NANO LETTERS
Microfocus X-ray Diffraction of Historical Parchment Reveals Variations in Structural Features through Parchment Cross Sections
2004 Vol. 4, No. 8 1373-1380
Craig J. Kennedy,*,† Jennifer C. Hiller,† Donna Lammie,† Michael Drakopoulos,‡,# Marie Vest,§ Martin Cooper,| W. Paul Adderley,⊥ and Timothy J. Wess† School of Optometry and Vision Sciences, UniVersity of Cardiff, Redwood Building, King Edward VII AVenue, Cathays Park, Cardiff CF10 3NB, Wales, UK, European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, 38043 Grenoble, France, School of ConserVation, Esplanaden 34, DK-1263, Copenhagen, Denmark, National Museums LiVerpool, Laser Technology, ConserVation Centre, LiVerpool L1 6HZ, UK, and School of Biological and EnVironmental Sciences, UniVersity of Stirling, Stirling FK9 4LA, Scotland, UK Received February 25, 2004
ABSTRACT We propose a new method of investigating variation of preservation within a parchment sample, which allows a more detailed analysis of alteration of the material structure. X-ray diffraction analysis of parchment typically involves the sample aligned with the plane of the parchment perpendicular to the direction of the X-ray beam, with a beam size of approximately 200 µm and an image consisting of the composite diffraction features from the entire thickness of the sample. Here we describe the use of microfocus X-ray beams, with a beam size of 1.5 µm vertically × 15 µm horizontally, to carry out surface-to-surface scans of thin sections of parchment. Up to 200 images can be taken in a single cross-sectional scan of a 300 µm thick parchment section. This allows for X-ray diffraction analysis of features present only in specific areas of the parchment, such as at the surface. The orientation of collagen fibrils in the plane of the parchment, the effects of laser cleaning (including possible laser induced damage), mineral phases and crystalline lipids present in samples, and parchment structure under an inked region are investigated. It is shown that the long collagen fibril axis lies parallel to the parchment surface throughout the sections. Laser cleaning appears not to damage the collagen in parchment, while laser-damaged samples display gelatinization of the collagen at the surface. Polymorphs of calcium carbonate were detected in several samples but in most cases were not confined to the surfaces, as would be expected if the chalk finishing process was the main source of mineral phases in parchment. Crystalline lipid is found in most samples and appears to exhibit a preferential alignment with the plane of the phospholipid bilayer arranged parallel to the long fibril axis of collagen. The d spacing of the lipid is variable throughout a parchment section, indicating fluctuations in the hydration state, phase, or biochemical composition of the lipid. Ink affects the parchment to a depth of approximately 90 µm, as measured by principal components analysis, disrupting the structure of the collagen to this depth. These features demonstrate the ability of this technique to examine diagenesis of individual components of parchment on a scale not previously studied.
Parchment is a collagen-based writing medium made from processed, untanned animal skins. While still produced today, the techniques of parchment preparation and finishing in a historical context are not fully understood.1 In addition to the text written on the parchment, the structure of the parchment material itself provides key historical information. * Corresponding Author: Tel: +44 29 2087 0203. Fax: +44 29 2087 4859. E-mail:
[email protected] † University of Cardiff. ‡ European Synchrotron Radiation Facility. § School of Conservation. | Laser Technology, Conservation Centre. ⊥ University of Stirling. # Present address: Diamond Light Source, Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX, UK. 10.1021/nl049696a CCC: $27.50 Published on Web 04/23/2004
© 2004 American Chemical Society
Parchment is believed to contain many structural features similar to skin and consequently is not uniform in cross section, but rather is made up of distinct layers.2 The parchment surface that was connected to the animal is known as the flesh layer; the surface that was previously the outer skin is known as the grain layer. As a biologically based material, parchment is subject to deterioration due to the effects of UV radiation, atmospheric sulfur dioxide, and microbial attack.3 The hierarchical structure of collagen in a feltwork, which provides mechanical strength to skin, is also responsible for maintaining the physical integrity of the parchment such that it is a viable long-term writing medium.
As the collagen deteriorates over time, the parchment loses strength, becomes brittle and degrades to the point where it can no longer be used. Analysis of this decay, and the development of novel methods to restore parchments, are key to hopes that many aspects of cultural heritage are not lost due to the biodegradation of collagen in parchment.3,4 In addition, cleaning of parchment to allow for the display and restoration of historically important documents has become a source of interest, as incorrectly applied cleaning methods may lead to irreparable damage to the parchment structure. The viability of techniques such as laser cleaning depends on an understanding of the impact, if any, that these have on the structure of collagen within parchment.5 X-ray diffraction is an analytical method capable of describing the structural hierarchy and molecular packing of collagen6 and can be used to detect changes which may occur upon decay or through cleaning treatments. The advent of microfocus X-ray technology has led to the development of intense micron sized X-ray beams, which can be used for X-ray microdiffraction experiments where small regions of textural variation can be probed in a sample. This technology also allows simultaneous measurements of fluorescence emission allowing elemental mapping. Microfocus X-ray diffraction can be used to make a detailed map of nanotexture within a biologically based material, such as bone;7 however, the technique can potentially be applied to all materials containing variation on the nanotextural level. In this study, we investigate a number of features present in parchment by microfocus X-ray diffraction: (i) the orientation of collagen fibrils in the plane of the parchment; (ii) the effects of laser cleaning and possible laser-induced damage such as gelatinization; (iii) the presence of mineral phases in samples; (iv) the state and presence of crystalline lipids; and (v) the effects of components of ink penetrating the parchment. Analysis of these attributes of parchment at this scale gives an indication of the usefulness of this technique when probing individual features that may be affected during the biodegradation of parchment over time. The National Archives for Scotland (Edinburgh, UK) and the National Museums Liverpool, UK, provided samples of parchment for this research. Table 1 outlines sample information. Samples provided by the National Archives for Scotland are historical samples; treated parchments provided by the National Museums Liverpool are modern samples (Z. H. de Groot, The Netherlands) that were produced in a manner similar to historic documents. Samples USH04 and MCP01 were laser cleaned, and sample MCP02 was deliberately damaged by laser treatment. Laser cleaning was carried out using a Q-switched Nd:YAG laser (Lynton Lasers Ltd.) at a wavelength of 1064 nm, a fluence level (energy density) of 0.34 J/cm2, and a pulse length of 5-10 ns.8 Laser cleaned samples received on average 22 pulses each. The parchment samples were mounted on a moveable computer-controlled X-Y table and irradiated from above; the laser beam was delivered from a fixed position.4b Exposing the parchment to the laser for an extended period of time at a fluence level in excess of its 1374
Table 1. Historical and Reference Parchment Samples Useda sample name
sample age (years)
sample source
USH01a USH01b USH02 USH03 USH04a USH04b
238 238 234 228 176 176
NAS NAS NAS NAS NAS NAS
USH05 USH08
179 186
NAS NAS
MCP01 MCP02
N/A N/A
NML NML
description horny, folded parchment as above; section under ink dirty with coarse feel large folded sheet large folded sheet as above; laser cleaned on upper surface large folded sheet tightly folded; several sheets tied together laser cleaned parchment laser damaged parchment
a NAS denotes National Archives for Scotland. NML denotes National Museums Liverpool. Sample age is estimated from dates inscribed on the parchment sheets at the time of initial parchment use.
Figure 1. X-ray transmission image of a cross section of sample USH01. The arrows indicate transects where the X-ray diffraction images were taken. Images were taken at 5 µm intervals, going beyond the outer boundaries of the parchment to ensure that a complete analysis of the cross section was undertaken.
damage threshold9b induced laser damage, until surface deformations became visibly apparent. X-ray diffraction data were collected at the microfocus beamline ID18F at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. A description of the instrumentation used is given by Wess et al.7 Samples were dissected to approximately 500 µm thick with a scalpel; a thickness where the effects of cutting were a relatively insignificant aspect of the texture of the sample and the cross section remained intact. The samples were mounted on to a goniometer, attached to a computer controlled motor, with the plane of the parchment parallel to the direction of the X-ray beam, allowing cross-sectional images to be taken. The sampleto-detector distance was 20 cm. The synchrotron beam was focused using a compound refractive lens9 to 1.5 µm vertically × 15 µm horizontally. Replicate scans were taken through each parchment sample. These were taken 1 mm apart by moving the goniometer head (Figure 1). For each scan, diffraction patterns were taken at 5 µm intervals over the entire thickness of the sample. Nano Lett., Vol. 4, No. 8, 2004
Figure 2. The expected X-ray diffraction patterns (top) are dependent on the orientation of the collagen fibril (bottom). (A) If X-ray beam (arrow) encounters a fibril with perpendicular orientation, then a pattern with clear meridional peaks (lines) and equatorial peaks (grey ovals) are seen. (B) If the sample is presented at more than 18° from a perpendicular orientation then the equatorial patterns are seen but not the meridional peaks. (C) If the X-ray beam travels down the length of a parallel fibril, then only the equatorial peaks appear, seen as a ring (c). The disks in the center of the expected X-ray diffraction patterns correspond to a beam-stop, which absorbs the direct beam from an X-ray source to avoid damage to the detector.
Data analysis was conducted using the methods described in Wess et al.10 The diffraction data were converted into polar plots, allowing the distribution and spacing of diffraction features to be analyzed by converting the two-dimensional detector images to a linear intensity distribution.10,11 The linear profile was corrected for the Lorenz effect, which occurs due to sample diffraction smearing with increased diffraction angle (2θ). This was applied as a linear function proportional to 2θ. The image was then integrated over a fixed angular range to obtain each profile. Mineral phase identifications were made using PCSIWIN software and the International Centre for Diffraction Data (ICDD) PDF-2 database. The principal equatorial reflection of dry collagen, which is indicative of the lateral interaction between collagen molecules, occurs in parchment samples at 0.85 nm-1 in reciprocal space, corresponding to a real spacing of 1.18 nm. This reflection was used to assess the orientation of the collagen fibrils relative to the plane parallel to the sample surface (Figure 2). Figure 3 shows a series of X-ray diffraction images through sample USH08; the reflections at 0.85 nm-1 and 2 nm-1 are clearly visible. The latter peak is due to amorphous interactions in polypeptide chains. Transforming the X-ray diffraction pattern from Cartesian coordinates to polar coordinates allows for the analysis of the orientation of the collagen fibrils.10 A reflection that occurs in an arc-like or circular fashion in a Cartesian system appears as a straight line at a constant radius from the center of the beam in a polar plot. The length of the line represents the angular range of the X-ray diffraction pattern (degrees) (Figure 4). Preferentially orientated collagen fibrils will show a peak in a plot of intensity of the 0.85 nm-1 reflection versus the angular span (Figure 5). Randomly orientated samples will not show this peak. Stacking these plots in accordance Nano Lett., Vol. 4, No. 8, 2004
with the position they were taken from in the parchment cross sections allows for the generation of orientation maps (Figure 6) which can then be compared from sample to sample. There is little evidence for variation in collagen fibril orientation; fibrils appear to be well aligned with the long fibril axis parallel to the surface of the parchment. Sharp peaks at 0.85 nm-1 (at 180 degrees on Figure 6) in the orientation maps of the parchment samples demonstrate this. The orientation of the collagen is less pronounced in some cases, which is indicated by a smearing of the equatorial peaks in the polar plot. When the 0.85 nm-1 reflection appears as a circle in the X-ray diffraction pattern, it is an indication that the X-ray beam has traveled down the long axis of a fibril (Figure 2) or that the fibrils are distributed randomly in the sample. When smearing occurs, it is not confined to one area of the parchment section such as the surfaces, but appears at different locations throughout the sections. Laser cleaning of the samples does not appear to have an effect on the distribution of the 0.85 nm-1 reflection at the surfaces of the parchment, indicating that the procedure does not induce reorientation of the collagen fibrils. Gelatinization occurs when the triple helix of collagen molecules unravels to form a random coil. This is an indication of the level of collagen degradation in historical parchments.12 The 0.85 nm-1 equatorial reflection of dry collagen was used to assess the degree of gelatinization of the collagen in parchment. As described by Weiner et al. (1980),12 the relative intensities of the peaks at 0.85 nm-1 and 2 nm-1 are capable of giving an indication of the relative amounts of collagen and gelatin in a parchment sample. The 2 nm-1 peak is consistent in terms of intensity and width in collagen and gelatin samples, whereas the 0.85 nm-1 peak displays increased intensity and reduced width if the amount of fibrillar collagen in a sample is high and the degree of gelatinization is low. 1375
Figure 5. Integration of the area of the X-ray diffraction image of parchment sample USH04, which encompasses the 0.85 nm-1 reflection. Two distances in θ are chosen that lie on either side of the 0.85 nm-1 reflection. Integration then occurs radially between the two distances, i.e., 0.7 nm-1 and 1.0 nm-1, producing a linear map of angular intensity distribution.
Figure 3. Composite of X-ray diffraction images (top) from a scan through a cross section of parchment sample USH08. The images are organized into rows from top left to bottom right. The images from the top surface of the scan (grain side) show sharp rings corresponding to minerals present in the sample. As the scan progresses toward the flesh side of the section, the collagenous component of the parchment sample becomes more apparent and displays prominent 0.85 nm-1 and 2 nm-1 reflections. At smaller angles, the meridional series can be observed. An individual image (bottom) clearly shows the 0.85 nm-1 and 2 nm-1 reflections.
Figure 4. Conversion from Cartesian (x,y) to polar (r,χ) coordinates. The patterns shown display an arc-like reflection in the Cartesian system; once converted to polar coordinates, the arc appears as a straight line at a constant distance, r, from the center of the beam. This allows for the intensity of the reflection to be measured and plotted against angular position (χ).
To quantify the 0.85 nm-1 and 2 nm-1 reflections in the samples, the one-dimensional peak-fitting program XFIT (Collaborative Computational Project 13 (CCP13)) was used. 1376
From linear profiles of the X-ray diffraction data, a polynomial background was removed, and the reflections were represented by Lorentzian distributions. Once the peaks were selected, a linearized least-squares algorithm was used to optimize the fit between the selected Lorentzian functions and the original linear profiles. This method was used to consider the effects of laser cleaning and deliberate laser damage at the surface of parchment samples. Laser cleaning appears not to induce gelatinization of the collagen at the parchment surface (Figure 7a). Intentional damage to the parchment surface with a laser, however, brings about a loss of intensity of the 0.85 nm-1 reflection at the surface of the parchment that indicates a degree of gelatinization (Figure 7b). Polymorphs of calcium carbonate, including aragonite, calcite, calcite hydrate, and vaterite, were present in some samples. Samples USH02, USH05, MCP01, and MCP02 showed traces of calcium carbonate phases at random locations through the sections, whereas sample USH08 showed strong levels of mineral deposits on the grain surface, which decreased with increasing distance from the surface. Samples USH01, USH03, and USH04 contained no detectable mineral phase. Figure 8 shows a representative trace from the center of the scan of sample MCP01, with prominent peak reflections indicated. Profiles of calcium carbonate morphologies varied from sample to sample. Lipids are clearly present in the majority of these samples and are identified by their characteristic diffraction pattern displaying reflections at a real d spacing of 4.4 nm to 4.8 nm, the distance between the polar headgroups in phospholipid bilayers. In some samples the lipid diffraction pattern is persistent throughout the depth of the section; in others, the lipid is present in part of the section, or is absent altogether. The lipid diffraction peaks appear in an equatorial position relative to the direction of the collagen long fibril axis, indicating that plane of the phospholipid bilayer is Nano Lett., Vol. 4, No. 8, 2004
Figure 7. (a) The integrated intensity of the Lorentzian representation of the 0.85 nm-1 peak divided by that of the 2 nm-1 peak (I/I). For reference sample USH04a (_9_9_9_) and laser cleaned sample USH04b (_____), these display a similar trend: toward the surface of the parchment (grain side) the I/I values are low, indicating a reduced level of fibrillar collagen at the surface; toward the center of the parchment the I/I values increase, indicating a greater degree of fibrillar collagen present. From this it can be concluded that laser cleaning does not induce gelatinization of the collagen at the surface of a parchment sample. (b) I/I values for laser cleaned and laser damaged parchments. The laser cleaned sample MCP01 (_9_9_9_) displays the trend observed in Figure 7a, indicating a reduced level of fibrillar collagen at the grain side surface of a parchment sample relative to the center of the parchment sample. The laser damaged sample MCP02 (_____), however, displays negative I/I values up to 20 µm into the parchment sample, indicating that the 1 nm peak is not present in those areas of the sample. This indicates that gelatinization is induced at the parchment surface after laser damaging; this is not observed after laser cleaning.
Figure 6. Angular orientation of samples USH01 (A) and MCP01 (B). The plots are intensity versus angular position of the 0.85 nm-1 equatorial reflection for each image through the cross section, taken at 5-µm intervals. Each line is normalized to allow for comparison of orientation regardless of the intensity of the 0.85 nm-1 reflections. The sample orientation appears well aligned throughout the cross section of the parchment, as shown by the increased intensity (darker regions) at the center and edge. The mean full width half-maximum (fwhm) values for the peaks is measured at 75.9° for USH01, and 81.2° for MCP01; these samples are representative of the whole sample set. The smeared images at the top and bottom of the maps are the first and last few measurements, which occur beyond the boundaries of the samples and do not encounter any collagen.
orientated parallel to the collagen long fibril axis. This suggests the possibility of specific collagen-lipid interactions and molecular packing, such as interactions between the polar headgroups from the lipid bilayers and the side chains of the collagen molecules. In some cases the lipid appears to Nano Lett., Vol. 4, No. 8, 2004
evince a great degree of crystallinity, displaying a number of orders of diffraction (Figure 9). The X-ray diffraction pattern under surfaces with ink present shows that the collagen structure of the section is disrupted immediately underneath the ink. The 0.85 nm-1 equatorial peak and meridional series that are characteristic of fibrillar collagen X-ray diffraction are not observed until a depth of about 45 µm, compared with a depth of 10 µm in uninked samples. The presence of lipid under inked surfaces appears to be unaffected, however, with the lipid reflection present throughout the parchment section. Principal components analysis (PCA) of the linear profiles of the X-ray diffraction data allows the effect of ink on the interiors of parchment to be examined more over a wider set of measured variables. PCA is a data reduction technique that aims to reduce the dimensionality of any given data set and to identify new underlying variables. The statistical basis for PCA has been well documented.13 PCA rotates these 1377
Figure 8. Representative trace from the center of the scan of MCP01, showing examples of mineral phases observed. Each peak is labeled and identified with the mineral name and respective (h k l) index that relates to the peak. PDF files 87-1863 (calcite), 71-2392 (aragonite), and 72-1616 (vaterite) were used to identify the minerals present.25
Figure 9. X-ray microdiffraction images from sample USH04. The image on the left shows crystalline lipid diffraction, with three orders of diffraction observed at a d spacing of 4.6 nm. The image on the right, taken 10 µm away from the image on the left in the same sample, displays the first order of lipid diffraction only. In both cases, the lipid diffraction appears to be orientated equatorial to the direction of the collagen fibril.
original data into a new set of axes, such that the first few axes reflect most of the variance. By plotting these data, major underlying patterns may be seen. In addition, PCA generates basis functions that can explain variance in these data. There are as many basis functions as there are initial variables, and they are sorted in decreasing order of relevance to the major patterns present in the data. For each original variable, PCA generates coefficients that describe how much that variable contributes to the basis functions. Plotting these coefficients allows correlations or trends to be assessed. Plots of the coefficients of the first principle component against sample position show that areas of sample USH01 with and without ink present exhibit different trends (Figure 10). Up to a depth of 90 µm, the coefficients from the two samples differ, indicating that there is a significant difference between the inked and uninked samples. As the two scans 1378
Figure 10. First principal component from analysis of parchment sample USH01 results. The solid line denotes the a scan taken under ink. The dashed line denotes a scan with no ink present. The sample with writing on the upper surface (solid line) displays positive coefficients at the surface of the parchment, until approximately 90 µm into the sample. At this depth, the sample under ink displays the same characteristics as defined by the first principal component as the sample where no ink is present. This indicated that the ink on the surface of the parchment has penetrated the parchment to a depth of approximately 90 µm.
were taken only a millimeter apart, it can be assumed that the characteristics of the parchment itself are consistent, suggesting that the presence of ink has altered the collagen structure to a depth of 90 µm. While conventional X-ray diffraction is a useful tool when assessing the structure of collagen within parchment;14 this technique is not capable of distinguishing between the layers within parchment, or elucidating localized effects that exist in parchment cross sections. This study demonstrates how microfocus X-ray diffraction analysis of cross sections of parchment can be used to reveal variations in a number of Nano Lett., Vol. 4, No. 8, 2004
components on a scale and dimension not previously examined. Well aligned collagenous systems such as rat tail tendon analyzed by conventional X-ray diffraction give images with clear meridional and equatorial features.15 In a feltwork such as parchment or skin, in contrast, diffraction profiles are normally radially smeared so that meridional peaks often appear as rings and the equatorial scattering is no longer prominent as a single diffraction feature.11 In a twodimensional feltwork such as parchment, the collagen fibrils are believed to be aligned in the plane of the sample surface.16 Microdiffraction of cross sections of parchment allows for the examination of the collagen structure aligned along an axis rather than radially smeared, and so can provide molecular and structural information of high quality similar to that obtained conventionally for rat tail tendon collagen. If the collagen fibrils are aligned in one direction, the lateral interaction between collagen molecules would therefore exist only in one direction, perpendicular to the collagen long fibril axis. If the collagen fibrils assume more than one orientation, for instance, with each fibril tilted relative to other fibrils in the sample, then the lateral interaction between molecules will occur over an increased angular range. A smearing of the 0.85 nm-1 equatorial reflection is indicative of this;17 the meridional series of fibrillar collagen diffraction is more difficult to use in this regard, as it can disappear if the sample is not aligned perpendicular to the X-ray beam (Figure 2). The orientation of the collagen fibrils throughout the sections of parchment appears to be relatively uniform. Surface cleaning of parchment is routinely carried out in a conservation context. One novel method that has emerged is laser cleaning. The impact of laser cleaning on parchment structure has been investigated by a variety of methods.5,8,18 Microfocus X-ray diffraction analysis of laser-cleaned parchment can provide detailed information concerning the impact, if any, of laser cleaning on the molecular structure or organization of collagen at the parchment surface. It may be speculated that laser cleaning will bring about gelatinization of the collagen at the surface of the parchment by heating the collagen molecules to the point where they unravel. Gelatinization causes a loss of the 0.85 nm-1 peak as the regular lateral interactions between collagen molecules are lost.12 The laser cleaned samples did not display gelatinization of the collagen, as might have been expected. Rather, in these samples the intensity and position of the 0.85 nm-1 reflection remain consistent at the surface after laser cleaning in comparison to control samples, indicating that the collagen has retained its molecular and fibrillar architecture. In addition, this indicates that laser cleaning did not induce a reorientation of the collagen fibrils relative to the plane of the sample surface. At the surface of parchment that was deliberately damaged with the laser, the 0.85 nm-1 reflection was not present. It is likely that prolonged exposure to the laser induced gelatinization of the collagen at the surface, which would bring about a loss of intensity of the 0.85 nm-1 reflection relative to the 2 nm-1 reflection.12 Nano Lett., Vol. 4, No. 8, 2004
Mineral phases in parchment have been previously investigated using FT-IR,19 revealing calcium carbonates such as calcite on the surface of parchment. This could arise from the treatment of finished parchments with chalk. Edwards et al.,20 using FT-Raman spectroscopy, have also detected calcite and ascribed its presence to residues from the slaked lime bath used to prepare parchments. Of the samples tested here, only USH08 displays the trend of mineral at the surface of the parchment; the other samples that contain calcium carbonate phases show a distribution of mineral throughout the sample. The presence of mineral at the surface in sample USH08 may be explained by a finishing chalk treatment. The random distribution of mineral throughout other samples is, however, more indicative of a residual mineral phase from the slaked lime bath. The mineral may be embedded in areas of lower fiber density in the samples; mineral in calcifying collagenous tissue can deposit on the fibril surface as well as the gap and overlap regions of collagen fibrils.21 The presence of several polymorphs of calcium carbonate is an interesting feature of these samples, since previous experimental studies have concluded that only calcite crystals of calcium carbonate can form in association with collagen.22 The presence of lipid in a sample of parchment suggests that the process of converting skin to parchment does not remove the lipid fraction entirely. The lipid present in parchment, however, may be diagenetic in origin rather than persisting from the original skin (Ghioni et al., in preparation). It is interesting to note that the lipid reflections observed in the samples in this study appear in an equatorial position relative to the collagen long fibril axis. It can be inferred that the lipid in this case is packed laterally between the collagen fibrils with the plane of the phospholipid bilayer orientated parallel to the collagen long fibril axis. Within a sample, the d spacing of the lipid appears variable, between 4.4 and 4.8 nm; this may be an indication of the state of hydration of the lipid.23 The PCA analyses showed differences in trends between ink-covered and bare parchment material to a depth of approximately 90 µm. The 0.85 nm-1 reflection and meridional series that are characteristic of fibrillar collagen in parchment are absent in the diffraction profiles where ink is present, indicating that the ink has damaged the structure of the collagen and wrought a conformational change. The presence of lipid in the samples, however, appears unaffected by the presence of ink. Ink is a known cause of corrosion to parchment, as inks are often acidic;24 from the results presented here, it can be speculated that the mechanism of ink corrosion in parchments involves interaction with and disruption of the collagen structure that gives the parchment its strength and durability. In summary, X-ray microdiffraction has been demonstrated to be a technique capable of examining a number of nanotextural features in parchment on a micron scale. This technique was employed to examine the characteristics of a set of parchment samples in cross section. It is clear that parchment is not a uniform biomaterial throughout its thickness. Analysis of cross-sections of parchment by X-ray microdiffraction allows for the observation of variations in 1379
collagen structure and orientation, as well as the position and nature of minerals and lipids, the effect of cleaning techniques, and ink corrosion. The orientation of the collagen was examined, and for all samples was found to be relatively uniform, indicating that the collagen lies parallel to the plane of the parchment surface. The effect of laser cleaning on the parchment surface was also investigated. It was found that areas that had undergone laser cleaning showed no alteration to the structure or orientation of the collagen. When deliberately laser damaged, the collagen at the parchment surface appeared to have undergone a degree of gelatinization. The primary mineral phase in many samples was identified as calcium carbonate, in multiple morphological conformations; this is present to varying degrees and at several locations throughout the sections. The lipid observed within parchment appears to be packed between collagen fibrils, as the lipid diffraction appears equatorial relative to the collagen diffraction. The d spacing of lipid is variable within parchment samples, which may be an indication of fluctuations in the hydration state of lipid. Finally, the presence of ink on the surface of a sample was shown to disrupt the collagen structure essential for parchment durability. The collagen in inked areas of parchment appeared to have lost its characteristic structure and become gelatinized. The ink was found to penetrate the parchment to a depth of 90 µm. Acknowledgment. This research is funded by European Union Fifth Framework project EVK4-2001-00099, Improved Damage Assessment of Parchment (IDAP), and a grant from the National Archives for Scotland to C.J.K. This work was undertaken as part of a long-term beamtime proposal (2002-2004) at the ESRF awarded to T.J.W. References (1) Poole, J. B.; Reed R. Technol. Culture 1962, 3, 1-26 (2) Horie, C. V. Polym. Degrad. Stab. 1990, 29, 109-133. (3) Larsen, R., Ed.; Microanalysis of Parchment; Archetype Publications: London, 2002. (4) (a) Faccini, A.; Fancinelli, M. F.; Bairati, A.; Bottani, C. E.; Cavallotti, P. L.; Fessas, D.; Schiraldi, A.; Zerbi, G. Quinio 2001, 3, 51-70. (b) Fessas, D.; Schiraldi, A.; Tenni, R.; Vitellaro Zuccarello, L.; Bairati, A.; Facchini, A. Thermochim. Acta 2000, 348, 129-137. (5) Kennedy, C. J.; Vest, M.; Cooper, M.; Wess, T. J. Appl. Surf. Sci. 2004, 227, 151-163. (6) Kennedy, C. J.; Wess, T. J. Restaurator 2003, 24, 61-80.
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