Formation of Poly (3-hydroxybutyrate)(PHB) Inclusion Compound with

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Formation of Poly(3-hydroxybutyrate) (PHB) Inclusion Compound with Urea and Unusual Crystallization Behavior of Coalesced PHB Pavithran Ravindran and Nadarajah Vasanthan* Department of Chemistry, Long Island University, One University Plaza, Brooklyn, New York 11201, United States ABSTRACT: PHB-U-IC has been prepared for the first time by the cocrystallization method. The structure and conformation of poly(3hydroxybutyrate) (PHB) chains encapsulated in urea (U) channels formed in the PHB-U inclusion complex (PHB-U-IC) were studied by DSC, FTIR, and solid-state NMR spectroscopy. The XRD pattern and FTIR spectroscopy demonstrated that PHB-U-IC takes a different crystal structure than the well-reported hexagonal and trigonal crystal structures of polymer urea inclusion compounds. PHB-U-IC takes an expanded tetragonal crystal structure. The conformation of PHB in the confined environment has been characterized by solid-state NMR spectroscopy and shown that PHB adopts a conformation similar to bulk PHB. The crystallization and thermal properties of as-received PHB and PHB coalesced from its U-IC were compared and demonstrated that the coalesced PHB crystallizes much slower than as-received PHB. Crystallinity of nonisothermally melt crystallized as-received PHB was found to be significantly higher than the crystallinity of nonisothermally melt crystallized coalesced PHB, indicating that coalesced PHB is not easily converted into semicrystalline PHB.

1. INTRODUCTION Urea inclusion compounds (ICs) with small molecule guests have been made and characterized extensively, and they exhibit specific physical and chemical properties.1−9 These U-ICs typically adopt a hexagonal crystal structure with linear tunnels containing disordered guest molecules. Urea is also able to form inclusion compounds with either hydrophilic or hydrophobic polymers depending on the cross section of the polymer chain.10−22 Three different crystal forms were reported for polymer urea inclusion compounds, and they are hexagonal,10,11,14,18 trigonal,12,13 and expanded tetragonal.13,15,22 It has been shown that there is no covalent bonding between the guest polymers and host urea, and the attraction is generally due to van der Waals forces and/or hydrogen bonding. Polymers occupying U-IC channels are extended and often adopt all-trans conformations. It has been suggested that polymer inclusion compounds can serve as model systems to study isolated chains in a confined environment. Polymer urea inclusion compounds were characterized extensively using various physical techniques, such as X-ray diffraction, FTIR spectroscopy, DSC, and solid-state NMR spectroscopy. These studies provided structural information, guest ordering, and dynamics of isolated polymer chains in the I-IC channels.10−22 Biodegradable polymers that can be produced from renewable resources by bacterial fermentation are of great interest as a new class of thermoplastics. Poly(3-hydroxybutyrate) (PHB), a member of this important class of poly(3-hydroxyalkonates), is a biopolymer that is gaining considerable interest due to its biodegradability and biocompatibility.23−30 The structure of PHB is displayed in Scheme 1. PHB is an optically active polymer that has an isotactic sequence. PHB is synthesized and © 2015 American Chemical Society

Scheme 1

accumulated by bacteria as an energy reservoir. The electron diffraction studies of PHB single crystals have been carried out, and it was found that a PHB single crystal has crystallographic “a”-axis, forming a lath-shaped crystal with dimensions of around 0.3−2 and 5−10 μm along the short and long axes, respectively.31 The thickness of PHB single crystals is around 4−10 μm, which depends on molecular weight, crystallization temperature, and solvent used. Yokouchi et al.31 have shown that PHB crystallizes in an orthorhombic crystal structure P212121 unit cell with lattice parameters a = 0.576 nm, b = 1.320 nm, and c = 0.596 nm with the chain in the left-handed 2/1 helix. It has been shown that PHB adopts a ttgg conformation. In this paper, we report for the first time formation of the PHB-U-IC by cocrystallization from solution. The PHB-U-IC was characterized using XRD, DSC, FTIR, and solid-state NMR spectroscopy. PHB crystals were coalesced by washing the PHB-U-IC sample with warm methanol, and nonisothermal Received: February 24, 2015 Revised: April 20, 2015 Published: April 30, 2015 3080

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Figure 1. DSC heating scan of (a) urea, (b) PHB, and (c) PHB-U-IC.

Figure 2. Wide-angle X-ray diffraction pattern of (a) urea, (b) PHB, and (c) PHB-U-IC. calibrated using an indium and zinc standards. Samples with mass about 2.0−5.0 mg were sealed in an aluminum pan and heated or cooled at a rate of 10 °C/min under a nitrogen atmosphere. The onset temperatures were taken as melting and crystallization temperatures. FTIR Spectroscopy. FTIR analysis was performed on a Nicolet 760 Magna spectrometer, equipped with a KBr beam splitter and DTGS detector at wavenumbers ranging from 4000 to 500 cm−1. The FTIR spectra were collected using 64 scans with a resolution of 2 cm−1. X-ray Powder Diffraction. Wide-angle X-ray diffraction of powder samples were obtained using a SCINTAG XGEN-400 at room temperature. Cu Kα radiation was used at 40 kV and 40 mA. The diffracting intensities were recorded in 0.02° 2θ steps over the range of 5° < 2θ < 80°. Solid-State NMR Experiment. High-resolution solid-state 13C NMR experiments were performed on a Varian Unity-Plus-200 spectrometer at Process NMR Associates operating at a 13C resonance frequency of 50.2 MHz. The cross-polarization (CP) and magic angle spinning (MAS) experiments were performed utilizing the variable amplitude cross-polarization pulse sequence in order to reduce the effects of spin modulation on the quantitative nature of the

melt crystallization behaviors of as-received and coalesced PHBs were compared.

2. EXPERIMENTAL SECTION Materials. Poly(3-hydroxbutyrate) (PHB) samples with weightaverage molecular weight 100 000 were purchased from Sigma-Aldrich Chemical Co. Urea was obtained from Fisher Scientific, and ACS grade methanol and dichloromethane were also obtained from Fisher Scientific. PHB-U-IC Preparation. Three grams of urea and 0.5 g of PHB were dissolved in 50.0 mL of methanol and 50.0 mL of dichloromethane, respectively, in two separate beakers by heating and stirring until complete dissolution. The PHB solution was slowly added into the urea solution while the solution was continuously heated and stirred. The resulting solution was later cooled to room temperature, and a white precipitate formed over a couple of hours. The precipitate was then filtered out and saved in a desiccator for characterization. Thermal Analysis. Differential scanning calorimetry (DSC) measurements were performed with a PerkinElmer DSC 7 differential scanning calorimeter. Temperatures and heats of fusion were 3081

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Figure 3. FTIR spectra of the (a) pure urea, (b) PHB, and (c) PHB-U-IC in the region between 600 and 1800 cm−1 and the region between 2800 and 3800 cm−1.

be seen that urea melts at ∼133 °C, PHB melts around ∼170 °C, and the PHB-U-IC melts at ∼140 °C. The DSC scan of PHB-U-IC shows no peak at 133 °C, suggesting that there is no uncomplexed urea in the sample. The melting temperature difference between PHB-U-IC and pure urea clearly confirms the formation of the U-IC and is consistent with other urea polymer ICs. In the second heating (not shown here), the melting peak of PHB-U-IC is shifted to slightly lower temperature, and a shoulder was apparent at 133 °C, indicating a portion of the IC decomplexing into free PHB and free urea. Several polymer urea ICs were examined previously using DSC and showed that the melting temperature of these ICs varied

experiment. Magic angle spinning was employed at a rate of 6 kHz. The CP/MAS experiments were performed with a variable contact time (100 μs−40 ms) and an 8 s relaxation delay. SP-MAS (single pulse with magic angle spinning and high power proton decoupling) was performed with a 30° tip angle to reduce the necessary relaxation delay and allow full relaxation of all 13C spins.

3. RESULTS AND DISCUSSION Structure Characterization of PHB-U-IC. DSC scans of urea, PHB, and PHB-U-IC were obtained in order to confirm the formation of inclusion compound. The DSC thermograms of urea, PHB, and PHB-U-IC recorded at a heating rate of 10 °C/min between 30 and 150 °C are shown in Figure 1. It can 3082

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Figure 4. 13C NMR CP/MAS/DD spectra of (a) bulk PHB and (b) PHB-U-IC.

from 137 to 143 °C depending on the polymer incorporated in the urea channel.10−22 However, no correlation between the crystal structures of ICs and melting temperatures was established. The melting temperature of PHB-U-IC also falls into the same melting temperature range as other polymer urea inclusion compounds. Figure 2 shows the XRD patterns of urea, PHB, and PHB-UIC. Major reflections at 2θ = 20.5, 22.7, 25.1, 29.8, and 36.1° were observed for pure urea. PHB shows two strong reflections at 2θ = 13.9 and 16.8°. The XRD pattern of PHB-U-IC is different from that of either pure urea or pure PHB, suggesting the formation of PHB-U-IC. The XRD pattern of PHB-U-IC displays major reflections at 2θ = 20.3, 22.4, 25.1. 29.7, 37.4, and 42.2° and shows no reflections associated with free PHB, confirming the purity of our sample. The XRD pattern of PHBU-IC was compared with XRD patterns of hexagonal, trigonal, and expanded tetragonal polymer U-ICs, and the PHB-U-IC XRD pattern closely resembles the expanded tetragonal U-IC XRD powder pattern. It was therefore concluded that PHB-UIC adopts an expanded tetragonal crystal structure similar to the polypropylene urea inclusion compound (PP-U-IC).10−22 Figure 3 displays FTIR spectra in the regions from 2000 to 500 and 2800 to 3800 cm−1 obtained for urea, PHB, and PHBU-IC. The bands associated with urea were previously reported.10,11 Tetragonal urea has strong bands at 3447 and 3347 cm−1 (N−H stretching vibrations), 1682 cm−1 (CO stretching), 1628 and 1600 cm−1 (N−H bending vibrations), and 1467 cm−1 (N−C−N antisymetric stretching vibration). The PHB-U-IC spectrum consists of bands associated with urea and PHB. The bands due to tetragonal urea are significantly affected by the formation of PHB-U-IC, whereas the bands due to PHB appear not to be affected that much by the formation of IC. New IR bands are present in the PHB-U-IC spectrum that are not present in either the spectra of pure PHB or free

uncomplexed tetragonal urea, confirming the formation of PHB-U-IC. It has been shown previously that IR bands in the region from 1400 to 1800 and 3000 to 3500 cm−1 are sensitive to the changes in crystal structure of urea, and therefore both regions are shown in Figure 3. The bands at 1683, 1628, 1600, and 1467 cm−1 due to tetragonal urea are shifted to 1664, 1623, 1613, and 1456 cm−1, respectively, in the PHB-U-IC. Several polymer urea inclusion compounds were prepared with poly(ε-caprolactone) (PEC-U-IC),10 poly(L-lactic acid) (PLLA-U-IC),16 polytetrahydrofuran (PTHF-U-IC),11 poly(ethylene oxide) (PEO-U-IC),13 poly(ethylene glycol) (PEGU-IC),12,13 polypropylene (PP-U-IC),15 and trans-1,4-polyisoprene (TPI-U-IC).22 It has been shown that PEC-U-IC, PLLAU-IC, and PTHF-U-IC adopt hexagonal crystal structures, PEO-U-IC takes a trigonal crystal structure, and PEG-U-IC, PP-U-IC, and TPI-U-IC adopt expanded tetragonal crystal structures. The band assignments for hexagonal, trigonal, and tetragonal assignments were reported previously.10−22 In the case of hexagonal urea the IR bands were shifted to 1658, 1639, 1600, and 1491 cm−1, for trigonal urea the IR bands were shifted to 1694, 1654, 1639, 1577, and 1457 cm−1, and for expanded tetrahedral urea IR bands were shifted to 1663, 1626, 1617, and 1454 cm−1. It is clear that the IR bands observed for PHB-U-IC are different from IR bands assigned to hexagonal and trigonal ICs but are closer to the expanded tetragonal crystal structure. FTIR spectra of urea, PHB, and PHB-U-IC in the region from 2800 to 3800 cm−1 are also shown in Figure 3, and this region is the most sensitive to difference in hydrogen bonding. The neat urea spectrum shows two broad bands centered at 3340 and 3440 cm−1, and they are attributed to symmetric and asymmetric stretching vibrations. In the case of PHB-U-IC, these bands are shifted to 3350 and 3457 cm−1, suggesting that hydrogen bonding within urea is weekend. On the other hand, 3083

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Figure 5. DSC heating scan of (a) as-received and (b) coalesced PHBs.

several times. DSC heating scans of neat PHB and coalesced PHB are compared in Figure 5. It should be noted that neat PHB was dissolved in dichloromethane and precipitated using methanol to have similarly treated as-received and coalesced PHBs. It appears that the melting temperature of neat PHB (∼167 °C) is slightly higher than the melting temperature of coalesced PHB (∼164 °C). The ΔHm values obtained for both PHB samples were found to be close. The crystallinity of both PHBs was calculated using the ΔHm° value reported for 100% PHB (146 J/g). The crystallinity of coalesced PHB was estimated as 48%, while the crystallinity of as-received PHB was estimated as 46%. Nonisothermal melt crystallization of as-received and coalesced PHBs were carried out by heating the samples to 200 °C to delete their previous thermal history and cooled down at the rate of 10 °C/min to room temperature. Figure 6 shows DSC cooling curves for as-received and coalesced PHBs from the melt as well as heating curves of nonisothermally melt crystallized as-received and coalesced PHBs. As-received PHB exhibits a strong exothermic peak (nonisothermal melt crystallization) at ∼90 °C, whereas the nonisothermal crystallization of coalesced PHB takes place at ∼70 °C. On the basis of this observation, we can reasonably deduce that the nonisothermal crystallization rate of coalesced PHB is much slower than as-received PHB. In order to examine the difference, the second cooling curves were obtained for both PHBs, and the difference in crystallization temperature was still apparent. Crystallization exotherms observed for as-received and coalesced PHBs are noticeably different. Crystallization enthalpies of as-received and coalesced PHBs ΔHcs were found to be 63.0 and 43.0 J/g, respectively, indicating that coalesced PHB is not as easily converted into semicrystalline PHB as is as-received PHB. To investigate nonisothermal melt crystallization kinetics of as-received PHB and coalesced PHB, relative crystallinity versus time is plotted in Figure 7 as a function of crystallization time. It can be clearly seen that crystallization rate of coalesced PHB is much slower than as-received PHB. Figure 8 shows optical micrographs obtained for nonisothermally melt crystallized asreceived PHB and coalesced PHB. Both PHBs show spherulitic

in the case of hexagonal and trigonal U-IC, N−H stretching bands are shifted to lower wavenumbers due to stronger hydrogen bonding within the urea matrix. FTIR spectra of neat PHB are compared to the bands associated with guest PHB in PHB-U-IC. The IR bands of guest PHB are similar to IR bands in neat bulk PHB, suggesting that conformation of PHB in the urea channel is similar to bulk PHB. CP/MAS/DD and single-pulse 13C NMR experiments were performed on PHB and PHB-U-IC and are shown in Figure 4. The PHB spectrum shows four 13C resonances at 169.6, 68.4, 42.6, and 21.0 ppm, and they are attributed to CO, CH, CH2, and CH3 carbons. The ratios of integral intensities of the lines associated with the CO, CH, CH2, and CH3 groups should be theoretically equal but differ slightly from the theoretical value. The difference in integrated intensities can be explained by an insufficient delay time (T1) used in our experiments. The CP/ MAS/DD 13C NMR of PHB-U-IC shows similar resonances for PHB as well as a chemical shift at 163 ppm, attributed to the urea carbonyl group. The urea carbonyl resonance is very weak, and that may be due to its very long relaxation time. Similar observations were made for other polymer urea inclusion compounds that adopt expanded tetragonal crystal structure.15,22 It should be noted that the NMR peaks are broad for PHB compared to PHB-U-IC. Neat PHB consists of both crystalline and amorphous phases whereas PHB-U-IC consists of a single phase. The chemical shifts of the crystalline phase in bulk PHB are virtually identical to PHB in the urea channels, suggesting that it adopts a conformation similar to bulk crystalline PHB. Modeling of PLLA chains in narrow cylinders has shown that only the trans conformation can fit in a cylinder diameter below 7 Å.32 In order for the bulk crystalline ttgg PHB conformation to fit into the urea channel, the channel diameter has to be more than 7 Å. Since from solid-state NMR we observe the bulk crystalline conformation for PHB in the urea channel, we concluded that the guest PHB chains in PHB-U-IC adopt an expanded tetragonal crystal structure with channel diameters more than 7 Å. Crystallization Behavior of Bulk As-Received PHB and PHB Coalesced from PHB-U-IC. Coalesced PHB samples were obtained by washing PHB-U-IC with warm methanol 3084

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Figure 6. A) DSC heating scans of nonisothermally melt crystallized a) neat and b) coalesced PHBs. B) DSC of the nonisothermal melt crystallization curves of a) neat and b) coalesced PHBs.

than as-received polymers with the same crystallinity. It was inferred from this observation that densities of coalesced polymer melt may be higher than as-received bulk melt. The physical and mechanical properties of PCL coalesced from its cyclodextrin inclusion compound have been studied recently32 and compared with as-received PCL. It has been suggested that elongated and unentangled PCL chains formed after coalescing significantly increased the nonisothermal melt crystallization temperature. Since we observed contradictory results for coalesced PHB compared to other polymers coalesced from their inclusion compounds, we have formed PHB inclusion compounds with α-CD and thiourea (TU) and coalesced the guest PHBs from these ICs. The crystallization and the melting behavior of PHB coalesced from both TU and α-CD were investigated, and results are given in Table 1, along with PHB coalesced from the U-IC. It appears that PHB coalesced from TU and α-CD

morphology, but the spherulite size of as-received PHB is apparently smaller and less developed than coalesced PHB, again suggesting nonisothermal crystallization rate of coalesced PHB is slower than as-received PHB. It has been shown previously that biodegradable polymers such as PLLA and PCL and other semicrystalline polymers coalesced from either hexagonal urea or cyclodextrin inclusion compounds remain largely extended and unentangled.33−37 These coalesced polymers showed quite distinct properties from bulk polymers crystallized from either the melt or solution. For example, coalesced polymers showed enhanced crystallization from the melt by having higher crystallization temperatures than their as-received polymers. It has also been shown that these properties remain distinct even after annealing above the melting temperature where polymer chains are mobile. It has been shown that the densities of the noncrystalline regions in solid coalesced polymers were higher 3085

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Table 1. Thermal Properties of As-Received and Coalesced PHB sample

melting temp (oC)

nonisothermal melt crystallization temp (oC)

crystallinity (%)

163

74

48

163

95

44

167

95

46

167

91

46

coalesced PHB from U-IC coalesced PHB from TU-IC coalesced PHB from a-CD-IC control neat PHB

polymer chains with gauche conformations to occupy in these narrow IC channels with diameter less than 7 Å, and therefore the PLLA and PCL chains adopt largely the extended all-trans conformation. This ultimately increases the crystallizability of their coalesced polymers. On the other hand, PHB-U-IC forms an expanded tetragonal crystal structure with diameter more than 7 Å, where the PHB chains adopt the bulk crystalline ttgg conformation, as shown by solid-state 13C NMR spectroscopy. PHB coalesced from expanded tetragonal PHB-U-IC channels appears to be less crystallizable compared to bulk PHB. After all, TU-ICs have a slightly larger channel diameter than hexagonal U-ICs, and αCD-IC has almost the same size as hexagonal U-ICs. PHB coalesced from both α-CD-IC and TU-IC behave as all other coalesced polymers.33−35 In addition to the conformation, intermolecular forces such as dipole−dipole forces favor crystallizability. We believe the intermolecular forces are weaker in coalesced PHB compared to bulk PHB even though both PHBs adopt same conformation. Therefore, coalesced PHB crystallizes slower than bulk PHB. Further study is required in order to understand this unusual crystallization behavior. We plan to address this issue by forming PHB-IC with α-CD, βCD, and γ-CD and studying coalesced PHB from these ICs. In conclusion, PHB-U-IC was successfully prepared for the first time by a cocrystallization method, and its structure and the conformation of included PHB chains were studied by DSC, X-ray diffraction, FTIR, and solid-state NMR spectroscopy. The XRD pattern and FTIR spectroscopy show PHB-UIC takes a different crystal structure than the well-reported hexagonal and trigonal crystal structures of polymer urea inclusion compounds. The guest PHB in the confined U-IC environment has been shown to adopt a similar conformation to bulk crystalline PHB. The crystallization and thermal properties of as-received PHB and PHB coalesced from its U-IC were compared and demonstrated that recovered coalesced PHB crystallizes much slower than as-received PHB. The coalesced PHB from U-IC exhibited completely different behavior compared with other coalesced polymers from various ICs including that of PHB coalesced from its TU and α-CD-ICs.

Figure 7. Relative crystallinity versus time for nonisothermally crystallized from the melt for (a) neat PHB and (b) coalesced PHB at a cooling rate of 10 °C/min.

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Figure 8. Optical micrographs of nonisothermally melt crystallized neat PHB (top) and PHB coalesced from its U-IC (bottom).

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph 718-246-6328; Fax 718-488-1465 (N.V.).

behave similar to PLLA and PCL coalesced from both α-CD and U. The question still remains why the PHB coalesced from urea crystallizes slowly even though PHB adopts bulk crystalline ttgg conformation. The answer is probably due to the expanded tetragonal structure formed by PHB-U-IC. All previously observed coalesced polymers showed enhanced crystallization behavior, and these α-CD and U-ICs have a channel diameter less than 7 Å.33−37 It is not possible for

Notes

The authors declare no competing financial interest.

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