Impact of Drying on Wood Ultrastructure Observed by Deuterium Exchange and Photoacoustic FT-IR Spectroscopy

Impact of Drying on Wood Ultrastructure Observed byDeuterium Exchange and Photoacoustic FT-IR Spectroscopy

Miro Suchy, Jenni Virtanen, Eero Kontturi,* and Tapani Vuorinen

Department of Forest Products Technology, Helsinki University of Technology, P.O. Box 6300,FIN-02150 TKK, Finland

Received November 9, 2009; Revised Manuscript Received December 5, 2009

The impact of drying on the ultrastructure of fresh wood was studied by deuterium exchange coupled with FT-IRanalysis. This fundamental investigation demonstrated that water removal leads to irreversible alterations of thewood structure, namely, supramolecular rearrangements between wood polymers. The deuteration of fresh woodwas shown to be fully reversible by a subsequent exposure of the deuterated sample to water (reprotonation).Therefore, the presence of any OD groups in deuterated and then dried wood samples after reprotonation is aclear indicator of reduced accessibility. The extent of changes was affected by drying temperature and relativehumidity. Application of this methodology for the evaluation of chemical pulp sample (reference material) resultedin similar response, only more pronounced. Two hypothetical alternatives were proposed for accessibility reductionin dried wood: (i) irreversible aggregation of cellulose microfibrils and (ii) irreversible stiffening of thehemicellulose/lignin matrix that extensively swells when exposed to water.

1. Introduction

Wood is a native composite of polymers: semicrystallinecellulose microfibrils are embedded in a matrix of hemicelluloseand lignin to form the cell wall of a wood fiber.1 The materialpotential of wood is enormous and it has been effectively utilizedby humans for thousands of years. Traditional applications in-clude papermaking and building materials among many others.On the other hand, modern materials science looks onto woodas a raw material for biofuels, a variety of specialty chemicalsand endemic, functionally versatile nano-objects.2-6 In view ofboth the traditional and the modern applications, fundamentalunderstanding of basic properties of the wood polymer com-posite is of great importance.

One of the fundamental issues that has received surprisinglylittle attention is the alteration of the wood cell wall ultrastructureupon drying of wood. Trees grow in water-swollen conditions,and once a tree is felled, the wood quickly undergoes clearlyperceptible changes in its texture, which are mostly associatedwith water escape from wood structure. A number of studieshave previously demonstrated that this loss of water has a directimpact on mechanical properties of wood.7-11 On the otherhand, several processes are determined to use wood in its“green” form (prior to its drying) because of easier pliabilityand higher accessibility in chemical reactions. As an example,alterations of wood cell wall ultrastructure and characteristicsby water removal can have an impact on wood bioconversionprocesses. It has been shown that in addition to chemicalcomponents and their interactions, physical features of the woodultrastructure can affect the efficiency of enzymatic hydrolysisof wood carbohydrates.12-14 In this paper, we intend todemonstrate that drying at moderate temperatures inducesfundamental, irreversible changes in the properties of the woodpolymers and wood ultrastructure.

One wood-derived material, chemical pulp, has receivedconsiderable attention with respect to its behavior during drying.

Chemical pulp is prepared by breaking the anisotropic networkwith individualization of fibers and removing the lignin fromwood cell wall, resulting in excessively porous fibers. Upondrying, the swelling potential of chemical pulp fibers is greatlyreduced due to irreversible pore closure. This phenomenon,termed hornification, directly affects the fiber properties, whichcannot be restored by rewetting.15-18 Hornification has beenconfirmed and described for chemical pulps only, and many ofits characterization methods, water retention value (WRV)measurement, in particular, are suited exclusively for chemicalpulp fibers. Therefore, to investigate the fundamentals of drying-induced phenomena in wood fibers, we have chosen to utilizea simple concept of deuteration coupled with photoacousticFourier transform infrared (PAS FT-IR) spectroscopy. Theconcept makes use of the exchange of accessible OH groups incellulose to OD groups upon exposure to D2O.19-22 Thedeuterium present in wood can then be easily monitored by IRspectroscopy because the OD stretch signal is located in an areaof spectrum with no interference from other signals, circum-venting the common problem in interpreting the complex IRspectrum of wood.

Deuteration in combination with IR spectroscopy has beenexploited previously to study accessibility of cellulose andcellulose derivatives,22-26 including native cellulose in wood.27,28

However, the fundamental changes in wood ultrastructure thattake place during drying have never been properly evaluatedfrom this point of view. In this study, the accessible OH groupswere converted to OD groups by deuteration and the sampleswere subsequently dried under controlled conditions (temper-ature and relative humidity). The conversion of accessible ODgroups to inaccessible ones, those retained in the sample afterflushing with an excess of H2O, was a clear indicator ofalteration in the wood sample ultrastructure. In addition,experiments with chemical pulps, a substrate known to undergoirreversible changes in fiber cell wall ultrastructure (hornifica-tion), were carried out by using similar experimental conditionsand the results were compared to each other.

* To whom correspondence should be addressed. Tel.: +358 9 451 4250.Fax: +358 9 4514 259. E-mail: [email protected].

Biomacromolecules 2010, 11, 515–520 515

10.1021/bm901268j 2010 American Chemical SocietyPublished on Web 12/21/2009

2. Experimental Section

Materials. Freshly felled pine (Pinus sylVestris) and spruce (Piceaabies) wood samples from Eastern Finland were supplied in the formof discs, 7-10 cm thick and 20-40 cm in diameter. The samplepreparation is depicted in Scheme 1.

The discs were cut with a saw to form a rectangle, from which sliversof ∼1 mm thickness were sliced with a chisel. Using a sharp knife,small squares of approximately 5 × 5 mm were cut from the slivers.The dimensions of the specimen were selected to agree with the sizerequirements of the FT-IR photoacoustic detection cell.

For pulp experiments, never-dried lignin-free (bleached) pulp wasused. The pulp, initially delignified by kraft pulping process and thenbleached, was obtained from paper mill in Sunila, (Finland). The pulpwas made from spruce and pine wood (approximately 60% spruce and40% pine).

Deuterium oxide (99.9 atom % D, Sigma-Aldrich) was used fordeuteration and relative humidity control. The salts used for humiditycontrol were NaCl (99.5%, J. T. Baker) and NaOH (p.a. Merck). Thesaturated solution of these salts placed in a closed environment createdapproximately 7 and 75% D2O relative humidity, respectively. Althoughthe approximations are based on literature data for water,29 the D2Orelative humidity values reported for saturated solutions of these saltsin deuterium at 20 °C did not differ significantly from the valuesreported for water.30

Deuteration and Controlled Drying Experiments. The deuterationwas carried out in 10 mL glass vials by immersing wood/pulp samplesin an excess of D2O for 2 × 20 min (pulp) or 60 min (wood). After thetreatment, the samples were dried under different conditions and thenflushed with an excess of water for identical period of time asdeuteration (2 × 20 and 60 min for pulp and wood, respectively). Bothdeuteration and flushing were carried out at room temperature. Allsamples were then dried in a convection oven at 40 °C prior tomeasurement with the FT-IR spectrometer.

The drying at controlled D2O relative humidity was carried out invacuum desiccators. A schematic of the experiments is shown inScheme 2.

The samples, put in perforated aluminum containers, were placedon the porcelain plate in the desiccators containing D2O saturatedsolutions at the bottom. The desiccators were then evacuated and placedinto oven (25 and 80 °C) for conditioning (7 days). Pulp samples forwater retention value measurements were dried in similar manner;except the relative humidity was achieved using saturated aqueoussolutions instead of solutions of D2O.

Pulp Analysis. The water retention value (WRV) of the pulps wasdetermined according to the standard ISO 23714:2007 with a JouanGR 4 22 centrifuge.

FT-IR Spectroscopy. The spectra were collected using a Bio-RadFTS 6000 spectrometer (Cambridge, MA) with a MTEC 300 photo-acoustic detector (Ames, IA) at a constant mirror velocity of 5 kHz,1.2 kHz filter, and 8 cm-1 resolution. First, a background spectrumwith standard carbon black was measured. After collecting thebackground spectrum, the wood or pulp sample was put into a detectioncell that was placed into the detector. After flushing with helium gasfor 5 min, the cell was sealed, and the actual spectrum of the samplewas recorded. The background measurement was carried out at thebeginning of each set of measurements.

For each measurement a minimum of 400 scans per spectrum werecollected and processed using the Win-IR Pro 3.4 software (Digilab,Randolph, MA). Each spectrum was normalized to have the same valueat 1200 cm-1. The spectra presented throughout this article are averagesof at least four measurements. Each treatment and drying scenario wascarried out in triplicate. A minimum of two samples were measured(both sides), with an additional sample measured if a noticeabledifference between the two measurement were observed.

3. Results and Discussion

3.1. Wood Deuteration and Drying at Different Temper-atures. The studies of cellulose deuteration have demonstratedthat the rate and extent of OH f OD exchange depend onseveral factors, including the type and crystallinity of thecellulose sample,31 temperature, and relative D2O vaporpressure,21,23 as well as mode of deuteration (liquid or vaporphase).22 Depending on the cellulose type and conditions, thedeuterium exchange in accessible regions has been shown tobe complete from less than or close to 1 h20,31 up to severalhours.32 The deuteration of cellulose in wood samples showedthe majority of OH f OD exchange completed at early stagesof the process (less than 100 min), in all accessible regions.27,28

Therefore, 60 min deuteration was assumed sufficient for thepurpose of this investigation. In addition to deuteration, it hasbeen previously shown that the majority of the exchanged ODgroups in cellulose samples can be readily reversed back to OHwhen exposed to water or water vapors. The published datawere obtained for pure cellulose-viscose films22 and sheetsmade from wood pulp,26 and, thus, it was important for thisinvestigation to establish the extent of reprotonation, or revers-

Scheme 1. Schematic of Wood Sample Preparationa

a Dimensions are not to scale.

Scheme 2. Schematic of Wood Drying Experiments at ControlledTemperature and D2O Relative Humidity

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ibility of deuteration, for the wood samples under the conditionsused. Ideally, complete reversibility of the exchange would bedesirable, and if achieved, the OD groups remained in the woodstructure after reprotonation would be a clear indicator of thechanges in wood ultrastructure. The detailed analysis of thereversibility studies are shown in the Supporting Information(Figure S1). The main outcome of the testing was that noresidual OD groups were retained within wood samples afterexposure to an excess of water immediately following deutera-tion. This demonstrates a complete reversal of the OH f ODexchange for wood samples, and therefore, the presence of ODgroups in dried samples after flushing would indicate occurrenceof irreversible changes in wood structure taking place betweenthe deuteration and the flushing steps.

The deuterated fresh wood samples were dried for seven daysunder controlled D2O relative humidity (7 and 75%) at 25 and80 °C. The controlled D2O environment during drying anddifferent relative humidity levels were designed to prevent theundesirable impact of water on deuteration reversal occurringbefore any possible structural changes, and to evaluate theimpact of RH on the exchange. After drying, the samples wereflushed with an excess of water and then dried in a convectionoven prior to the FT-IR measurement. The measured FT-IRspectra for spruce and pine samples are shown in Figure 1. Adetailed image of the spectral region of interest is included inthe lower part of the figure.

Although the OD stretch peak was clearly visible in thespectra of all deuterated samples, a marked difference in thepeak size was observed between samples dried at differenttemperatures. The samples dried at 25 °C exhibited noticeablysmaller peaks than those observed for the samples dried at 80°C. In addition, at 25 °C the relative humidity of D2O did notappear to have an effect on the amount of deuterium retainedin the dried wood samples after flushing with water. This wasindicated by the equal size of the OD peaks of the samples driedat low and high relative humidity. In contrast, during drying at80 °C, the D2O relative humidity appeared to have an impacton the deuterium retention. The OD stretch peaks for samplesdried at 75% RH were noticeably greater compared to the peaksof the samples dried at 7% RH.

The chemical composition and ultrastructure of both spruceand pine wood are comparable and the IR spectra are similar.The response to drying represented by the size of the peaksmeasured for spruce and pine samples was practically identical.A direct comparison of both spruce and pine samples is shownin Supporting Information (Figure S2).

3.2. Deuterium Retention Stability. Photoacoustic (PAS)FT-IR measurement requires the tested materials to be dry, thus,all wood and pulp samples had to be dried prior to themeasurement. To minimize the possible impact of this dryingon wood samples, particularly since the effect of actual dryingon the wood structure was investigated, the drying was carriedout at rather moderate temperature of 40 °C. The samples hadto be dried in the vicinity of the IR spectrometer and theavailable convection oven used for the drying was not equippedwith sufficient humidity control. The humidity (5.5% RH)present in the oven during premeasurement drying had asignificant impact on the deuterium retention in the wood controlsamples, as shown in the Supporting Information (Figure S1).Although the interaction of humidity with deuterated samplesshould preferably be prevented, no significant additional repro-tonation was expected, assuming all accessible OD groupsavailable after drying already reprotonated in the flushing stage.However, it was important to realize and potentially quantifythe impact of slightly elevated temperature coupled with watervapor present in the drying prior to the FT-IR measurement onthe deuterium retention, particularly considering the reductionsobserved for the control samples. The actual evaluation wascarried out by conducting parallel experiments, drying ofdeuterated spruce and pine species at 25 and 80 °C, with 75%D2O relative humidity at both drying temperatures. After thesubsequent flushing, the samples were dried before the FT-IRmeasurement at 40 °C. While one set was dried in the ovenwithout RH control, the other set was dried at 0% RH, achievedby drying the samples in the same oven in a desiccatorcontaining drying agent. The comparison of treated samplesdried prior to the measurement at 0% RH and without humiditycontrol is shown in Figure 2.

Despite reprotonation of the deuterated and dried samplesby flushing with an excess of water, the impact of water vaporon additional protonation is evident (Figure 2). The reductionin retained deuterium is mainly observed for the samples driedat 25 °C. Based on the OD peak area comparison, a decreaseof 69 and 67% for spruce and pine samples, respectively, wasobserved. The overall reduction was much less for the samplesdried at 80 °C, with the corresponding reductions measured at34% for both spruce and pine samples. A complete comparisonof the OD peak areas and relative retained deuterium values inpercentage for spruce and pine samples is shown in Figure 3.

Figure 1. Top: FT-IR spectra of deuterated spruce (lower) and pine(upper) wood samples dried under different conditions and flushedwith water. Spectra in black indicate drying at 75% D2O RH; grayspectra indicate drying at 7% D2O RH. Below: Direct peak sizecomparison of samples dried at different temperature-enlargementof the spectrum segment of interest indicated by frames in originalspectra.

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It appeared that even after exposure to water during theflushing stage, further reprotonation occurred during the pre-measurement drying. The majority of OD sites within thestructure of the sample dried at 25 °C that were inaccessible towater during flushing became accessible to water vapor atslightly elevated temperature (40 °C). This could indicate thatthe structure alterations induced by drying at 25 °C can bealmost completely reversed by applying water vapor at slightlyelevated temperature. This is to a certain extent in agreementwith the work of Hofstetter and co-workers who demonstrated

nearly complete reprotonation of OD groups in cellulose byexposure to water vapor.26 In those experiments, however, thetesting was carried out using pure cellulose from wood pulpthat was dried before deuteration.

In contrast, the impact of water vapor on samples previouslydried at 80 °C was lesser and the retained OD groups afterflushing were markedly more resistant to reprotonation. Itappeared that the alterations taking place in the wood structureduring drying at 80 °C are for the most part irreversible.

This decrease of the retained deuterium in the samples flushedwith water indicated that the rate and extent of reprotonationmay differ depending on whether it is carried out in liquid orgaseous phase. Although the slightly elevated temperature hasto be considered, it appeared that the water vapor could reachregions inaccessible by liquid water. Preliminary testing doneprior to this investigation showed that the extent of reprotonationof dried deuterated wood samples in liquid water is the sameafter 24 h exposure as that of 60 min flushing. The exposure towater vapor in excess of 24 h during the premeasurement dryingwas sufficient to reprotonate all accessible OD groups and thusthe measured spectra indicate the final extent of reprotonationat the given conditions. Even though various aspects of cellulosedeuteration have been studied previously, a systematic studyon the impact of temperature on stability or accessibility of theexchanged OD groups has not been carried out. In conclusion,the impact of drying temperature on the extent of wood structurealterations is clearly evident; however it appears that the dryingtemperature may also have an effect on the reversibility of thechanges.

3.3. Pulp Testing. The structure of the cell wall of chemicalpulp fibers bears a similarity to the cell wall of the fibers presentin native wood. The major distinction between the two is lignin,which is present in wood but is removed during the pulpingprocess, ensuing the presence of larger pores within the matrixof the pulp fiber.33 This fundamental similarity coupled withthe previous extensive studies on the impact of drying makesthe chemical pulp fibers a suitable reference substrate forevaluation of the analytical concept. In addition, the correlationof the behavior of pulp fibers and wood during deuteration anddrying can help better understand the alteration within theultrastructure during initial drying of fresh wood.

The pulp drying evaluation was carried out in a similarmanner as on the wood samples. Deuterated pulp samples weredried at two different temperatures (25 and 80 °C), in a 7%D2O relative humidity environment. The lower RH level wasselected to reflect the superior OH group accessibility and greaterextent of ultrastructure alteration expected for pulp fiberscompared to wood samples. An additional set of deuterated pulpsamples was dried overnight at 105 °C. After drying, the sampleswere flushed with an excess of water. The measured spectrafor the pulp samples are shown in Figure 4.

Compared to the original pulp sample (control), the deuter-ated, dried, and flushed pulp samples exhibited a distinctive peakin the OD stretch region of the IR spectrum. Similarly, asobserved in wood sample testing, the deuterated pulp samplesdried for a shorter time at 105 °C (without conditioning) andflushed showed a distinct peak in the OD region, althoughvisibly smaller compared to OD peaks of the conditionedsamples. The lower amount of deuterium retained in the samplesdried at 105 °C agrees with findings for the wood controlsamples in this investigation (Supporting Information, FigureS1). Although a reduction of retained deuterium during dryingat this temperature was previously described for deuterated filmsof regenerated cellulose,21 the low amount of deuterium was

Figure 2. Effect of water vapors present in drying before FT-IRmeasurement. Comparison of deuterated spruce (top) and pine(bottom) samples dried at 25 (left)/80 °C (right) and 75% D2O RH.Black lines (also indicated by 0% RH) represent peaks of samplesdried in desiccator with a drying agent; gray lines represent the peaksof samples dried without humidity control.

Figure 3. Effect of water vapor present in drying before FT-IRmeasurement. OD peak area comparison of spectra shown in Figure2. Darker (dotted) columns represent samples dried at 0% RH; emptycolumns indicate samples dried without RH control (black and graypeaks in Figure 2, respectively). Relative values (peak areas of thesample dried without RH control vs sample dried at 0% RH) inpercentage are shown on the right.

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not indicative of the extent of hornification the pulp is expectedto undergo when dried at this temperature. In contrast, the impactof temperature on retention of inaccessible OD groups resistingreprotonation was clearly evident for the samples dried at lowertemperatures in D2O relative humidity environment. The ODpeak size comparison indicated that the extent of changes inthe pulp samples dried at 80 °C was significantly greater thanthe alterations in the samples dried at 25 °C. This extent ofchanges was confirmed by measurement of water retentionvalues of the pulp samples under similar condition but watervapor environment. The comparison of the OD peak size andWRV of the dried samples is shown in Table 1.

The impact of drying and of drying temperature on thedecrease in WRV of dried samples is clearly evident. Thissuggests that the presence of the inaccessible OD groups afterdrying is an indicator of hornification. Although only twotemperature data points were measured, it is interesting to notethat the ratio of the WRV reduction at 25 and 80 °C (16 and62%, respectively) is identical to the relative OD peak areaincrease, both calculated to be 1:3.8.

3.4. Wood and Pulp Testing Comparison. In the currentstudy, chemical wood pulps behaved as expected: a decreasein WRV was accompanied by a comparable reduction in theaccessibility of water as indicated by the irreversible retentionof OD groups upon drying. The subsequent alterations in thepulp fiber properties have acquired the term hornification in theprevious literature.15-18 Although the origins of hornificationare still under debate, most recent studies agree that “irreversibleaggregation of cellulose microfibrils” is the ostensible phenom-enon leading to fiber stiffening.16,34-36 The aggregation hy-

pothesis has been strengthened by the studies, showing that anincreased hemicellulose content in pulps clearly reduces theextent of hornification, which is a logical causality consideringthat the hemicellulose matrix between the cellulose microfibrilswould hinder their inherent tendency to aggregate.37 Untreatedwood, on the other hand, boasts an extensive matrix of bothhemicellulose and lignin between the microfibrils and themicrofibril aggregation, therefore, appears like a remote pos-sibility. Nevertheless, in the light of the current results (Figures1-3), it is indisputable that the accessibility of water is alteredduring drying also in the case of fresh wood samples. This mustbe indicative of supramolecular rearrangements between thewood polymers, something that is smaller in scale than thepreviously reported microcracks in the cell wall after drying.11,38

We propose two hypothetical alternatives for the ultrastruc-tural rearrangements: (i) irreversible aggregation of cellulosemicrofibrils in a similar manner (but to a smaller extent) thatoccurs during hornification of pulp fibers and (ii) irreversiblestiffening of the hemicellulose/lignin matrix that extensivelyswells when exposed to water. The first hypothetical alternativeis partially backed up by literature accounts. Microfibrils tendto form lamellar bundles in wood and rigorous freeze-dryingprocedures in the sample preparation are required to visualizesingle microfibrils in high resolution microscopy images.39

Elazzouzi-Hafraoui et al. recently found aggregated microfibrilseven after severe acid hydrolysis, which was intended toindividualize cellulose crystallites, and they suggested theassociation of adjacent cellulose microfibrils may take placealready during biosynthesis.40 Similarly, Paakko et al. and Abeet al. ended up with larger units than individual microfibrilsafter mechanical disintegration of fibers into nanocellulosicobjects.5,41 Only Saito et al. have managed to truly individualizenative microfibrils by the so-called TEMPO-mediated oxida-tion, which is a process that was shown not to be affected bythe drying history of the fibers.42 According to our hypothesis,this association of microfibrils could be an artifact of drying. Itis very seldom that plant cell walls are subjected to, for example,hydrolysis, enzymatic treatment, or microscopic analysis beforeat least partial dehydration, which results from removing theplant from its native growth environment.

The second hypothetical alternative of the stiffening hemi-cellulose matrix is a known phenomenon: it is bound to takeplace during water removal, which elevates the glass transitiontemperature of dry hemicellulose to over 200 °C.43 However,the possible irreversible character of stiffening has not beenreported and its verification would require testing of materialproperties by, for example, differential scanning calorimetry ordynamic mechanic analyzer, which is outside the scope of thisintroductory article. We emphasize that these two hypotheticalmechanisms are not mutually exclusive.

4. Conclusions

The presence of inaccessible OD groups in the deuteratedand then dried wood samples after reprotonation with water wasdetected by FT-IR. This change in accessibility indicated thatsupramolecular rearrangements between wood polymers areoccurring within the cell wall. The observed changes in woodultrastructure were somewhat similar to the well-reportedreduction of swelling properties in chemical pulps upon drying.As with chemical pulp, the effect was enhanced with elevatedtemperature during drying. Two hypothetical alternatives wereproposed for the accessibility decrease in wood upon drying:(i) irreversible aggregation of cellulose microfibrils in a similar

Figure 4. FT-IR spectra of deuterated softwood bleached kraft pulpsamples dried under different conditions and flushed with water.

Table 1. Comparison of OD Peak Area and Water RetentionValues (WRV) of Pulp Samples Dried at Different Temperaturesa

sample OD peak area WRV (%) MC (%)

control 142 66.1dried at 25 °C 219 126 5.1dried at 80 °C 831 80 0.2a The drying conditions were similar except the samples for OD peak

area measurement were dried at 7% D2O relative humidity and thesamples for WRV and moisture content (MC) were dried in 7% H2O relativehumidity environment.

Drying of Wood Observed by D2O Exchange and FT-IR Biomacromolecules, Vol. 11, No. 2, 2010 519

manner (but to a smaller extent) that occurs during hornificationof pulp fibers and (ii) irreversible stiffening of the hemicellulose/lignin matrix that extensively swells when exposed to water.

Acknowledgment. Prof. Mark Hughes and Lauri Rautkari(M.S.) are thanked for help with sampling and deuterationexperiments. Rita Hatakka is acknowledged for assisting withFT-IR measurements and data transfer. The authors acknowl-edge the support by UPM-Kymmene corporation.

Supporting Information Available. Deuteration reversibilitytesting of spruce wood samples (S1), comparison of pine andspruce wood samples (S2), reproducibility comparison (S3), anduniformity of deuteration throughout the tested wood specimen(S4). This material is available free of charge via the Internetat http://pubs.acs.org.

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