Molar Masses and Molar Mass Distributions of Chitin and Acid

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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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Biomacromolecules

1

Molar Masses and Molar Mass Distributions of Chitin

2

and Acid-Hydrolyzed Chitin

3 4

Ryunosuke Funahashi, Yuko Ono, Zi-Dong Qi, Tsuguyuki Saito, and Akira Isogai*

5 6

Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, The

7

University of Tokyo, Tokyo 113-8657, Japan

8 9

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.

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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

24 25

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

31

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,

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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

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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

44

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

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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

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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

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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

71

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

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obtained from local sea food stores. The samples were cut into small pieces of ~3 cm in length

80

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

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in acetone/water (9:1 v/v), 1 M HCl, 1 M NaOH, and 0.3% (w/v) NaClO2. The isolated chitin

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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

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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

99

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

107

Technologies, USA).

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Other Analyses. The original and acid-hydrolyzed chitin samples (~0.1 g each) were pressed

109

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,

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RINT 2000, Tokyo, Japan) with Ni-filtered Cu Kα radiation (λ = 0.1548 nm) at 40 kV and 40

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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

115

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

122

measurements were performed using a JNM-ECA II 500 spectrometer (JEOL, Japan) at 125.77

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MHz for

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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

130

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.

135

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

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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

143

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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Biomacromolecules

1.5

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

164

never-dried squid pen chitin has a large polydispersity (Mw/Mn) value because of the wide

165

molar mass distribution, as shown in Figure 1.

166 167

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


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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13

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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


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


0.6

2

1.0

12

1.0

8

220

0.5


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


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


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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228

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


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


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.

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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


271


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)


Never-dried squid pen chitin

2.5

280 281 282 283


279


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

Biomacromolecules

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

Page 14 of 21

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

Page 15 of 21

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


Biomacromolecules

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

Page 16 of 21

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

443 444

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

Biomacromolecules

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


454 455


Molar Masses and Molar Mass Distributions of Chitin and Acid

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