Photon Factory Activity Report 2019 #37 (2020)
BL-6A/2017G672

Structural Evolution in Isothermal Crystallization Process of Poly(L-lactic acid)Enhanced by Silk Fibroin Nano-Disc
Amit Kumar PANDEY1, Vimal KATIYAR2, Hideaki TAKAGI3, Nobutaka SHIMIZU3,Noriyuki IGARASHI3, Sono SASAKI1 and Shinichi SAKURAI1,*
1
Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Kyoto,606-8585, Japan2
Department of Chemical Engineering, Indian Institute of Technology Guwahati,Guwahati, Assam 781039, India
3
Photon Factory, Institute of Materials Structure Science,High Energy Research Organization, 1-1 Oho, Tsukuba, 305-0801, Japan
1 Introduction
The nucleating effect of silk fibroin nano-disc (SFN)
on the crystallization behavior of poly(L-lactic acid)(PLLA) was investigated by simultaneous synchrotron
small- and wide-angle X-ray scattering measurements.
For the isothermal crystallization at 110 ̊C from the melt,
the induction period of the PLLA specimens containing
1% SFN was reduced compared to that of the neat
specimens, indicating the acceleration of the nucleation ofPLLA. The final degree of crystallinity was also
increased, and the crystallization half-time was decreased,
which indicates that the overall crystallization process
was accelerated. Furthermore, the final value of thecrystallite size (the lateral size of the crystalline lamella)
was slightly lower for the specimens containing 1% SFN
than that for the PLLA neat specimen, although thecrystallites started growing much earlier. However, it was
found that there was no effect of SFN on the growth rateof the crystallite size. The lamellar thickening process
was also accelerated with a clear overshooting
phenomenon with the inclusion of 1% SFN. As for thepolymorphism, the α’ phase is dominant with about 96%,
but a small amount of the phase (4%) is found to exist. Itwas found that the SFN can also accelerate the formationof the minor α phase as well as the major α’ phase.
2 Experimental
The PLLA samples used in this study are obtained fromNatureWorks LLC. As shown in Table 1, one of thembears 1.4% D content (PLLA4032D) and the other 0.5%
D content (PLLA2500HP), which enabled us to study theeffects of the optical impurity on the crystallization of
PLLA with or without the presence of SFN. The SFN is ananoparticle that was extracted from wastes of the muga
silk (Antheraea assama) cocoon. The crystalline portion(the sheets) of the silk fibroin was isolated by using theacid-hydrolysis method. The obtained extract comprises
83.8% L-alanine, and the well-defined disc-like nanoparticles were obtained with the average diameter andthickness of ~45 nm and ~3 nm, respectively. The
composites of PLLA and SFN were prepared by thesolution-casting method by using dichloromethane as asolvent. The specimens are labeled as D1.4/SFN(x) orTable 1: Sample characteristics

D0.5/SFN(x), where the numbers after D denote the % ofD moiety in PLLA, and x stands for % of SFN inclusion.
The time-resolved SWAXS (small- and wide-angle Xray scattering) measurements were carried out by using
the synchrotron radiation as an X-ray source at thebeamline BL-6A of Photon Factory at the KEK (HighEnergy Accelerator Research Organization) in Tsukuba,
Japan. The wavelength of the incident X-ray beam was
0.150 nm. The sample was packed into an aluminum cell,which had a diameter of 4 mm and thickness of 1 mm.
The specimens were sandwiched by a couple of pieces ofthe polyimide (Kapton) film, obtained from DuPontToray Co., Ltd., Tokyo, Japan. Firstly, the specimens weremelted at 200 ̊C for approximately 5 min, and then thesample cell was quickly moved to another heater block,which was maintained at 110 ̊C, and then thesimultaneous SWAXS measurement was performed.
Note here that this isothermal crystallization temperature(110 ̊C) can be considered to be located in regime III.
This is because Song et al. (2018) have reported thatPLLA specimens with 100% and 98% L moieties show
the regime II at higher temperature and regime III atlower temperature with the transition temperature of 120
̊C [1]. The actual sample temperature was monitored by atemperature sensor, which was directly attached to thesample. The stabilization time for the temperatureequilibration after the T-jump was 15s and thetemperature fluctuation was less than 0.3 ̊C. The
scattering vector q was calibrated by using collagen for
SAXS (small-angle X-ray scattering) and polyethylene
for WAXS (wide-angle X-ray scattering). The SAXS andWAXS patterns were recorded at every 5 s with the X-rayexposure time of 5s. The background scattering wassubtracted. The one-dimensional SAXS and WAXS
profiles were obtained by taking the circular average ofthe 2d-SAXS pattern and the sector average of the 2dWAXS pattern, respectively.

Photon Factory Activity Report 2019 #37 (2020)
3 Results and Discussion
The changes in the WAXS profiles were measured in
the isothermal crystallization process at 110 °C from themelt (200 °C). Figure 1 shows the WAXS profiles for theD1.4 neat and D1.4/SFN(1.0) specimens as a function oftime. Here, q denotes the magnitude of the scatteringvector, as defined by q = (4π/λ) sin(θ/2), with λ and θ
being the wavelength of X-ray and the scattering angle,respectively. As shown in Figure 1, in the early stage,
there is no crystalline peak, which shows the presence of
100% amorphous phase. As time goes on, a crystalline
peak appears (which has been shown by the red arrow inFigure 1). The induction period (t0) of the crystallization
is evaluated from the first detection of the crystalline peak.
It can be seen from Figure 1 that the loading 1% SFNcaused the reduction of the induction period from 90s to40s, which shows the enhancement of the crystallizability
by SFN. The time evolution of the degree of crystallinity
was calculated from the WAXS profiles. The peakdecomposition was conducted, and the degree ofcrystallinity (ϕWAXS) was evaluated. The calculated ϕWAXS
plotted as a function of time in Figure 2. As can be seenfrom Figure 2, the final degree of the crystallinity has
been increased by the inclusion of 1% SFN. Judging
from the slope of the curve at t = t0.5 (where t0.5 is thecrystallization half-time at the 50% of the finalcrystallinity attained) in Figure 2, it can be consideredthat the crystallizabilty of PLLA is increased by adding
1% SFN. The induction period, the final degree ofcrystallinity and the crystallization half-time of all thespecimens were calculated from the results of Figure 2. It
is clearly observed that t0.5 is decreased by loading of SFN,
indicating the acceleration of the crystallization rate.
Furthermore, it is important to check the effect of SFN on
the formation of the crystal polymorph (α and α’ phases).
For this purpose, the evaluation of the fraction of α and α’phases formed during the isothermal crystallization at
110 °C was conducted by analyzing the (200)/(110)reflection. Since there observed a small shoulder aroundq = 12.2 nm-1, we tried to decompose the shoulder peak(α phase) from the main peak (α’ phase). Figure 3 shows
an example of the peak decomposition in the range of 11< q < 13 nm-1, ensuring the perfect peak decomposition.
Figure 4 shows the changes in the Lorentz-corrected
SAXS profiles as a function of time for the D1.4 neat andD1.4/SFN(1.0) specimens. Here, the scattering intensity,
I(q), is corrected as q2I(q) by multiplying q2. In the earlystage of the crystallization, there was no observation ofthe peak. At t = 180 s for the D1.4 neat or t = 90 s for theD1.4/SFN(1.0) specimen, a clear scattering peak wasobserved, which indicates the development of thelamellar stacking with sandwiching the amorphous layers.
It is notable to observe that the WAXS peak appears
earlier than the SAXS peak (Figures 1 and 4). Such a
result indicates that during the early stage ofcrystallization, the lamellar stacks are incomplete bynoting t0 = 90 s and 40 s for the D1.4 neat andD1.4/SFN(1.0) specimens (see Figure 1). In other words,

Fig. 1 Time-resolved WAXS profiles of (a) D1.4 neat(b) D1.4/SFN(1.0) specimens. The red arrow
indicates the first detection of the peak.

Fig. 2 Degree of crystallinity as a function of time forthe isothermal crystallization at 110 °C.

Fig. 3 Decomposition of the (200)/(110) reflection
peak in the range of 11 < q < 13 nm-1.
single lamellae (without stacking) are generated in theearly stage of the crystallization.
The intensity of the peak observed at q = 0.29 nm-1
increases as a function of time. From the peak position(q*), the long period (D) of the lamellar stacks wasevaluated as D = 2π/q*. As seen in Figure 4, the SAXS
peak moves towards the higher q as the crystallization
proceeds. Increase in q suggests the decrease in D. As
shown in Figure 5a, the repeating distance of the lamellarstacks (D) decreases as a function of the crystallization
time. This result seemed to be opposed to the process ofPhoton Factory Activity Report 2019 #37 (2020)
crystallization. In order to understand this behavior, weevaluated the average thickness of the crystalline lamella(L). To evaluate L, the correlation function γ(r) wascalculated from the 1d-SAXS profile through thefollowing equation (inverse Fourier transform method):

Here, γ(r) is the correlation function and r is the distancein the real space. Figure 5 shows thus-evaluated L and D
and the ratio (L/D) as a function of time during theisothermal crystallization upon T-jump from 200 °C to110 °C. As a result (Figure 5b), the average lamellar
thickness increases with time, which is reasonable as acrystallization behavior. Therefore, the decreasing
behavior of D is also reasonable, as schematically shownin Figure 6. Upon crystallization, shrinkage takes place.
Since the lamella thickens with time, this results in thedecrease of D (Figure 6b,c), as an amorphous layer
thickness is decreased to a greater extent as compared tothe increasing extent of L (lamellar thickness).
It should be noted here that the average lamellar
thickness (L) did not monotonically increase as a functionof time, as the increasing tendency turns over around 7
min for the D0.5 neat specimen or 4 min for theD0.5/SFN(1.0) specimen. The reason why L decreased a
bit before reaching a constant value may be explained by
the formation of new lamellae. It may be explained that inprior to 7 min for the D0.5 specimen or 4 min for theD0.5/SFN(1.0) specimen, a new thin lamellae may beformed from the amorphous region. This argument has
been recently reported by our group [2,3] for theisothermal crystallization of the D0.5 specimen in theFig. 4 Changes in the Lorentz-corrected SAXS
profiles as a function of time for (a) D1.4 neat and(b) D1.4/SFN(1.0) specimens during the isothermal
crystallization at 110 ̊C from melt. The red arrow
indicates the first detection of the peak.

Fig. 5 (a) Long period (D), (b) average lamellar
thickness (L), and (c) L/D as a function of (t - t0),
where t0 is the induction period.

Fig. 6 Schematic illustrations showing the change in the nanostructure upon crystallization of PLLA (a) at theamorphous state before crystallization of the polymer melt and (b) in an early stage of crystallization. (c)Lamellar thickening in the subsequent stage of the crystallization. Note that the illustrations are focusing on thechange in the long period (D) of the lamellar stacks. (b’) and (c’) correspond to the states of (b) and (c),respectively. The illustrations of (b’) and (c’) are intended to explain the reason why D decreases as a functionof time along the proceeding of crystallization, where L increases with time [3,4].

Photon Factory Activity Report 2019 #37 (2020)
presence of a special plasticizer (OMG) at 100 °C fromthe melt, where a higher order peak was observed in theLorentz-corrected SAXS profiles. Furthermore, as shownin Figure 5b, the initial average thickness of the lamella isthinner for the case of 1% addition of SFN as comparedto the neat specimen. This result may explain themechanism of acceleration of the crystallization by SFN
to lower the activation energy of the crystallization withlowering of the thickness of the critical nucleus [3]. Notehere that L = 6.3 nm is the maximum available for theD1.4 sample because of the D moiety (optical impurity),
which is distributed with every 70 repeating units of L
moieties when the homogeneous distribution of the Dmoieties in the main chain of PLLA is assumed.
Therefore, the final value of L for the D1.4 specimens issomewhat larger. This would imply the random
distribution of the D moieties in the main chain of PLLA,
further meaning that the Gaussian distribution of the Lunit repeating length gives its larger value as compared tothe average value which results in L = 6.3 nm.
The ratio L/D seems to be the crystallinity in the stacksof the lamella. To check the increasing tendency of thisratio as a function of time during the isothermal
crystallization, L/D is plotted as a function of time in
Figure 5c. It is then found that the monotonic increase in
L/D is then leveled off without the overshooting, as isseen in the plot of L in Figure 5b. Moreover, there isobserved no effect of the SFN. This is very muchcontrasted with the crystallization behavior shown inFigure 2. As shown in Figure 5c, L/D is larger than ϕWAXS(Figure 2). The reason why L/D is larger than ϕWAXS is
because the lamellar stacks are sparsely dispersed in thematrix of polymer melts, and they do not completely fill
the specimen space in the early stage. Then, the lamellar
stacks completely fill the space in the specimen in the latestage, where the L/D is almost identical to ϕWAXS. It isnoteworthy that there is no effect of SFN on the behaviorof L/D as shown in Figure 5c for the D1.4 neat andD1.4/SFN(1.0) specimens. At this moment, it is unclearthat this result is universal, irrespective of the amount ofloading. More detailed study is required. As for the D0.5
neat and D0.5/SFN(1.0) specimens, the SFN even reduces
the L/D value, which is opposed to the overall behavior
shown in Figure 2. This result may suggest that the SFN
enhances the formation of the isolated lamellae without
stacking.

4 Conclusion
The crystallization behavior of the neat PLLA andPLLA/SFN(1.0) specimens was investigated by using thetime-resolved SWAXS techniques. The results showedthat the loading SFN shortens the induction period andthe increase in the ultimate degree of crystallinity. Thecrystallization half-time was reduced, which suggests thatthe overall crystallization process was accelerated.
Furthermore, the crystallite size was very slightly reduced,
but the growth rate of the crystallites was unchanged.
The amount of the α’ and α phases were increased as afunction of time, while the fraction was constant (onlyabout 4% of α phase). The SAXS results suggest that thelamellar thickening process was accelerated, and thethickness of the initial lamella was decreased by loading
1% SFN in PLLA. This result clearly indicates that thethickness of the critical nucleus is reduced by the SFN.
References[1] Song, P.; Sang, L.; Jin, C.; Wei, Z., Polymer, 134, 163(2018).
[2] P. T. N. Diep, H. Takagi, N. Shimizu, N. Igarashi, S.
Sasaki, S. Sakurai, J. Fiber Sci. Technol., 75, 99(2019).
[3] A. K. Pandey, P. T. N. Diep, R. Patwa, V. Katiyar, S.
Sasaki, S. Sakurai, in Advances in SustainablePolymers
Synthesis,
Fabrication
andCharacterization (Editers: V. Katiyar, A. Kumar, N.
Mulchandani), Springer Nature Singapore, Chapter 14(2019). DOI: 10.1007/978-981-15-1251-3[4] A. K. Pandey, V. Katiyar, H. Takagi, N. Shimizu, N.
Igarashi, S. Sasaki, S. Sakurai, Materials, 12, 1872
(2019).
Research Achievements
1. Amit Kumar Pandey, Best Oral English Presentation
Awards of the Society of Rubber Science andTechnology, Japan (2019).
* shin@kit.ac.jp