Nanostructure of Biogenic Calcite and Its Modification under Annealing

Sep 10, 2014 - ABSTRACT: We apply advanced X-ray diffraction techniques at synchrotron microfocus beamlines in order to study the local ultrastructure...
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Nanostructure of Biogenic Calcite and Its Modification under Annealing: Study by High-Resolution X‑ray Diffraction and Nanoindentation Till H. Metzger,† Yael Politi,† Gerardina Carbone,‡ Bernd Bayerlein,† Igor Zlotnikov,† Emil Zolotoyabko,*,§ and Peter Fratzl† †

Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany European Synchrotron Radiation Facility, 38043 Grenoble Cedex, France § Department of Materials Science and Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel ‡

ABSTRACT: We apply advanced X-ray diffraction techniques at synchrotron microfocus beamlines in order to study the local ultrastructure of biogenic calcite with high spatial and angular resolution. Specifically, we investigate individual calcitic prisms extracted from Pinna nobilis mollusk shells with an aim to shed additional light on the structural aspects of organic/inorganic interfaces. We use annealing at elevated temperatures to destroy intracrystalline organics and measure the same prisms before and after annealing to achieve deeper understanding of the internal organization of these nanobiocomposites. Complementary nanoindentation measurements (also performed before and after annealing) allow us to elucidate the role of intracrystalline organics in increased hardness in pristine prisms and hardness reduction after annealing. We found that removal of intracrystalline organics during annealing facilitates generation of well-oriented lattice defects, which reduce the (006) diffraction intensity and are responsible for the [001]-elongated diffuse streaks nearby diffraction spots. These findings indicate the formation of internal material’s discontinuities with smooth and flat interfaces. Such nanodiscontinuities facilitate microcrack propagation under load that explains the reduced hardness of calcitic prisms after annealing.

1. INTRODUCTION The ultrastructure of biogenic (or biotic) crystals, i.e., crystals produced by organisms, has attracted growing attention of numerous research groups worldwide. One of key issues is uncovering the recipes used by nature for fabricating rather complicated composite architectures. The latter, in turn, allows controlling mechanical, optical, and other physical properties of natural biomaterials.1−3 It is widely accepted that for this purpose organisms use specific organic macromolecules, around which minerals are assembled, thus producing organic/ inorganic composite materials with superior characteristics.4,5 For example, in Pinna nobilis shell, which we investigate in the current paper, the outer prismatic layer consists of well-oriented calcite crystals, “glued” together by the interprismatic (intercrystalline) organic matrix. The latter mainly comprises sulfated polysaccharides and acidic proteins.6,7 Organic macromolecules are also located within the prisms (the intraprismatic or intracrystalline organics), although the manner in which they are intercalated is still poorly understood and is the subject of an ongoing discussion. The existence of organic macromolecules within individual crystallites of biogenic calcite and their potential effect on its mechanical characteristics have been discussed in refs 8−10, based on the measured widths of X-ray diffraction profiles in © 2014 American Chemical Society

sea urchin skeletal parts. Intracrystalline organic macromolecules in different species of biogenic calcite have been identified and further studied (see e.g., refs 6, 7, and 11−14). Later, it was found that intracrystalline organics is the source of substantial anisotropic lattice distortions and changes in atomic bond lengths in biogenic calcite.15,16 Under mild annealing at 200−250 °C, lattice distortions disappear due to the heatassisted defragmentation of the organic macromolecules and their partial removal from ceramic crystallites. The presence of intraprismatic organics, seemingly, causes subdivision of calcite crystals onto smaller blocks clearly visible by scanning electron microscopy (SEM) or atomic force microscopy (AFM), being applied to polished and etched samples. For example, in such a way crystalline formations with sizes in the 100 nm range have been visualized in P. nobilis6 and Atrina rigida.14 At the same time, high-resolution X-ray powder diffraction measurements of the widths of diffraction profiles, in both shells revealed larger sizes (up to 700 nm in maximum) of crystal blocks, which coherently scatter X-rays.17 This fact, together with earlier observations of sharp Bragg spots Received: July 16, 2014 Revised: August 27, 2014 Published: September 10, 2014 5275

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2. EXPERIMENTAL SECTION

produced by calcitic prisms in single-crystal X-ray diffraction,9,10,18,19 was the driving force for suggesting a certain topology of growing mineral with structural units being coherently connected along complicated trajectories defined by organic macromolecules.20 Upon annealing at about 250 °C, substantial additional broadening of X-ray diffraction peaks occurs, which was treated in terms of diminishing the crystallite coherence length down to 200−300 nm.17,21 Despite all these studies, the main questions, namely, how exactly these organic macromolecules are incorporated into growing mineral, what types of calcite planes they are attached, and how exactly the calcite structure is modified under annealing, remain not fully answered yet. Similar questions push forward intensive investigations of the nanostructure of aragonitic nacre (see, e.g., refs 22−24). Visualization of individual organic macromolecules within calcite crystallites in a nondestructive manner (with no etching) is, seemingly, beyond the capabilities of the existing characterization methods. Very recently, much larger objects, i.e., disklike organic inclusions (20−50 nm in size), have been directly visualized in individual calcitic prisms extracted from A. rigida shells25 and in nacre tablets extracted from Perna canaliculus shells.24 By using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) in the tomography mode, it was shown that organic inclusions in both species are mostly aligned with the (001)-plane, being nearly randomly distributed within the latter. This result fits our expectations to find acidic proteins attached to the charged calcite planes, such as the (001)-plane. Preferential alignment of organic macromolecules with the (001)-planes in calcite prisms, extracted from P. nobilis shells, was deduced from the threedimensional (3D) synchrotron small-angle X-ray scattering (SAXS) measurements.19 These observations are also in agreement with earlier experimental results, which showed that acidic proteins extracted from A. rigida11 and P. nobilis13 induce the formation of rather flat (001) faces in calcite. However, in scattering experiments,19 additional streak of diffuse scattering was observed, being concentrated toward the (104)-diffraction spot. This was unexpected, since “neutral” (104)-planes of calcite are not considered as containing appropriate binding sites for proteins with negatively charged residues. Nevertheless, similar SAXS results were reported for biomimetic synthetic calcite crystals grown in the presence (and incorporation) of the negatively charged polymer additives.26 In order to shed additional light on the nanostructure of biogenic calcite, in this work we carried out firsthand highresolution X-ray diffraction measurements by using micron- and submicron-sized X-ray beams at synchrotron facilities BESSY II (Helmholtz-Zentrum, Berlin, Germany) and ESRF (Grenoble, France). As before,19 we study calcite prisms extracted from P. nobilis shell, both pristine and annealed at elevated temperatures toward removal the intracrystalline organics. Therefore, we use annealing as a tool to destroy intracrystalline organics and, by monitoring the induced structural modifications, to achieve deeper understanding of the initial organization of these nanobiocomposites. Keeping experimental details for relevant section (see below), we stress that it is of uppermost importance to measure the same prisms in situ, i.e., before and after annealing. This is also valid for complementary nanoindentation measurements, which were performed after the Xray diffraction experiments.

Individual calcitic prisms were extracted from the outer calcitic layer of P. nobilis bivalve shells by sonication in sodium hypochlorite, as described in ref 7. The investigated prisms were typically between 30 and 80 μm in diameter and up to several millimeters long in the [001] growth direction. Prism microstructure is described in detail in refs 27 and 28. Thermal gravimetric analysis (TGA) performed with the aid of a Netzsch TG 209 F1 Iris instrument showed weight loss of about 0.25 wt % at an annealing temperature of 300 °C (marked by the solid arrow in Figure 1). This weight loss is due to the heat-assisted removal

Figure 1. Thermal gravimetric analysis (TGA) of calcitic prisms showing the weight loss in the temperature range close to the annealing temperature of 300 °C (solid arrow). Dashed arrow indicates the beginning of the calcite decomposition into CaO and CO2. of intracrystalline organics from the prisms. Massive weight loss, which starts at about 400 °C (dashed arrow in Figure 1) and becomes much more pronounced at higher temperatures, reflects decomposition of calcium carbonate into CaO and CO2 (see, e.g., ref 29). In order to investigate crystallite misorientation (mosaicity) with high spatial and angular resolution, we used the X-ray scanning microdiffraction set up (see Figure 2) at beamline ID01 of European Synchrotron Radiation Facility (ESRF, Grenoble). The sample is mounted with the vertically oriented [001]-axis on a x,y,z-piezo stage, allowing for translations of the sample with a stroke of 100 μm and an accuracy of 10 nm. In the diffraction instrument used, the piezo-drives in the z- and x-directions are named, respectively, as “piz” and “pix”, the names being kept throughout the paper. The piezo-stage is placed on a hexapod serving to accurately align the investigated sample with respect to the incident X-ray beam. The whole sample stage can be rotated around the vertical and horizontal axis with a precision of 0.001°. The incoming X-ray beam is focused by a Fresnel zone plate (FZP)30 to a size of 400 nm by 300 nm in the horizontal and vertical directions, respectively. The FZP made of gold has a diameter of 200 μm and an outermost zone width of 70 nm. The slit aperture in front of the FZP is used to define the divergence of the focused beam. In the case of a full illumination of the FZP (slit opening 200 × 200 μm2), the resulting divergence is about 0.1°, and the beam size becomes 300 × 400 nm2 in vertical and horizontal directions, respectively. For high angular resolution, the entrance slit is closed typically to 20 × 20 μm2, decreasing the divergence down to 0.008° but increasing the focus size to 1.2 × 1.2 μm2. In this case, the aperture slit is moved vertically by 50 μm to avoid the illumination of the central beam stop. An order sorting aperture (OSA) with a diameter of 50 μm is introduced in the focused beam to avoid direct beam contribution and to block higher diffraction orders. The measurements of the direct beam in the forward direction and the diffracted beam are performed in the far-field regime, using a Maxipix 2D pixel detector with 256 × 256 pixels, each being 55 × 55 μm2 in size.31 This photon counting detector produces zero readout noise and is installed at a distance of 1.1 m downstream 5276

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Figure 2. Nanodiffraction imaging set up at beamline ID01 of ESRF. For most of the measurements, the high resolution setting (an aperture of 20 × 20 μm2) has been utilized. For details, see the main text. from the focal plane (the sample position), the latter being located about 10 cm after the FZP (see Figure 2). A detailed characterization of the X-ray wavefront produced by a partially illuminated FZP is discussed elsewhere.32 During the measurements, the X-ray energy varied between 7 and 10 keV, and the focal distance of FZP has changed accordingly. The 2D detector is positioned at the detector arm, 2θ, either in the vertical plane for the (006) reflection or in the horizontal plane for the (110) reflection (see Figure 3). In most cases, the intensity recorded by the 2D detector was integrated over a region of interest (ROI) containing all Bragg scattered intensity. Visualization of the diffuse intensity streaks in the proximity to the diffraction spots was accomplished at the μ-Spot beamline of

synchrotron BESSY-II (Helmholtz-Zentrum, Berlin), using X-ray energy of 15 keV. The prisms were mounted on a goniometer, allowing positioning of the long axis of the prism roughly perpendicular to the goniometer’s rotation axis. Measurements were performed for the (006)- and (104)-reflections, by applying 0.2°rotation steps and acquisition times 600 s per frame. Since the streak intensity is many orders of magnitude weaker than the Bragg diffraction intensity, a beam stop was used to attenuate the latter. For measuring mechanical properties of pristine and annealed prisms, we carried out nanoindentation experiments. Sample for these measurements, 1 × 1 cm2 in size, was cut from the outer prismatic layer of P. nobilis. To attain a completely flat surface, the nonembedded sample was hand-polished perpendicular to the prism’s vertical axis, by using SiC polishing papers of different grades (1200, 2400, and 4000) and, finally, a 3 and 1 μm diamond paste. The nanoindentation studies were carried out at ambient conditions using a Triboindenter TI950 nanoindentation instrument (Hysitron, Inc.) with a Berkovich diamond tip. To obtain hardness and reduced modulus, all load−displacement curves were analyzed by the method described by Oliver and Pharr.33 All measurements were performed with a loading function comprising a 5 s loading segment up to a maximal load of 2000 μN, followed by a 30 s holding segment and finally a 5 s unloading segment. Mechanical characterization was performed in two steps. First, the entire sample was viewed by an optical microscope installed within the Triboindenter system, and eight randomly selected prisms were indented. Second, the sample was annealed in a furnace for 1 h at 300 °C. After cooling, it was mounted back into the Triboindenter system in the same orientation as in the first step, and the same eight prisms were characterized again. The results from at least 10 indents on each prism before and after annealing were averaged.

3. EXPERIMENTAL RESULTS 3.1. Prism Ultrastructure Revealed by High-Resolution Diffraction Mapping. In order to resolve the fine structure of individual crystallites within calcitic prisms, we first mapped the (006) diffraction intensity by using a setup which provided X-rays with a beam size of 1.2 × 1.2 μm2 and angular resolution of 0.008° (see section 2). One such map (in coordinates, piz-pix, with piz-axis being nearly parallel to the axial prism’s axis), taken at constant angular Bragg position of the investigated individual prism, is presented in Figure 4a. The intensity map (about 80 μm vertically and 40 μm

Figure 3. Scattering geometry, illustrating the rocking curve measurements in close proximity to two nodes (filled red circles) of reciprocal space, i.e., the (006)-node and (110)-node. Yellow rod represents the investigated calcite prism. 5277

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Figure 4. Spatially resolved diffraction intensity mapping along pixand piz-directions at a constant Bragg angle for the (006)-reflection. The sample size is about 40 μm in diameter. (a) Aperture is open to 20 × 20 μm2; the angular resolution is 0.008° and the beam size is 1.2 × 1.2 μm2; (b) aperture is open to 200 × 200 μm2; diffraction intensity is integrated over angular resolution of 0.1° and a beam size of 300 × 400 nm2.

Figure 5. (006) Rocking curve mapping, i.e., measuring the angular positions, θ, of the maximum intensity (colored) vs measurement position, pix, at constant height, piz. The collected maps show very different ultrastructures in the investigated calcitic prisms: (a) the prism with homogeneous crystalline orientation (in the limits of angular resolution used, i.e., of about 0.01°); (b) the prism, composed of several crystallites, which are mutually misoriented by a few tenths of degree. Yellow lines indicate the external borders of the prisms in the pix-direction.

horizontally) clearly shows substantial spatial inhomogeneity of crystalline blocks. Some of them are in the exact Bragg angular position and the scatter is rather strong (red color). These domains are a few microns in size in both directions. Other blocks are out of Bragg position (turned by more than 0.01°) and then scatter much more weakly (blue color). In order to emphasize the importance of high angular resolution, we show in Figure 4b another map taken from the same prism, using a 300 × 400 nm2 X-ray beam, but with a much lower angular resolution of about 0.1°. We see that in this map the above-mentioned fine structure is practically absent, indicating that the angular distribution of crystalline blocks (mosaicity) in this specific prism is well within 0.1°. This is in agreement with earlier results,9,10 in which some mosaicity in calcite crystals was resolved when using highresolution single-crystal synchrotron X-ray diffraction with an unfocused X-ray beam. With a focused X-ray beam and 0.008° angular resolution, we can further proceed toward obtaining quantitative information on local mosaicity spreads in our prisms. For this purpose, we are measuring the (006) rocking curves at each point (pix) at certain height (piz) and find the specimen angle, θ, at which maximum local diffraction intensity, Im, is achieved. We plot the Im magnitude, as a function of θ and pix-position, thus producing the mosaicity map along the pixdirection. Examples of such maps, taken from selected prisms with a 1 μm step size in the pix-direction, are shown in Figure 5. We see that individual prisms are very different, revealing mosaicity spreads between a few hundredths of degree (Figure 5a) and about one degree (Figure 5b). This finding emphasizes very clearly that the annealing effect on prisms’ ultrastructure should be studied in situ with the same specimen measured before and after annealing. Moreover, to ensure that diffraction measurements before and after annealing are performed at the same spatial positions of the specimen, its bottom end was determined by observing the vanishing of Bragg diffraction intensity in a piz-scan, and subsequently this position was used as a positional reference in further measurements. 3.2. Effect of Annealing on Crystal Structure and Mechanical Properties. With this knowledge, we carried out detailed rocking curve measurements aimed at obtaining quantitative information on the changes induced in calcite ultrastructure by annealing at elevated temperatures. For this purpose, a prism was put into glass capillary and placed at the rotation center of the multicircle diffractometer. After the rocking scan (diffraction intensity measurements under changing the entrance angle of the incident X-rays with respect to atomic planes) was performed at room temperature, the prism was placed in the focus of a halogen lamp and annealed at 300 °C for 1 h. The temperature was measured with a thermocouple positioned in the capillary about

10 μm above the specimen. Particular annealing temperature and duration were chosen on the basis of our TGA measurements (Figure 1) and earlier results for biogenic calcite,15,17 to ensure the removal (at least partially) of the intracrystalline organics from our prisms. After the specimen was cooled back to room temperature, the rocking curve was taken again from the same local region of the prism. These measurements were performed for two systems of atomic planes, (006) and (110), being perpendicular to each other (see Figure 3). The results of such rocking curve measurements for pristine and annealed prisms are shown in Figures 6 and 7 for (006)- and (110)reflections, respectively. Analyzing rocking curves taken at (006)-reflection (Figure 6), we can fit them well by three Gaussians. For both pristine and annealed specimens, the Gaussian centers are within 0.04°, whereas their full widths at half-maximum (fwhm, Γ1) are between 0.013° < Γ1 < 0.021°. It means that irradiated volume comprises three crystallites slightly inclined by Δθ ≤ 0.04° with respect to each other in the plane of specimen’s rotation. According to the measured fwhm (corrected by instrumental broadening, Γ0 = 0.008°), the thickness, L1, of these crystallites perpendicular to the vertical direction of the prism (in the plane of crystal rotation, see Figure 3) is L1 = (d006/(Γ12 −Γ02)1/2) ≥ 840 nm (where d006 is the (006)-lattice spacing) and stays almost unchanged after annealing. Rocking curves for the (110)-reflection can be fitted by two Gaussians, with their centers being separated by only Δθ ≈ 0.01° in the plane of specimen’s rotation. It implies the presence of two almost co-oriented crystallites within the irradiated volume. For both pristine and annealed specimens, the Gaussian fwhm’s are very close to instrumental broadening. Taking the largest value of Γ2 = 0.013° yields the thickness of these crystallites, perpendicular to the vector of reciprocal lattice, g = (110) (in the plane of this particular crystal rotation), to be L2 = (d110/(Γ22 −Γ02)1/2) ≥ 1400 nm (where d110 is the (110)-lattice spacing). Also for this reflection, the modifications of the discussed parameters are marginal under annealing. Concerning intensity changes, we can say that some intensity redistribution between individual peaks after annealing occurs (see Figures 6 and 7), clearly visible for the (110)-reflection. Apparently these modifications (not very substantial) reflect slight changes in the crystal orientations out of rotation plane. What really is changed, as a result of annealing, is the integrated diffraction intensity, I, i.e., total area under diffraction peaks, for the (006)-reflection. Taking into account somewhat different apertures used in these measurements (20 × 20 μm2 and 20 × 10 μm2 for pristine and annealed prism, respectively) and the obtained Gaussian fit parameters, we calculated 5278

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Figure 6. (006) rocking curves taken from one and the same prism before (a) and after (b) in situ annealing. The intensity distributions (filled squares) are well fitted by three Gaussians (red, green, and blue curves).

Figure 7. (110) rocking curves taken from one and the same prism before (a) and after (b) in situ annealing. The intensity distributions (filled squares) are well fitted by two Gaussians (red and green lines).

Figure 8. Diffuse intensity streaks along the [001]-direction, which appear after annealing in the proximity to the measured diffraction spots: (a) the (006)-reflection; (b) the (104)-reflection.

the ratio, Ia/Ip, between the integrated (006)-intensities for pristine, Ip, and annealed prisms, Ia, to be Ia/Ip ≈ 0.53. At the same time, this ratio for the (110)-reflection is close to 1; i.e., the integrated intensity remains almost unchanged after annealing. In addition to the intensities, angular positions, and widths of the Bragg reflections, we studied the diffuse scattering close to the selected diffraction spots at the μ-Spot beamline at BESSY II (see section 2). Under X-ray irradiation, we rotated the investigated individual prism (mounted horizontally) around the vertical goniometer axis until the desired diffraction spot has appeared, and then we followed its shape modification, slightly changing the diffraction angle in steps of 0.2°. In pristine prisms, the diffraction spots were round-shaped (not shown here) with no traces of anisotropic intensity distribution close to the Bragg position. In contrast, after annealing, pronounced diffuse streaks were observed in the proximity to (006) and (104) diffraction spots (Figure 8), in both cases streaks being elongated parallel to the vector of reciprocal lattice, g = (001). In fact, the appearance of the diffuse streaks close to all reflections is expected from the X-ray scattering theory, if crystal blocks, which coherently scatter X-rays, are truncated by extended flat surfaces.34 In the theory, a streak appears perpendicularly to the truncated plane and its intensity is decaying as ∼q−2 (where q = Q − 2πg), when moving away from the exact Bragg position, q = 0. This dependence was verified experimentally by us (not shown here). Finally, the reduced modulus and hardness of the prisms were measured before and after annealing. Note that natural biocomposites, comprising ceramic minerals and only few weight percent of organic substance, often reveal superior mechanical properties, especially those strongly affected by the mechanisms of plastic deformation, i.e., toughness and hardness (see, e.g., refs 35−38). In particular, a 50− 70% increase in hardness, as compared with geological calcite, has been reported39 for biogenic calcite extracted from the prismatic layer of the A. rigida shells, containing about 0.3 wt % of intracrystalline

organics.14 Similar results (about 50% increase in hardness) have been reported for Placuna placenta shell,40 which is composed of a layered assembly of elongated diamond-shaped calcite crystals, containing about 1 wt % of organics. According to ref 40, this gain in hardness is achieved because of 1 order of magnitude smaller volume of plastic deformation zone (where energy dissipation processes proceed), than in geological calcite. On the basis of these results and bearing in mind that annealing facilitates the removal of intracrystalline organics from biogenic calcite, we carried out nanoindentation measurements with our prisms, expecting to observe an opposite effect, i.e., some hardness reduction after annealing. Though the measurement details are given in section 2, we additionally stress the upmost importance of comparing the same individual prisms before and after annealing. Note also that the relative orientation of the Berkovich tip (possessing 3-fold symmetry) with respect to the crystallographic orientation of calcite prism may significantly influence the obtained results.39 Therefore, we have kept this mutual orientation even during measurements. The obtained results, showing rather modest changes upon annealing, are summarized in Figure 9. The hardness reduction is on average by 10%, while reduced modulus practically remained with no change.

4. DISCUSSION We begin this section by summarizing the most important experimental observations, which in our view are (a) small-angle inclinations of the c-axis in individual calcite crystallites about the prism’s growth direction; (b) in the investigated prism, the crystallite size in lateral directions, i.e., perpendicular to the c-axis (as estimated from the Bragg peak widths) remains practically unchanged after annealing; 5279

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linearly proportional to L(g). The same behavior is expected for the integrated intensity of the rocking curve, taken along a particular direction perpendicular to vector g = (hkl), since the width of such rocking curve does not depend on L(g). Therefore, when the coherent domain size L(g) is reduced by a factor 2 (keeping the same irradiation area), one expects equivalent reduction of the rocking curve intensity that is experimentally obtained for the (006)-reflection (observation (c)). The fact that the coherent domain size in the lateral directions, i.e., perpendicular to the c-axis, remains practically unchanged upon annealing (observation (b)) correlates well with almost constant intensity of the (110) rocking curve (observation (c)). These findings are also in general agreement with the results of previous diffraction studies of P. nobilis powders, showing a 3-fold reduction of coherent crystal size upon annealing in the (001)-direction and much smaller effects for the directions situated in the perpendicular plane.17 The obtained X-ray diffraction results can also be explained by using the “defect” language, i.e., in the framework of X-ray scattering by particular lattice defects generated during annealing. Such defects will reduce the diffraction intensity due to the static Debye−Waller factor, exp(−Ws).45 Parameter, Ws = CQ2UQ2, is determined by the dimensionless defect concentration, C (i.e., by the ratio of the number of the defected unit cells to total number of unit cells in the irradiated volume) and average defect-induced atomic displacements, UQ, along diffraction vector, Q = 2πg. We assume that organics removal from the (001) atomic planes will produce lattice displacements, U, mostly along the vector of reciprocal lattice, g = (001), and, if so, will strongly affect the (006) diffraction intensity, since vectors Q ≈ 2πg and U, in this case, are parallel. Assuming that proteins in P. nobilis form antiparallel folded βsheet structures, we can take for estimations, UQ = 0.47 nm, i.e., typical size of an individual β-strand (see, e.g., ref 46). In this case, we find that defect concentration, C = 0.006, created as a result of annealing, is enough for providing two times reduction of the (006) diffraction intensity. For the (110)-reflection (see Figure 3), this influence will be negligible because vectors Q and U are perpendicular to each other. In general, static deformation fields also cause broadenings of diffraction profiles, if the deviation vector, q = Q − 2πg, in the diffraction scan has nonzero projection on vector U.45 This is not the case for present rocking curve measurements with the (006) and (110) reflections since in both cases vector q is perpendicular to g = (001) (see Figure 3). Observation (d): The above-mentioned defects should be “visible” in the (006) theta/2theta scans, in which vector q is parallel to g = (hkl). In fact, substantial (triplicate) broadening of the (006) diffraction profiles after annealing was found in high-resolution powder diffraction measurements17 and was treated in terms of the reduced crystallite coherence length. Profile broadening in perpendicular directions was much less pronounced. For example, in the (100) and equivalent directions, (010) and (1̅10), in reciprocal space, additional broadening of diffraction profiles after annealing was only by 20%. Our rocking curve analysis confirms that lattice defects potentially arising as a result of annealing are most influential in the (001)-direction in reciprocal space. Imaging of the diffuse scattering nearby the Bragg diffraction spots that we describe below sheds additional light on this issue. Note that anisotropy of diffuse scattering in individual prisms of P. nobilis was studied before by SAXS, and a substantial redistribution of intensity after annealing toward the (001)-

Figure 9. Mechanical properties of eight individual prisms before and after annealing at 300 °C, as measured by nanoindentation; (a) reduced modulus and (b) hardness.

(c) the integrated intensity of the (006) Bragg peak decreases nearly two times upon annealing, while the intensity of the (110) Bragg peak, associated with one of the perpendicular directions in reciprocal space, remains practically unchanged; (d) the (001)-oriented flat internal interfaces are created by annealing, as is evidenced by the [001]-elongated diffuse streaks nearby Bragg diffraction spots; (e) reduced modulus does not significantly change upon annealing, while the prism hardness is reduced by about 10%. Observation (a) reflects the prism microstructure, which consists of calcite sublayers with a typical thickness in the micron range stacked along the growth axis.14,27 In fact, the possibility of slight misalignment between successive sublayers in the stack has been previously mentioned.27 Our findings indicate that this misalignment occurs within individual calcitic prism on a submicron scale as well. Note that much larger misalignments (tens of degrees) between individual prisms are also well documented (see, e.g., refs 41−44). Observations (b) and (c) are most likely related to the fact that large amounts of intracrystalline organics, initially concentrated within the (001) atomic planes,19,25 are removed under annealing. While the associated change in the effective crystallite size, L, is minimal in the directions perpendicular to the prism’s axis (observation (b)), it may be very significant in the (001)-direction. Indeed, in the kinematic diffraction theory, the peak intensity, I, of reflection g = (hkl) is proportional to L2(g) (where L(g) is the size of crystal block along vector g, participating in the coherent X-ray scattering) and to the number, T/L(g), of coherent domains which add up incoherently (T being the sample’s size along g irradiated by X-ray beam). Hence I ∝ L2(g)·(T/L(g)) = TL(g), i.e., is 5280

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node of reciprocal lattice has been reported.19 This result was explained at the expense of the massive removal of intracrystalline organics from (001)-planes and respective enhancement of SAXS contrast, being sensitive to local changes of electron density. Besides that the azimuthally integrated SAXS intensity, I, revealed regular decaying behavior, I ≈ q−v, with increasing the q-magnitude, but with dissimilar power indices, v, before and after annealing. These measurements, carried out at different annealing temperatures, showed the presence of some kind of “phase transition” at about 200 °C, during which the initially rough organic/inorganic interfaces become smoother.19 However, uncovering the details of this transformation remained beyond the abilities of the SAXS technique being sensitive to the electron density variation. The latter is close to zero, e.g., for ordered/disordered calcitic interfaces. In this context, observing diffuse streaks in the coherent wide-angle X-ray scattering (WAXS) is of uppermost importance since the WAXS diffraction technique, in contrast to SAXS, indeed is sensitive to crystalline/noncrystalline interfaces. Specifically, observation of diffuse streaks along the [001]-crystallographic direction allows us to conclude that internal crystallite (CaCO3) surfaces of the (001)-type extend and become flat, as a result of annealing, rather than remaining rough. This process takes place in tight connection with the removal of organic substance and, as we think, as the result of it. In fact, smoothing internal interfaces implies considerable mass transfer of CaCO3. Bulk diffusion of Ca (the slowest and most important diffusant) in calcite is negligible at an annealing temperature of 300 °C, providing diffusion lengths of about 0.0001 nm.47 Grain boundary diffusion is certainly faster; however, the available data have been received for much higher temperatures, when microcrack formation can strongly influence the obtained results.48 In any case, in order to achieve reasonable diffusion lengths of about 1 nm (the average size of calcite unit cell), grain boundary diffusion at 300 °C should be 8 orders of magnitude faster than bulk diffusion that looks not very realistic. Most probably, mass transfer proceeds during annealing by a joint removal from interfaces some CaCO3 molecules attached to the organic macromolecules, together with releasing the latter. Anyway, during annealing the (001)-oriented organic/inorganic interfaces become empty of organics, extended, and flat and, namely, this process is behind the observed intensity reduction of the (006) rocking curves. Lateral merging of arising material’s discontinuities produces smaller crystalline grains, participating in the coherent X-ray scattering, and consequently causes additional broadening of the (006) diffraction profiles found in ref 17. Possible impact of the described interface transformation process on mechanical characteristics in biogenic calcite is discussed below. Observation (e): Nanoindentation measurements provide us with both reduced modulus and hardness,33 whose magnitudes for eight pristine and annealed prisms are summarized in Figure 9. Concerning reduced moduli, Er (see Figure 9a), we did not find a clear-cut annealing effect on these data. After annealing, the values of Er in individual prisms are a little lower or higher than before annealing, but these fluctuations are mostly within experimental error bars. In principle, such result is expected because of the little amount (about 0.2 wt %) of intracrystalline organics in our prisms. The average value of Er = 65.3 GPa is somewhat lower than that reported in ref 39, obtained in A. rigida for the [001] load direction: 74.9 or 70.1 GPa, depending on azimuthal angle between the prism and the tip. Perhaps, the

differences between elastic moduli of P. nobilis and A. rigida are due to chitin fibers, which are present only in the latter shell.14 Besides that slight deviation of loading direction from the [001]-crystallographic direction can introduce shear deformations, which will cause the lowering of the measured reduced modulus values. Considering our hardness results (see Figure 9b), we notice systematic hardness reduction, as a result of annealing, which fits our expectations. In fact, the average hardness before annealing, Hb = 3.61 GPa, agrees well with previous results for Placuna placenta (3.5 GPa40) and A. rigida (3.5−4.2 GPa39). After annealing, the average hardness drops down to Ha = 3.28 GPa. This value is still higher than in geological calcite (2.3 GPa according to ref 40 and 2.3−2.5 GPa according to ref 39). We can speculate that after annealing, some amount of organic macromolecules in our prisms remains to be attached to atomic planes, other than (001). Anyway, the reduction of hardness by factor, ((Ha− Hb)/Hb) ≈ 10%, due to the removal of less than 0.25 wt % of intracrystalline organics, is very impressive. We think that the annealing-mediated smoothing and flattening of the (001)-interfaces, free of organics, as discovered by X-ray diffraction imaging, plays an important role in this process. Extended flat interfaces facilitate microcrack propagation under nanoindentation load, which results in the expanding of the plastic deformation zone and, consequently, the observed decreasing in the magnitude of hardness. To strengthen this point, we quote nanoindentation results of Xi and Li49 who observed substantial reduction of elastic modules and hardness in nacre layer heat-treated at higher temperatures of 500 and 1000 °C. The authors concluded that this reduction is due to burning out organic substance and subsequent nanohole formation at organic/inorganic interfaces.

5. CONCLUSIONS By using a micron-sized focused X-ray beam and a setup providing a 0.008° angular resolution, we mapped the ultrastructure of individual calcitic prisms extracted from P. nobilis mollusk shells. On this spatial and angular scale, the ultrastructure looks strongly nonhomogeneous, comprising regions (crystalline blocks, a few microns in size), which are slightly misoriented with respect to each other. The measured misorientation angles in different prisms are spread between a few hundredths of a degree and about one degree. Rocking curve analysis, performed in an individual prism before and after annealing, has revealed only subtle changes in mutual misorientation of crystallite blocks, while substantial reduction (by a factor of 2) occurred in the integrated intensity of the (006) rocking curves. We link the latter result to massive removal of intracrystalline organics from the (001) calcite planes during annealing and subsequent appearance of related lattice defects with displacement vectors mostly parallel to the vector of reciprocal lattice, g = (001). This conclusion is strongly supported by the measurements of the intensity distributions nearby the Bragg diffraction spots, revealing diffuse scattering streaks elongated in the [001]-direction. Streak appearance, as a result of annealing, indicates smoothing, flattening, and extension of the (001)-interfaces which become empty of organics. Such nanodiscontinuities facilitate microcrack propagation under load, which is revealed in the reduced hardness of calcitic prisms after annealing. 5281

dx.doi.org/10.1021/cg501068e | Cryst. Growth Des. 2014, 14, 5275−5282

Crystal Growth & Design

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Article

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AUTHOR INFORMATION

Corresponding Author

*Tel: 972-4-8294545. Fax: 972-4-8295677. Website: http:// materials.technion.ac.il/emil-zolotoyabko-microstructurestructure/. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are greatly indebted to F. Marin for supplying calcitic prisms for this research, Ch. Gilow for his help in diffraction measurements at BESSY II, V. Chamard for participation at the initial stage of this project, and I. Shekova for her help with TGA measurements. We thank the German Research Foundation (DFG) for financial support (FR 2190/4-1 Gottfried Wilhelm Leibniz Prize 2010). E.Z. also thanks the Shore Fund in Advanced Composites (Technion) for partial financial support.

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