argon was allowed to flow over the solution at all times.

2.2. Cell and electrodes For both electrochemical and in situ FTIR experiments, a Au(111) single-crystal electrode which was grown, cut, and polished in our laboratory was used as the working electrode. Both the working electrode and the gold coil counter electrode used in the electrochemical measurements were flame annealed before each experiment. The reference electrode was an external saturated calomel electrode (SCE) connected to the cell through a salt bridge. A syringe type IR cell with a 60° CaF2 prism window was used for the in situ FTIR studies. The working electrode was pushed against the CaF2 window to form a thin-layer configuration. The angle of the incident photon beam at the electrode solution interface was about 80°, ensuring maximum enhancement [40]. The thickness of the thin layer was determined to be less than 10 mm, which minimized absorption by the solvent. A cylindrical platinum foil was used as the counter electrode, and the reference electrode was a Ag AgCl 3 M KCl electrode connected to the cell through a salt bridge. All potentials measured with respect to Ag AgCl were converted to the saturated calomel electrode (SCE) scale. All experiments were carried out at 209 2°C.

2.3. Experimental procedures The electrochemical experimental methods and procedures have been described elsewhere [38,39]. The electrochemical experiments were made using a PAR model 173 potentiostat controlled by a computer. All data were acquired by a plug-in acquisition board (RC Electronics, model IS-16). Custom software was used to record cyclic voltammograms (CVs), for chronocoulometric experiments and data processing. A 25 Hz sinusoidal wave (amplitude 5 mV rms) was superimposed onto a 5 mV s − 1 voltage ramp to measure differential capacity curves. The output from the potentiostat was fed into the lock-in analyzer (PAR model 5204) where the ac current was separated into its in-phase and out-of phase components. The output signals from the lock-in analyzer were stored in the computer and used to calculate the interfacial capacity. A series RC circuit was assumed in these calculations. The in situ FTIR experiments were carried out on a Nicolet 20SX/C FTIR apparatus equipped with a

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MCT-B detector cooled with liquid nitrogen. The sample compartment of the FTIR apparatus was purged throughout the experiment using CO2 and H2O free air provided by a Puregas heatless dryer. The IR spectra were recorded using the subtractively normalized interfacial Fourier transform infrared (SNIFTIR) technique [2,17 –19]. A multiple potential step (MPS) procedure was used to step the electrode potential between two values, E1, the reference potential where complete desorption of the adsorbate occurs, and E2, the sample potential which governs the orientation and coordination behaviour of the adsorbate molecule. The electrode potential was controlled by a PAR 173 potentiostat. During one cycle, the same number n of interferograms (n= 100) were recorded at each potential, E1 and E2. This cycle was repeated m= 20 times for each variable potential (E2) with the acquisition delayed for a 10 s interval after each potential change to allow the interface to reach thermodynamic equilibrium at that potential. The change of the electrode potential was synchronized with the acquisition of the interferograms by connecting the external trigger port of the PAR 173 potentiostat to the communication port of the DX 486 computer of the FTIR instrument. This procedure was repeated to give the total number N = m ×n (20× 100=2000) interferograms acquired at each of the two potentials. The interferograms were added to improve the signal-to-noise ratio, Fourier transformed, and used to calculate a relative change of the electrode reflectivity, defined as:

Fig. 1. For a Au(111) electrode in 0.1 M KClO4 (dashed lines) and 0.1 M KClO4 +1.06 × 10 − 2 M Phe solutions (solid line); (a) cyclic voltammetry curves recorded using the sweep rate 20 mV s − 1; (b) differential capacity recorded using a 5 mV (rms) sine wave modulated at 25 Hz and a 5 mV s − 1 sweep rate.

301

DR (R(E2)− R(E1)) (1) = R(E1) R The values of DR/R are reported in absorbance units (a.u.). By subtracting the reflection spectrum at potential E2 (R(E2)) from the reflection spectrum at potential E1 (R(E1)), the background due to the absorption of the solution species is eliminated. The potential for E1 was maintained at −600 mV (SCE) throughout the experiments while the potential for E2 was increased in a stepwise manner over the range of potentials from −100 to 600 mV (SCE). The spectra were recorded with a resolution of 4 cm − 1. The transmission spectra were recorded using a thin layer of a solution of phenylalanine in D2O between two flat ZnSe windows or solid phenylalanine immobilized in a KBr pellet. 3. Results and discussion

3.1. Electrochemical results Cyclic voltammetry (CV), differential capacity (DC) and chronocoulometric measurements provide information concerning the properties and characteristics of the electrochemical processes of phenylalanine adsorption at the Au(111) surface. This is particularly important in order to determine suitable reference and sample potentials for the FTIR studies and for the interpretation of the spectroscopic data. Fig. 1(a) shows the cyclic voltammetry curves recorded in the double layer region of Au(111) in 0.1 M KClO4 and in the presence of 1.06× 10 − 2 M Phe. Three pairs of quasi-reversible peaks appear between − 300 and 600 mV resulting from the adsorption of Phe. The peaks recorded in the positive voltage scan are quite broad relative to those associated with the adsorption of inorganic ions such as Cl− [41]. Fig. 1(b) shows differential capacity curves recorded for the supporting electrolyte alone and in the presence of 1.06×10 − 2 M Phe. Consistent with the CV curves, the capacities display three peaks suggesting that Phe adsorption is a three state process. The peaks on the differential capacity curves are observed at potentials,  − 100,  300 and  500 mV. The curves recorded in the presence of Phe merge with that of the pure electrolyte, at − 600 mV, indicating total desorption of Phe at this potential. Thus, − 600 mV was chosen as the initial potential for capacitive chronocoulometry and in situ FTIR measurements. In addition, the CV and differential capacity curves were helpful in establishing the upper potential limits for the in situ FTIR studies. Capacitive chronocoulometry was used to measure the difference between the charge density at the potential of adsorption (Ei) and that of total desorption (Ef) [38,42 –44]:

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port rate and to ensure that the state of adsorption equilibrium was established. The potential of zero charge (pzc) was determined from the position of the diffuse layer minimum of a differential capacity curve measured separately with a dilute solution of the supporting electrolyte. It was equal to 280 mV versus SCE. The absolute charge density at Ei was calculated from D|M(pzc)= |M(Ef)− |M(pzc)= |M(Ef)

Fig. 2. Charge density curves determined from chronocoulometry in 0.1 M KClO4 (dotted line) and, for clarity, a selected set of concentrations of Phe are shown ranging from 1.83 ×10 − 5 to 1.06 × 10 − 2 M.

(3)

Since there is no adsorption at Ef, the charge, |M(Ef), is independent of the presence of the molecules in solution. Thus D|M(Ei) and |M(Ef) were calculated using the above equations for the complete set of potential ranges and solutions investigated. The charge density (|M) versus potential plots, determined for the pure electrolyte and selected concentrations of Phe are chosen in Fig. 2. All charge density curves merge at − 500 mV, indicating that Phe molecules are already desorbed at this potential. The charge density curves also display multiple-step characteristics of a multi-state adsorption process of Phe. The potentials of these steps correspond to the position of peaks observed in both the CV (Fig. 1(a)) and DC (Fig. 1(b)) curves. The pzc shifts to more negative values in the presence of Phe. This feature indicates that the adsorbed molecules assume an orientation in which the negative pole of the permanent dipole moment is directed towards the Au(111) surface and the positive pole towards the solution.

3.2. Gibbs excess and gibbs energy of adsorption The film pressures (y) of adsorbed phenylalanine were calculated using y=

&

Ei

Ef

|M qdE −

&

Ei

Ef

|M q = 0dE

(4)

where q and q= 0 represent the presence and absence of the amino acid in the bulk solution, respectively. The relative Gibbs excesses were then calculated from Y= Fig. 3. Gibbs excess versus potential plots for Phe on Au(111) at 6.52× 10 − 4 M (filled circle); 3.14 × 10−4 M (open circle); 1.26 × 10 − 3 M (filled square); 2.27 ×10 − 3 M (open square); 3.52 × 10 − 3 M (filled up-triangle); 6.36 × 10 − 3 M (filled down-triangle); 1.06 ×10 − 2 M (filled down-triangle). Inset, for 1.06 × 10 − 2 M Phe solution, comparison of the Gibbs excess and the integrated intensities of the 1574 cm − 1 (open triangle) and 1410 cm − 1 (open circle) SNIFTIR bands in the spectrum of Phe in D2O recorded using s-polarized photons.

D|M(Ef)= |M(Ef)− |M(Ei)

(2)

Solutions of phenylalanine were prepared over the concentration range of 10 − 5 – 10 − 2 M in 0.1 M KClO4. When the Phe concentration was lower than 10 − 4 M, solutions were stirred in order to enhance mass trans-



(y RT( ln c



(5)

T,P,E

Fig. 3 shows the Gibbs excess (Y) versus potential curves for various concentrations of Phe in 0.1 M KClO4 solution. The Gibbs excess increases with potential until it reaches a maximum at 300 mV followed by a decrease at higher potentials. The maximum Gibbs excess of Phe is about 2× 10 − 10 mol cm − 2. This number is somewhat lower than the maximum excess of Phe determined at the Bi(111) and Hg electrodes [7,8]. Qualitatively, adsorption of Phe on Au(111) and Bi(111) as well as on Hg electrodes displays many common features. Somewhat lower values of Y observed for the Au(111) electrode may be explained by the eight times lower concentration of Phe used in our work.

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The Gibbs energy of adsorption DGads is usually determined from a fit of the experimental data to an equation of a particular adsorption isotherm. The adsorption of Phe apparently has a multiple state character and no simple isotherm can be used to describe the change of its surface concentration. However, in the limit of zero coverage, all isotherms simplify to the Henry isotherm [42]:

2 kJ mol − 1) and this feature indicates that the surface properties of Phe do not deviate too much from those of the system described by the Langmuir isotherm. The Gibbs energy values for Phe have comparable magnitude to the values of Gibbs energies of other weakly chemisorbed aromatic molecules [42].

y = RTYmaxic/55.5

3.3.1. Choice of sol6ent The infrared frequency range of interest is 1300 to 2000 cm − 1. A strong OH bending deformation of water occurs at 1635 cm − 1 and it adversely affects the investigation of Phe bands that are close to this region. In contrast, the corresponding OD bending mode of deuterated water is shifted to  1200 cm − 1. In addition, D2O gives a very flat background in the frequency region of interest [3]. For these reasons D2O was chosen as the solvent in the present studies. However, isotopic exchange of hydrogen by deuterium can occur from the interaction of D2O with compounds containing labile hydrogen, such as, for example, the hydrogen bonded to nitrogen in amines and amides [45]. Under normal conditions, hydrogen atoms attached to the benzene ring are not readily exchanged.

(6)

where y is the surface pressure, Ymax is the limiting surface concentration, c is the bulk Phe concentration, and i is the equilibrium constant which is related to the Gibbs energy of adsorption through the equation DGads = − RT ln i. The equilibrium constant can be evaluated from the initial slopes of the surface pressure versus the bulk concentration plots. The value of Ymax equal to 2× 10 − 10 mol cm − 2 was taken to calculate i. The Gibbs energies of adsorption determined in this way are plotted against the potential in Fig. 4. The standard state corresponds to unit mole fraction of the organic species in the bulk of the solution and to monolayer coverage by the noninteracting adsorbates. One can also use the value of − RT ln(cq = 0.5/55.5) as an additional measure of the energetics of Phe adsorption. When adsorption is described by the Langmuir isotherm, then RT ln(cq = 0.5/55.5) = DGads. Therefore, the difference between RT ln(cq = 0.5/55.5) and DGads determined from Henry’s Law can be used as a measure of the deviation of the behavior of a given system from that described by the Langmuir isotherm. The values of RT ln(cq = 0.5/55.5) are plotted along the DGads determined from Henry’s Law in Fig. 4. The differences between the two sets of data are quite small (less than

Fig. 4. For Phe on Au(111), Gibbs energy of adsorption and RT ln(cq = 0.5/55.5) plotted versus the electrode potential.

3.3. FTIR studies

3.3.2. General properties of IR spectra Amino acids are amphoteric. At low pH values, both functional groups are fully protonated so that the amino acid molecule assumes the cationic form; (C) RCH(NH+ 3 )COOH. At high pH values, all the acidic protons have been removed so that it assumes the anionic form (A); RCH(NH2)COO−. At pH near the isoelectric point (pI), the amino acid exists as the − zwitterionic form (Z), RCH(NH+ 3 )COO . The pK1 and pK2 for Phe are 2.20 and 9.31, respectively [37], and the pI is 5.75. The zwitterionic form will therefore be present in varying amounts in the pH range of 4–10. To facilitate interpretation of the electroreflectance spectra, we will first discus the transmission spectra of Phe shown in Fig. 5. Curve a shows the spectrum of pure, solid Phe pressed in a KBr pellet. The molecule of Phe in the KBr pellet is in the zwitterionic form, Z [46]. Table 1 gives the assignment of major bands, based on the band assignment for alanine [46 –49]. The band at 1625 cm − 1 is assigned to the NH+ 3 asymmetric deformation (Amino Acid I band). The band at 1562 cm − 1 corresponds to the symmetric NH+ deformation 3 (Amino Acid II band). Its broad envelope contains also the asymmetric COO− stretch. Bands at 1495 and 1458 cm − 1 correspond to CH2 scissoring deformations and the band at 1410 cm − 1 is the symmetric COO− stretch band. Curve b in Fig. 5 shows the spectrum of Phe in a neutral solution of D2O (pH  7). At this pH the molecule is predominantly in the zwitterionic form. There are significant differences between the spectra of

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Fig. 5. Transmission spectra of Phe and related compounds; (a) spectrum of Phe in a KBr pellet; (b) spectrum of Phe in neutral solution of D2O, pH 7; (c) spectrum of Phe in alkaline solution of D2O, pH 11; (d) spectrum of phenylacetate in D2O.

form Z in the KBr pellet and in the D2O solution. Spectrum b contains only two strong bands at 1616 and 1410 cm − 1 that may be assigned to the asymmetric and symmetric COO− stretch, respectively [46,47]. The weak bands are lost in the relatively high noise observed for this spectrum. Curve c in Fig. 5 shows the spectrum of Phe in alkaline (pH 11) solution of D2O, where the molecule is in the anionic form A [46]. This spectrum is also dominated by the two COO− stretch bands. For the anionic form, the asymmetric COO− stretch appears at 1574 cm − 1 and is red shifted with respect to the position of the same band in the zwitterionic form [47]. Characteristically, the 1625 cm − 1 band seen in spectrum a is absent in spectra b and c. The

absence of this band suggests that the use of D2O as a solvent results in deuteration of the amino group due to the presence of labile hydrogens attached to the nitrogen. Hence, form Z assumes a structure RCH(ND+ 3 )COO− and form A is RCH(ND2)COO−. Because of the change in the reduced mass from NH to ND, the deformational modes shift to lower frequencies by an amount wND/wNH  0.72 [46]. This corresponds to frequencies below 1200 cm − 1, outside the spectral range examined in this work. Consequently, in D2O the IR spectra of forms Z and A consist chiefly of two strong bands corresponding to symmetric and asymmetric COO− stretches and two small CH2 scissoring deformations. To verify this assignment, a transmission spectrum for phenylacetate in D2O (spectrum d) is also plotted in Fig. 5. Indeed, the IR spectrum of the A form of Phe resembles closely the spectrum of phenylacetate. Fig. 6 compares the transmission spectra of Phe in D2O recorded in the alkaline solution (pH 11)-spectrum a and in the acidic solution (pH 2)-spectrum b. The differences between the two spectra are very significant. In acidic solution, the IR spectrum of Phe consists of two broad bands at 1732 and 1458 cm − 1 that may be assigned to the CO stretch and the symmetric COOD stretch of the deuterated carboxylic group. Apparently, the use of D2O as the solvent in acidic electrolyte results in deuteration of the NH+ 3 group as well. The structure of the phenylalanine molecule becomes RCH(ND+ 3 )COOD (form C) [48]. The net result of these changes is that the IR spectrum of phenylalanine in acidic D2O resembles the spectrum of the phenylacetic acid shown as spectrum c in Fig. 6. The SNIFTIR spectra described below were recorded in unbuffered solution of 0.1 M KClO4. In this solution, reduction of traces of oxygen or water reduction at negative potentials may cause an increase of pH in

Table 1 Vibrational frequencies and assignments for transmission and SNIFTIR measurements a Vibrational frequency/cm−1 Zwitterionic form in KBr 1341 1410 1458, 1495 1562

Assignment of transmission spectra

Zwitterionic form in neutral D2O

Anionic form in alkaline Cationic form in acidic D2O D2O

1410

1341 1412 1458, 1495

1616

1574

1458

1625 1732

CH bend COO− symmetric stretch or COOD CH2 scissoring deformation NH+ 3 symmetric deformation vibrations COO− asymmetric stretch NH+ 3 asymmetric deformation vibrations CO asymmetric stretch of COOD group

a − − + Z, zwitterionic form, RCH(NH+ (or RCH(ND+ in D2O); C, cationic form, RCH(NH+ 3 )COO 3 )COO 3 )COOH (or RCH(ND3 )COOD in D2O); A, anionic form, RCH(NH2)COO− (or RCH(ND2)COO− in D2O).

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of pH 11 and here only the band at 1574 cm − 1 is observed. At intermediate pH values, the concentrations of zwitterionic and anionic forms are of comparable magnitude and here the IR spectra contain the two COO− stretch bands in the  1600 cm − 1 region.

Fig. 6. Transmission spectra of; (a) Phe in alkaline solution of D2O, pH 11; (b) Phe in acidic solution of D2O (pH 2); (c) phenylacetic acid in D2O.

3.3.3. SNIFTIR spectra for s-polarized photons For s-polarized light, Fig. 8 shows the evolution of SNIFTIR spectra of Phe as a function of the sample potential E2 in unbuffered 0.1M KClO4 in D2O. Since for s-polarized light with SNIFTIR the spectra are those of solution species, therefore in principle, their shape should not vary with E2. Only the amplitude of bands in these spectra should change with E2, due to the change of Y with the potential [3,5,6,20]. This is indeed the case for the spectral region below 1500 cm − 1. Integrated intensities of the 1410 cm − 1 bands are plotted versus E2, along the surface concentrations in the inset to Fig. 3. Clearly, the spectroscopic data track well the surface concentration plot. However, the relative amplitudes of the 1616 and 1574 cm − 1 bands change quite dramatically with the electrode potential. Using Fig. 7, these changes may well be explained in terms of a variable pH in the thin layer cavity. At the most negative potentials the amplitudes of the 1616 and 1574 cm − 1 bands have comparable magnitude. This feature suggests that the pH within

Fig. 7. Transmission spectra of Phe in solutions of D2O with pH ranging from 7 to 11. The pH values are indicated at the corresponding spectra.

the thin layer cell. Therefore, we have examined changes in the character of the transmission IR spectra of Phe in the pH range from 7 to 11. These spectra are plotted in Fig. 7. The ratio of the zwitterionic to anionic form changes significantly in this region of pH. In the IR spectrum of Phe, this could be seen as a change of the amplitude of the band corresponding to the asymmetric COO− stretch for the Z and A forms of the molecule. At pH 7 the zwitterionic form dominates and only the COO− stretch at 1616 cm − 1 characteristic for form Z is seen in the spectrum. In contrast, the anionic form predominates in a solution

Fig. 8. SNIFTIR spectra over the range of frequencies, 1300–1800 cm − 1 acquired using the s-polarized infrared beam, for the Au(111) electrode in 0.1 M KClO4 +1.06 ×10 − 2 M Phe solution in D2O. For each spectrum, the reference potential E1 was equal to − 0.60 V/SCE, and the value of the E2 is indicated in the figure.

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3.3.4. Orientation of adsorbed molecules The orientation of adsorbed molecules can be conveniently studied using p-polarized radiation. The electric field of p-polarized light has nonzero strength both at the metal surface and in the solution. For this photon polarization, the SNIFTIR spectrum is a difference between the spectra of molecules adsorbed at the electrode surface at potential E2 and spectra of molecules desorbed into the thin layer cavity at E1. The measured (DR/R) can be expressed as: (DR/R)=2.3Y[cos2 q(E1)m(E1)− cos2 q(E2)m(E2)]

Fig. 9. SNIFTIR spectra for Phe adsorbed at the Au(111) electrode from 0.1 M KClO4 +1.06× 10 − 2 M Phe solution in D2O using a p-polarized infrared beam. For each spectrum, the reference potential E1 was equal to − 0.60 V/SCE, and the value of the E2 is indicated in the figure.

the thin layer cavity is about 8.5. By moving the potential in the positive direction, the amplitude of the 1616 cm − 1 band decreases and of the 1574 cm − 1 band increases, suggesting that the pH of the solution increases and that Z is progressively converted into the A form. When E2 \300 mV, the 1616 cm − 1 starts to grow again at the expense of the 1574 cm − 1 band, suggesting that the pH in the thin layer cavity begins to decrease. We have already mentioned earlier that the initial increase of pH may be explained by the reduction of residual oxygen or reduction of water at the reference potential. Since the spectra were acquired sequentially, moving from negative limit in the positive direction, the increase of pH may actually be caused by the increasing time of the electrolysis rather than by the change of the potential. For future experiments, a reverse order of the potential change would be recommended. We will show later that the decrease of pH observed at E\300 mV correlates well with the onset of Phe oxidation and formation of a weak acid such as CO2 in the oxidation reaction. The changes of the spectral features seen in Fig. 8 illustrate difficulties in studying surface properties of amino acids adsorbed at the solid surface from unbuffered solutions. Unfortunately, anions present in suitable buffers adsorb more strongly than Phe, preventing their use in this study.

(7)

where R is the electrode reflectivity, q is the angle between directions of the electric field of the photon and the direction in which the dipole moment of the molecule changes, m is the molar absorption coefficient, Y is the surface concentration of the adsorbed molecules. At potential E1, Phe molecules are desorbed from the electrode surface and are randomly oriented. The function cos q has to be averaged over all possible orientations and the result is B cos2 q(E1)\ random =1/ 3. At potential E2 the electric field of the photon is nearly normal to the surface and hence the magnitude of cos q(E2) depends on the direction of the transition dipole with respect to the normal to the surface. Fig. 9 shows the family of SNIFTIR spectra of 0.01 M Phe in 0.1 M KClO4 in D2O with p-polarized light. Consistent with Eq. (7), the SNIFTIR spectrum for p-polarized photons consists of negative and positive bands. The negative bands correspond to adsorbed and positive bands to desorbed molecules. The strength of the electric field of the p-polarized photon on the electrode surface is enhanced (m(E2)\ m(E1)) [50]. Hence, bands of adsorbed molecules may be stronger than bands of the solution species. However, the amplitude of the band for adsorbed molecules also depends on the magnitude of angle q(E2). Vibrational modes that have a large component of the transition dipole in the direction perpendicular to the surface are strong, while vibrational modes with the transition dipole parallel to the surface are inactive. In contrast, for molecules desorbed into the thin layer cavity at E1, all IR modes are active. In the region of 1725 –1732 cm − 1, a bipolar peak appears normally assigned to the CO stretch in protonated (deuterated) carboxylic acids [48]. In addition, a positive peak at 1574 cm − 1 assigned to an asymmetric carboxylate stretch and a bipolar peak at 1410 cm − 1 due to the symmetric carboxylate stretch also are observed in the spectra. The simultaneous presence of the band due to CO stretch of a protonated (deuterated) carboxylic group and the asymmetric and symmetric COO− bands of the dissociated carboxylic group indicates that the molecule of Phe is undissociated when it is adsorbed at the surface (i.e. as the neutral molecule) and is dissociated (A form) when it is

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desorbed into the solution. In fact, it is well documented [51] that, in the adsorbed state, pKa of a weak acid may differ from the pKa in the bulk. It is therefore reasonable to expect that an adsorbed molecule is protonated at the surface but dissociates when desorbed into the bulk. We note that the symmetric COOD stretch, seen at 1450 cm − 1 in the transmission spectrum of the protonated molecule in Fig. 6, is absent in the SNIFTIR spectrum. This suggests that in the adsorbed molecule the transition dipole of the symmetric COOD stretch is oriented in the direction parallel to the surface and consistent with the surface selection rules is IR inactive. We also note some differences between SNIFTIR spectra recorded using s- and ppolarized light. In the spectra acquired using s-polarized photons (Fig. 8) the 1616 cm − 1 band was present when EB0 mV. In the spectra for p-polarized light (Fig. 9) this band is absent at E B 300 mV. These differences suggest that in addition to the species adsorbed in the neutral form, as discussed previously, a certain amount of Phe molecules resides on the surface − on in the zwitterionic form with the ND+ 3 and COO − the surface and the plane of the COO group being normal to the surface. In this orientation the permanent dipole of the zwitterion and the transition dipole of the symmetric COO− stretch are parallel to the surface

Fig. 10. For a Au(111) electrode in 0.1 M KClO4 + 1.06× 10 − 2 M Phe solutions; (a) integrated intensities of 1725 cm − 1 band (square, CO stretch of the deuterated carboxylic group), 1410 cm − 1 band (circle, symmetric COO− stretch) and 2343 cm − 1 (triangle, CO2 band); (b) Gibbs excess versus electrode potential plot.

307

while the transition dipole of the asymmetric COO− stretch has a significant component in the direction normal to the surface. When p-polarized light is used, the bands due to the solution (at E1) and adsorbed species (at E2) cancel each other and this band does not appear in the SNIFTIR spectrum. A close inspection of the spectra in Fig. 9 reveals that amplitudes of the CO stretch band and the two COO− bands display different dependences on the electrode potential. In Fig. 10(a), the peak-to-peak amplitudes of bipolar bands at  1725 cm − 1 (CO stretch) and 1410 cm − 1 (symmetric COO− stretch) are plotted against the electrode potential. It is useful to recall at that point that the pzc in the 1.06×10 − 2 M Phe solution is equal to 150 mV. Clearly, the amplitude of the CO stretch is large at the negatively charged surface, attains a maximum at E= −100 mV and decreases at E\ − 100 mV. In contrast, the amplitude of the symmetric COO− stretch is very small at negative potentials and begins to increase to a maximum at 200 mV, just positive to the pzc. At these potentials there is a progressive change in the orientation of the COO− group towards the surface, with the ND+ 3 rotating off the surface. In this orientation, the transition dipole of the symmetric COO− stretch and the permanent dipole of the molecule become aligned in the direction normal to the surface while the transition dipole of the asymmetric COO− stretch becomes parallel to the surface. This behavior suggests that at a negatively charged surface, Phe molecules display a tendency for preferential adsorption in the undissociated –neutral form as C6H5CH2CH(ND2)COOD which is mixed with the zwitterionic form. By moving from the negative end of potentials in the positive direction, the neutral form progressively transforms into the zwitterion and the zwitterion changes orientation so that the direction of its permanent dipole rotates from being parallel to being normal with respect to the surface. For comparison with spectroscopic data, the surface concentration of Phe determined from chronocoulometric data is plotted in Fig. 10(b). One can clearly see that the first step on the surface concentration plot correlates well with the maximum of the band intensity for the neutral species and the second step on the Y versus E plot correlates well with the step on the band intensity for the zwitterionic form. Apparently, the spectroscopic data explain the complex character of the adsorption isotherm determined from electrochemical measurements. The bipolar character of SNIFTIR bands indicates that the IR bands for the adsorbed species are shifted to lower frequencies with respect to the IR bands of molecules in the solution. This shift depends on the electrode potential. In Fig. 11, frequencies corresponding to the minimum of the negative lobe of the CO stretch and the symmetric COO− stretch are plotted

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Fig. 11. Dependence of the frequency of the minimum on the negative lobe of the bipolar bands (closed points) at 1410 cm − 1 and (open points) at 1725 cm − 1 on the electrode potential.

confirm this hypothesis, a series of SNIFTIR measurements were made to determine whether CO2 is formed as a product. CO2 has an asymmetric stretching mode [36] at 2343 cm − 1. This band should appear as a negative band in SNIFTIR, because CO2 is produced only at the potential (E2). The broad absorption band of D2O centered at 2500 cm − 1 [45] interferes with the 2343 cm − 1 band of CO2. Therefore, it was necessary to use H2O as a solvent for these measurements. Fig. 12 shows the SNIFTIR spectra of Phe in the range from 2000 to 2600 cm − 1. A small negative peak at 2343 cm − 1 appears at E2 = 300 mV indicating the onset of the oxidation process. The intensity of this peak is plotted against potential in Fig. 10. It increases rapidly with E indicating substantial oxidation of Phe at the electrode surface. The increase of the CO2 band intensity correlates very well with the decrease of the surface concentration of Phe seen in Fig. 10(b). The appearance of the 1616 cm − 1 band observed at E\300 mV in the SNIFTIR spectra for both s- and p-polarized light, may be explained by the decrease of pH in the thin layer cavity, due to the production of CO2, which shifts the equilibrium between the A and Z forms towards the zwitterions.

4. Summary and conclusions

Fig. 12. SNIFTIR spectra in the CO2 asymmetric stretch region resulting from the oxidation of Phe adsorbed at the Au(111) electrode in 0.1 M KClO4 + 1.06 ×10 − 2 M Phe solution in H2O, acquired using an s-polarized infrared beam. For each spectrum, the reference potential E1 was equal to − 0.60 V/SCE, and the value of the E2 is indicated in the figure.

against the electrode potential. Apparently, the frequencies of these two bands move in opposite directions with the electrode potential. We do not know whether this shift is caused by the Stark effect or is due to the mixing of the molecular orbitals of the adsorbate with the electronic states in the metal. We suspected that the decrease of Y seen in electrochemical measurements at E \300 mV may be due to the oxidation of Phe at these positive potentials. To

The adsorption of Phe at the Au(111) electrode surface has been described in terms of Gibbs excesses and Gibbs energies of adsorption. In addition, SNIFTIR has been employed to acquire molecular level information concerning the nature of the adsorbed species and the orientation of the adsorbed molecule at the surface. Fig. 10(a) and (b) illustrates that the electrochemical and spectroscopic measurements are complementary. The surface concentration of Phe increases with potential up to 200 mV and then drops down. The rising section on the surface concentration plot displays two inflections (two overlapping steps) characteristic of a two state character of adsorption. The spectroscopic data explain very well this shape of the surface concentration plot. They show that the first (most negative) step corresponds to adsorption of the neutral form of the molecule. They also show that the second step is due to the transformation of the neutral into the zwitterionic form and reorientation of the zwitterionic form. The decrease of the surface concentration at E \300 mV correlates well with the intensity of the CO2 band and is explained well by the decarboxylation of Phe molecules at these positive potentials. On the basis of information extracted from combined thermodynamic and spectroscopic studies we can propose the model of the adsorbed molecule shown in Fig. 13. At negative potentials (negatively charged surface) Phe is adsorbed in the neutral form with the phenyl

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Fig. 13. Models describing orientation of Phe at the Au(111) electrode surface; (a) at negative potentials; (b) at positive potentials.

ring oriented parallel to the surface and with both the ND2 and the COOD groups interacting with the surface. The plane of the COOD group is oriented in the direction normal to the surface. This surface geometry is very similar to the structure proposed for glycine and a-alanine adsorbed at a silver particle by Suh and Moskovits [52]. In this orientation the transition dipole of the CO stretch should have a strong component in the direction normal to the surface while the transition dipole of the symmetric COOD stretch should be nearly parallel to the surface, consistent with the SNIFTIR data. We do not have spectroscopic evidence to support a flat orientation of the phenyl ring, since the phenyl ring bands were either weak or hidden within the envelope of the asymmetric COO− stretch bands. However, at the negatively charged Au(111) surface, aromatic molecules are known to assume a flat p-bonded surface geometry [2,3,5] and these data suggest that such an orientation is energetically favorable. At positive potentials, the neutral molecule is converted into the zwitterion. The molecule is rotated so that the ammonium group and the phenyl ring are directed towards the solution and the carboxylate group towards the metal surface. The plane of the COO− group is nearly normal with respect to the surface. In this orientation, the transition dipole of the symmetric COO− stretch has a strong component in the direction normal to the surface. Simultaneously, the transition dipole of the asymmetric COO− is parallel to the surface, consistent with the IR data. The present results illustrate a need for a concerted use of thermodynamic and spectroscopic techniques to give a complete description of the adsorption of molecules at the metal solution interface.

Acknowledgements This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada.

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