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Local Electronic Structure of Functional Groups in Glycine As Anion, Zwitterion, and Cation in Aqueous Solution Johan Gråsjö,† Egil Andersson,‡ Johan Forsberg,‡ Laurent Duda,‡ Ev Henke,‡ Wandared Pokapanich,‡ Olle Bjo¨rneholm,‡ Joakim Andersson,‡ Annette Pietzsch,§ Franz Hennies,§ and Jan-Erik Rubensson*,‡ Department of Pharmacy, Uppsala UniVersity, Box 580, SE-751 23 Uppsala, Sweden, Department of Physics and Materials Science, Uppsala UniVersity, Box 530, SE-751 21 Uppsala, Sweden, MAX-lab, Box 118, SE-221 00 Lund, Sweden ReceiVed: June 26, 2009; ReVised Manuscript ReceiVed: October 28, 2009
Nitrogen and oxygen K emission spectra of glycine in the form of anions, zwitterions, and cations in aqueous solution are presented. It is shown that protonation has a dramatic influence on the local electronic structure and that the functional groups give a distinct spectral fingerprint. 1. Introduction Amino acids dissolve in water preferentially as cations at low, neutral zwitterions at intermediate, and anions at high pH. The variations in local environment will alter the shape and reactivity of the molecules, which in turn can have dramatic effects on their biological function. The complexity of the interaction of the molecules with each other and their aqueous environment hampers a closer look on the changes in the electronic structure of the single molecules. Resonant soft X-ray spectroscopy, monitoring transitions between local quasi-atomic core levels and the outermost orbitals, gives local atom-specific electronic-structure information. Especially, details in the local electronic structure of the functional carboxylic and amino groups can be selectively probed by spectroscopy at the O K and N K edges, respectively. Consequently, amino acids have been studied by soft X-ray spectroscopic methods over the years (see refs 1-8 and refs therein). With recent technical developments, it has been made possible to study the molecules in their biologically relevant chemical surroundings; for example, in aqueous solution. Here, we present soft X-ray emission (SXE) spectra of glycine in aqueous solutions of various pHs, selectively excited at different oxygen and nitrogen sites. Nitrogen K emission spectra reflect the local electronic structure at the -NH3+ group in the zwitterion and at the -NH2 group of the anion. Oxygen K emission spectra reflect the local electronic structure at the -COO- group of the zwitterion, and the -COOH group of the cation. Using selective excitation, the oxygen spectra of glycine can be separated from the oxygen spectra of the surrounding water molecules. We demonstrate that protonation mediated by the aqueous environment has a strong influence on the local electronic structure and that the overall shape of the spectra is specific to the functional group. 2. Materials and Methods Glycine (NH2-CH2-COOH) in aqueous solution takes zwitterionic, cationic, and anionic forms (Figure 1). The * Corresponding author. E-mail:
[email protected]. † Department of Pharmacy, Uppsala University. ‡ Department of Physics and Materials Science, Uppsala University. § MAX-lab.
Figure 1. Structural formulas of neutral gas-phase glycine and the various forms of glycine appearing in water solution.
distribution is determined by the pH and the pKa of the carboxylic group (pKa ) 2.34) and the amino group (pKa ) 9.6)9 and can be determined by the Henderson-Hasselbach equation.10 Thus, in the pH range between 2.34 and 9.6, the majority of molecules are zwitterionic with both ends charged (-NH3+) and (-COO-). At a pH lower than 2.34, a proton is added to a majority of the molecules to make a cation terminated with a -COOH group, whereas at pH higher than 9.6, a proton is removed from a majority of the molecules to make an anion terminated by -NH2 (see Figure 1). 2.1. Samples Glycine, HCl (37%), and NaOH (solid) were purchased from Sigma Aldrich, sodium polyacrylate (NaPA, MW 170 000, purity > 99%) was purchased from Fluka, and pH indicator paper with 0.3 pH resolution was purchased from Kebo. Water used in the aqueous solutions was deionized (18 MΩ) in a Millipore deionizer before mixing. Glycine was prepared in a 0.48 M aqueous solution at pH 6, 12, and 1, values at which one specific ionic form of glycine is completely dominating. The intermediate pH is obtained by dissolving glycine in water, whereas high and low pHs were set by adding an appropriate amount of NaOH (0.48 mol/L) and HCl (0.56 mol/L) to the solution. The pH was monitored
10.1021/jp905998x CCC: $40.75 2009 American Chemical Society Published on Web 11/16/2009
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Figure 2. Structural formula of the monomer unit of the polyacrylate polyion in water solution.
using indicator paper with a resolution of 0.3 pH units. Sodium polyacrylate (Figure 2) was prepared in 0.3 M water solution. 2.2. Experimental Section The experiments were carried out at beamline I511-311 at MAX-lab. The sample solutions were kept in a cell equipped with an ultrathin carbon membrane12 separating the solution from the ultra high vacuum in the experimental chamber. The solutions were continuously flowed through the cell, to avoid measuring on radiation-degraded substance and to prevent substance or degradation products from adsorbing onto the carbon membrane. The flow was maintained by a peristaltic pump on a level giving an average residence time in the cell on the order of ∼1 s for each part of the solution. Soft X-ray absorption (SXA) spectra were measured by fluorescence yield using a multichannel plate detector positioned at an angle of 45° relative to the direction of propagation of the incident beam in the plane of the incident polarization The sample cell was aligned with the normal of the carbon window at 22.5° relative the incident beam direction and in the incident polarization plane. SXE spectra were measured using a Gammadata Scienta XES-300 Rowland spectrometer13 equipped with a grating of 5 m radius and a groove density of 1200 lines/mm. The spectrometer measured in the direction of the polarization of the incoming beam. Measuring the oxygen SXE spectra, the spectrometer was operated with a 10 µm slit to reach a nominal resolution of 0.4 eV. At the N K edge, the spectrometer was run in slitless mode using the incident radiation spot on the sample as the source for the spectrometer optics, giving a nominal resolution of around 0.5 eV. SXE spectra of liquid water14-16 and of NH4Cl (in solution (10 g/40 mL)) were used for energy calibration at the oxygen and nitrogen K edge, respectively. The nitrogen K emission spectrum of NH4Cl was, in turn, accurately referred to the K emission of gas-phase N217 3. Results and Discussion In Figure 3, we show SXE spectra of glycine resonantly excited at the oxygen K edge. For excitations above 534 eV, the emission spectra simulate the SXE spectra of liquid water.14-16 The resonance at around 532-533 eV in the absorption spectra cannot be related to water molecules, and we can therefore unambiguously assign it to absorption in the glycine molecule. Consequently, the spectra excited at this resonance show little resemblance to water spectra, demonstrating that selective excitation facilitates SXE studies of the local electronic structure at such oxygen sites in molecules also in aqueous solution. At both pH 6 and pH 1, the resonantly excited SXE spectra are dominated by a sharp (fwhm < 1 eV) peak at 526.5 eV. At pH 6, where glycine is terminated by the -COOgroup, there is a second sharp peak on the low energy flank at 525.5 eV. There is also a broader structure with a maximum at 522.3 eV. At pH 1, where glycine is terminated by -COOH, the spectrum shows a very broad structure at lower energies with faint structure only.
Figure 3. Oxygen K emission spectra of glycine in water solution at pH 1 (solid black line) and pH 6 (dashed red line). Above 534 eV, the absorption in water dominates the SXA spectrum, and the SXE spectra excited at higher energies are identical to the spectra of pure water (blue dots) within our experimental accuracy. Spectra excited at the resonance around 532-533 eV can be associated with excitations localized at the oxygen atom at the -CdO group of glycine. The absorption spectrum in the inset is not corrected for self-absorption.
To interpret these observations, we use results from the literature and general SXE phenomenology. SXE spectra are generated in transitions of electrons from the valence orbitals to localized quasi-atomic nitrogen and oxygen 1s vacancies. Within the dipole approximation and the one-center model,18 the intensity is proportional to local nitrogen p and oxygen p character of the molecular orbitals, respectively. Accompanying vibrational excitations are the same as in valence level photoemission, provided that the core-excited state has the same equilibrium geometry as the ground state. When this is not the case, the effects of nuclear rearrangement during the excitationemission process are observed. At resonant excitation, the coupling to the excited electron may influence the spectrum. The resonance at around 532 eV in the SXA spectrum of gas-phase glycine has been assigned to transitions to π *CdO orbitals.2,5 This resonance prevails also in low-pH aqueous solution, and Messer et al.4 have assigned it accordingly. Following this assignment, we expect that the oxygen atom of the -CdO group located at the -COOH termination (pH 1) is excited and that the spectrum in the first approximation exhibits the local electronic structure of that group. A comparison to the valence electronic structure of gas-phase glycine analyzed by photoelectron spectroscopy (PES)1,6 supports this assumption. In Figure 4, the photoelectron spectrum of gas-phase glycine is shown along with oxygen K SXE spectra of glycine in aqueous solution at pH 1, resonantly excited at the -CdO group, and nitrogen K SXE spectra of glycine at pH 12. At pH 1, the -COOH group is unchanged, as compared to the neutral free molecule, and at pH 12, the -NH2 group is intact. The highest occupied orbital of gas-phase glycine is the nitrogen lone pair, nN, at a binding energy of 10.0 eV. This state therefore has only intensity in the nitrogen K spectra and is suppressed in the oxygen K emission. At 11.2 eV follows an in-plane lone-pair orbital localized at the oxygen atom of the carbonyl group, nO. As expected, this state is clearly pronounced in the oxygen K SXE spectrum. At 12.2 eV binding energy, the lone pair associated with the hydroxyl group, πOO, perpen-
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Figure 4. Oxygen K emission spectrum (solid red line) of glycine in water solution at pH 1 (-COOH terminated) excited at 531.1 eV and the nitrogen K emission spectrum (solid blue line) of glycine in water solution at pH 12 (-NH2 terminated) excited at 403.3 eV. The energy scale of the SXE spectra is shifted so that relevant features are aligned with the photoemission spectrum of gas-phase glycine6 (dashed black line).
dicular to the molecular plane,1,6 is found. The selectively excited oxygen K SXE spectrum of glycine in solution shows no significant feature at the corresponding emission energy because the hydroxyl oxygen is not excited. At higher binding energies, there are several close-lying orbitals where, for example, one out-of-plane orbital at 15.8 eV has a large C-O weight.1,6 The core level binding energy for the carbonyl oxygen in gas-phase glycine is 538.4 eV. For a carbonyl-oxygen-selected SXE spectrum of gas-phase glycine, we would thus expect the single dominating peak arising from the nO final state to appear at an emission energy given by the difference between the core and the valence level binding energies: 527.2 eV. The observed sharp peak in the spectrum recorded for pH 1, corresponding to an intact -COOH group, is found at 526.5 eV. The energy shift between the observed peak position and the transition energy expected for gas-phase SXE may be a shift due to the polarization of the surrounding medium, but it may also be related to the screening associated with the excited electron. In addition, we find that the spectrum shows large similarities with the resonantly excited spectra of liquid acetic acid,19 demonstrating that the local electronic structure at the carboxyl oxygen site of a -COOH group in these two molecules is similar and suggesting the use of the resonantly excited SXE spectrum as a -COOH group fingerprint. We now turn back to the interpretation of the SXA spectra (Figure 3). The absorption resonance around 532 eV remains visible for a glycine solution at intermediate pH 6 (Figure 3). This observation is in concordance with the results of Messer et al.4 and the spectrum of solid glycine,2 where the zwitterionic form dominates. In this situation, the SXA peak is shifted by 0.3 eV toward higher energy, confirming the literature findings, and reflecting the deprotonation of the -COOH group. After deprotonation, the two oxygen atoms in the resulting -COOgroup are quasi-equivalent. Excitation at this resonance is therefore fundamentally different as compared to the low-pH case, since both oxygen atoms may contribute to the spectra. Comparing the SXE spectra of the pH 1 and the pH 6 solution (Figure 3) that are selectively excited at this resonance shows that the local electronic structure changes significantly upon deprotonation. An additional sharp peak appears where the final
state at 12.2 eV in the free molecule is expected. It seems natural to assign the two sharp peaks accordingly: the high-energy peak in the selectively excited SXE spectrum at pH 6 is due to the in-plane oxygen lone-pair orbital, and intensity from the outof-plane orbital follows around 1 eV below. The latter intensity was lacking at pH 1 because the out-of-plane lone-pair was associated with the hydroxyl oxygen, and the assignment would follow if deprotonation did not radically change the orbital structure. The assignment is also corroborated by a quantum chemical calculation.20 At lower emission energies, the spectrum has a maximum where the C-O dominant orbital at 15.8 eV should contribute to the spectrum. Again, at low pH, these final states were suppressed in the spectra since they were associated with the hydroxyl oxygen. Note, however, that this comparison between the neutral free molecule, and the zwitterionic form in solution has limitations. A more sophisticated comparison between the two cases has to await further computational aid. Further information about the situation at pH 6 can be obtained from a comparison with an aqueous solution of sodium polyacrylate, comprising several -COO- groups (Figure 2). In Figure 5, we demonstrate that the corresponding resonantly excited SXE spectra show large similarities with glycine at pH 6, suggesting that such spectra can be used as a -COOfingerprint. Note, however, that there are significant differences when it comes to the finer details in the spectra. These reflect the various environments of the -COO- groups. Primarily, the difference is due to the remaining molecule of which they are part and because the nearest neighbor in both cases is the same; this implies long-range sensitivity. The corresponding variation in electronic structure may be related to the large difference in pKa value for the molecules. We speculate that also the surrounding water molecules influence the spectra, but further systematic studies are needed for quantitative conclusions. A series of nitrogen K emission spectra of glycine in solution at pH 12, where the -NH2 termination completely dominates, and at pH 6, where most of the molecules have -NH3+ termination, are shown in Figure 6. All spectra have their maximum close to 395 eV and show broader structures toward lower energies, ending around 385 eV. At both pHs, there is
Electronic Structure of Functional Groups
Figure 5. Resonantly excited and detuned oxygen K emission spectra of glycine at pH 6 (red dashed line) and sodium polyacrylate (solid blue line) in water solution. In both systems, the carboxylic group takes the -COO- form.
J. Phys. Chem. B, Vol. 113, No. 49, 2009 16005 There is no profound excitation-energy dependence, but we note that excitation at the lowest resonance leads to a small red shift of the spectrum and broadening and increased intensity on the low-energy side of the main peak. The two resonances responsible for the preedge features at 401.3 and 402.5 eV in the absorption spectrum have been assigned to N1s f σ* transitions in different acceptor hydrogen bond environments.4 Note, however, that two resonances at similar excitation energy are found also in the SXA spectrum of gas-phase glycine2,5 and that the first gas-phase resonances in the spectrum of ammonia are retained when ammonia is adsorbed on Cu.21 Therefore, we find it reasonable that the two features should be assigned similarly to the gas-phase resonances found at 401.2 and 402.4 eV, to NH-σ* and CN-π* antibonding orbitals, respectively.5 We speculate that the intensity increase on the low-energy flank of the main peak in the SXE spectrum upon resonant excitation is due to the acceptor hydrogen bond. Conversely, the decrease of intensity at the low-energy flank upon core ionization may be due to the associated change in charge state. This change will dramatically weaken the acceptor hydrogen bond, and one may anticipate a dynamic change during the core hole lifetime. Since the excitations under consideration directly affect the bonding to hydrogen atoms, such dynamic vibronic effects are generally expected to be large. Effects of ultrafast nuclear rearrangements are all the more obvious at pH 6, where glycine in its zwitterionic form is terminated by -NH3+. In the equivalent core model, the nitrogen core ionization leads to a -OH32+ group, which is highly unstable. Therefore, violent nuclear rearrangement during the scattering duration time leads to smearing out of all spectral features. At resonant excitation, this smearing-out effect is even larger, which may be intriguing because the excited electron probably delocalizes very fast, as upon postedge excitation in liquid water.22 We tentatively attribute the variation to selective excitation of molecules in different surroundings. 4. Conclusions Oxygen and nitrogen K emission spectra of cationic, zwitterionic and anionic glycine in aqueous solution have been measured. Our analysis shows that the molecular electronic structure is significantly changed upon protonation/deprotonation of the amino and carboxylic groups. Thereby, the modified functional groups exhibit a characteristic local electronic structure similar to that of these groups in other chemical environments. Resonantly excited SXE provides a good probe for the occurrence of these functional groups in a complex liquid environment, using their spectral signature as a fingerprint.
Figure 6. Nitrogen K emission spectra of glycine at pH 12 (solid blue line) and pH 6 (red dashed line).
small but significant excitation energy dependence. The main peak is markedly sharper in the high-pH case. A comparison to gas-phase PES is appropriate for the spectrum excited above the ionization limit, at 410 eV, at pH 12 (see Figure 4). For the free molecule, the nitrogen 1s binding energy is 405.4 eV, and the binding energy corresponding to the outermost nitrogen lone-pair orbital, nN, is 10.0 eV. A sharp peak is thus expected in the SXE spectrum due to transitions between these states at 395.4 eV, very close to the observed narrowest peak in the spectrum. An intensity minimum is expected because states not primarily localized on the nitrogen site follow a few electronvolts below, and further down, many delocalized states contribute to the broad spectral feature peaking around 389 eV.
Acknowledgment. This work was supported by Swedish Research Council. We thank L. G. M. Pettersson for valuable discussions. We are grateful for the support by the MAX-lab staff. References and Notes (1) Cannington, P. H.; Ham, N. S. J. Electron Spectrosc. Relat. Phenom. 1983, 32, 139. (2) Gordon, M. L.; Cooper, G.; Morin, C.; Araki, T.; Turci, C. C.; Kaznatcheev, K.; Hitchcock, A. P. J. Phys. Chem. A 2003, 107, 6144. (3) Nilsson, A.; Pettersson, L. G. M. Surf. Sci. Rep. 2004, 55, 49. (4) Messer, B. M.; Cappa, C. D.; Smith, J. D.; Wilson1, K. R.; Gilles, M. K.; Cohen, R. C.; Saykally, R. J. J. Phys. Chem. B 2005, 109, 5375. (5) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.; Carravetta, V. J. Electron Spectrosc. Relat. Phenom. 2007, 155, 47. (6) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.; Carravetta, V. J. Phys. Chem. A 2007, 111, 10998.
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(7) Aziz, E. F.; Ottosson, N.; Eisebitt, S.; Eberhardt, W.; JagodaCwiklik, B.; Vacha, R.; Jungwirth, P.; Winter, B. J. Phys. Chem. B 2008, 112, 12567. (8) Zubavichus, Y.; Shaporenko, A.; Grunze, M.; Zharnikov, M. Nucl. Instrum. Methods Phys. Res., Sect. A 2009, 603, 111. (9) Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 12th ed.; Budavari, S. Ed.; Merck Research Laboratories, Merck & Co. Inc.: Whitehouse Station, NJ, 1996, ISBN: 0911010-12-3. (10) Atkins, P.; de Paula, J. Physical Chemistry, 7th ed.; Oxford University Press: Oxford, U.K., 2002, ISBN: 0-19-879285-9. (11) Denecke, R.; Va¨terlein, P.; Ba¨ssler, M.; Wassdahl, N.; Butorin, S.; Nilsson, A.; Rubensson, J.-E.; Nordgren, J.; Mårtensson, N.; Nyholm, R. J. Electron Spectrosc. Relat. Phenom. 1999, 101-103, 971. (12) Forsberg, J.; Duda, L.; Olsson, A.; Schmitt, T.; Andersson, J.; Nordgren, J.; Hedberg, J.; Leygraf, C.; Aastrup, T.; Wallinder, D.; et al. ReV. Sci. Instrum. 2007, 78, 083110. (13) Nordgren, J.; Bray, G.; Cramm, S.; Nyholm, R.; Rubensson, J.-E.; Wassdahl, N. ReV. Sci. Instrum. 1989, 60, 1690. (14) Fuchs, O.; Zharnikov, M.; Weinhardt, L.; Blum, M.; Weigand, M.; Zubavichus, Y.; Ba¨r, M.; Maier, F.; Denlinger, J. D.; Heske, C.; Grunze, M.; Umbach, E. Phys. ReV. Lett. 2008, 100, 027801.
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JP905998X
