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Journal of Molecular Structure 799 (2006) 102–110

Drying process of microcrystalline cellulose studied by attenuatedtotal reflection IR spectroscopy with two-dimensional correlation

spectroscopy and principal component analysis

Akihiko Watanabe a,b, Shigeaki Morita a, Serge Kokot c, Mika Matsubara b,Katsuhiko Fukai b, Yukihiro Ozaki a,*

a Department of Chemistry, Research Center for Near Infrared Spectroscopy, School of Science and Technology, Kwansei Gakuin University,

2-1 Gakuen, Sanda 669-1337, Japanb R&D Unit, Yasuma Co. LTD., 2100 Nakagawa, Morimachi 437-0223, Japan

c Centre for Instrumental and Developmental Chemistry, Queensland University of Technology, 2 George Street, G.P.O. BOX 2434,

Brisbane, Qld. 4001, Australia

Received 8 November 2005, received in revised form 8 December 2005; accepted 6 March 2006Available online 24 April 2006

Abstract

Molecular interactions between microcrystalline cellulose (MCC) and water were investigated by attenuated total reflection infrared(ATR/IR) spectroscopy. Moisture-content-dependent IR spectra during a drying process of wet MCC were measured. In order to dis-tinguish overlapping O–H stretching bands arising from both cellulose and water, principal component analysis (PCA) and, generalizedtwo-dimensional correlation spectroscopy (2DCOS) and second derivative analysis were applied to the obtained spectra. Four typicaldrying stages were clearly separated by PCA, and spectral variations in each stage were analyzed by 2DCOS. In the drying time rangeof 0–41 min, a decrease in the broad band around 3390 cm�1 was observed, indicating that bulk water was evaporated. In the drying timerange of 49–195 min, decreases in the bands at 3412, 3344 and 3286 cm�1 assigned to the O6H6� � �O3 0 interchain hydrogen bonds(H-bonds), the O3H3� � �O5 intrachain H-bonds and the H-bonds in Ib phase in MCC, respectively, were observed. The result of thesecond derivative analysis suggests that water molecules mainly interact with the O6H6� � �O3 0 interchain H-bonds. Thus, the H-bondingnetwork in MCC is stabilized by H-bonds between OH groups constructing O6H6� � �O3 0 interchain H-bonds and water, and the removalof the water molecules induces changes in the H-bonding network in MCC.� 2006 Elsevier B.V. All rights reserved.

Keywords: Two-dimensional correlation spectroscopy; Principal component analysis (PCA); Attenuated total reflection infrared (ATR/IR) spectroscopy;Cellulose; Hydrogen bond

1. Introduction

Cellulose, a linear 1,4-b-glucan, is the most commoncomponent found in the cell walls of higher plants [1–4].This polysaccharide constitutes the most abundant, renew-able polymer resource available today worldwide. Celluloseis used as a raw material in various fields such as textile,chemical, food and pharmaceutical industries [1–6].

0022-2860/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.molstruc.2006.03.018

* Corresponding author. Tel.: +81 79 565 8349; fax: +81 79 565 9077.E-mail address: ozaki@ksc.kwansei.ac.jp (Y. Ozaki).

Interactions between cellulose and water have been con-sidered to play important roles not only in chemistry andphysics of cellulose but also in the technology of celluloseisolation and processing such as papermaking [1,3–6].Therefore, the interactions between cellulose and waterhave been studied by using various techniques like sorptionisotherm [7], differential scanning calorimetry (DSC)[8–10], nuclear magnetic resonance (NMR) [10–12], infra-red spectroscopy [13,14] and so on [10,15,16]. Based onthe results obtained by DSC [8], Nakamura et al. suggestedthat three types of water, free water, freezing bound water

A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110 103

and nonfreezing bound water, exist in cellulose. They [9]also showed that tensile strength, elongation and elasticityof cotton cellulose vary with water content. According tothe studies of the molecular dynamics simulation by Heineret al. [15,16], it is suggested that hydroxymethyl conforma-tion on the cellulose Ib–water surface is a mixture ofgauche–gauche and gauche–trans conformations. On theother hand, from the 13C NMR study of various crystallineglucose and glucosides, and crystalline cellulose, Newmanand Davidson [12] proposed that the hydroxymethylconformation on the cellulose–water interface assumesgauche–gauche conformation. According to the IR studyof the H/D exchange process of cellulose Ib by Marechaland Chanzy [14], water molecules do not penetrate thehydrogen bonds (H-bonds) network of cellulose Ib exceptat crystal surfaces and interfaces where the crystalline net-work is interrupted. In this way, it has been revealed thatwater molecules interacting with cellulose influence thecellulose property and cellulose conformation. Nativecellulose, such as wood and cotton cellulose, contain cellu-lose I form in the crystal structure [1,2,17–21]. Both interand intrachain H-bonds are involved in cellulose I andthese are considered to be important for the properties ofnative cellulose [17–21]. However, despite the importanceof the H-bonds in cellulose I, the influences of water ontheir structural changes have not been explored sufficiently.

Infrared spectroscopy is sensitive to the conformationand local molecular environment of molecules includingbiopolymers. It has been employed to elucidate the struc-ture of cellulose [14,22–28]. There is much informationabout the inter and intrachain H-bonds in the OH stretch-ing region of an infrared spectrum of cellulose. IR spectros-copy has also been used for the studies of water structure[29–31] and dynamics of water in polymers [31–34]. Despiteits great ability mentioned above, however, IR spectrosco-py has rarely been used for the investigation of interactionbetween cellulose and water [14]. Since informative O–Hstretching bands, arising from inter and intrachainH-bonded cellulose, such as due to cellulose H-bondedby water, water H-bonded by cellulose and bulk water,are severely overlapped, it is not straightforward to distin-guish them. In other words, much information about cellu-lose–water interaction still remains hidden in the IR spectraof cellulose–water system. Therefore, one must look for apowerful spectral analysis method that allows one tounravel the overlapping bands.

Generalized two-dimensional correlation spectroscopy(2DCOS) proposed by Noda in 1993 [35–39] has becomea powerful and versatile tool for elucidating subtle spectralchanges induced by an external perturbation such as tem-perature, concentration and time. 2DCOS has widely beenapplied to various spectroscopic data for two major rea-sons. One is that 2DCOS enhances apparent spectral reso-lution by deconvoluting highly overlapped bands intoindividual components. Another reason is that it givesthe information about specific order of the spectral intensi-ty changes from the analysis. We studied the thermal

behavior of cotton cellulose by temperature-dependent IRspectra combined with 2DCOS and PCA [26]. In our study,PCA showed very good ability as a pattern recognitionmethod to provide primary information about the thermaldegradation of cotton cellulose, and 2DCOS disclosed itsstructural changes in the heating process.

The purpose of the present study was to explore interac-tions between cellulose and water at the functional grouplevel. To facilitate this task, the drying process of theMCC and water mixture was investigated by IR spectros-copy with the aid of PCA, 2DCOS and second derivativeanalysis. The new insights of this article are that (1) thecombination of PCA and 2DCOS, and second derivativeanalysis show excellent ability for analysis of the compli-cated spectral changes in the O–H stretching region wheremany spectral features due to MCC and water are heavilyoverlapped, (2) as a result, they provide useful informationabout structural changes in inter and intrachain H-bondsin MCC in the drying process.

2. Experimental

2.1. Samples

Wood pulp origin MCC of Avicel FD-F20 (Lot. P353)with a mean particle size of 20 lm was purchased fromAsahi Kasei Chemicals Corp. (Tokyo, Japan) and usedwithout further purification. Water was distilled and puri-fied by an Ultrapure Water System Model CPW-101(Advantec Toyo Kaisha, Ltd, Tokyo, Japan).

2.2. Methods

A 2.5 g of MCC was mixed well with 5 ml of water. Themixture was stuck into an ATR cell and it was left underthe blowing of nitrogen gas for 3 h. Subsequently, time-de-pendent IR spectra were measured for a drying process ofthe wet MCC under the purge of nitrogen gas. All the spectrawere measured with a NEXUS 470 Fourier transform IRspectrometer (Thermo Nicolet) equipped with a liquid nitro-gen cooled mercury–cadmium–telluride (MCT) detector. A10 bounces ATR cell was made of a horizontal Germaniumcrystal with an incidence angle of 45�. A total of 256 scanswas coadded for each spectrum at a spectral resolution of4 cm�1. All the spectra were given in absorbance unit definedas�log10 (R/R0), where R and R0 are the intensities of ATRsignals from MCC on the Germanium crystal at an ambienttemperature of ca. 21 �C (±0.5 �C) and that from the samecrystal without MCC at the same temperature, respectively.ATR correction was not applied to these spectra.

2.3. Spectral analysis

The second derivative spectra were calculated by theSavitzky–Golay method [40] after the spectra were subject-ed to Kawata–Minami smoothing [41] by using homemadesoftware.

3600 3500 3400 3300 3200 3100 3000

Wavenumber /cm-1

3524

3446

3412

33443278

3223

195 min.

101 min.

14 min.

49 min.

0 min.

Seco

nd d

eriv

ativ

e

Fig. 2. Second derivative spectra of the moisture-content-dependent IRspectra of the MCC–water mixture (3600–3000 cm�1).

104 A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110

Before the calculation of PCA and 2D correlation anal-yses, all the spectra were subjected to Kawata–Minamismoothing and flattened in the 3700–2700 and 1750–1567 cm�1 regions. A total of 20 spectra over a drying timerange of 0–195 min was measured, and a total of 614 datapoints consisting the spectral ranges of 3700–2700 and1750–1567 cm�1 was picked up as spectral variables. Thedata set was y-centered and the pretreated matrix was thenimported into commercially available chemometric soft-ware (The Unscrambler, Ver.8.0, CAMO, Norway). Allthe generalized 2D correlation spectra were calculated bythe software named ‘‘2DShige’’ composed by S. Morita(Kwansei Gakuin University).

3. Results and discussion

3.1. IR spectra

Fig. 1 shows moisture-content-dependent IR spectra ofMCC–water mixture in the 3700–2800 and 1750–1567 cm�1 regions after smoothing and baseline correction.Bands in the 3700–3000 cm�1 region are assigned to theOH stretching modes of cellulose [14,22–28] and water[29–33]. The absorbance in the 3700–3000 cm�1 regiondecreases with the elapse of drying time of MCC. Bandscentered at 2900 cm�1 are due to the CH and CH2 stretch-ing modes of cellulose [22,26]. Spectral features observed inthe 1700–1600 cm�1 region are attributed to the OH bend-ing mode of water. The absorbance in the 1700–1600 cm�1

region also decreases with the progress of drying time. Theintensity of this region, which is free from the contributionfrom cellulose, can be used for an index of moisture con-tents in MCC. Both the 3700–2800 and 1750–1567 cm�1

regions are used for PCA and 2DCOS.

3.2. Second derivative analysis

Fig. 2 shows second derivative spectra of the moisture-content-dependent IR spectra (3600–3000 cm�1). Only fiverepresentative spectra are shown here. At least six peakscan be identified in this region. According to the literature[14,26], all of these peaks are assigned to the OH stretchingmodes of cellulose.

3600 3400 3200 3000 2800 1700 16000.00

0.02

0.04

0.06

Low moisture content

High moisture content

Abs

orba

nce

Wavenumber /cm-1

Fig. 1. Moisture-content-dependent IR spectra of the MCC–watermixture (0–195 min) after smoothing and baseline correction in the3700–2800 and 1750–1567 cm�1 regions.

Fig. 3 illustrates the H-bond network in cellulose I pro-posed by Gardner and Blackwell [17] on the basis of theresults from X-ray diffraction. According to their idea,there are two classes of H-bonds. The H-bonds formedbetween atoms of the same chain comprise one class whilethose connecting neighbor chains of the same sheet formthe second class. The O2H2� � �O6 and O3H3� � �O5 linksare the intrachain H-bonds while the O6H6� � �O3 0 connec-tions are the interchain H-bonds [17]. Although thedetailed structure of H-bonds network is still subjected todispute, the idea of the presence of these inter and intra-chain H-bonds has generally been accepted [14,19–21],even if the structure given by Gardner and Blackwell isnot definitive.

Band assignments of the peaks observed in the secondderivative spectra are summarized in Table 1. Theseassignments are mainly based on the assignments givenby Marechal and Chanzy [14] and Kokot et al. [26]. Thepeak at 3524 cm�1 is due to the free OH group. The peakat 3446 cm�1 is assigned to the O2H2� � �O6 intrachainH-bonds. According to the assignment by Marechal andChanzy [14], these intrachain H-bonds are weak. The peakat 3412 cm�1 is attributed to the O6H6� � �O3 0 interchainH-bonds. Notice that the assignments by Marechal et al.are different from those by Kokot et al. for an interpreta-tion of the band at 3278 cm�1. Marechal and Chanzy [14]have assigned the band at 3275 cm�1 to the O2H2� � �O6intrachain H-bonds (in Ib phase) because the band at3275 cm�1 in the polarized spectra of Ib cellulose preparedfrom Valonia microcrystals showed almost entire polariza-tion parallel to the cellulose chain axis. On the other hand,on the basis of the molecular dynamics simulation byHeiner et al. [19], Kokot et al. have assigned the band at3268 cm�1 to the O6H6� � �O3 0 interchain H-bonds (Ib)from the temperature-dependent changes in IR spectra ofcotton fiber. Recently, a detailed crystal and molecular

O5O5

O5

O5

n

O5'

O5'O5'

O5'

C1

C2C3

C4

C5O2

O6

O2

O2

O2

O3

O3

O3

O3

O6

O6

O6C6

O4

O4

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O4

C1'

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C4'

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C6'

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O4'

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O4'

O4'

O4'

O4'

O2'

O2'O2'

O3'

O3'O3'

O6'O6'

O6'

H2H3

H6

H3'

H6'

H2

H2

H2

H6

H6

H6

H3

H3

H3

H2'

H2'

H2'

H2'

H3'

H3'

H6'

H6'H6'

H3'

Fig. 3. A pattern of H-bonds in the inter- and intrachain structures of cellulose I, based on a scheme proposed by Gardner and Blackwell [17].

Table 1Band assignments for O–H stretching region in MCC

Wavenumber (cm�1)a Assignments given in this study Literature

3524 Free OH group Free OH group Marechal and Chanzy (Ib) [14]Kokot et al. (cotton) [26]

3446 O2H2� � �O6 intrachain O2H2� � �O6 intrachain Marechal and Chanzy (Ib) [14]Kokot et al. (cotton) [26]

3412 O6H6� � �O3 0 interchain O6H6� � �O3 0 interchain Marechal and Chanzy (Ib) [14]

3344 O3H3� � �O5 intrachain O3H3� � �O5 intrachain Marechal and Chanzy (Ib) [14]

3278 Ib phase O2H2� � �O6 intrachain Marechal and Chanzy (Ib) [14]O6H6� � �O3 0 interchain (Ib) Kokot et al. (cotton) [26]Ib phase Sugiyama et al. [23]

3223 Ia phase O6H6� � �O3 0 interchain (Ia) Kokot et al. (cotton) [26]Ia phase Sugiyama et al. [23]

a Wavenumbers observed in the second derivative spectrum (drying time of 0 min).

A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110 105

structure together with H-bonding systems in native cellu-lose of Ia [21] and Ib [20] has been proposed by Nishiyamaet al. According to their study, the multiple possibilities forthe H-bonds at O2 and O6 have been shown for cellulose I(both Ia and Ib). Therefore, it seems reasonable that thebands in the region of 3280–3265 cm�1 contain muchinformation about OH groups, both O2H2 and O6H6, incellulose. Sugiyama et al. [23] have assigned bands near3270 and near 3240 cm�1 to the Ib and Ia phase, respec-tively. As mentioned above, Marechal et al. have assignedthe band around 3270 cm�1 to the O2H2� � �O6 intrachainH-bonds in Ib phase while Kokot et al. have assigned tothe O6H6� � �O3 0 interchain H-bonds in Ib phase. Thus, inthe point that the band near 3270 cm�1 is attributed tothe H-bonds in Ib phase, the interpretation by Marechalet al. and that by Kokot et al. are the same. From these

reasons, we adopt the assignments for the bands in theregion of 3280–3265 cm�1 and that near 3230 cm�1 by Sug-iyama et al. in the present study.

Fig. 4 shows the peak positions identified in the secondderivative spectra as a function of the drying time. Thepeaks at 3524, 3412 and 3278 cm�1 shift to a higher wave-number with the elapse of drying time. This is probably dueto the disruption of H-bonds constructed between OHgroups in cellulose and water (OHcellulose� � �OHwater). Thehigher wavenumber shift of the peak at 3412 cm�1 is signif-icant. This result suggests that water molecules interactmore easily with the O6H6� � �O3 0 interchain H-bonds(3412 cm�1) rather than the O3H3� � �O5 intrachainH-bonds (3344 cm�1) and the H-bonds in Ib phase(3278 cm�1). In other words, water molecules mayapproach the interchain H-bonds more easily. The shift

0 50 100 150 2003522

3524

3526

3528

0 50 100 150 2003444

3446

3448

3450

3452

0 50 100 150 200341034123414341634183420

0 50 100 150 2003346

3344

3342

3340

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3282

3284

0 50 100 150 2003216

3218

3220

3222

3224

Drying time (min)

Drying time (min)

Drying time (min)

Drying time (min)

Drying time (min)

Drying time (min)

Wav

enum

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-1W

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enum

ber

/cm

-1

Wav

enum

ber

/cm-1

a

c

e

b

d

f

Fig. 4. Peak positions of the second derivative spectra (Fig. 2) as a function of the drying time, (a) near 3524 cm�1, (b) near 3446 cm�1, (c) near 3412 cm�1,(d) near 3344 cm�1, (e) near 3278 cm�1, (f) near 3223 cm�1.

-0.2 -0.1 0.0 0.1 0.2 0.3

-0.006

-0.004

-0.002

0.000

0.002

0.004

195

175155

141

101

80

7460

49

52

413330

25

14

118

6

3

0

PC2

PC1

Fig. 5. PC1 versus PC2 scores plots of the IR spectra shown in Fig. 1. Thenumber close to each symbol corresponds to the drying time in units ofminutes.

106 A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110

of the peak at 3412 cm�1 is pronounced in the drying timerange of 80–195 min. From this result, it is probable thatevaporation of adsorbed water occurs in the drying range.The peak at 3344 cm�1 shows no shift. This result suggeststhat water molecules do not influence the O3H3� � �O5interchain H-bonds directly. The ambiguous shift of thepeak at 3446 cm�1 indicates that the interaction betweenthe O2H2� � �O6 intrachain H-bonds and water is verycomplicated. The peak at 3223 cm�1, which is assigned tothe H-bonds in Ia phase, shows a shift to a lowerwavenumber with the increase in the drying time. Becausecellulose Ia structure is unstable [23], it seems that theH-bonds in Ia phase are weakened by the penetration ofwater molecules.

3.3. Principal component analysis

At the initial stage of ca. 0–60 min, a somewhat wetMCC sample was observed by the naked eye, while at thelate stage of ca. 60–195 min, almost dried powder sampleappeared. Fig. 5 shows a PCA scores plot obtained fromthe moisture-content-dependent IR spectra in the regionof 3700–2700 and 1750–1567 cm�1. The first and secondPC account for 99.0% and 1.0% of the data variance ofthe examined spectra, respectively. PC1 scores decreasewith the increase in the drying time. PC2 scores increasein the region of positive PC1 scores with the elapse of dry-ing time while PC2 scores in the region of negative PC1scores decrease. This result suggests that an interactionbetween MCC and water in the drying time range of0–41 min significantly differs from that in the drying time

range of 49–195 min. According to the result of the secondderivative analysis, the shifts of the peaks at 3412 and3223 cm�1 are remarkable in the drying time range of49–195 min (Fig. 4). Therefore, it seems that the decreasingof the PC2 scores reflects the structural change in the interand intrachain H-bonds in MCC.

The spectra in the drying time regions of 0–11 and14–41 min show (PC1 > 0, PC2 < 0) and (PC1 > 0,PC2 > 0), respectively. The spectra in the regions of49–80 and 101–195 min indicate (PC1 < 0, PC2 > 0) and(PC1 < 0, PC2 < 0), respectively. It is expected from thisresult that the spectral changes in these four drying time

A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110 107

ranges are different. In other words, the drying process ofMCC can be separated into above four drying time regions.

PC1 and PC2 loadings plots for the PCA scores plot inFig. 5 are presented in Fig. 6a and b, respectively. PC1loading plots show a positive broad region of 3700–2700 cm�1 with a peak at 3382 cm�1 and another positiveregion of 1750–1567 cm�1 with a peak at 1641 cm�1. Sincethe PC1 scores decrease with the drying time and peakpositions of the PC1 loadings are close to that of anATR/IR spectrum of water [30,31], these high positiveloadings values of PC1 are strongly related to changes withthe moisture contents in MCC. Thus, PC1 is considered asthe moisture content axis.

Although the second PC account for only 1.0% of thespectral data variance, it is expected to possess useful infor-mation about interaction between MCC and water. A posi-tive peak at 3558 cm�1 in the PC2 loadings impliesintensity changes in bands due to the free OH groups incellulose [28] or water molecules forming weakOHwater� � �OHwater H-bonds [30,32,34] in drying processof MCC. A negative peak at 3428 cm�1 suggests structuralchanges in H-bonded OH groups in cellulose [26]. Anegative shoulder near 3392 cm�1 arises from the waterconstructing strong H-bonds network (i.e., bulk water)[30,32,34] and positive peak tops at 3342 and 3268 cm�1

ascribed to the O3H3� � �O5 intrachain H-bonds and theH-bonds in Ib phase, respectively. The peaks at 3342 and3268 cm�1 imply that the behavior of bulk water differsfrom those of O3H3� � �O5 intrachain H-bonds and theH-bonds in Ib phase in the drying process. In the regionof the water O–H bending mode, a positive peak at1633 cm�1 and a negative peak at 1693 cm�1 are noticed.

3600 3400 3200 3000 2800 1700 16000.00

0.05

0.10

3600 3400 3200 3000 2800 1700 1600-0.15

0.00

0.15

Loa

ding

s (P

C2)

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16932886

3428

3558

Wavenumber /cm-1

1641

3382

Wavenumber /cm-1

1633

32683342

3315

3392

a

b

Fig. 6. (a) PC1 and (b) PC2 loadings plot of the IR spectra shown inFig. 1.

Libnau et al. [30] investigated temperature-dependentIR spectra of pure water over a temperature range of2–80 �C. They found that isosbestic points are located at1655 and 1595 cm�1 in the H–O–H bending region andthat the absorbance of water in the region above1665 cm�1 decreases with increasing temperature. Theirresults indicate that the band observed in the region above1665 cm�1 is caused by the water constructing strongOHwater� � �OHwater H-bonds (i.e., bulk water) and that theband observed between 1655 and 1595 cm�1 is attributedto the water forming weak OHwater� � �OHwater H-bonds.Therefore, it seems that the peaks at 1693 and 1633 cm�1

observed in the PC2 loadings reflect two types of waterin equilibrium; the bands at 1693 and 1633 cm�1 areassigned to the bulk water and the water constructing weakOHwater� � �OHwater H-bonds, respectively. In the samestudy by Libnau et al. [30], an isosbestic point is locatedat 3460 cm�1 in the O–H stretching region and the absor-bance in the region above 3460 cm�1 increases while thatin the region below the point decreases with increasingtemperature. Therefore, it is reasonable that the peaks at1693 and 1633 cm�1 due to water in the bending regioncorrespond to the peaks at 3558 cm�1 and around3428–3392 cm�1 in the O–H stretching region, respectively.Further details about these two bands at 1693 and1633 cm�1 are discussed in the next section. Thus, PC2may be considered as the axis representing the structuralchanges in MCC and water, especially those in theH-bonds of MCC and water.

Because of the excellent ability for pattern recognitionof PCA (PCA divides the drying process into the fourstages), it provides a convenient objective guide to selectspectra for 2DCOS. 2DCOS linked with PCA can yieldadditional information about the detailed structuralchanges in MCC and water.

3.4. 2D correlation analysis of moisture content-dependent

IR spectra

As described above, the four typical score plot regionsof (a) 0–11 (PC1 > 0, PC2 < 0), 14–41 (PC1 > 0,PC2 > 0), 49–80 (PC1 < 0, PC2 > 0), and 101–195 min(PC1 < 0, PC2 < 0) can be distinguished (Fig. 5), and it isexpected that these regions show specific spectral changes.The 2DCOS was applied to each region. Fig. 7(A) showssynchronous 2D correlation spectra generated from themoisture-content-dependent IR spectral variations in thedrying time ranges of 0–11 (a), 14–41 (b), 49–80 (c) and101–195 min (d). Fig. 7(B) depicts the corresponding auto-correlation spectra extracted from the synchronous 2D cor-relation spectra shown in Fig. 7(A). The intensities in the1750–1567 cm�1 region of the autocorrelation spectra aredisplayed by 10-fold values. In the synchronous 2D corre-lation spectrum of the time range of (a) 0–11 min, twoauto-peaks of U(3382,3382) > 0 and U(1643,1643) > 0,and one pair of cross-peaks of U(3382,1643) > 0 andU(1643, 3382) > 0 are observed, implying that both bands

3600 3200 2800 17001600

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3100

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A Ba

b

c

d

Fig. 7. (A) Synchronous 2D correlation spectra and (B) those auto-correlation spectra in the 3700–2700 and 1750–1567 cm�1 regionsconstructed from the moisture-content-dependent IR spectra. Dryingtime region of (a) 0–11 min, (b) 14–41 min, (c) 49–80 min and (d) 101–195 min.

108 A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110

around 3382 and 1643 cm�1 decrease with the increase inthe drying time. Similar tendencies are observed for thesynchronous 2D correlation spectra of other drying timeregions.

Auto-peaks around 3380–3400 cm�1 observed in thedrying time ranges of 0–11, 14–41 and 49–80 min areascribed to bulk water [30,31]. The shifts to a higher wave-number of these auto-peaks around 3380–3400 cm�1 withthe progress of the drying process indicate that theOHwater� � �OHwater H-bonds are weakened with thedecrease in the moisture content in MCC. Since the shapesof the autocorrelation spectra in the drying time ranges of0–11 and 14–41 min are similar to that of an ATR/IR spec-trum of water [30,31], it is suggested that the evaporationof bulk water is pronounced in these drying time ranges.The auto-peaks of the O–H bending mode of water around1640 cm�1 are observed for all the drying time ranges.These auto-peaks show a lower wavenumber shift withincreasing the drying time. As described in the previoussection, the bands at 1693 and 1633 cm�1 are due to bulkwater and water forming weak OHwater� � �OHwater

H-bonds, respectively. The lower wavenumber shift ofauto-peaks in the 1750–1567 cm�1 region of autocorrela-tion spectra (Fig. 7) may be caused by a change in theequilibrium of two bands of water. Thus, it is indicatednot only from the higher wavenumber shift of auto-peaksin the stretching region but also from the lower wavenum-ber shift of auto-peaks in the bending region that theOHwater� � �OHwater H-bonds are weakened with thedecrease in the moisture content in MCC. This result is

in a good agreement with the proposal that the cluster ofwater molecules are broken in the surface of cellulose mol-ecules by Goring [5], i.e., OHwater� � �OHwater H-bonds ofthe adsorbed water are weakened. Therefore, it is reason-able that the peak at 1633 cm�1 observed in the PC2 load-ings is related to the changes in adsorbed water.

Since the intensities of the bands both in the regions of3700–2700 and 1750–1567 cm�1 decrease with decreasingthe moisture content (Fig. 1), the decrease of the intensityin the negative wavenumber regions of PC2 loadings induc-es the increase in PC2 scores and the decrease of the inten-sity in the positive wavenumber regions of PC2 loadingsinduces the decrease in PC2 scores. Therefore, it may beconcluded that a loss of bulk water mainly occurs in thedrying time range of 0–41 min, while a loss of adsorbedwater starts in the drying time range of 49–195 min. Theresults that asynchronous 2D correlation spectra in thedrying ranges of 0–11 and 14–41 min show noisy spectraaround zero correlation intensities (results are not shown)make sure that evaporation of bulk water is dominant inthe drying time range of 0–41 min.

Two auto-peaks at 3396 and 3276 cm�1 are observed inthe autocorrelation spectrum of the drying time range of (c)49–80 min. One is caused by the bulk water band discussedabove and the other may be due to the H-bonds in Ibphase. Three auto-peaks at 3496, 3344 and 3286 cm�1,and two shoulder peaks near 3412 and 3223 cm�1 areobserved in the autocorrelation spectrum of the dryingtime range of (d) 101–195 min. The positions of the auto-peaks at 3412, 3344, 3286 and 3223 cm�1 almost complete-ly correspond to the positions of the peaks identified in thesecond derivative spectra (Fig. 2). The bands at 3286 and3276 cm�1 are attributed to the H-bonds in Ib phase andthe band at 3223 cm�1 is assigned to the H-bonds in Iaphase (Table 1). The bands at 3412 and 3344 cm�1 aredue to the O6H6� � �O3 0 interchain H-bonds and theO3H3� � �O5 intrachain H-bonds, respectively (Table 1).These results indicate that the disruption of the inter andintrachain H-bonds in cellulose takes place in the dryingtime region of 49–80 and 101–195 min. As discussed above,it has been suggested that the evaporation of adsorbedwater occurs and OHwater� � �OHwater H-bonds of adsorbedwater are weaker in this drying time region. It is expectedthat the band of adsorbed water in cellulose shifts to ahigher wavenumber than that of bulk water (3382–3396 cm�1). Therefore, the auto-peak at 3496 cm�1 indi-cates the evaporation of the adsorbed water in cellulose.In summary, (1) the auto-peaks presented in the bendingregion shift to lower wavenumbers in the drying timeranges of 49–80 and 101–195 min and this indicates that theevaporation of adsorbed water occurs in these drying timesranges, (2) the auto-peaks due to the breaking of the interand intrachain H-bonds in cellulose are observed in thisdrying time range. These results suggest that the structuralchanges in MCC occur with the loss of adsorbed water inthe drying time of 49–195 min, especially after 101 min.In other words, it is suggested that the evaporation of

A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110 109

water embedded in cellulose induces breaking of the interand intrachain H-bonds of cellulose. On the basis of IRand Raman studies of hydrothermally degraded cellulose,Proniewicz et al. [42] revealed that the removal of waterfrom cellulose is an important factor for accelerating theaging process of paper. Therefore, it seems reasonable thatadsorbed water in cellulose plays a critical role for the sta-bilization of H-bonds in cellulose. Based on the result ofthe second derivative analysis (Fig. 4), water moleculesmainly interact with O6H6� � �O3 0 interchain H-bonds,whereas they do not influence O3H3� � �O5 intrachainH-bonds directly. It is well known that cooperativity ofH-bonds plays a key role in the cellulose conformation[14,17–21,26]; the formation of one H-bond in H-bondedchains enhances the formation of another H-bond. Theschematic visualization of the cellulose chains in Fig. 3clearly demonstrates the existence of the cooperative effectin the H-bonding network of cellulose. Therefore, it may beconcluded that the disruptions of the H-bonds in cellulosearise in the drying process as follows, (1) the evaporation ofwater molecules induces the breaking of O6H6� � �O3 0 inter-chain H-bonds (3412 cm�1), (2) the breaking of theO6H6� � �O3 0 interchain H-bonds induces the disruptionsof the O3H3� � �O5 intrachain H-bonds (3344 cm�1) andthe H-bonds in Ib phase (3278 cm�1) immediately. Accord-ing to the IR study of cellulose Ib in H/D exchange processby Marechal and Chanzy [14], water molecules do not pen-etrate the hydrogen network of cellulose Ib except at crys-tal surfaces and interfaces where the crystalline network isinterrupted. It is suggested that the structural changesdescribed above mainly take place at the crystal surfaceof MCC. No negative peak that indicates the constitutionof free OH groups in cellulose with the disruption ofH-bonds can be observed in the 2D correlation spectra inFig. 7(A-c) and (A-d). According to the literature[14,26,28], the bands correlated to the free OH groups in

170031003500

-4.0

4.0

3459

3436 -4.0

2.0

170031003500

3500

3100

1700

1700

3100

3500

0.0

0.0

Wavenumber /cm-1

Ψ/ ×

10-8

Wavenumber /cm-1

Ψ/ ×

10-8

Wav

enum

ber

/cm

-1W

aven

umbe

r /c

m -1

Aa

b

Fig. 8. (A) Asynchronous 2D correlation spectra in the 3700–2700 and 1750spectra. Drying time region of (a) 49–80 min and (b) 101–195 min. (B) Horizorespective asynchronous correlation spectrum.

cellulose are observed approximately in the 3550–3450 cm�1 region. This is because the increase in the absor-bance attributed to the free OH groups is likely to bemasked by the decrease in the absorbance with the lossof water that has a broad and relatively intense band near3496 cm�1.

Fig. 8(A) shows asynchronous 2D correlation spectraconstructed from the moisture-content-dependent IR spec-tral variations in the drying time ranges of 49–80 (a) and101–195 (b) min, and Fig. 8(B) depicts slice spectra extract-ed from the asynchronous spectra at 3436 and 3459 cm�1,respectively. The negative correlation areas in the 2D cor-relation spectra are given by gray color. Although, the slicespectrum extracted at 3436 cm�1 is somewhat noisy, thepeak at 3436 cm�1 has asynchronicity with the peaksappearing between 3583–3570 and 3350–3000 cm�1. Theslice position at 3436 cm�1 corresponds to the positionfor the O2H2� � �O6 intrachain H-bonds in cellulose[14,26]. A negative area around 3583–3570 cm�1 mayreflect the changes in free OH groups in cellulose or thatin water molecules forming weak OHwater� � �OHwater

H-bonds network. Negative peaks at 3344 and 3236 cm�1

are correlated to the O3H3� � �O5 intrachain H-bonds [14]and the H-bonds in Ia phase [23] in cellulose, respectively.In the range of 101–195 min, the slice spectrum extractedat 3459 cm�1 has a positive peak at 1699 cm�1 and negativepeaks at 3529, 3340, 3282, 3225 and 1637 cm�1

(Fig. 8(B-b)). The slice point at 3459 cm�1 is attributedto the position for the O2H2� � �O6 intrachain H-bonds incellulose [14,26]. The negative peaks at 3340, 3382 and3225 cm�1 are due to the O3H3� � �O5 intrachain H-bonds,the H-bonds in Ib phase and the H-bonds in Ia phase,respectively. Based on the fundamental rule of an asyn-chronous spectrum [35–37], the spectral intensity changeat 3436 cm�1 occurs before those at 3344 and 3286 cm�1

in the drying time range of 49–80 min, and the intensity

Slice@3459 cm-1

Slice@3436 cm-1

16001700280032003600

1637

1699

322532823340

3344

3571

3583

16001700280032003600

Wavenumber /cm-1

Wavenumber /cm-1

3529

3236

B

–1567 cm�1 regions constructed from the moisture-content-dependent IRntal slice spectra extracted along (a) 3436 cm�1 and (b) 3459 cm�1 in the

110 A. Watanabe et al. / Journal of Molecular Structure 799 (2006) 102–110

change in the band at 3459 cm�1 appears before that at3340 cm�1 in the drying time range of 101–195 min. Theseresults imply that the structural change in the O2H2� � �O6intrachain H-bonds proceeds faster than that of the otherH-bonded structures such as the O3H3� � �O5 intrachianH-bonds and H-bonds in Ib phase. In addition, the slicespectrum in the drying time range of 101–195 min indicatesthat the decrease in the absorbance at 1637 cm�1 takesplace later than the decrease in that at 1699 cm�1. This isin good agreement with the interpretation of the PCAresult that the band around 1637–1633 cm�1 is related tothe adsorbed water.

4. Conclusion

The present study has demonstrated that moisture-de-pendent ATR/IR spectra of MCC–water mixture com-bined with PCA and 2D correlation analyses, and theirsecond derivative analysis can provide detailed informationabout the interactions in the microcrystalline cellulose. 2Dcorrelation spectra linked with PCA were able to explorethe structural changes in cellulose and water in the fourstages of drying process. In the first two drying stages of0–11 and 14–41 min, respectively, evaporation of bulkwater is dominant. The H-bonding network of bulk waterin MCC is weakened with the evaporation. In the dryingstages at 49–80 and 101–195 min, the loss of adsorbedwater in MCC occurs. Especially, in the last drying stageof 101–195 min, the disruptions of the O6H6� � �O3 0 inter-chain H-bonds, O3H3� � �O5 intrachain H-bonds, andH-bonds in Ib and Ia phases are distinctly observed. Fromthe second derivative analysis in the O–H stretching region,it is suggested that water molecules mainly interact with theO6H6� � �O3 0 interchain H-bonds in MCC. These resultssuggest that the H-bonding network of MCC is stabilizedby H-bonds forming between water molecules and OHgroups constructing interchain H-bonds in MCC, and thatthe loss of the adsorbed water causes changes in structuralfeatures of MCC.

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