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Molar Masses and Molar Mass Distributions of Chitin and Acid-Hydrolyzed Chitin Ryunosuke Funahashi, Yuko Ono, Zi-Dong Qi, Tsuguyuki Saito, and Akira Isogai Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01413 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017
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Molar Masses and Molar Mass Distributions of Chitin
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and Acid-Hydrolyzed Chitin
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Ryunosuke Funahashi, Yuko Ono, Zi-Dong Qi, Tsuguyuki Saito, and Akira Isogai*
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Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, The
7
University of Tokyo, Tokyo 113-8657, Japan
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ABSTRACT: Never-dried and dried crab shell chitin and squid pen chitin samples were
10
acid-hydrolyzed in 1M HCl at 85 °C for up to 2 h. The crystallinities, crystal sizes, and degrees
11
of N-acetylation of the acid-hydrolyzed chitin samples are almost unchanged the same before
12
and after acid hydrolysis. The original and acid-hydrolyzed chitin samples were dissolved in
13
8% (w/w) lithium chloride/N,N,-dimethylacetamide and the solutions were subjected to
14
size-exclusion chromatography with multi-angle laser-light scattering analysis to determine
15
their molar masses and molar mass distributions. The molar mass of each chitin sample
16
decreases with increasing acid hydrolysis time, and the weight-average degree of
17
polymerization (DPw) becomes constant after acid hydrolysis for 0.5–2 h. However, the DPw
18
values of the chitin samples after acid hydrolysis for 2 h (DPw-2h) are different: never-dried
19
squid pen chitin has the highest DPw-2h of 1530, whereas the DPw-2h values of other chitin
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samples are in the range 220–410.
21 22
KEYWORDS:
Chitin,
acid
hydrolysis,
LiCl/N,N-dimethylacetamide,
size-exclusion
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chromatography with multi-angle laser-light scattering, leveling-off degree of polymerization
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INTRODUCTION
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Chitin is the second most abundant biopolymer after cellulose. It consists of
27
N-acetyl-D-glucosamine and D-glucosamine units with various molar ratios linked by
28
β-(1→4)-glycoside bonds.1 The degrees of N-acetylation of commercial chitin is at least ~0.9,
29
depending on the isolation and purification conditions. Crab shell and squid pen chitin form
30
nanosized fibrils in living bodies, similar to plant celluloses, in which fully extended chitin
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molecules are regularly packed in each fibril, forming multiple intra- and inter-molecular
32
hydrogen bonds and hydrophobic interactions.1–3 Based on the fibril structures of native chitin,
33
chitin nanofibers and nanocrystals (i.e., nanosized chitins) are prepared by mechanical
34
disintegration in water under various conditions with or without chemical pretreatment to
35
improve the nanofibrillation efficiencies and yields. Characterization and various applications
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of nanosized chitin have been reported, similar to nanocelluloses.4–8
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Because nanosized chitin consists of chitin polymers, its molar mass and molar mass
38
distribution are significant factors that influence the properties and morphologies of nanosized
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chitins as well as end products containing nanosized chitin. There are no convenient solvents
40
for chitin to determine its viscosity-average degree of polymerization (DPv), so the DP values
41
of various chitin sources and their acid-hydrolyzed products have not been comprehensively
42
investigated. In some cases, chitin samples have been deacetylated under strongly alkaline
43
conditions to prepare acidic water-soluble chitosans The DPv values and molar mass
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parameters of aqueous chitosan solutions can be determined using a viscometer and
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size-exclusion chromatography with multi-angle laser-light scattering (SEC/MALLS),
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respectively.1,9–12 The molar mass and DP value of the original chitin samples have then been
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deduced from those obtained for the corresponding chitosans.1 However, depolymerization is
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inevitable during deacetylation of chitin to prepare chitosan under harsh alkaline conditions.13
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Moreover, the residual N-acetyl groups in chitosans often cause aggregation of chitosan
50
molecules in aqueous solution, which gives inaccurate molar masses.12
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Dilute acid hydrolysis of chitin has not been investigated in terms of the relationships
52
between the acid hydrolysis conditions and the molar mass of the hydrolyzed chitin samples
53
because of the lack of suitable and convenient solvents to determine the molar masses of
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acid-hydrolyzed chitin samples. However, dilute acid hydrolysis of plant celluloses to prepare
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microcrystalline celluloses has been extensively studied, and microcrystalline celluloses with
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DPv values of 200–300 have been obtained.14,15 To determine the DPv values of
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acid-hydrolyzed celluloses, 0.5M copper ethylenediamine (cuen) solution is commonly used as
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the cellulose solvent, although cuen cannot dissolve chitin.15
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Chitin is soluble in 8% (w/w) lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) at
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room temperature (RT) without any pretreatment,13,16–18 whereas pretreatment is always
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required for complete dissolution of celluloses in 8% (w/w) LiCl/DMAc.19 SEC analysis of
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chitin using LiCl/DMAc solvent and a series of poly(styrene) standards for SEC analysis has
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been performed,13 although it is difficult to determine whether all of the chitin molecules are
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dispersed at the individual molecular level without any molecular aggregation. In a previous
65
study, we obtained the accurate specific refractive index increment (dn/dc) of chitin, which is
66
required for calculation of molar mass parameters by SEC/MALLS, using a similar method to
67
those used for various celluloses.18
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In this study, we used never-dried and dried crab shell chitin and squid pen chitin as starting
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materials. Crab shell chitin and squid pen chitin have different crystalline allomorphs: crab 3 ACS Paragon Plus Environment
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shells are α-chitin with antiparallel chain packing while squid pens are β-chitin with parallel
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chain packing.1–3 The never-dried and dried chitin samples were subjected to dilute acid
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hydrolysis, and the hydrolyzed products and original chitin samples were dissolved in 8%
73
(w/w) LiCl/DMAc. The solutions were analyzed by SEC/MALLS to determine the molar mass
74
parameters of the original and acid-hydrolyzed chitin samples.
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MATERIALS AND METHODS
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Materials. Never-dried crab (Paralithodes camtschaticus) shell was provided by Nippon
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Kayaku Food Techno Co. (Gunma, Japan). Never-dried squid pen (Sepioteuthis lessoniana) was
79
obtained from local sea food stores. The samples were cut into small pieces of ~3 cm in length
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using scissors and chitin was isolated and purified from these samples according to previously
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described procedures.20 In brief, the crab shell and squid pen samples were sequentially soaked
82
in acetone/water (9:1 v/v), 1 M HCl, 1 M NaOH, and 0.3% (w/v) NaClO2. The isolated chitin
83
was further deproteinized with 10% (w/w) NaOH at RT for 12 h. The purified crab shell and
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squid pen chitin samples were not dried and stored at 4 °C before use. Dried crab shell and squid
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pen chitin powders were provided by Dainichiseika Color & Chemicals Co., Ltd. (Tokyo,
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Japan). The chitin powders were soaked in 1 M HCl at RT for 6 h followed by thorough washing
87
with water by filtration. All the chitin samples used in this study contained no calcium, when
88
determined using an X-ray fluorescence analyzer. No residual protein contents were determined
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for the chitin samples. All of the chemicals and solvents were laboratory grade (Wako Pure
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Chemical Ind., Osaka, Japan) and used as received.
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Dilute Acid Hydrolysis. The chitin samples (0.1 g on dry weight) were magnetically stirred
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in 1 M HCl (100 mL) at 85 °C for 0.25–2 h. The mixtures were neutralized with 1 M NaOH (100 4 ACS Paragon Plus Environment
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mL) after acid hydrolysis. The water-insoluble acid-hydrolyzed products were filtered on a glass
94
filter and thoroughly washed with water.
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SEC/MALLS Analysis. The original and acid-hydrolyzed chitin samples were soaked in
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ethanol and then centrifuged at 12,000×g for 10 min. This process was repeated three times. The
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ethanol-washed chitin samples were soaked in tert-butanol and centrifuged. This process was
98
repeated three times followed by freeze-drying. The freeze-dried chitin samples were dissolved
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in 8% (w/w) LiCl/DMAc by magnetic stirring at RT. The chitin solutions were diluted to 1%
100
(w/w) LiCl/DMAc with fresh DMAc.17 All of the chitin solutions were filtered through a 0.45,
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0.20, or 0.02 µm poly(tetrafluoroethylene) disposable membrane (Millipore, USA). SEC and
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guard columns (KD-806M and KD-G, respectively, Shodex, Tokyo, Japan) were set in the
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SEC/MALLS system and 1% (w/w) LiCl/DMAc was used as the eluent. The details of the
104
SEC/MALLS system and operation conditions are described elsewhere.18,19 A dn/dc value of
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0.138 mL/g was used for chitin in 1% (w/w) LiCl/DMAc.18 The molar mass parameters of the
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chitin samples were calculated using the included software (ASTRA VI software, Wyatt
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Technologies, USA).
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Other Analyses. The original and acid-hydrolyzed chitin samples (~0.1 g each) were pressed
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at ~600 MPa for 1 min to make disk pellets. The X-ray diffraction (XRD) patterns for the pellets
110
were recorded from 2θ = 5° to 35° in reflection mode using a X-ray diffractometer (Rigaku,
111
RINT 2000, Tokyo, Japan) with Ni-filtered Cu Kα radiation (λ = 0.1548 nm) at 40 kV and 40
112
mA. The crystallinity indices were calculated from the peak intensities of Itotal and Iam at ~19.6°
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and ~16.0°, respectively. The two diffraction peaks centered at ~9.6° and ~19.6° in the XRD
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patterns of the crab shell α-chitin were separated by deconvolution using a pseudo-Voigt
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function.21,22 The crystal sizes of the (0 2 0) and (1 1 0) planes corresponding to the diffraction
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angles at ~9.6° and ~19.6°, respectively, were measured from the full widths at half maximums
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using Scherrer’s equation.23 The diffraction peak centered at ~9.0° in the XRD patterns of the
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squid pen β-chitin samples was also separated by deconvolution, and the crystal size of the (0 1
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0) plane was measured from the full width at half maximum using Scherrer’s equation.22
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Solid-state 13C nuclear magnetic resonance (NMR) was performed to determine the degrees of
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N-acetylation of the samples.24,25 Cross-polarization/magic angle spinning (CP/MAS) 13C NMR
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measurements were performed using a JNM-ECA II 500 spectrometer (JEOL, Japan) at 125.77
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MHz for
124
performed at 298 K with the following conditions: sample spinning frequency of 15 kHz, 90°
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pulse time of 2.8 µs, and relaxation delay of 5 s. A linear CP ramp was used with a contact time
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of 2 ms. Adamantane was used as the external standard for the chemical shifts.
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C with a 3.2 mm HXMAS probe and ZrO2 rotors. All of the measurements were
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RESULTS AND DISCUSSION
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Molar Mass and Molar Mass Distributions of the Chitin Samples. All of the never-dried
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and dried crab shell and squid pen chitin samples were completely dissolved in 8% (w/w)
131
LiCl/DMAc after magnetic stirring at RT for 0.5–1 month. The concentrations of the chitin
132
samples in 1% (w/w) LiCl/DMAc were diluted from 50 to ~25 µg/mL because of the
133
extremely high viscosities of the solutions. The SEC-elution patterns and the corresponding
134
molar mass plots, and molar mass distributions of the chitin samples are shown in Figure 1.
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The molar mass parameters, such as the weight- and number-average molar masses (Mw and
136
Mn, respectively), and the corresponding DPw and DPn values of the four chitin samples
137
obtained by SEC/MALLS are listed in Table 1. The chitin samples have different SEC-elution
138
patterns and correspondingly different molar mass distributions. The dried crab shell chitin has
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a symmetric and normal molar mass distribution, whereas the molar mass distribution of
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never-dried squid pen chitin contains two peaks. The molar mass distribution of dried squid
141
pen chitin also contains two broad peaks. The molar mass plots linearly decrease with
142
increasing SEC-elution volume, showing that the chitin molecules were separated in the SEC
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column, depending on their molecular sizes. However, the molar mass plots of the four chitin
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samples are slightly different, probably because their different degrees of N-acetylation.
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107
146
106
0.015
105
0.010
104
0.005
Concentration (mg/mL)
148
Molar mass (g/mol)
147
0.020
A
149 103 5
150
6
7
8
9
10
0.000 11
Elution volume (mL) 2.0
151 152 153 154 155 156
Differential weight fraction
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B
Never-dried crab shell chitin Dried crab shell chitin Never-dried squid pen chitin Dried squid pen chitin
1.0
0.5
0.0 103
104
105
106
107
Molar mass
157
Figure 1. Relationship between the elusion volume and the SEC-elution pattern, and the
158
corresponding molar mass plots (A) and their molar mass distributions (B).
159 160
We constructed a double logarithmic plot of the molar mass against the root-means-square
161
radius of each sample. All of the chitin samples have slopes of 0.54–0.67 (Table S1), showing
162
that the chitin molecules have random-coil conformations without forming any molecular 7 ACS Paragon Plus Environment
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aggregates or branched structures in 1% (w/w) LiCl/DMAc. It is noticeable that the
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never-dried squid pen chitin has a large polydispersity (Mw/Mn) value because of the wide
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molar mass distribution, as shown in Figure 1.
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Table 1. Molar Mass Parameters of the Chitin Samples, Determined by SEC/MALLS
168
DPna
Mw/Mn
Slopeb
3770
1720
2.2
0.59
70400
560
340
1.7
0.58
907000
64700
4380
313
14.0
0.54
312000
61000
1580
309
5.1
0.67
Mw
Mn
Never-dried crab shell chitin
780000
357000
Dried crab shell chitin
116000
Never-dried squid pen chitin Dried squid pen chitin 169
a
170
sample.
171
b
172
root-mean-square radii.
DPwa
The DP value was calculated from Mw or Mn based on the degree of N-acetylation of each The slopes were obtained from double logarithmic plots of the molar masses against the
173 174
Dilute Acid Hydrolysis of the Chitin Samples. The four chitin samples were subjected to
175
dilute acid hydrolysis at 85 °C for 0.25–2 h. The solid recovery ratios of the water-insoluble
176
acid-hydrolyzed products were greater than 90% and most of the yield losses were caused by
177
handling during filtration and the washing processes. Solid-state
178
performed to determine the degrees of N-acetylation of the chitin samples before and after
179
dilute acid hydrolysis (Figure S1), and the results are listed in Table 2. After dilute acid
180
hydrolysis for 2 h, there is almost no deacetylation of chitin. This is because the acid
181
hydrolysis conditions used in this study are more moderate than those used for preparation of
182
chitin nanocrystals, where harsher acid hydrolysis conditions are used, such as treatment with 3
13
C NMR analysis was
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M HCl at 104 °C for 1 h.4–7,26
184 185
Table 2. Degrees of N-Acetylation of the Chitin Samples Before and After Dilute Acid
186
Hydrolysis for 2 h
187
Never-dried crab shell chitin
Dried crab shell chitin
Never-dried squid pen chitin
Dried squid pen chitin
Before acid hydrolysis
0.97
0.95
0.95
0.87
After acid hydrolysis for 2 h
0.98
0.95
0.92
0.88
188 189
The XRD patterns of the original and acid-hydrolyzed chitin samples are shown in Figure S2.
190
The crystallinity indices and crystal sizes were calculated from the XRD patterns, and the
191
crystal sizes and crystallinity indices of the acid-hydrolyzed chitin samples are shown in Figure
192
2. The crystallinity indices of the four chitin samples are almost the same before and after
193
dilute acid hydrolysis. Although the crystallinity indices of the original squid pen chitin
194
samples are ~0.7 and lower than those of the crab shell chitin samples (>0.95), there is no
195
significant change in the crystallinity indices of the acid-hydrolyzed squid pen chitin samples
196
compared with the original samples. This result indicates that for the squid pen chitin samples,
197
there is almost no removal of the disordered regions during dilute acid hydrolysis under the
198
conditions used in this study.
199
The crystal sizes of the (0 2 0) and (1 1 0) planes of the never-dried crab shell samples and
200
the (0 1 0) plane of the two squid pen chitin samples are almost unchanged with dilute acid
201
hydrolysis. The crystal size of the (0 2 0) plane of the dried crab shell chitin sample increases
202
by 1 nm with hydrolysis for 0.25 h. This size is then constant during acid hydrolysis for 0.25–2
203
h. It is not clear why the (0 2 0) plane size of the dried crab shell chitin sample increases in the 9 ACS Paragon Plus Environment
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initial stage of acid hydrolysis. These solid-state
205
chemical structures and crystallinities/crystal sizes of the original chitin samples are almost
206
unchanged with dilute acid hydrolysis for 0.25–2 h under the conditions used in this study.
207
C NMR and XRD results show that the
Dried crab shell chitin
Never-dried crab shell chitin 12
1.0
12
1.0
208
0.4 4
213
0 0.5
214
0.4 4
0.0 0.0
Crystallinity index Crystal size of the (0 1 0) plane
0.2
0.0 0.0
0.5
1.0
1.5
2.0
Acid hydrolysis time (h)
222
Crystal size (nm)
0.4 4
0
2.0
1.0 Crystallinity index Crystal size of the (0 1 0) plane
10
0.8
6
221
1.5
Dried squid pen chitin
0.6
2
1.0
12
1.0
8
220
0.5
Acid hydrolysis time (h)
Never-dried squid pen chitin 10
0.2
Crystallinity index Crystal size of the (0 2 0) plane Crystal size of the (1 1 0) plane
0
2.0
12
216
219
1.5
6
Acid hydrolysis time (h)
215
218
1.0
0.6
2
0.0 0.0
217
0.2
Crystallinity index Crystal size of the (0 2 0) plane Crystal size of the (1 1 0) plane
2
8
Crystallinity index
6
Crystal size (nm)
0.6
0.8
0.8
8 0.6 6 0.4 4
Crystallinity index
212
8
Crystallinity index
211
10
0.8
Crystallinity index
210
Crystal size (nm)
10
209
Crystal size (nm)
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0.2
2 0
0.0 0.0
0.5
1.0
1.5
2.0
Acid hydrolysis time (h)
223
Figure 2. Crystal size and crystallinity index of the chitin samples with dilute acid hydrolysis
224
for 0.25–2 h.
225 226
SEC/MALLS Analysis of the Acid-Hydrolyzed Chitin Samples. The SEC-elution patterns
227
and the corresponding molar mass plots of the original and acid-hydrolyzed chitin samples are 10 ACS Paragon Plus Environment
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shown in Figure S3, and their Mw, Mn, and Mw/Mn values are listed in Table 3. The average
229
ratio of the mass of chitins detected by the RI detector in the SEC/MALLS analysis to that
230
used in preparation of chitin solutions in 8% LiCl/DMAc for chitin samples shown in Table 3
231
was 96.3 ± 6.6%. Thus, more than 90 wt % of chitin samples using in preparation of their 8%
232
LiCl/DMAc solutions were recovered and detected by SEC/MALLS analysis. From the results
233
in Table 3, changes in the DPw values of the chitin samples during dilute acid hydrolysis are
234
shown in Figure 3.
235 236
Table 3. Mw, Mn, and Mw/Mn Values of the Chitin Samples During Acid Hydrolysis for 0.25–2
237
h Determined by SEC/MALLS
238 Acid hydrolysis time (h)
Never-dried crab shell chitin Mw
Mn
Dried crab shell chitin
Mw/Mn
Mw
Mn
Mw/Mn
Never-dried squid pen chitin Mw
Mn
Mw/Mn
Dried squid pen chitin Mw
Mn
Mw/Mn
0
780000 357000
2.2
116000 70400
1.6
907000 64700
14.0
312000 61000
5.1
0.25
116000 71900
1.6
91400 62000
1.5
528000 26200
20.2
77600 16900
4.6
0.5
100000 57200
1.7
70600 45900
1.5
345000 22500
15.3
34800
9200
3.8
1
86300
54400
1.6
63300 44100
1.4
342000 24400
14.0
39000
8200
4.7
2
85000
55500
1.5
65600 50200
1.3
317000 28100
11.3
43500
9200
4.7
239 240
In Figure 3, the DPw values decrease with increasing acid hydrolysis time from 0 to 0.5 h.
241
Because the DPw values of the chitin samples are almost constant after dilute acid hydrolysis
242
for 1–2 h, the chitin samples seem to have so-called leveling-off DP values (LODPs), although
243
the DPw values after acid hydrolysis for 2 h are different for the four chitin samples (Figure 3).
244
The never-dried squid pen chitin sample has a remarkably high DPw of ~1530, whereas the
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245
other chitin samples have DPw values in the range 220–410. The latter DPw values after acid
246
hydrolysis for 2 h are similar to those of plant celluloses after dilute acid hydrolysis.
247 248 5000 Never-dried crab shell chitin Dried crab shell chitin Never-dried squid pen chitin Dried squid pen chitin
4000
249
3000
250 251 252
2000
DPw
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1000
500
253 0 0.0
254 255
0.5
1.0
1.5
2.0
Acid hydrolysis time (h)
Figure 3. Changes in the DPw values of the chitin samples with acid hydrolysis time.
256 257
The reason for formation of microcrystalline celluloses with constant DPv values of 200–300
258
by dilute acid hydrolysis of plant celluloses is thought to be based on the alternating
259
distributions of disordered/crystalline regions along each cellulose microfibril.14,15 The molar
260
mass distributions of the original and acid-hydrolyzed chitin samples are shown in Figure 4.
261
The acid-hydrolyzed crab shell chitin samples have LODPs similar to those of acid-hydrolyzed
262
plant celluloses because the acid-hydrolyzed samples have sufficiently narrow molar mass
263
distributions, or small Mw/Mn values of 1.3–1.7. However, after dilute acid hydrolysis for 0.5–2
264
h, the never-dried and dried squid pen chitin samples have wide molar mass distributions, or
265
large Mw/Mn values of 11–15 and 4–5, respectively. Although the DPw values of the squid pen
266
chitin samples become constant during dilute acid hydrolysis (Table 3 and Figure 3), the squid
267
pen chitin samples have no LODPs, especially the never-dried squid pen chitin sample.
268 12 ACS Paragon Plus Environment
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269 270
273 274 275 276
0h 0.25 h 0.5 h 1h 2h
2.0
Differetial weight fraction
272
2.5 Acid hydrolysis time
Differetial weight fraction
271
Dried crab shell chitin
Never-dried crab shell chitin 2.5
1.5
1.0
0.5
0.0 10 3
10 4
277
10 5
2.0
1.5
1.0
0.5
0.0 10 3
10 6
278
2.5
10 4
10 5
10 6
Molar mass (g/mol)
Molar mass (g/mol)
Dried squid pen chitin
Never-dried squid pen chitin
2.5
280 281 282 283
Differetial weight fraction
279
Differetial weight fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
2.0
1.5
1.0
0.5
2.0
1.5
1.0
0.5
284 285 286 287
0.0 10 3
10 4
10 5
10 6
0.0 10 3
Molar mass (g/mol)
10 4
10 5
10 6
Molar mass (g/mol)
Figure 4. Molar mass distributions of the original and acid-hydrolyzed chitin samples.
288 289
According to the hypothesis of formation of microcrystalline plant celluloses, the never-dried
290
and dried crab shell chitin samples may have disordered regions periodically located along
291
each chitin fibril similar to plant celluloses. In contrast, the never-dried squid pen chitin sample
292
may have much longer intervals between two disordered regions or longer crystalline region 13 ACS Paragon Plus Environment
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293
lengths. However, further investigation of the distributions of the disordered regions of native
294
chitin is required to obtain more detailed distributions and structures of the disordered regions
295
and to make clear the formation mechanism of the disordered regions during biosynthesis
296
and/or isolation/purification processes, as with plant celluloses.14,27,28 As a result, both
297
never-dried and dried crab shell chitin samples may be convertible to microcrystalline chitin by
298
dilute acid hydrolysis at high temperature. The obtained microcrystalline chitin may be used as
299
a functional additive in the food and pharmaceutical fields, similar to microcrystalline plant
300
celluloses.
301
Kinetics of Dilute Acid Hydrolysis of Chitin Samples. The depolymerization rate
302
constants, or chitin kinetics, during dilute acid hydrolysis were determined according to the
303
heterogeneous acid hydrolysis model, in which disordered/crystalline two phase structures are
304
assumed to exist in native chitin, similar to plant celluloses.29 From the data shown in Table 3
305
and Figure 3, the (1/DPwt ‒ 1/DPw0) values are plotted with respect to the acid-hydrolysis time,
306
where DPwt and DPw0 are the DPw values after acid hydrolysis for t and 0 h, respectively
307
(Figure S4). The depolymerization rate constants k were then obtained from curves fitted to the
308
experimental data.29
309
The results are shown in Figure 5 together with the DPw values after dilute acid hydrolysis
310
for 2 h. Never-dried crab shell chitin has the highest k value of ~3.5 h−1 and the other chitin
311
samples have values in the range 1.5–2 h−1, although the crab shell chitin samples have higher
312
crystallinity indices than the squid pen chitin samples (Figure 2). Therefore, there is no direct
313
relationship between the amount of disordered regions in the chitin samples determined by
314
XRD and the depolymerization rate constant. The detailed distributions and structures of the
315
disordered regions susceptible to dilute acid hydrolysis are probably different for crab shell and
316
squid pen chitin samples. The k values of the chitin samples in Figure 5 are higher than those 14 ACS Paragon Plus Environment
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of wood celluloses under similar acid hydrolysis conditions (0.8–1 h−1).15 indicating that chitin
318
molecules are more susceptible to depolymerization during dilute acid hydrolysis.
319 320 321 322 323
1600
4
1200
3
800
2
400
1
0
324
Ne ve r-d rie d
325
Dr ie d c ra bs
c ra bs
he ll
Ne ve r-d ri
he ll
ch itin
ch itin
Dr ie d ed
Sq
uid p
Depolymerization rate content (1/h)
317
DPw after acid hydrolysis for 2 h
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
0 sq en
uid p
en
ch itin
ch itin
326
Figure 5. DPw values of the chitin samples after dilute acid hydrolysis for 2 h and the
327
depolymerization rate constants of the chitin samples during dilute acid hydrolysis.
328 329
CONCLUSION
330
In this study, we dissolved crab shell chitin, squid pen chitin, and their acid-hydrolyzed
331
products in 8% (w/w) LiCl/DMAc and then determined their molar mass parameters by
332
SEC/MALLS using 1% (w/w) LiCl/DMAc as the solvent and SEC eluent. Under the acid
333
hydrolysis conditions used in this study, neither deacetylation nor changes in the
334
crystallinity/crystal size occur in the chitin samples. Dried and never-dried crab shell chitin
335
samples show typical LODP patterns in dilute acid hydrolysis. After acid hydrolysis for 0.5–2
336
h, acid-hydrolyzed dried and never-dried crab shell chitin samples have almost constant DPw
337
values of ~220 and ~410, respectively, with narrow DP distributions. In contrast, both
338
acid-hydrolyzed never-dried and dried squid pen chitin samples have wide molar mass
339
distributions and no LODPs after dilute acid hydrolysis. Thus, the distributions of the
15 ACS Paragon Plus Environment
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340
disordered regions susceptible to dilute acid hydrolysis and their structures may be different for
341
crab shell and squid pen chitin samples. Never-dried crab shell chitin has the highest
342
depolymerization rate constant (~3.5 h−1). The depolymerization rate constants of other chitin
343
samples are lower and in the range 1.5–2 h−1. These chitin depolymerization constants are
344
higher than those of plant celluloses under similar acid hydrolysis conditions (0.8–1 h−1). Thus,
345
the disordered regions in the chitin samples are more susceptible to depolymerization under
346
acid hydrolysis than those of plant celluloses. The results obtained in this study suggest that
347
microcrystalline chitin can be prepared from crab shell chitin and used as a functional additive
348
in the food and pharmaceutical fields, similar to microcrystalline celluloses.
349 350
ASSOCIATED CONTENT
351
Supporting Information
352
SEC elution patterns of the original and acid-hydrolyzed chitin samples, kinetic plots of
353
depolymerization of the chitin samples during acid hydrolysis, solid-state
354
and XRD patterns of the acid-hydrolyzed chitin samples.
13
C NMR spectra,
355 356
AUTHOR INFORMATION
357
Corresponding Author
358
*Tel: +81 3 5841 5538. E-mail:
[email protected].
359 360
Notes
361
The authors declare no competing financial interest.
362 363
ACKNOWLEDGMENTS 16 ACS Paragon Plus Environment
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Biomacromolecules
364
This research was supported by Core Research for Evolutional Science and Technology
365
(CREST, Grant number JPMJCR13B2) of the Japan Science and Technology Agency (JST). We
366
thank Tim Cooper, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of
367
this manuscript.
368 369
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Table of Contents
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Molar Masses and Molar Mass Distributions of Chitin and
445
Acid-Hydrolyzed Chitin
446 447
Ryunosuke Funahashi, Yuko Ono, Zi-Dong Qi, Tsuguyuki Saito, and Akira Isogai*
448 449 5000
450 451
Chitin Dilute acid hydrolysis
452 453
Acid-hydrolyzed Chitin
Degree of polymerization
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Never-dried crab shell chitin Dried crab shell chitin Never-dried squid pen chitin Dried squid pen chitin
4000 3000 2000 1000
Leveling-off degree of polymerization? 500
0 XRD, 13C NMR Dissolution in 0.0 0.5 analyses LiC/DMAc SEC/MALLS analysis to determine molecular mass parameters
1.0
1.5
2.0
Acid hydrolysis time (h)
454 455
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