Soft X-ray spectromicroscopy of humic acid europium(III) complexation ...

Journal of Electron Spectroscopy and Related Phenomena 135 (2004) 53–62

Soft X-ray spectromicroscopy of humic acid europium(III) complexation by comparison to model substances Markus Plaschke, Jörg Rothe∗ , Melissa A. Denecke, Thomas Fanghänel Institut für Nukleare Entsorgung, Forschungszentrum Karlsruhe, Postfach 3640, D-76021 Karlsruhe, Germany Received 28 March 2003; received in revised form 10 December 2003; accepted 12 December 2003

Abstract To provide the experimental basis for a deeper understanding of humic acid (HA) C 1s-near edge X-ray absorption fine structure (NEXAFS) spectra with and without metal ion complexation, a set of model compounds is investigated. Phthalic acid and hydroxyl substituted benzoic acids (benzoic, 4-hydroxy benzoic, protocatechuic, and gallic acid) are examined in an attempt to describe the C 1s-NEXAFS spectra of more complex organic acids—including HA—based on the building block principle. While the peak assignments and observed oscillator strengths for aromatic and carboxylic groups in these spectra are in good agreement with common assumptions, the spectral intensity in the range of the phenolic band is not readily explained with a simple building block model. The spectral signature observed for HA and Eu(III)–HA complexes is compared to the one obtained for polyacrylic acid (PAA) and Eu(III)–PAA, chosen as model substances for the complexation. In both systems strikingly similar spectral changes going from the uncomplexed to the metal-loaded macromolecules are evident. Therefore, a distinct complexation effect on the C 1s-NEXAFS is postulated. Possible effects of radiation damage on the C 1s-NEXAFS are found to be negligible. © 2004 Elsevier B.V. All rights reserved. Keywords: Humic acid; Polyacrylic acid; Scanning transmission X-ray microscopy (STXM); C 1s-NEXAFS; Building block principle

1. Introduction Humic acids (HA) are potentially important in binding traces of actinides or lanthanides, thus affecting their transport in aquatic systems [1]. HA are natural colloidal biopolymers with a variety of chemical functional groups, which may serve as metal ion binding sites [2]. Although HA have been studied extensively for their environmental and biochemical importance, their high chemical and morphological heterogeneity has been a limiting factor in the search of their chemical structure [3]. Therefore, a spectromicroscopy technique such as scanning transmission X-ray microscopy (STXM) is a promising tool for unraveling HA chemical functionality in addition to visualizing HA morphology on a sub-␮m scale [4–8]. Moreover, STXM benefits from the ability to characterize organic samples in a thin film of aqueous solution. Both morphological and microchemical information can be obtained at the same time in situ on aqueous colloid species within the ‘water window’ (i.e., between the ∗ Corresponding author. Tel.: +49-7247-82-4390; fax: +49-7247-82-3927. E-mail address: [email protected] (J. Rothe).

0368-2048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.elspec.2003.12.007

C 1s and O 1s absorption edges at 284 and 537 eV, respectively). This ensures that complex hydrated structures of HA are kept in their native state. Additionally, high resolution C 1s-near edge X-ray absorption fine structure (NEXAFS) provides direct speciation of carbon containing macromolecules from their characteristic 1s → ␲∗ , ␴∗ resonances. Several concepts may contribute to the analysis of C 1s-NEXAFS spectra: ‘functional group fingerprinting,’ the ‘building block principle,’ and ‘molecular modeling’ [9]. ‘Functional group fingerprinting’ is a qualitative method trying to assign spectral features by comparison to spectra of well-characterized reference compounds. Generally, the spectroscopic signature of the chemical functional groups (e.g., carboxylic, aromatic, phenolic) is fingerprinted by comparison to spectra of known organic compounds reported in the literature, e.g., [10]. In a semi-quantitative way, the analysis based on the ‘building block principle’ attempts to explain NEXAFS spectra of complex molecules as a combination of spectra derived from simpler sub-units (e.g., benzene and formic acid as building blocks for benzoic acid). These approaches have been especially successful in the analysis of NEXAFS data obtained for polymer systems (e.g., [11,12]). Attempts to establish a theoretical foundation

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M. Plaschke et al. / Journal of Electron Spectroscopy and Related Phenomena 135 (2004) 53–62

for the ‘building block principle’ by ‘molecular modeling’ calculations are reported for selected systems, such as fluorine substituted benzenes, phenol, and aniline [13,14]. Assignment of NEXAFS spectral characteristics of HA–actinide/lanthanide complexes is difficult due to the inherent heterogeneity and polydispersity of HA. As known from previous STXM investigations, the aggregation of Aldrich HA induced by Eu(III) is accompanied by the segregation into zones with different morphologies and optical densities [5]. It was observed that the spectrum of the uncomplexed HA cannot be reproduced from a superposition of any spectra extracted from the segregated fractions of the Eu(III)–HA complexes [15]. Therefore, a distinct complexation effect on the molecular states, influencing the strong carboxyl ␲∗ -transition as well as the resonances commonly associated with aliphatic or phenolic carbon, was assumed. The ultimate goal of our investigation is to provide an experimental basis for understanding the observed NEXAFS of HA with and without metal ion complexation. In the literature the structure of HA has been described as assembly of covalently linked aromatic and aliphatic residues with carboxyl and phenolic functional groups as possible binding sites for metal ions. Previous XAFS investigations of actinide binding properties of HA implicate carboxyl groups as the primary functional groups with high affinity to metal cations [16,17]. It has also been pointed out that even identical functional groups may have different complexing characteristics and affinities effected by their surrounding steric and electronic environment [18]. In the present study, the NEXAFS spectra of a set of model substances of carboxyl and hydroxyl substituted benzenes is examined with regard to the peak assignments and the applicability of the building block principle in NEXAFS spectroscopy of HA. The water-soluble polymer polyacrylic acid (PAA) is used as a model system for the study of Eu(III)–HA complexation. The C 1s-NEXAFS spectra are correlated with those obtained from the segregated regions in the HA aggregates.

2. Experimental 2.1. Sample preparation All chemicals from Sigma–Aldrich (Deisenhofen, Germany) are of analytical grade. A droplet of 1 ␮l sample solution of phthalic, (0.281 mol/l, pH 4.0), benzoic (0.4 mol/l, pH 5.6), 4-hydroxybenzoic (0.235 mol/l, pH 5.4), protocatechuic (0.284 mol/l, pH 5.3), gallic acid (0.235 mol/l, pH 5.3) and polyacrylic acid sodium salt (calibration standard, molecular weight MW ∼ 83,400, 6.1 g/l, pH 2.0) is pipetted onto a silicon nitride window and air-dried. For the STXM investigation of Eu(III)–PAA complexation, 3.1E−2 mol/l Eu(III) is added to a 1.9 g/l PAA solution, pH adjusted to 4.9. Rapid flocculation and sedimentation of the colloidal suspensions is observed. A portion of 1 ␮l of the super-

natant not containing any large flocs is used for sample preparation. Additional time-resolved laser fluorescence spectroscopy (TRLFS) studies of Eu(III)–PAA complexation are performed in aqueous solution using 1 cm quartz cuvettes (Eu(III) solution: 1.0E−4 mol/l, NaCl 0.1 mol/l, pH 2; Eu(III)–PAA solution: Eu(III) 2.5E−4 mol/l, PAA 410 mg/l—yielding an excess ligand concentration with [COO− ]/[Eu(III)] ∼ 3, NaCl 0.1 mol/l, pH 5.4). Commercial Aldrich HA (Deisenhofen, Germany) is purified according to a procedure described by Kim et al. [19]. The HA proton exchange capacity, determined by potentiometric pH titration, is 5.43 ± 0.16 meq/g [19]. A stock solution of 200 mg/l HA, with an electrolyte content of 0.1 mol/l NaCl (Merck suprapur, Darmstadt, Germany), is prepared, adjusted to pH 6.0 and stored at 5 ◦ C. The amount of Eu(III) (ICP-AES standard, Johnson Matthey GmbH, Karlsruhe, Germany), diluted to 6.3E−3 mol/l, pH adjusted to 4.2) added to aliquots of the HA stock solutions for STXM samples is calculated to saturate the total loading capacity of Aldrich HA [20]. After addition of Eu(III), rapid flocculation, and sedimentation of the colloidal suspensions is observed. As described for Eu(III)–PAA, the supernatant is taken for sample preparation. The HA, Eu(III)–HA, and Eu(III)–PAA samples are investigated as thin films of colloid suspension in fully hydrated state using silicon nitride wet-cells. The sample preparation technique for STXM measurements has been described elsewhere [21]. For the estimation of the radiation dose according to [22] (Fig. 3c) a sample thickness of 300 nm is assumed. 2.2. Scanning transmission X-ray microscopy Soft X-ray spectromicroscopy investigations of HA and reference compounds are performed at the STXM endstation (X-1A Outboard-STXM) at the National Synchrotron Light Source, Brookhaven, NY [23]. At the X-1A Outboard-STXM, a Fresnel zone plate is used to focus the undulator beam into a spot of soft X-rays. The zone plate has a diameter of 160 ␮m and a width of 45 nm of the outer most ring segment, giving a resolution (Rayleigh criterion) of 55 nm [24]. The STXM endstation provides a flux of about 107 photons per second, with an energy bandwidth of about 0.1 eV at the C 1s-energy (see, e.g., [21] and references therein). The spherical grating monochromator is calibrated against the C 1s-absorption threshold resonance of carbon dioxide at 292.76 eV [10]. Pseudo-Voigt profile curve-fitting of this transition peak indicates that spectral line widths down to 0.5–0.6 eV FWHM (including contributions from natural line-width and instrumental broadening) can be resolved with the experimental set-up. Stacks of images from selected sample regions are recorded as a function of incident photon energy E. C 1s-NEXAFS spectra are extracted through the analysis of the absorption signal, µ(E)d = ln(I0 (E)/I(E)), of vertical projections onto aligned image stacks. Image regions free of particles supply information on the I0 signal. The


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Table 1 Peak assignments and energy positions (in eV) for 1s → ␲∗ transitions in humic acid and reference compounds Peak assignment energy ranges in Figs. 1–4

C=C (A)

C−OH/complexationa (B)

Phthalic acid Benzoic acid 4-Hydroxo benzoic acid Protocatechuic acid Gallic acid Polyacrylic acid (PAA) Eu(III)–PAA Humic acid (HA), average Eu(III)–HA, dark zones

284.9 284.9 285.0 285.0 285.0 285.0 285.0 284.8 284.9

– – 286.8 286.9 286.7 – 286.6a – 286.5a

a

C=O (C) – – – – 287.4 – 287.5a – 287.4a

288.5 288.2 288.3 288.3 288.3 288.4 288.5 288.4/288.9 288.4

These transitions are assumed to be affected by metal ion complexation.

transmitted intensity (I) is derived from image regions containing structures of interest. Details of an X-ray absorption spectrum such as sharp features in the C 1s-NEXAFS may be dependent upon the thickness of the absorber [25]. Hence, sample zones with 40–60% transmission were selected for extraction of the spectra, minimizing possible thickness effects [26]. It has to be emphasized that normalization artifacts in the spectral region of about 282–286 eV, due to a strong dip in the I0 signal (caused by carbon deposits on mirrors and DCM grating), cannot be ruled out. For a review of the procedure for image stack data analysis, see [27]. All C 1s-NEXAFS spectra are obtained from stacks of 210 images, recorded at E ranging between ∼275 and 310 eV. E is varied for recording images in 1 eV steps in the range 275–282 eV, in 0.1 eV steps for 282–300 eV, and in 0.5 eV steps at energies 300–309 eV. A linear pre-edge background (275–280 eV) is subtracted from the extracted spectra, followed by normalization (µd = 1) at 305 eV. Semi-quantitative analysis of the C 1s-NEXAFS extracted from the organic acids and HA is attempted by curve-fitting of the normalized spectra. C 1s-NEXAFS peak positions and energy assignments are summarized in Table 1. The spatial distribution of spectral features can be visualized by calculating ratio images −log[I( En )/I( E0 )], where I( E0 ) and I( En ) are average images of the energy interval E0 before the edge (e.g., 270–283 eV) and En at a distinct absorption feature (e.g., E4 = 288.3–289.5 eV for the carboxylic band), respectively (see Fig. 4a). In the ratio images shown in Figs. 5b–e and 6b–e high brightness corresponds to enrichment of the corresponding spectral feature. A constant grey scale indicates a constant distribution of chemical functionalities within a region of interest. 2.3. Time-resolved laser fluorescence spectroscopy (TRLFS) TRLFS luminescence lifetime measurements of Eu(III) (5 D0 → 7 F1,2 transitions) are used to determine the number of water molecules coordinated to Eu(III) in the PAA complex. The Nd:YAG third harmonic (355 nm) laser line

(source: Continuum Powerlite 9030, ND 6000) with a pulse energy of 1 mJ is used to promote Eu(III) from the ground 7 F state into the 5 L state, which decays to the emitting 5 D 0 7 0 level through non-radiative relaxation. Fluorescence emitted in the 540–740 nm range from the 5 D0 → 7 Fx transitions is detected by an optical multi-channel analyzer (polychromator Chromex 250) with 300 lines/mm grating. The WINSPEC data acquisition software is used to track the time dependent emission by recording 51 spectra with increasing delay time between laser pulse and camera gating from 1 to 1.5 ms in 30 ␮s intervals.

3. Results and discussion 3.1. NEXAFS spectra of model compounds The C 1s-NEXAFS spectra of phthalic, benzoic, and hydroxyl substituted benzoic acids (from top to bottom: 4-hydroxy benzoic, protocatechuic, and gallic acid) are displayed in Fig. 1, an enlarged section emphasizing the NEXAFS energy region of discrete transitions is shown in the right panel. The spectra exhibit sharp peaks due to localized 1s → ␲∗ transitions between about 284 and 289 eV, i.e., below the C 1s ionization potential, followed by broad features representing multielectron excitations as well as ‘shape resonances’ (1s → ␴∗ transitions), leading to the continuum part beyond 290 eV. The following discussion will be restricted to the most prominent transitions in the discrete part of the spectra. In agreement with the literature these transitions are assigned as, e.g., aromatic, phenolic, and carboxylic peaks (energy ranges A, B, and C in Table 1 and Figs. 1–4) [10]. We begin the discussion of NEXAFS spectra with the set of compounds containing a single carboxyl group, i.e., benzoic, 4-hydroxy benzoic, protocatechuic, and gallic acid. The C=O double bond in these molecules is visible by the C 1s (COOH) → ␲∗C=O transition at ∼288.3 eV (carboxyl peak). Peak location, intensity, and width are nearly identical in all four spectra (Fig. 1b–e). The carbon atoms at the unsubstituted benzene ring sites are reflected by their C 1s (C–H) → ␲∗C=C transition at about 285.0 eV

56


A COOH

2

COOH

B

C

phthalic acid

a

benzoic acid

b

1

normalized absorption (a.u.)

0 2

COOH

1

0 2

COOH

1

OH

0 2

COOH

1

OH

0 2

COOH

4-hydroxy benzoic acid

c

protocatechuic acid

d

gallic acid

e

OH

HO OH

1

0

OH

285

290

295

300

305

282

284

286

288

290

292

photon energy (eV) Fig. 1. Normalized C 1s-NEXAFS spectra of phthalic acid, benzoic acid, and hydroxylated benzoic acids (the right panel is an enlargement of the spectra in the 282–292 eV range; see Table 1 for assignments).

(aromatic peak). The observed line width of this peak (>1 eV) is increased compared to the line width obtained for CO2 (0.5–0.6 eV, as a combined effect of natural line width and spectrometer resolution). This is explained by the fact that each carbon atom of the benzene ring has its own C 1s → ␲∗ transition; those for the unsubstituted ring carbons are grouped in an experimentally unresolved energy range, leading to the broad ‘aromatic’ peak [13]. Based on comparison to results obtained for poly(ethylene terephthalate) and related compounds [11], we assume that the C 1s (C–COOH) → ␲∗C=O transition is located at the tailing edge of the aromatic peak. As for benzoic acid, a high energy tailing of the aromatic peak is also found in the spectra of phthalic, protocatechuic, and gallic acids. The trend of decreasing intensity of the aromatic peak from 4-hydroxy benzoic acid to gallic acid is in accordance with the decreasing number of C–H units and is in agreement with calculations for fluorine substituted benzenes [13]. The aromatic peak observed for benzoic acid shows a reduced intensity, increased width and a peak maximum at a slightly lower energy compared to 4-hydroxy benzoic acid (see discussion of phthalic acid below). The

peak associated with the C 1s (C–OH) → ␲∗C=C transition (phenolic peak) is observed to vary with increasing number of hydroxyl groups. This peak appears at ∼286.8 eV in the spectrum of 4-hydroxy benzoic acid. It is comparatively broadened for protocatechuic acid and becomes asymmetric with a distinct low energy shoulder in the spectrum of gallic acid. This observation indicates that the increasing ring substitution with hydroxyl groups does not simply lead to an additive growth of the corresponding transition band. The neighboring phenolic groups exert an electronic influence on the C 1s (C–OH) → ␲∗C=C transitions, probably leading to a further splitting of the C=C ␲∗ final state molecular orbitals. Ring substitution by two neighboring carboxyl groups in phthalic acid leads to increased intensity in the energy region of the C 1s (COOH) → ␲∗C=O transition compared to benzoic acid. Additionally, the peak is significantly broadened and shifted to higher energies (peak maximum ∼288.5 eV). As for the hydroxyl substituted benzoic acids, the neighboring carboxyl groups influence each other electronically, probably leading to the increase of oscillator strength and the splitting of the C=O ␲∗ final state molecular orbitals.


57

Fig. 2. STXM images and normalized C 1s-NEXAFS spectra of (a) PAA (dried); (b) Eu(III)–PAA (wet-cell); and (c) comparison of the NEXAFS regions of discrete transitions for PAA and Eu(III)–PAA (see Table 1 for assignments).

Fig. 3. (a) STXM image of Eu(III)–PAA (wet-cell); (b) normalized C 1s-NEXAFS spectra obtained from 12 images, 10 of these spectra are sequentially recorded and plotted on top of each other; and (c) optical density relative to the value in the first spectrum of this sequence at 288.5 eV (filled squares) and 287.5 eV (open squares) as a function of the accumulated radiation dose (and irradiation time) during the acquisition of the spectra (see discussion).

58


2.0 1.5

a

∆E2

∆E1

∆E0

∆E3

humic acid (HA)

∆E4

1.0

I1

0.5

normalized absorption (a.u.)

0.0 2.0

I2 Eu(III)-HA

b

1.5 1.0

I3

0.5

I4

0.0 284 2.0

288

292

296

300

c C' C''

1.5

A

B'

B''

1.0 0.5 0.0 284

286

288

290

292

photon energy (eV)

Fig. 4. Normalized C 1s-NEXAFS spectra of (a) HA (spectra extracted from different sample regions I1 and I2 in Fig. 5a)—grey bars representing intervals chosen for calculation of ratio images in Figs. 5 and 6; (b) Eu(III)–HA light and dark (spectrum vertically shifted) zones (spectra are extracted from regions I3 and I4 in Fig. 6a) and (c) comparison of the discrete NEXAFS transitions of HA (average spectrum of whole aggregate in Fig. 5a) and the light and dark zones of Eu(III)–HA (see Table 1 for assignments).

The qualitative spectral trends for these compounds are in agreement with previous NEXAFS spectral ‘functional group fingerprinting.’ The carboxyl peak changes in intensity, form, and energy position for phthalic and benzoic acids but remains nearly constant for the benzoic and hydroxylated benzoic acids. The intensity of the aromatic band decreases with increasing hydroxyl substitution, while a phenolic band with complex structure simultaneously develops. These results illustrate the limitation of the ‘building block principle.’ The analysis of NEXAFS spectra in terms of ‘building blocks’ requires, that the spectra of the sub-units are little perturbed by the specific electronic environment. This is, e.g., the case for the C 1s (C–H) → ␲∗C=C and C 1s (C–F) → ␲∗C=C transitions in fluorobenzenes (C6 H6−n Fn , n = 0–6) as reported in [13]. The carboxyl and hydroxyl substituted benzenes in this study exhibit a more complex behavior (compared with fluorine) probably due to effects of symmetry, electronegativity of the substituents, possible intra- and intermolecular hydrogen bonding and conjugation with the aromatic ring. The carboxylic group in the hydroxyl substituted benzoic acids is apparently sufficiently electronically separated from the substituted benzene ring to be conceived as an appropriate building block. However, the successive hydroxylation results in a slight

shift of the aromatic and splitting of the phenolic C=C ␲∗ final state molecular orbitals. Likewise, the presence of an additional COOH group in phthalic acid shifts the carboxyl peak and splits the C=O ␲∗ final state molecular orbitals compared to that observed for benzoic acid. A quantitative understanding of these spectral fingerprints might be obtained by theoretical calculations of core electron excitations for each carbon atom in the substituted benzenes as, e.g., performed for the fluorobenzenes [13]. 3.2. NEXAFS spectra of polyacrylic acid before and after Eu(III) complexation The water-soluble polyelectrolyte PAA is an appropriate model compound for studying the role of the carboxyl group in metal ion complexation of HA [18]. As reported in the literature, Eu(III)/PAA complexation can be detected by TRLFS using Eu(III) luminescence as a probe [28,29]. The Eu(III) 5 D0 → 7 F2 transition at 616 nm is sensitive to complexation by organic ligands [30]. As a result of reduced energy transfer from the excited 5 D0 state to the coordinated water molecules, replacement of H2 O by the carboxyl groups in a Eu(III)–PAA complex enhances the luminescence emission intensity of the 7 F2 transition (not


shown) and increases the Eu(III) luminescence lifetime. The average number of water molecules coordinated to Eu(III) is determined by measuring the excited-state lifetime, τ, for the observed mono-exponential decay of the 5D → 7F 0 1,2 transitions [31]. The measurements show that about four to five of nine water molecules in the aquo ion are replaced by the monofunctional organic ligand. A Eu(III)(COO− )x ·yH2 O (x = 2–3, y = 3–5) species can thus be postulated. No photodegradation of the complex after lifetime measurements is observed. In Fig. 2, the C 1s-NEXAFS spectra from PAA and Eu(III)–PAA are displayed. The corresponding STXM image of PAA is taken from a dried droplet (inset of Fig. 2a), whereas Eu(III)–PAA aggregates are measured in their hydrated state using a wet-cell (inset of Fig. 2b). The Eu(III)–PAA aggregates exhibit a fractal morphology. The signature of the NEXAFS spectra discussed below is nearly independent of the sample regions, which points to the chemical homogeneity of the sample. The discrete part of the C 1s-NEXAFS spectrum of PAA is dominated by a strong C 1s (COOH) → ␲∗C=O transition at ∼288.4 eV (carboxylic peak). A relatively weak and broad absorption feature (∼285.0 eV) is found in the region commonly assigned to carbon in C=C double bonds, e.g., the aromatic C 1s (C–H) → ␲∗C=C transition. This weak band is equally found in Eu(III)–PAA (Fig. 2c). Weak features in this spectral region are also visible in literature spectra of PAA [32]. In PAA, consisting of an aliphatic chain and carboxyl groups, C=C double bonds can only occur at the end of the aliphatic chain in end-standing groups of acrylic acid. Most likely, this feature is due to normalization artifacts as described in the experimental section. The sharp feature at 290.8 eV visible in the spectrum of PAA (dried sample, Fig. 2) is not detected in the spectrum of Eu(III)–PAA (wet-cell). This feature is assigned to precipitated carbonate impurities. In Eu(III)–PAA, a strong decrease of the C 1s (COOH) → ␲∗C=O transition is observed (C in Fig. 2c). At the same time, two additional absorption features appear at 286.6 and 287.5 eV (B and B

in Fig. 2c). As for the hydroxylated benzoic acids (see discussion above), the 286.6 eV peak is typical for phenolic carbon (C 1s (C–OH) → ␲∗C=C ) and the latter is commonly associated with weak transitions to Rydberg orbitals (C 1s (CH3 , CH2 , CH) → 3p/␴∗C–H ) of aliphatic carbon (e.g., [33]). However, these groups are neither present in PAA (phenolic groups) nor likely involved in the Eu(III) complexation (aliphatics). There are two conceivable mechanisms that might explain this change of spectral signature in the Eu(III)–PAA sample: (i) a distinct effect of the Eu(III) complexation on the carboxyl group due to the change of the carboxyl carbon molecular environment; (ii) radiation damage resulting in changes of the PAA chemical structure. The loss of mass and carbonyl functionality and the appearance of additional spectral features were observed for a series of carbonyl group containing polymers in [22]. Radiation damage was found to be

59

enhanced in the presence of oxygen and may, therefore, be more significant for hydrated specimens [22,34]. However, for hydrated PAA embedded in a polypropylene membrane only