Supramolecular polymer networks as nextgeneration consolidants for archaeological

wood conservation

Zarah Walsh1, 2, Emma-Rose Janecek1, 2, James T. Hodgkinson2, 3,Julia Sedlmair4, 5, 6, Alexandros Koutsioubas7, David R. Spring2,

Martin Welch3, Carol J. Hirschmugl5, 8, Chris Toprakcioglu9,Jonathan R. Nitschke2, Mark Jones10, and Oren A. Scherman*1, 2

1Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge,Lensfield Road, Cambridge CB2 1EW, UK. Email: oas23@cam.ac.uk

2Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK3Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW,

UK4US Forest Service, USDA, Forest Products Lab, Madison, WI, 53726, USA

5Synchrotron Radiation Center, 3731 Schneider Drive, Stoughton, WI 53589-3097, USA6Pennsylvania State University, Department of Agriculture and Biological Engineering, University

Park, PA 16802, USA7Forschungszentrum Jülich GmbH, JCNS at Heinz Maier-Leibnitz Zentrum, Lichtenbergstrasse 1,

D-85747 Garching, Germany8Department of Physics, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.

9Department of Physics, University of Patras, Patras 26500, Greece.10The Mary Rose Trust, HM Naval Base, College Road, Portsmouth, PO1 3LX, UK.

S.1 Instrumentation and Materials

Initial scanning electron microscopy (SEM) of archaeological wood samples was performed on a fieldemission FEI XL30 Environmental Scanning Electron Microscope (ESEM) (FEI Company, Hillsboro,Oregon, USA). Further images with energy dispersive X-ray characterisation were performed on aStereoscan 420 SEM from Leica (Jena, Germany). Sulfur, iron, calcium and zinc were detectedby accumulating counts over 100 s per spectrum using a RonTec EDX detector incorporated in theSEM. WinShell software was used to graph x-ray energy, while WinTool software was used to makea quantitative analysis of the presence of elements in the first approx. 1 µm depth of the timbersamples. Scanvision software was used to convert the quantitative data from the WinTool software

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into a dot-mapping image to show the approximate location of the elements under investigation in thesample.

1H NMR (500 MHz) spectra was recorded using a Bruker Avance 500 MHz Triple Resonance In-verse spectrometer. Chemical shifts are recorded in ppm (δ ) in D2O with the internal reference set toδ 4.79 ppm. Solid-state NMR samples were, where required, prepared by ball milling using a Sarto-rius Mikro-Dismembrator S at 3000 rpm until a fine powder was achieved, typically 30 seconds. Thepowdered sample was then packed into a 4 mm diameter zirconia rotor. High resolution 13C ssNMRwas recorded using magic angle spinning (MAS) and cross polarization (CP) at ambient temperatureusing a Bruker Avance 400 WB 3-channel solid state spectrometer operating at 100.5 MHz with a4 mm dual resonance probe. Spinning rate for each experiment was set to 12.5 MHz and chemicalshifts measured in ppm (δ ) relative to tetramethylsilane via glycine as a secondary reference materialwith the Cα signal set to 43.1 ppm. The delay time was set such that maximum signal intensity wasachieved across the spectra.

ATR FT-IR spectroscopy was performed using a Perkin-Elmer Spectrum 100 series FT-IR spec-trometer equipped with a universal ATR sampling accessory.

Rheological characterization was performed using an AR-G2 controlled stress rheometer fitted witha Peltier stage set to 20◦C. Strain sweep measurements were performed at a frequency of 10 rad/s.Frequency sweep measurements were performed at a 1% strain amplitude. All measurements wereperformed using a 40 mm parallel plate geometry set to a gap height of 0.50 mm and analyzed usingTA Instruments TA Orchestrator software.

SAXS experiments were performed at the SWING beamline of the French synchrotron facilitySOLEIL. The x-ray wavelength and the sample-detector distance were λ = 1.03 and D=7000 mm,respectively. The polymer networks were placed inside quartz capillary tubes with an internal diam-eter of 1.5 mm. The beam path was held under vacuum conditions, except for a path length of about1 cm, around the sample. Then scattering data (40 frames of 1 s duration for each sample) were col-lected with a 17 x 17 cm2 low-noise AVIEX CCD detector. The 2D scattering images were radiallyaveraged, divided by the transmitted intensity and finally averaged for each sample. The scatteringcurves that correspond to the gel form and structure factor were obtained by subtracting the quartzscattering signal.

For biological experiments, samples were agitated using a Heidolph Titramax shaker at 37 oC. Theoptical density of the bacterial broths were measured on a micro plate reader model 680 from Biorad.

All other materials were purchased from Aldrich and used as received.

S.2 General Synthetic Protocols

Synthesis of 1-(4-boronobenzyl)-1’-methyl-[4,4’-bipyridine]-1,1’-diium (MV-BA). 1-methyl-4,4’-bi-pyridinium was synthesized from 4,4’-bipyridine from a standard literature method .1 Followingthis, (4-(bromomethyl)phenyl)boronic acid (100 mg, 0.47 mmol) and 1-methyl-4,4’-bipyridinium(120 mg, 0.40 mmol) were dissolved in acetonitrile (4 ml) and the solution heated to reflux overnight.The resulting red solid was isolated by filtration and recrystallized from ethanol, containing a fewdrops of water, to yield a red solid (132.2 mg, 0.28 mmol, 70%). 1H NMR (400 MHz, D2O): δ = 9.21(d, 2H, 7 Hz), 9.09 (d, 2H, 7 Hz), 8.59 (d, 2H, 7 Hz), 8.55 (d, 2H, 7 Hz), 7.91 (d, 2H, 8 Hz), 7.59 (d,2H, 8 Hz), 6.00 (s, 2H), 4.54 (s, 3H) ppm. 11B NMR (160.48 MHz, D2O): δ = 28.81 ppm. FT-IR: ν

= 3200 cm−1 (Broad, OH), 1635 cm−1 (aromatic), 1366 cm−1 and 1333 cm−1 (B-O).

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Synthesis of 3,4-dihydroxyphenyl acetic acid functionalized chitosan (PolyCat). In the first stage3,4-dihydroxyphenylacetic acid (HPAA) (59.4 mg, 0.35 mmol) is stirred for 24 h with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (54.8 mg, 0.35 mmol) in an equimolar ratio, in order toaminate the carboxylic acid of the HPAA. After 24 h a 3 wt.% solution of chitosan in 1 vol.% aceticacid is added to the guest solution in a molar ratio of 0.2:1 (functional group:chitosan) and stirredfor a further 72 h to attach the aminated guest molecule to the free amine on the chitosan backbone.After 72 h the polymer is precipitated from solution using a 10 wt.% solution of NaOH, which is thenfiltered and washed with copious amounts of water (approx. 3 L) to return the pH to neutral. Thematerial is collected, frozen in dry ice and then lyophilized for 72 h before use. A schematic for thepreparation of the material is shown in Figure S1.FT-IR: ν = 3380 cm−1 (Broad, OH), 1662 cm−1 (amide), 1643 cm−1 (aromatic), 1382 cm−1, 1368 cm−1

and 1155 cm−1 (amine)

Synthesis of 3,4-dihydroxyphenyl acetic acid functionalized chitosan (PolyNap). For the attach-ment of the 2-naphthylacetic acid (NPAA) to the chitosan backbone, the same procedure as for HPAAis followed. In this case 71.7 mg NPAA (0.35 mmol) is reacted with the EDC in the first step, beforereacting with the chitosan polymer.FT-IR: ν = 3395 cm−1 (Broad, OH), 1663 cm−1 (amide), 1643 cm−1 (aromatic), 1383 cm−1, 1368 cm−1

and 1155 cm−1 (amine)

General preparation of supramolecular crosslinked materials. Gels are prepared by weighing10 mg of guar, 1 mg of MV-BA (which forms a dynamic covalent linkage to the guar and there-fore no pretreatment of the guar is required, Figure S1), 30 mg of the PolyCat or PolyNap (or a 1:1mixture of the two polymers) and 4 mg of cucurbit[8]uril into a clean vial and solvating in 1 ml of a1 vol.% solution of acetic acid by stirring overnight on a magnetic stirring plate. Once prepared thePolyCat containing materials appear beige/orange in color, while the PolyNap containing materialsare white, PolyCatNap materials are an opaque, beige color.

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I-

N N

B

HO

OH

OOO OHO

H

OH

OH

HOOH

HO

O

O

OH

HO

OH OH

n

I-

OOO OHO

H

OH

OH

HOOH

HO

O

O

OH

O

O OH

B

N

N

HO

n

I-

A

O

NH2

O

OH

HO O

O

NH2

HO

OH

n

HO

OH

OH

O

OO

OOO

OH

HONH

OH

OH

NH2HO

OH

HOHO

NH2nO

HO

HO

where OH

O

OH

O

Alternatively some can be replaced by or

B

Figure S1: Schematic representation of the functionalization of the natural polymers A. dynamiccovalent functionalization of guar with MV-BA and B. covalent attachment of catechol to a chitosanbackbone, with the structures of other second guests examined in this study.

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S.3 Scanning electron microscopy with energy dispersive X-ray(SEM-EDX)

Scanning electron microscopy (SEM) was used to visually determine the extent of degradation in theoutermost areas of the timbers where degradative processes are maximized due to proximity to airand moisture. Using an environmental SEM (FEI XL30 ESEM), a 5 mm cut from the exposed endof a 15 cm core sample from the Mary Rose was imaged without sputter coating with a conductivematerial. This image (Figure S2) illustrates the extent of degradation of the outer surfaces of thetimbers in which the majority of the cellulose is degraded and the wood is mainly supported by thelignin skeleton, being more resistant to both biological and chemical degradation.

Figure S2: Scanning electron micrograph of degraded timber taken from the hull of the Mary Rose

Energy dispersive X-ray on the SEM was used to understand the issue of iron saturation and howhigh concentrations of iron overlapped with concentrations of sulfur are problematic in that the ironcan catalyze the formation of sulfuric acid in the timbers leading to further degradation of cellulose,Figure S3.

In addition to catalyzing acid production, the iron can also contribute to the propagation of bac-terial action in the timbers by acting as a promoter. Contributing to both chemical and biologicaldegradation of the cellulose and causing structural instability in the wood.

Figure S4 shows the dot-maps of SEM images taken over 30 µm2 sections of the 5 mm core sample.It can be seen clearly that there is a reasonably high distribution of Fe (of unknown oxidation state)and S in the timbers, which overlap with one another, which is known to be disadvantageous to thetimbers. It is now clear that trapping iron in the timbers and preventing their catalytic activity andtheir ability to promote bacterial growth is extremely important to the conservation effort.

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DC

BA

Figure S3: X-ray spectra of (A) fresh oak, (B) a main deck plank core, (C) a port-side core, (D) coresample B4/2, all taken 60-65 mm below the surface with the exception of the fresh oak sample whichwas taken at 10 mm

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B4/2 0-5 mm B4/2 60-65 mm MDP 60-65 mm PS 60-65 mm

Fe

S

Overlay

over SEM

Figure S4: Dot-mapping images where point-to-point X-ray spectra are overlaid over SEM imagesof the samples

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S.4 Evidence of cellulose degradation by solid state NMR

Solid state NMR using the methods described above was carried out to show that the carbohydratecontent of the Mary Rose timbers is reduced compared to that of fresh oak of the same species,Quercus rober, Figure S5. While the lignin peaks remain almost identical in shape in both samples(peaks at ∼ 153 ppm, 147 ppm, 136 ppm, 134 ppm) there is a significant decrease in the magnitudeof those signals from the carbohydrate content (∼ 105 ppm, 72 ppm), illustrating the extent of thedegradation of the polysaccharides of the timber.

200 150 100 50 0 ppm

Fresh Oak

Mary Rose Oak

Figure S5: Solid state NMR of fresh oak (black) and oak from the hull of the Mary Rose

S.5 Characterization of second guest binding kinetics throughUV-Vis spectroscopy.

The binding constant of the napthyl unit inside the CB[8] cavity in the presence of methyl viologenhas previously been measured experimentally within the group and is reported to be in the region of(6.1 ± 0.5) x 105 M−1,2 the same paper reports the binding constant of catechol as (1.0 ± 0.3) x104 M−1. For this reason experiments to determine the binding constant of the ternary complexeswere not carried out.

UV-Vis absorbance spectroscopy was used to observe the interaction of catechol with Fe3+ andFe2+ and naphthol with Fe3+ and Fe2+, to show that only catechol can interact appreciably with Feand specifically Fe3+. Figure S6 shows the absorbance spectra of catechol with Fe3+ (A) and Fe2+

(B), the same spectra are shown for naphthol (C, D). It is clear from the spectra that only (A) showsa specific interaction, represented by the eventual disappearance of the catechol peak with increasingconcentrations of Fe3+ and the appearance of a complex peak at 400 nm. In each of the other casesthere is no appreciable change to the spectra indicating binding. This shows that only the interaction ofcatechol and Fe3+ should be able to cause any changes in the assembly of the polymer network, whichis important for the action of the material as a conservation treatment. Non-specific or competitiveinteractions between components of the system could be detrimental to the function of the treatment.Using procedures described by Shao3 and Yang,4 a rough estimate of the stoichiometry and Ka ofcatechol with Fe3+ was obtained from the absorbance of the complex peak at 400 nm, data shown inFigure S6E Ka was estimated to be in the region of 2 x 108 M−2 with a stoichiometric ratio of 0.3:1(catechol:Fe3+).

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A

C

B

D

E

Figure S6: UV-Vis titrations of catechol against increasing concentrations of Fe3+ (A), Fe2+ (B) andnaphthol against Fe3+ (C), Fe2+ (D)

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S.6 SAXS Data Analysis

For gels composed of flexible polymer chains, the scattering intensity is usually described by the com-bination of two terms, a dynamic and a static component.5,6 The dynamic term follows an Ornstein-Zernike law, as for polymer semi-dilute θ -solutions in a good solvent, leading to a Lorentzian formof the scattering function given by:

I(q)L =I(0)L

1+q2ξ(1)

where IL(0) is the extrapolated structure factor at zero q and ξ , a thermal correlation length, whichcan be assimilated to a ‘mesh size’ of the network. In good solvents, where the coils are swollenon length scales smaller than ξ , this expression takes a slightly different form to reflect the fractaldimension of the system, D = 1/ν ≈ 1.7 :

I(q)L =I(0)L

1+(ξ q)1ν

(2)

where ν is the Flory exponent. In cross-linked gels, however, the presence of mechanical con-straints may restrict the movement of polymer segments thereby causing the emergence of additionaland essentially permanent spatial concentration fluctuations, which give rise to excess scattering atlow q. If the mean size of these static inhomogeneities is large compared to the correlation length ξ ,then the two contributions may be regarded as decoupled and added together to give the total intensity:

I(q) = I(q)L + I(q)ex (3)

where the second term, I(q)ex, corresponds to the static concentration fluctuations causing excessscattering at low q, and can be described either by the Debye-Bueche model6–8 or by a Gaussianspatial distribution of the inhomogeneities.5,9

Within the q-range accessed in the current experiments there is no evidence of excess scattering atlow q that can be accounted for either by the Debye-Bueche expression or by a Gaussian distributionof spatial concentration fluctuations. The SAXS intensity of PolyCatNap with no iron present followsa q−3 dependence, which is consistent with a self-similar structure of fractal dimension D=3, sugges-tive of a micro-particulate gel, likely containing large gel particles solubilized in a polymer matrix.Upon addition of Fe+3, however, significant changes occur in the SAXS pattern, with the q−3 behav-ior now confined to low q, while at higher q a clear transition to a q−1.7 dependence is observed, inline with eq. 3 above. This can be attributed to Fe+3 chelation, via interaction with pendant catecholgroups, and the emergence of additional cross-links in the system.

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S.7 Chelation behavior of polymer networks

The full text of the paper describes the behavior of the PolyCatNap in the presence of both Fe2+ andFe3+. For control purposes the behavior of the PolyCat and PolyNap in the presence of the two Fesalts was also monitored in order to show that the combination of the two second guest polymers in thenetwork produced a more significant Fe3+ chelation effect than either of the second guest polymersalone. Images of the control interactions are shown in Figure S7. Although purely qualitative, thepolymers alone appear of a lower viscosity, which is reduced further in the presence of Fe2+. Amines,like those on the chitosan backbone, can chelate with Fe2+ and it is known that chitosan can havehydrophobic and H-bonding interactions within its chains,10 it is therefore likely that the presence ofthe Fe2+ binds to the amines and encourages further interactions between the chitosan chains reducinginteractions with the functionalized guar. Thus, the polymers in the presence of Fe2+ become micro-particulate gels solubilized in aqueous solutions of guar, reducing their viscosity. In the PolyCat, thegel appears less viscous on account of the strengthening of the gel by catechol-Fe3+ interactions inaddition to the ternary-complex already present. This is not visible in the PolyNap material as thereare no ligands present which interact significantly with Fe3+.

PolyCat + Fe2+

PolyCatNap PolyCatNap + Fe3+PolyCatNap + Fe2+

PolyNapPolyNap + Fe2+

PolyCat + Fe3+PolyCat

PolyNap+ Fe3+

Figure S7: Inverted vial test showing the response of PolyCat, PolyNap and PolyCatNap to the addi-tion of Fe2+ and Fe3+

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S.8 Rheological data controls

To understand the need, rheologically, for the presence of two second guest polymers in the finalfunctional supramolecular network rather than using a single second guest polymer, the rheologicalcontrol data for the PolyCat and PolyNap, unmodified and in the presence of Fe2+ and Fe3+ is shown,Figure S8. The data clearly shows that both PolyCat and PolyNap are required to be present simulta-neously to improve the structural stability of the supramolecular material. In all cases the stability ofthe material is significantly improved compared to PEG200.11

BA

FE

DC

Figure S8: Plots of angular frequency (rad/s) vs. G’ and G” (Pa) (A, C, E) and shear rate (1/s)vs. complex viscosity (Pa.s) (B, D, F) for untreated natural polymers and polymer mixtures (A, B),PolyCat with Fe2+ and Fe3+ (C, D) and PolyNap with Fe2+ and Fe3+ (E, F)

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S.9 Biological activity experiments

Colonies of the desired strain were grown on a Luria-Bertani (LB) agar plate at 37 oC overnight.The colonies were then used to inoculate 10 mL fresh Mueller-Hinton broth and grown overnightat 37 oC. The overnight cultures were used to inoculate fresh Mueller-Hinton broth media to aninitial OD600 nm of 0.05. The inoculated media (135 µL) was then transferred to 96 well microtiterplates in the presence of each polymer (15 µL), cultures were grown for 8 h (600 rpm - Heidolphtitramax shaker) at 37 oC. Each polymer was grown in the presence of each bacterial species intriplicate. The OD600 nm was measured at t=0 and at 2 h intervals using a microplate reader (bioradmodel 680). The ability to hinder bacterial growth was measured for pure guar, pure chitosan, purePEG200 and PolyCatNap. Pure samples were prepared by simply dissolving in water at 10 mg/mlfor guar and 0.5 ml/ml for PEG200, as chitosan does not dissolve in pure water this was prepared ata concentration of 30 mg/ml in 1 vol.% acetic acid in water. PolyCatNap was prepared as outlinedin S.2 above. Samples were then diluted to either 10% or 20% of their original concentration for theOD600nm measurements.

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S.10 Shrinkage and uptake of polymer

Shrinkage of the wood samples was determined after 1 week of treatment and 1 week of freeze-drying and was calculated relative to both the initial weight and the weight after treatment but beforefreeze-drying. Shrinkage data was obtained for samples which were treated with polymer at ambientconditions and under vacuum, and in both cases this was compared with data obtained for PEG treatedsamples, Figure S9. For comparison, in a worst case scenario, an untreated piece of timber from theMary Rose could be expected to shrink up to 59%, according to studies carried out at the Mary Rosemuseum.

PEG200 PolyCatNap0

10

20

30

40

50

60

70

80

90

100 % uptake (ambient) % shrinkage (wrt initial wt, ambient) % shrinkage (wrt uptake wt, ambient) % uptake (vacuum) % shrinkage (wrt initial wt, vacuum) % shrinkage (wrt uptake wt, vacuum)

Upt

ake/

Shrin

kage

(%)

Performance of PolyCatNap vs. PEG200

Figure S9: Uptake and shrinkage data obtained for samples treated with PolyCatNap and PEG200

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S.11 Infrared environmental imaging to determine polymerpresence in wood

High-resolution FTIR images were acquired at the IRENI (InfraRed Environmental Imaging) beam-line located at the Synchrotron Radiation Center in Madison, WI, USA. A uniquely shaped syn-chrotron beam is used as IR-source for a Bruker Hyperion 3000 FTIR microscope. A section of320 mrad (horizontal) x 25 mrad (vertical), is extracted from a dedicated bending magnet, collimatedand rearranged into a matrix of 3 x 4 beams, by a set of mirrors. Slight defocussing at the sampleposition results in a homogeneously illuminated area of 40 µm x 60 µm.12,13

The microscope combines a 74 x objective (NA=0.65) with a condenser of matching NA, with aneffective pixel size of 0.54 µm, allowing for diffraction limited imaging at all wavelength recordedwith a focal plane array detector (FPA).14,15

For the measurements, chemical images of 10 µm thick cross sections of samples were recordedover mid-IR range (4000 cm−1 - 800 cm−1), with a resolution of 4 cm−1, accumulating over 128 scans.For the analysis, the programs IRidys (www.iryidys.com), running on IGOR Pro, and Bruker’s ownsoftware OPUS were used.

The full text of the paper shows a comparison image between the untreated archaeological woodand wood after 1 week treatment with PolyCatNap with subsequent freeze-drying. The images arespectral images integrated over 5 different wavenumber ranges in the IR spectrum. In order to showthat the material was truly impregnated and not spectral anomaly due to inhomogeneities in the wood,samples which had been treated with unfunctionalized guar and an unfunctionalized guar/chitosan(1:3) were also measured and integrated over the same spectral ranges, shown in Figure S10 - Fig-ure S13, below.

DACB

0 20 40 60

0

20

40

60

Figure S10: (C) Optical image of untreated Mary Rose wood, 10 µm thick, (A) integration image ofthe same area imaged in the optical microscope integrated over the entire spectral range, (B) averageIR spectrum of the native wood sample, (D) IR spectrum of native Mary Rose wood, taken at severalpoints on the wood surface, marked by an x in the integrated image (A)

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A B C D

0 30

0

50

100

150

Figure S11: (A) Optical image of Mary Rose wood treated with a 10 mg/ml solution of guar, 10 µmthick, (B) integration image of the same area imaged in the optical microscope integrated over theentire spectral range, (C) average IR spectrum of the guar-treated wood sample, (D) IR spectrum ofthe guar-treated Mary Rose wood, taken at several points on the wood surface, marked by an x in theintegrated image (B)

CA

DB

0 20

50

100

0

150

Figure S12: (A) Optical image of Mary Rose wood treated with a 40 mg/ml solution of guar (10 mg)and chitosan (30 mg), 10 µm thick, (B) integration image of the same area imaged in the opticalmicroscope integrated over the entire spectral range, (C) average IR spectrum of the guar/chitosan-treated wood sample, (D) IR spectrum of the guar/chitosan-treated Mary Rose wood, taken at severalpoint on the wood surface, marked by an x in the integrated image (B)

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C

B DA

0 20 40 60

0

20

40

60

Figure S13: (C) Optical image of Mary Rose wood treated with a 40 mg/ml solution of PolyCatNap,10 µm thick, (A) integrated image of the same area imaged in the optical microscope integrated overthe entire spectral range, (B) average IR spectrum of the PolyCatNap-treated wood sample, (D) IRspectrum of the PolyCatNap-treated Mary Rose wood, taken at several point on the wood surface,marked by an x in the integrated image (A)

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