An Optical Microscopy and Microfocus X-ray Diffraction Study

Two-dimensional spatially resolved microfocus X-ray diffraction has been used to investigate spherulites of pure bacterial poly(3-hydroxybutyrate) (PH...
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Biomacromolecules 2000, 1, 604-608

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Bacterial Poly(3-hydroxybutyrate): An Optical Microscopy and Microfocus X-ray Diffraction Study Massimo Gazzano,† Maria Letizia Focarete,† Christian Riekel,‡ and Mariastella Scandola*,† Department of Chemistry “G. Ciamician” and Centro di Studio per la Fisica delle Macromolecole del CNR, University of Bologna, via Selmi 2, 40126 Bologna, Italy; and European Synchrotron Radiation Facility, B.P. 220, F-38043 Grenoble Cedex, France Received May 5, 2000; Revised Manuscript Received September 4, 2000

Two-dimensional spatially resolved microfocus X-ray diffraction has been used to investigate spherulites of pure bacterial poly(3-hydroxybutyrate) (PHB) and of a blend of natural and synthetic atactic PHB (aPHB) crystallized at a relatively high temperature (Tc ) 140 °C). Both samples investigated contained practically two-dimensional spherulites, characterized by wide extinction bands (band spacing > 80 µm). The X-ray diffraction patterns confirmed that the unit cell a-axis is oriented along the spherulite radius in PHB and that the same is true for the a-PHB containing blend. Comparison of the matrix of diffraction patterns with the polarized optical micrograph of the scanned sample area indicated a very clear correlation between pattern changes and banding, yielding a straightforward picture of the structural variations within the spherulite. Introduction Poly(3-hydroxybutyrate), PHB, is a natural polyester produced by a number of microorganisms as an endogenous carbon and energy source.1,2 PHB biodegrades in a broad range of natural environments, and during the past decade, it has attracted much attention in connection with plastic waste management issues. Upon solidification from the melt, PHB easily crystallizes. Owing to its biosynthetic origin, it is free from any residual contaminating catalyst, and spherulites of very large dimensions are formed during crystallization from the melt, the latter aspect being related to a particularly low nucleation density.3,4 PHB spherulites show typical concentric alternating extinction bands when viewed between the crossed polars of an optical microscope. Band spacing increases with crystallization temperature, and under appropriate crystallization conditions PHB spherulites with band spacings on the order of 100 µm have been reported.5,6 Earlier studies have shown that natural PHB7,8 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV9 form miscible blends with a synthetic PHB obtained from the polymerization of racemic β-butyrolactone, atactic PHB (aPHB). In blends containing up to 50% a-PHB, space-filling spherulites were found to grow from the melt at a constant rate at all crystallization temperatures investigated.7-9 It was concluded that upon crystallization of the bacterial polyester the second blend component remained trapped within the spherulites (interlamellarly or between bundles of lamellae). In the PHBV/a-PHB system,10 over a broad range of crystallization temperatures (Tc) the spherulites showed clear * Corresponding author. Telephone: +39+051+2099577. Fax: +39+ 051+2099456. E-mail: [email protected]. † University of Bologna. ‡ European Synchrotron Radiation Facility.

extinction bands, whose spacing increased with Tc and decreased with a-PHB content. Similar results were obtained in miscible blends of natural PHB and other amorphous polymers, i.e., cellulose esters11 or polyepichlorohydrin.12 The present paper reports an investigation of twisting crystal orientation in spherulites of pure bacterial PHB and of a miscible blend of natural PHB with 10% atactic PHB. The study was carried out by means of two-dimensional spatially resolved microfocus X-ray diffraction, using a 3 µm diameter beam. All samples under investigation were characterized by wide extinction bands. The particular experimental setting adopted in this study allowed collection of a large number of diffraction patterns, while bidimensionally step-scanning the sample. Comparison of the matrix of collected images with the polarized optical micrograph of the scanned sample area, allowed immediate visual correlation between the occurrence of spherulite banding and changes of the X-ray diffraction pattern. Experimental Section Sample Preparation. Bacterial poly(3-hydroxybutyrate), PHB, was an ICI product (GO8, Mn ) 5.39 × 105, Mw/Mn ) 4.11) provided in powder form. A PHB film was obtained by compression molding the as-received powder between Teflon plates with an appropriate spacer, at 195 °C for 1 min under a pressure of 2 ton/m2 (Carver C12 laboratory press). Atactic PHB (a-PHB; Mn ) 31 000; Mw/Mn ) 1.1), synthesized as described previously,13,14 was shown to be atactic and amorphous by NMR spectroscopy15 and DSC analysis, respectively. A PHB/a-PHB blend (90/10 weight ratio) was obtained by dissolving weighed amounts of the two polymers in dichloromethane, followed by solution

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Figure 1. (a) Polarized optical micrograph of the PHB spherulite investigated by microfocus X-ray diffraction (marked area: 300 × 300 µm). (b) X-ray diffraction patterns recorded from the square area highlighted in part a. The arrows in selected frames indicate the a-axis direction.

casting, solvent evaporation, and vacuum-drying of the obtained film overnight. Samples for X-ray investigation were prepared as follows: a small film fragment, inserted between two microscope cover glasses, was placed in the hot stage (Linkham TH 600) of a polarizing optical microscope (Zeiss Axioscop), heated at 20 °C/min to 195 °C and held at this temperature for 1 min. The melt was squeezed into a film through a gentle pressure applied to the upper glass. The isotherm was then followed by rapid quenching to 140 °C (cooling rate > 250 °C/min), and the sample was allowed to crystallize isothermally (Tc ) 140 °C). Spherulite dimensions and band spacing were measured, after calibration with a micrometric reticule, using the video cameraLinkham VTO232 interface attached to the optical microscope. Microfocus X-ray Diffraction Experiments. The X-ray diffraction patterns were recorded using the scanning diffractometry setup of the microfocus beamline ID13 of the European Synchrotron Radiation Facility (Grenoble, France). The beam with a wavelength of 0.782 Å was monochromatized by a Si(111) monochromator and focused to 3 µm (fullwidth at half-maximum) by a glass capillary. The sample was scanned through the beam by a computer-controlled x/y gantry. The isothermally crystallized films (∼20-25 µm thick) were recovered from the microscope cover glass by means of a scalpel, cut in a circular portion of about 3 mm in

diameter and mounted on a goniometer head. The surface of the samples was set perpendicular to the X-ray beam and therefore parallel to the detector. In the case of pure PHB a square area of 300 × 300 µm was scanned, tracking the sample in two dimensions perpendicular to the X-ray beam. Then, 121 diffraction photographs were collected as the specimen was scanned in horizontal and vertical steps of 30 µm. For the blend sample a square area of 150 × 150 µm was scanned with a spatial resolution of 10 µm. Then 256 diffraction photographs were collected while moving the sample along the horizontal and vertical axes. The diffraction patterns were recorded with a MarrCCD detector with exposure times of 10 s. Features of the detector: 2048 × 2048 pixels; 64.45 µm × 64.45 µm pixel size; 16 bit readout. The graphical display of single and multiple patterns and the calculations of the angular spread of the reflections along the Debye ring was performed with the FIT2D software package.16 Results Figure 1a shows the optical micrograph of the PHB spherulite that was investigated by spatially resolved X-ray diffraction. The evident double-ringed concentric extinction bands indicate that PHB spherulites grown at 140 °C have

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Figure 2. (a) Polarized optical micrograph of a blend of bacterial PHB with 10% a-PHB before investigation by microfocus X-ray diffraction (marked area: 150 × 150 µm) (see text). (b) Optical micrograph of the same spherulite after X-ray data collection where the black spots caused by beam damage are clearly visible. (c) X-ray diffraction patterns recorded from the square area highlighted in part a. The arrows in selected frames indicate the a-axis direction.

a biaxial optical character,17 as expected for a polymer crystallizing with an orthorhombic structure.18 Another typical feature of high-temperature crystallized PHB are the circumferential cracks,19 that can be easily observed in the spherulite shown in Figure 1a. Figure 1b shows the X-ray diffraction photographs collected from the square area highlighted in Figure 1a. Comparison of the X-ray results with the optical microscope picture clearly shows that the X-ray diffraction pattern changes while investigating different spherulite portions, i.e., when moving from one band to the next. At least two kinds of different patterns that lie along concentric arcs can be easily identified in Figure 1b, rather closely corresponding to the different extinction bands of Figure 1a. Figure 2 shows (a) the polarized optical micrograph of a PHB sample containing 10% of atactic PHB (a-PHB), (b) a picture of the same sample taken after X-ray data collection (clearly showing the effects of beam damage on the sample surface), and (c) the matrix of X-ray diffraction patterns obtained. As already pointed out for plain PHB, also in the blend the distribution of patterns in the diffraction-array (Figure 2c) shows a correspondence with the extinction bands in the optical photograph (Figure 2a). The arrow drawn in Figure 2a indicates the presence of a slight irregularity (a sort of “bump”) in the banding pattern. The same irregularity is also appreciable in the matrix of the X-ray diffraction

Figure 3. Magnification of two X-ray diffraction patterns: (a) bacterial PHB, from frame G1 of Figure 1b; (b) PHB/a-PHB blend, from frame F7 of Figure 2b.

images of Figure 2c. This observation shows the potentiality of the microfocus technique to evidence morphological details associated with structural modifications. Two frames taken from Figures 1b (pure PHB) and 2c (blend with a-PHB) are magnified in Figure 3. In Figure 3a (PHB) the spots are clearly arranged along layer lines, reminiscent of the typology of the X-ray patterns obtained in flat-plate oscillation photographs of single crystals. In the blend sample (Figure 3b), all reflections tend to spread along the Debye rings and location of the reflection layers is somewhat more difficult. The half width of the reflection corresponding to d ) 4.41 Å (indicated by an arrow in the two patterns of Figure 3), increases from 12° (PHB) to 23°

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(PHB/a-PHB blend). The angular distribution of this reflection yields an estimate of the parallelism (Π), that is a practical parameter indicating the degree of orientation.20 The calculated Π value decreases from 93 ( 1% to 87 ( 1% on going from pure PHB to the blend. Discussion The samples examined were crystallized from the melt under the spatial constraint of two parallel surfaces, which allowed an essentially bidimensional growth of the spherulite after nucleation. Spherulites with radius >500 µm and thickness 20-25 µm were obtained, which represent true thin sections through the spherulite center. All frames of Figures 1b and 2c were taken on still samples; nevertheless, they show oscillation-type patterns that originate from a slight misorientation of the unit cells with respect to one of the crystal axes. In each frame, the direction perpendicular to the layer lines is the axis around which the crystal oscillates. This is the a-axis, as can be directly calculated from the measured distance between the layer lines. In Figures 1b and 2c, the orientation of the layer lines within the X-ray matrix follows the circular path of the bands, indicating that the a-axis lies parallel to the film surface and is radially oriented (see arrows in selected frames of Figures 1b and 2c). This result agrees with earlier data on plain bacterial PHB5,21,22 and shows that the same is true also for a blend containing 10% of a miscible amorphous polymer. In Figures 1b and 2c there is an evident periodic variation of the reflection intensities while crossing the bands along a radius whereas the intensity variation is weak within each spherulite band. In the bidimensional array of Figure 1b, the radial distance between similar patterns (frames K4 and K9, for example) is about five frames, while after a radial interval of two to three frames the change of reflection intensities is maximum. Since the scanning step used was 30 µm it can be stated that the distance between regions of equal structural organization is 150 µm ((30 µm). When the same region of the spherulite is observed between crossed polars in the optical microscope (Figure 1a), the measured band spacing (125 µm) is in good agreement with the period resulting from the above calculation. This result shows that bidimensional data collection using microfocus X-ray diffraction provides unique evidence of the structural changes associated with the lamellar organization within banded spherulites. Analogously, when the blend with a-PHB is considered (Figure 2c), it is seen that similar patterns are eight frames apart (see, for example, frames D6 and L6). Since in this case the sample was scanned in 10 µm steps, the distance between regions with the same structure is 80 µm ((10 µm). Again this value matches the band spacing obtained from the optical measurements (80 µm, Figure 2a). The observed decrease of band spacing (from 125-150 µm in plain PHB to 80 µm in the blend) is in line with earlier evidence11,12,23 that in polyesters and polyamides spherulite banding narrows in the presence of increasing amounts of miscible polymeric diluents. Previous work by the Authors11 showed that in miscible blends of bacterial PHB with two different cellulose esters (CE) the bandwidth reduction was

rather independent of the type of the CE used in the blend. The present result that 10% a-PHB blended with the natural polymer leads to a 40-45% decrement of the band spacing well agrees with earlier results.11 Besides inducing a decrease of band spacing, the presence of a-PHB also broadens the X-ray reflections along Debye rings (Figure 3). This observationsquantified by the values of the parallelism (Π)sis attributed to the fact that during crystallization of bacterial PHB, the atactic component is rejected from the growing front of the lamellae and remains trapped in the interlamellar space. As a consequence, the overall PHB crystallization process is disturbed, the radial growth rate decreases (data not shown) and crystals somewhat less regularly oriented than in the pure polymer are formed (see Π values). The excellent agreement between spatially resolved microdiffraction results and optical microscope observations shows that the X-ray technique used in this work is particularly fit for the study of banded two-dimensionally structured objects. The matrix of X-ray frames offers an overall picture of the structural changes within the object, that can be straightforwardly correlated with the morphological features highlighted by polarized optical microscopy. Support to this conclusion is given by a spatially resolved microfocus X-ray investigation of the portion of the spherulite in Figure 1a that lies below the marked square area, where no regular banding is observed. The matrix of X-ray patterns obtained24 does not show any periodicity, indicating a random positioning of the unit cell b- and c-axes, while the a-axis remains roughly oriented in the radial direction. Acknowledgment. The authors wish to thank ESRF for beam time allocation, and Dr. Marek Kowalczuk for kindly supplying the atactic PHB used in the blend. This work was partially supported by the Italian Ministry for University and Research (MURST). Supporting Information Available. An additional figure showing the results of spatially resolved microfocus X-ray investigation of a PHB spherulite portion where no regular banding is observed: (a) polarized optical micrograph of the PHB spherulite investigated by microfocus X-ray diffraction (marked area: 300 × 300 µm); (b) X-ray diffraction patterns recorded from the square area highlighted in part (a) of the figure. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. Doi, Y. Microbial Polyesters; VCH Publishers: New York, 1990. Barham, P. J. J. Mater. Sci. 1984, 19, 3826. de Koning, G. J. M.; Lemstra, P. J. Polymer 1992, 33, 3292. Barham, P. J.; Keller, A.; Otun, E. L.; Holmes, P. A. J. Mater. Sci. 1984, 19, 2781. Scandola, M.; Ceccorulli, G.; Pizzoli, M.; Gazzano, M. Macromolecules 1992, 25, 1405. Abe, H.; Doi, Y.; Satkowski, M. M.; Noda, I. Macromolecules 1994, 27, 50. Pearce, R.; Brown, G. R.; Marchessault, R. H. Polymer 1994, 35, 3984. Scandola, M.; Focarete, M. L.; Adamus, G.; Sikorska, W.; Baranowska, I.; Swierczek, S.; Gnatowski, M.; Kowalczuk, M.; Jedlinski, Z. Macromolecules 1997, 30, 2568. Unpublished results.

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(11) Pizzoli, M.; Scandola, M.; Ceccorulli, G. Macromolecules 1994, 27, 4755. (12) Finelli, L.; Sarti, B.; Scandola, M. J. Macromol. Sci.sPure Appl. Chem. 1997, A34, 13. (13) Kurcok, P.; Kowalczuk, M.; Hennek, K.; Jedlinski, Z. Macromolecules 1992, 25, 2017. (14) Jedlinski, Z.; Kurcok, P.; Lenz, R. W. J. Macromol. Sci.sPure Appl. Chem. 1995, A32, 797. (15) Jedlinski, Z.; Kowalczuk, M.; Kurcok, P.; Adamus, G.; Matuszowicz, A.; Sikorska, W.; Gross, R.; Xu, J.; Lenz, R. W. Macromolecules 1996, 29, 3773. (16) Hammersley, A. FIT2D website: http://www.esrf.fr/computing/expg/ subgroups/data_analysis/FIT2D/index.html. (17) Keith, H. D.; Padden, F. J. J. Polym. Sci. 1959, 39, 101. (18) Saracovan, I.; Keith, H. D.; Manley, R. St. J.; Brown, G. R. Macromolecules 1999, 32, 8918.

Gazzano et al. (19) Barham, P. J.; Keller, A. J. Polym. Sci., Polym. Phys. Ed. 1986, 24, 69. (20) Kakudo, M.; Kasai, N. X-ray Diffraction by Polymers; Kodansha, Tokyo, 1972; Chapter 10. (21) Gazzano, M.; Tomasi, G.; Scandola, M. Macromol. Chem. Phys. 1997, 198, 71. (22) Mahendrasingam, A.; Martin, C.; Fuller, W.; Blundell, D. J.; MacKerron, D.; Rule, R. J.; Oldman, R. J.; Liggat, J.; Riekel, C.; Engstro¨m, P. J. Synchrotron Rad. 1995, 2, 308. (23) Keith, H. D.; Padden, F. J., Jr.; Russell, T. P. Macromolecules 1989, 22, 666. (24) Available as Supporting Information.

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