Effect of �-Cyclodextrin on the Crystallization of Poly(3-hydroxybutyrate)

YONG HE, YOSHIO INOUE

Tokyo Institute of Technology, Department of Biomolecular Engineering, Nagatsuta 4259, Midori-ku,Yokohama 226-8501, Japan

Received 2 December 2003; revised 27 May 2004; accepted 31 May 2004DOI: 10.1002/polb.20213Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: The effect of �-cyclodextrin (�-CD) on the crystallization behavior of poly(3-hydroxybutyrate) (PHB) was investigated with polarized optical microscopy, differen-tial scanning calorimetry, and wide-angle X-ray diffraction. We found that the additionof �-CD can greatly accelerate the crystallization of PHB and that �-CD has a potentialnot only to enhance the nucleation but also to accelerate the crystallization of PHB.Compared to a conventional nucleation agent, such as talc, �-CD is a natural productand has many advantages because it is environmentally friendly and safe to humans.© 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3461–3469, 2004

INTRODUCTION

Today, synthetic polymer materials are widelyused throughout the world and contribute enor-mously to the quality of life. Polymer materialsare strong, lightweight, easily processable, inex-pensive, and energy efficient. However, the irre-versible environmental buildup of the waste fromthese materials has resulted in worldwide con-cern about the consequences of polymeric wasteaccumulation.

Biodegradable aliphatic polyesters are an at-tractive solution to alleviate these environmentalproblems. The polymers can be easily convertedinto simpler compounds and mineralized in theenvironment. Hence, minimum environmentalpollution is expected when such materials aredisposed of after their use. Based on their origins,aliphatic polyesters are classified into two types,that is, natural polyesters and chemosyntheticpolyesters.

A representative of natural polyesters is bacte-rial poly(3-hydroxybutyrate) (PHB), which is ac-cumulated as intracellular energy reserves by avariety of microorganisms.1–6 It can be degradedinto water and carbon dioxide by a variety ofbacteria under normal environmental condi-tions.7 However, its application suffers from thedisadvantages of brittleness and a narrow pro-cessability window.8

The brittleness of PHB is because of the verybig size of its spherulites and secondary crystal-lization. Bacterial PHB are produced by a batchfermentation process to produce extremely purematerial that results in an unusually low nucle-ation density.6,9,10 Correspondingly, crystalliza-tion only occurs at comparatively low tempera-tures upon rapid cooling in a mold and is subse-quently followed by a long-term secondarycrystallization while in storage. This leads to brit-tleness because the degree of crystallinity increaseslogarithmically with the storage time.6,9,10 Further-more, the extremely low nucleation density leads toa low crystallization rate; even the growth rate of aPHB spherulite is not very slow, resulting in a PHBmaterials with low production efficiency.

Correspondence to: Y. Inoue (E-mail: yinoue@bio.titech.ac.jp)Journal of Polymer Science: Part B: Polymer Physics, Vol. 42, 3461–3469 (2004)© 2004 Wiley Periodicals, Inc.

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To inhibit brittleness and to improve the pro-cessing efficiency nucleating agents, such as bo-ron nitride or talc,11 must be added during theproduction of PHB materials. However, to pro-duce a more environmentally friendly product, itis important to seek alternative nucleatingagents.

In the past few years, our research has beenfocused on poly(hydroxyalkanoic acid)s (PHAs).12–16

Recently we noted, when we investigated the prop-erties of the of PHA inclusion complex and cyclodex-trin, that the nucleation of the PHA segment in itspartial inclusion complex with cyclodextrin was en-hanced and the crystallization was accelerated.17 Inthis study, we are interested in whether the directaddition of cyclodextrin (rather than as an inclusioncomplex) into PHB can enhance the nucleation andpromote the crystallization of PHB. Cyclodex-trins18–20 originated from a renewable natural ma-terial, starch, and are environmentally friendly;they should be good alternative nucleating agents iftheir effectiveness is comparable to conventionalnucleation agents, such as talc.

EXPERIMENTAL

Materials

The bacterial PHB sample (Mw � 5.86 � 105,Mw/Mn � 2.35), was supplied by Mitsubishi GasCo., Ltd. (Japan). Because some bacterial cellcomponents remained in the received sample andthey may have affected the crystallization behav-ior of the PHB,21 the sample was used after puri-fication from a 1,2-dichloroethane solution, byprecipitation in ethanol. The powder samples ofcyclodextrins (�, �, and �-CD) were kindly sup-plied by Nihon Shokuhinn Kagaku Co., Ltd.(Japan). The talc powder was purchased fromKanto Kagaku Co., Ltd. (Japan). Before use, cy-clodextrins were dried in vacuo at 80 °C for atleast 3 days.

Sample Preparation

The inclusion complex (IC) of PHB and �-CD (ab-breviated as IC-�-CD-PHB) was prepared as re-ported in the literature.17,22 After drying IC invacuo, a portion of the IC was added into the PHBas a nucleating agent. Another part of the IC ofthe �-CD/PHB was washed with excessive waterat 50 °C to dissolve almost all of the �-CD in theIC and the PHB was coalesced. A 1H NMR spec-

trum of the coalesced PHB (PHBc) in CDCl3/DMSO-d6 (7/3,v/v) solution at 30 °C was recordedon a JEOL GSX270 NMR spectrometer with a 90°pulse to estimate the content of �-CD in thePHBc.17 The spectrum only provides a very roughestimate, 0.8 � 0.3 wt %, of the �-CD content inPHBc because the the �-CD content is low and theCDCl3/DMSO-d6 mixed solvent does not workwell for PHB. The crystallization behavior of thecoalesced PHB (PHBc) was also investigated, as areference.

Considering the thermal instability of thePHB, the blends of PHB and nucleating agentswere prepared by admixing the nucleating agentinto a concentrated CHCl3 solution of PHB(0.1 g/mL); the solvent was then allowed to evap-orate during rigorous stirring. The resultant filmswere dried at room temperature under reducedpressure for 1 week before analysis.

Polarized Optical Microscopy (POM)

Polarized optical microscopy was performed on anOlympus BX90 polarizing microscope equippedwith a digital camera. The polymer sample wasplaced between a microscope glass slide and acover slip and heated with a Mettler FP82HT hotstage. The sample (ca. 0.2 mg) was first heated to190 °C and kept at that temperature for 1 min,then cooled to the desired crystallization temper-ature, with a cooling rate of 20 °C/min. The

Figure 1. DSC cooling curves. The cooling rate is10 °C/min. Before the cooling, the samples were meltedat 190 °C for 2 min to erase the thermal history.

3462 HE AND INOUE

growth rate of the spherulite was calculated asthe slope of the line, obtained by plotting theradius against the time.

Differential Scanning Calorimetry (DSC)

DSC measurements were performed on a Perkin-Elmer differential scanning calorimeter (Pyris Di-amond). The sample weight was between 5 and6 mg. An indium standard was used for the cali-bration, and nitrogen was used as the purge gas.A nonisothermal and an isothermal temperatureprogram were used to evaluate the effects of thenucleating agents on the PHB nucleation. In thenonisothermal temperature program, the samplewas melted at 190 °C for 2 min, cooled at10 °C/min to 0 °C, and kept at 0 °C for 2 min, thenheated at 10 °C/min to 190 °C. The temperature ofcrystallization (Tc) and the corresponding heat ofcrystallization (Hc) values were taken as the peakand area of the crystallization exotherm in thecooling run, respectively. In the isothermal tem-perature program, after melting at 190 °C for2 min, the sample was quenched to the desiredcrystallization temperature and the isothermalcrystallization curves were recorded. Based onthese curves, the crystallization kinetics of thePHB was analyzed and the effectiveness of thenucleation agent was evaluated.

Wide-Angle X-ray Diffraction (WAXD)

WAXD measurements were carried out on aRigaku RU-200 operated at 40 kV and 200 mA.Nickel-filtered Cu K� radiation (� � 0.154 nm)was used. The WAXD patterns were recorded inthe 2� range of 5–55° at a scan speed of 1.0°/minat room temperature.

RESULTS AND DISCUSSION

Nonisothermal Crystallization Behavior

The DSC cooling curves of the samples are sum-marized in Figure 1. The peak crystallizationtemperature (Tc) of pure PHB is about 79 °C andthe crystallization proceeds in a broad tempera-ture range. With the addition of the nucleation

Figure 2. DSC heating curves. The heating rate is10 °C/min.The heating run follows the dynamic coolingrun (see the Experimental section for details).

Table 1. The Crystallization and Melting Behavior of PHB and Its Blends

Sample Tc/°C �Hc/J g�1 Tm/°C �Hf/J g�1

Pure PHB 78.7 74.6 171.7 91.2PHB/�-CD 2 wt % 95.3 70.5 173.0 83.9PHB/�-CD 2 wt % 96.4 70.9 172.9 83.4PHB/�-CD 1 wt % 97.0 76.5 172.9 93.2PHB/IC-�-CD-PHB 2 wt % 99.8 73.8 173.9 92.2PHB/�-CD 1 wt % and talc 1 wt % 103.6 74.8 172.4 89.9PHB/�-CD 2 wt % 106.2 79.9 173.9 92.7PHB/talc 2 wt % 112.3 83.0 174.3 93.9PHBc (coalesced from IC) 112.5 85.8 174.4 96.6

EFFECT OF �-CD ON CRYSTALLIZATION OF PHB 3463

agents, the Tc of PHB greatly increased and thecrystallization process finished within a narrowtemperature range. After the addition of 2 wt % of�-CD, 2 wt % of �-CD, 1 wt % of �-CD, 2 wt % ofIC-�-CD-PHB, 1 wt % of �-CD and 1 wt % of talc,and 2 wt % of �-CD, the Tc of the PHB increasedfrom 79 °C to 95, 96, 97, 100, 104, and 106 °C,respectively (Fig. 1 and Table 1). An increase of 18and 27 °C in the Tc of the PHB, induced by theaddition of �-CD 1 and 2 wt %, respectively, sug-gested that �-CD was effective for enhancing thecrystallization of the PHB. The highest Tc, 112 °C,was observed for the PHB including 2 wt % talc. Itis noteworthy that the coalesced PHB from IC(PHBc) also shows a high Tc, 112 °C, the same asthat of the PHB including 2 wt % talc. Also, boththe PHBc and the PHB blends with 2 wt % talccrystallized in a similar narrower temperaturerange. This result may come from the fact that

some �-CD (0.8 � 0.3 wt %, as determined from1H NMR) was left over during the coalescence ofPHB from IC. The remaining �-CD might be inthe complex state and effectively enhanced thecrystallization of PHB as the nucleating agent oftalc. It is noteworthy that the crystallization be-havior of the PHBc is quite different from that ofthe IC-�-CD-PHB, in which the crystallization ofthe PHB segment is greatly restricted becausemost of the PHB units are included in the cavityof the �-CD.22

It is interesting that the Tc of the PHBc isabout 5 °C higher than that of PHB/�-CD 2 wt %.Considering that most of the �-CD in the PHBcshould be in the inclusion complex state, althoughmost of the �-CD in PHB/�-CD 2 wt % should bein the free state, this may suggest that �-CD inthe complex state is more effective for promotingthe crystallization of PHB than for promoting the

Figure 3. The isothermal crystallization curves of PHB, PHB/�-CD 2 wt % blend,PHBc, and PHB/talc 2 wt % blend at (a) 115, (b) 118, and (c) 120 °C.

3464 HE AND INOUE

free state �-CD. This suggestion is also indirectlysupported by the fact that the crystallization be-havior of the PHB is not as sensitive to the addi-tion of �-CD and �-CD as to the addition of �-CD,in accord with the fact that �-CD and �-CD can-not form stable inclusion complexes with PHB.22

The DSC curves of the samples in the heatingrun, which followed the dynamic cooling when thecrystallization temperatures were determined,

are depicted in Figure 2. Higher melting points(Tms) are detected for the PHBc and PHB/talc2 wt %. Also, the Tms of the PHB/�-CD blends arehigher than for that of pure PHB, correspondingto the fact that the Tc values of the PHB/�-CDblends are higher than for that of pure PHB(Fig. 2 and Table 1).

The crystallization enthalpy (�Hc) and theheat of fusion (�Hf) are not very sensitive to theaddition of the nucleation agents, as seen inTable 1. However, it also seems that high �Hc and�Hf values are related with a high Tc value.

Isothermal Crystallization Behaviors

The isothermal crystallization curves of the PHB,the PHB/nucleation agents, and the PHBc at 115,118, and 120 °C are plotted in Figure 3(a–c),respectively. Clearly, the isothermal crystalliza-tion rates of the PHB/�-CD blends are higherthan for that of pure PHB at each crystallizationtemperature, similar to the nucleation effects of�-CD on the crystallization of PHB.

The parameters of the crystallization kineticswere determined from the isothermal DSC exper-iments. The isothermal heat flow curves obtainedfrom the DSC experiments were integrated todetermine the sample crystallinity as a functionof crystallization time. The relative crystallinity[X(t)] at a given time, t, was calculated from thearea under the DSC curve, from t � 0 to t � t

Figure 4. Plots of ln{�ln[1 � X(t)]} versus ln(t) forPHB, PHB/�-CD 2 wt % blend, PHBc, and PHB/talc2 wt % blend isocrystallized at 118 °C.

Table 2. The Kinetic Parameters for the Isothermal Crystallization of PHB

Iso. Cry.Temp.(°C) Parameter

Sample

PHB PHB/�-CD 2 wt % PHBc PHB/talc 2 wt %

115 k � 106 7.31 11.3 35.1 55.3n 1.93 2.12 2.24 2.15G 0.88 0.87 0.88 0.89

N/106 2.56 4.09 12.3 18.8

118 k � 107 2.75 51.6 120 364n 2.45 2.42 2.50 2.50G 0.76 0.75 0.77 0.78

N/105 1.50 29.2 63.1 185

120 k � 107 1.27 24.8 55.0 88.4n 2.43 2.20 2.30 2.43G 0.61 0.63 0.62 0.60

N/105 1.33 23.7 55.0 96.8

The units for k, G, and N are s�n, �m s�1, and cm�3, respectively.

EFFECT OF �-CD ON CRYSTALLIZATION OF PHB 3465

Figure 5. POM photographs of PHB, PHB/�-CD 2 wt %, PHBc, and PHB/talc 2 wt %at 190 °C.

Figure 6. POM photographs of PHB, PHB/�-CD 2 wt % blend, PHBc, and PHB/talc2 wt % blend after isothermal crystallization at 118 °C.

divided by the whole area of the heat flow curve.The isothermal bulk crystallization kinetics areprimarily analyzed with the Avrami equa-tion,23–25 which is commonly written in the fol-lowing form:

X�t� � 1 � exp(�ktn) (1)

where t is the elapsed time during the course ofisothermal crystallization, k is the Avrami rateconstant, and n is the Avrami exponent. X(t) canbe obtained by integrating the crystallization exo-therms of DSC according to eq 2:

X�t� � �0

t

�dHc/dt�dt/�0

�

�dHc/dt�dt (2)

where dHc is the enthalpy of crystallization re-leased during an infinitesimal time interval dt.

Qualitative interpretation of the nucleationmechanism and morphology as well as the overallcrystallization growth rate of the sample can beattained from the n and k values. The linear formof eq 1 is given in eq 3:

ln{�ln[1 � X�t�]} � ln(k) n ln(t) (3)

Plotting ln{�ln[1 � X(t)]} versus ln(t) gives n (theslope) and k (the intercept). The linear form of theAvrami equation is inaccurate at both the highand low extents of crystallization,26 so only theintermediate range of crystallization [linear re-gion, with an X(t) range of about 30–70%] wasused in the analysis. The plots of ln{�ln[1 � X(t)]}; versus ln(t) for PHB, PHB/�-CD 2 wt% blend, PHBc, and PHB/talc 2 wt % blend isoc-rystallized at 118 °C are shown in Figure 4.

The results of the Avrami analysis are listed inTable 2. The Avrami exponent (n) increases withan increase of the isothermal crystallization tem-perature, whereas it is insensitive to the additionof nucleation agents. However, the Avrami rateconstant (k) greatly increases with the addition ofnucleation agents and decreases with an increaseof the isothermal crystallization temperature.

Employing a model of three-dimensionalspherulitic growth simultaneously initiated fromactive nuclei (n � 3), eq 4 relates the Avrami rateconstant, k, the nuclei density, N, and the crystallinear growth rate, G (determined by opticalmicroscopy).27,28

N 3k/�4 G3� (4)

In the sample, N was quantified with isothermalDSC experiments at 115, 118, and 120 °C with theAvrami theory and eq 4. The calculated N valuesare also shown in Table 2. It is clear that theaddition of �-CD results in an increase of N byone to two orders, direct evidence of the nucle-ation effects of �-CD on the crystallization ofPHB.

It appears that the nucleation effect of �-CD isless than that of talc because Tc in the cooling andthe N of the PHB with the addition of 2 wt % �-CDare lower than those with 2 wt % talc. However,two facts should be pointed out: one is that thecrystallization behavior of PHBc, in which thecontent of �-CD is less than 1 wt %, is similar tothat of PHB/talc 2 wt % blend; the other is thatthe particle size of �-CD powder used in thisstudy is much larger than that of talc (Fig. 5),which should indicate that �-CD has the potentialto act as effectively as talc to enhance the nucle-ation and accelerate the crystallization of PHB.The differences in the size and the distribution ofthe �-CD particles (Fig. 5) is probably the mainreason for the difference in the crystallizationbehavior between PHB/�-CD 2 wt % and PHBc. Itis probable that the fine and well-dispersed �-CDpowder possesses nucleation effects comparableto talc for PHB.

POM

POM was used to observe the distribution and thesize of nucleation agent particles in the moltenPHB, the growth of the spherulites, and the mor-phology after crystallization. From Figure 5, it isclear that the distribution of �-CD (and talc) inthe PHB matrix is basically uniform on the POMobservation scale. The size of the �-CD particles(ca. 50 �m) in the blend of PHB/�-CD 2 wt % ismuch larger than that of talc particles (ca. 8 �m)in PHB/talc 2 wt %. Correspondingly, the numberof talc particles is much higher than the numberof �-CD particles.

Considering that the Tc value of the PHB/�-CD2 wt % system is lower than that of the PHB/talc2 wt % system during dynamic cooling and the Nof the former is lower than that of the latter inisothermal crystallization, as demonstrated inthe previous sections, one may conclude that thenucleation effectiveness of �-CD is lower thanthat of talc. However, this conclusion is not defin-

EFFECT OF �-CD ON CRYSTALLIZATION OF PHB 3467

itive because the size and the number of the �-CDparticles is quite different from those of talc.

The POM photographs recorded after isother-mal crystallization at 118 °C are shown inFigure 6. During the isothermal crystal growth at118 °C, the diameters of the pure PHB spheru-lites reach up to 3000 �m before they impinge oneach other because the number of the nuclei is sosmall in the pure PHB. By adding �-CD (2 wt %)and talc (2 wt %), the number of nuclei increasedand the average diameter of the spherulitesdropped drastically to about 800 and 200 �m,respectively. The average diameter of the spheru-lites observed for PHBc was about 400 �m, be-tween those of the PHB/�-CD 2 wt % system andthe PHB/talc 2 wt % system. This observation iscompatible with the analysis of the N based onthe Avrami theory and eq 4. The growth rate, G,of the PHB spherulites is a function of tempera-ture, whereas it is insensitive to the addition of�-CD or talc (Table 2).

WAXD

The WAXD pattern was recorded to determine ifthe addition of the �-CD affects the crystal form ofthePHB. The results are shown in Figure 7. Thethree peaks, at about 9.6, 19.1, and 28.7° in thecurve of PHB/talc 2 wt % are diffraction patternsof talc crystals rather than PHB crystals. Obvi-

ously, the diffraction pattern of PHB in the blendsor in PHBc is the same as in the pure state,indicating that the addition of nucleation agentshas only a minor effect on the crystal form ofPHB.

CONCLUSIONS

The effect of �-CD on the crystallization behaviorof PHB was investigated with DSC and POM. Wefound that the addition of �-CD could greatlyaccelerate the crystallization of PHB, that is,�-CD, an environmentally safe compound, can beused as a nucleation agent for the biodegradablepolyester PHB. In this work, the nucleation effectof �-CD is less than that of talc because the aver-age particle size of �-CD used here was muchlarger than that of talc. However, we stronglysuggest that �-CD particles with much smallersizes may have nucleation effects comparable tothat of talc for PHB. Further studies are under-way in our laboratory.

We gratefully acknowledge a Research Institute of In-novative for the Earth, Japan (RITE) fellowship to Y.He. The authors are grateful to Mitsubishi Gas Co.,Ltd., Japan, for kindly supplying the PHB sample.

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EFFECT OF �-CD ON CRYSTALLIZATION OF PHB 3469