Acetylation and characterization of xylan from hardwood kraft pulp.pdf

Carbohydrate Polymers 87 (2012) 170– 176

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Carbohydrate Polymers

j ourna l home pag e: www.elsevier.com/locate/carbpol

Acetylation and characterization of xylan from hardwood kraft pulp

Noreen Grace V. Fundador a , b , Yukiko Enomoto-Rogers a , Akio Takemura a , Tadahisa Iwata a , ∗

a Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo

113-8657, Japan

b College of Science and Mathematics, University of the Philippines Mindanao, Mintal, Tugbok Dist., Davao City 8022, Philippines

a r t

i c

l e

i n f o

a b s t r a c t

Article history:

Received 29 March 2011

Received in revised form 12 July 2011

Accepted 20 July 2011

Available online 28 July 2011

Alkaline treatment of eucalyptus hardwood kraft pulp with 10% NaOH yielded 6–8% xylan. The acetylation

of the extracted xylan was carried in DMAC/LiCl/pyridine system to obtain a series of xylan acetates with

different degrees of substitution (DS). Structure elucidation of xylan and xylan acetate was obtained by

1 H and 13 C NMR spectroscopy and other homonuclear and heteronuclear 2D-NMR techniques. Inverse-

gated 13 C NMR was employed to determine the DS of xylan acetate. Furthermore, results also revealed

equal reactivities at the C-2 and C-3 positions of xylan towards acetylation. Thermal stability, solubility

behavior and nanoﬁber formation of xylan acetate were inﬂuenced by its DS values. The mechanical

properties of xylan acetate propionate were also investigated.

Keywords:

Hardwood xylan

Xylan acetate

Xylan acetate propionate

Films

Nanoﬁbers

1. Introduction

Xylan can be easily extracted by alkaline treatment with KOH,

NaOH or DMSO ( Janzon, Saake, & Puls, 2008 ). On a dry weight

basis, xylan comprises 10–15% in softwoods, 10–35% in hardwoods

and up to 40% of agricultural residues in annual plants ( Hettrich

et al., 2006 ). O-Acetyl-4-O-methyl glucuronoxylan and arabino-

4-O-methylglucoronoxylan are the main hemicelluloses found in

hardwood and in softwood, respectively ( Cacais, Silveira, & Ferreira,

2001 ). Hardwood xylan has 4-O-methylglucuronic acid (MeGlcA)

residues attached to the C-2 position of the d-xylp residues in the

main chain. Approximately 10% of the d-xylp residues in hard-

wood xylan have this substitution. In addition, 70% of the d-xylp

residues in hardwood xylan is acetylated at the C-2 and/or C-

3 positions. In contrast, for softwood xylan, 20% of the d-xylp

residues are branched with MeGlcA and 13% are branched with

Renewable materials have been the subject of much research

with the aim to replace non-biodegradable petroleum-based

polymers. Numerous studies have exploited the use of plant

polysaccharides (e.g. cellulose and starch) as substrates for chem-

ical modiﬁcation. These products were used to develop plastics,

ﬁlms and nanoﬁbers ( Amim, Kosaka, Petri, Maia, & Miranda, 2009;

Son, Youk, & Park, 2006; Yang & Montgomery, 2008 ). In addition,

they were also considered suitable for agricultural (e.g. controlled

release of fertilizers) and medical applications (e.g. hemodialysis

and blood ﬁltration) ( Aburto et al., 1999 ).

Aside from cellulose and starch, hemicelluloses also represent

one of the most abundant renewable polysaccharides found in

nature. They belong to the second largest class of lignocellulosic

materials which comprise about one-fourth to one-third of the

plant’s material ( Fang, Sun, Fowler, Tomkinson, & Hill, 1999 ). The

predominating polysaccharide belonging to this class is xylan. It

consists of d-xylp (d-xylopyranose) units in the backbone linked

-l-arabinofuranose at the C-3 position ( Coughlan & Hazlewood,

1993 ). Unlike hardwood xylan, softwood xylan is not acetylated.

The degree of polymerization of xylan varies depending on the

wood species from 150 to 200 in hardwoods and 70 to 130 in

softwoods ( Saha, 2003 ). The structure of xylan in annual plants is

more complex due to the presence of arabinofuranose, xylopyra-

nose, rhamnose, glucuronic acid and acetyl groups attached to the

main chain ( Hettrich & Fanter, 2010 ).

Despite xylan’s abundance in nature, cellulose and starch are

more commonly used as raw materials for derivatization due to

xylan’s inherent low molecular weight and heterogeneous struc-

ture. Recently, xylan is gaining importance for the basis of new

biopolymeric materials and functional biopolymers by chemical

modiﬁcation. Various studies on the esteriﬁcation of arabinoxylan

with different acyl compounds in homogeneous system had already

been explored ( Fang, Sun, Tomkinson, & Fowler, 2000; Peng et al.,

4) glycosidic bonds. Depending on the source, naturally

occurring xylan is usually substituted with sugar units and O-acetyl

groups. Homoxylans can be isolated from higher plants such as guar

seed husk ( Ebringerova & Heinze, 2000 ). On the other hand, xylan

obtained from seaweeds (Palmariales sp. and Bryopsis sp.) has a

backbone of d-xylp residues linked by

-(1

→

-(1

→

3) or mixed

-(1

→

and

-(1

→

4) ( Heinze, 2005 ).

∗ Corresponding author. Tel.: +81 3 5841 7888; fax: +81 3 5841 1304.

E-mail address: atiwata@mail.ecc.u-tokyo.ac.jp (T. Iwata).

0144-8617/$ – see front matter ©

doi: 10.1016/j.carbpol.2011.07.034

N.G.V. Fundador et al. / Carbohydrate Polymers 87 (2012) 170– 176

171

2008; Ren, Sun, Liu, Cao, & Luo, 2007; Sun, Fang, Tomkinson, & Jones,

1999; Sun, Sun, & Sun, 2004 ). The resulting derivatives offer poten-

tial applications for the production of biodegradable plastics, resins

and ﬁlms. Several reports have also been made on the ﬁlm forming

properties of xylan and its blends ( Buchanan et al., 2003; Gabrielii,

Gatenholm, Glasser, Jain, & Kenne, 2000; Goksu, Karamanlioglu,

Bakir, Yilmaz, & Yilmazer, 2007; Kayserilioglu, Bakir, Yilmaz, &

Akkas, 2003; Shaikh, Pandare, Nair, & Varma, 2009 ).

In this study, xylan from hardwood kraft pulp was isolated by

NaOH extraction. Structure elucidation of the xylan based on NMR

spectroscopy is discussed in this paper. This work also presents

the acetylation of the extracted xylan by homogeneous reaction

in DMAc/LiCl/pyridine system. Characterization of xylan acetate in

terms of degree of substitution (DS) and its structural features are

considered to be one of the important highlights of this research.

Thermal properties, solubility behavior and nanoﬁber formation of

xylan acetate were also investigated. In addition, the mechanical

properties of xylan acetate propionate ﬁlms with different DS were

discussed as well.

2.5. Determination of degree of substitution (DS)

2.5.1. Perpropionylation of xylan acetate

Fifty milligrams of xylan acetate were mixed in a solution of

pyridine (1 ml) and propionic anhydride (0.4 ml) for 16 h at 50 ◦ C.

The mixture was poured slowly in excess ethanol with stirring and

followed by ﬁltration. Reprecipitation was done three times and

dried as mentioned in Section 2.3 .

2.5.2. DS analysis

Perpropionylated samples dissolved in CDCl 3 (20 mg/ml) were

analyzed by 13 C NMR spectroscopy using inverse-gated technique.

The degree of substitution was calculated based on the area of

the carbonyl peaks of acetyl and propionyl groups. Peaks were

assigned by HMBC (heteronuclear multiple bond correlation) NMR

spectroscopy.

2.6. Solubility behavior

Twenty milligrams of xylan acetate was dissolved in 1 ml CHCl 3

at room temperature. The undissolved solids were separated by

centrifugation and dried. The percentage solubility was calculated

based on the weight difference divided by the original weight.

2. Materials and methods

2.1. Materials

Bleached hardwood kraft pulp sheets from Eucalyptus were

donated by the Pulp and Paper Laboratory, The University of Tokyo,

Tokyo, Japan. N,N-dimethylacetamide (DMAc), lithium chloride

(LiCl) and other reagents were purchased from Wako Chemicals.

2.7. Thermogravimetric analysis (TGA)

The thermal stability of xylan, xylan acetate and xylan acetate

propionate was observed using a Rigaku Thermoplus TG-8120.

Approximately 1 mg of sample was placed in an aluminum pan and

heated from 50 to 450 ◦ C at 20 ◦ C/min. Nitrogen was used as the

purge gas.

2.2. Extraction of hardwood xylan

Hardwood kraft pulp sheets were cut into pieces and placed in

a blender with deionized water. The mixture was blended for 30 s

and ﬁltered. The resulting pulp was then mixed with 10% NaOH

(1:20) at room temperature. After 2 h, the mixture was ﬁltered and

the ﬁltrate was neutralized with acetic acid. Ethanol was added

to the mixture until twice the original volume. The mixture was

allowed to stand overnight and the precipitate was collected by

centrifugation and washed three times with distilled water.

2.8. Electrospinning

A dope solution was prepared by dissolving xylan acetate

in 1,1,1,3,3,3-hexaﬂuoro-2-propanol to a ﬁnal concentration of

10 wt./vol.-%. Nanoﬁbers were made using an Esprayer ES-2000

electrospinning device (Fuence, Co. Ltd.). The dope solution (0.5 ml)

was drawn into a 1 ml syringe with a needle diameter of 0.5 mm.

The voltage applied to the needle was 30 kV and the dope solution

was extruded at a speed of 1.4 ml h − 1 . Nanoﬁbers were collected on

an aluminum substrate perpendicular to and 15 cm from the needle

and dried in vacuo overnight.

2.3. Synthesis of xylan acetate

Previously dried xylan (100 mg) and DMAc (2 ml) were placed

in a ﬂask equipped with a condenser. The mixture was heated at

120 ◦ C for 2 h with stirring. After cooling to 100 ◦ C, LiCl (0.175 mg)

was added. Stirring was continued at room temperature until all

the xylan has dissolved into the solution (ca. 5 h), after which, pyri-

dine (0.24 ml) and acetic anhydride (0.28 ml) were added to the

mixture and stirred for 0.5, 1, 2, 3, 4 and 6 h at 50 ◦ C. At the end of

the reaction, the mixture was poured slowly in excess ethanol and

ﬁltered. The precipitate was dissolved in a small amount of chlo-

roform and reprecipitated with ethanol three times. The resulting

xylan acetate was dried in vacuo for 12 h at room temperature.

2.9. Scanning electron microscopy

Nanoﬁber mats were transferred on a SEM stud and coated with

platinum. The images were captured using a Hitachi S-4000 SEM at

7 kV.

2.10. Preparation of xylan acetate propionate ﬁlms

Films were prepared by solvent casting method at room temper-

ature. Two hundred milligrams (200 mg) of xylan propionate were

dissolved in 10 ml chloroform. The solution was poured into a teﬂon

dish and covered with aluminum foil with tiny holes to allow slow

evaporation. The samples were dried in vacuo overnight prior to

mechanical testing.

2.4. Nuclear magnetic resonance (NMR) analysis

1 H, 13 C, DQF-COSY (double quantum-ﬁltered correlation spec-

troscopy), and HSQC (heteronuclear single-quantum correlation)

NMR spectra were obtained from a JEOL spectrophotometer oper-

ating at 500 MHz. The spectra were recorded at 25 ◦ C. Chemical

shifts (

2.11. Mechanical characterization

in ppm) were expressed relative to the resonance of Me 4 Si

(TMS;

= 0). Samples for NMR analysis were prepared by dissolving

20 mg xylan in 1 ml DMSO-d 6 . In the case of xylan acetate, CDCl 3

was used as solvent.

The mechanical

properties

xylan

ester

ﬁlms

(20 mm

1.5 mm) were carried out at room temperature using

a Shimadzu EZ test with a load cell of 10 N. A crosshead speed

172

N.G.V. Fundador et al. / Carbohydrate Polymers 87 (2012) 170– 176

of 20 mm/min and a 10 mm distance between grips were the

parameters used. Five replicates were tested on each sample.

assignment of the ring carbons was done by HSQC NMR anal-

ysis which showed the correlation of the ring protons and ring

carbons (data not shown). Furthermore, the signals at

169.3

and 169.8 ppm correspond to the carbonyl carbons at the C-3

and C-2 positions, respectively. The assignment of the signals was

based on the data obtained from the HMBC NMR analysis which

will be discussed later in this section. The absence of other sig-

nals indicates that the xylan acetate was free of any unreacted

reagents.

Each xylose unit in xylan has two hydroxyl groups available for

acetylation. In order to monitor the DS, a series of xylan acetate was

synthesized at varying reaction times. At 3 h reaction time, 50% of

the hydroxyl groups of xylan was acetylated (DS = 1.1). Complete

acetylation of xylan (DS = 2.0) was achieved within 6 h reaction time

as shown in Table 1 .

The DS of xylan acetate was

3. Results and discussion

3.1. Extraction of xylan

The presence of signiﬁcant amount of xylan and minor lignin

impurities makes hardwood paper grade pulp a good raw material

for the extraction of xylan. Solvents known to extract xylan include

DMSO, NaOH and KOH ( Janzon et al., 2008 ). In this current research,

10% NaOH was used to extract xylan from hardwood kraft pulp

and the yield was 6–8%. Higher alkaline concentration was also

employed. Although it produced higher yield, the xylan obtained

had lower M w (data not shown).

In this study, different NMR experiments were conducted to elu-

cidate the complete structure of the isolated xylan in DMSO-d 6 . The

1 H NMR spectrum of xylan is presented in Fig. 1 a. The

investigated after perpropi-

onylation of partially substituted xylan acetate

followed by

inverse-gated 13 C-NMR analysis. During perpropionylation, any

unreacted hydroxyl groups present in xylan acetate were esteriﬁed

with propionic anhydride. Based on the 13 C-NMR spectrum ( Fig. 3 ),

the signals found at

-(1

→

4)-

linked d-xylp units were characterized by the signals at

3.0, 3.2,

3.3, 3.5, 3.9 and 4.3 ppm, which correspond to H-2, H-5 a , H-3, H-4,

H-5 e and H-1, respectively. The DQF-COSY spectrum displayed in

Fig. 1 b conﬁrms the assignment. Furthermore, it is noted that the

two signals which are found downﬁeld at

9.1 and 27.5 ppm are assigned to the methyl

(–CH 3 ) and methylene (–CH 2 –) protons of the propionyl groups.

In addition, the carbonyl carbons of acetyl and propionyl groups

are seen at different chemical shifts. Based on the HMBC spectrum

shown in Fig. 3 , the carbonyl carbon signals of the acetyl group

at the C-2 and C-3 positions occurred at

5.1 and 5.2 ppm show

a weak correlation with H-3 and H-2, respectively. These signals

originated from the protons of the hydroxyl groups attached at C-

3 (–C–OH,

169.4 and 169.9 ppm,

respectively. The carbonyl carbon signals of the propionyl group

at the C-2 and C-3 positions are seen at

5.1 ppm) and C-2 (–C–OH , ı

5.2 ppm) positions of the

d-xylp units in xylan.

Fig. 1 c shows the 13 C NMR spectrum. The ﬁve major signals at

172.8 and 173.3 ppm,

63.3, 72.7, 74.0, 75.5 and 101.8 ppm are assigned to C-5, C-2, C-3, C-4

and C-1 of the d-xylp units in xylan. No other additional signals were

observed indicating that the isolated xylan does not contain any

acetyl groups, 4-O-methylglucuronic acid (MeGlcA), neutral sug-

ars and lignin. According to literature ( Teleman, Tenkanen, Jacobs,

& Dahlman, 2002 ), acetyl groups are cleaved from its main chain

during alkaline extraction. Likewise, the MeGlcA, neutral sugars

and lignin may have also been removed either during the pulp-

ing or extraction process. Thus, based on the above observations

the isolated hemicellulose was a homoxylan.

respectively.

The DS of xylan acetate was calculated based on the integral of

the carbonyl carbon peaks. Such method calculates not only for the

total DS but also for the partial DS at the C-2 and C-3 positions of

xylan acetate. The DS values are calculated applying Eqs. (1) and

(2) :

I A

DS Ac at C-2

I D ×

(1)

I A +

I B +

I C +

I B

DS Ac at C-3

I D ×

(2)

I A +

I B +

I C +

3.2. Characterization of xylan acetate

I: integral; A: C O Ac at C-2; B: C O Ac at C-3; C: C O Pr at C-2; D:

C O Pr at C-3.

Aside from the DS, the same method is used to determine the

regioselectvity of the acetylation reaction. Table 1 summarizes the

results of acetyl distribution for xylan acetate synthesized at differ-

ent reaction times. Data show that the DS values at the C-2 and C-3

positions of xylan acetate are almost the same. This implies that

the hydroxyl groups at the C-2 and C-3 positions of xylan towards

acetylation reaction have equal reactivities.

Synthesis of xylan acetate was carried out by homogeneous

reaction in DMAC/LiCl/pyridine system at 50 ◦ C. The structural

features of xylan acetate were analyzed by employing differ-

ent NMR experiments. A 1 H NMR spectrum of a fully acetylated

xylan (DS = 2.0) is presented in Fig. 2 a. The signals within the

range of

3.3–5.0 ppm are assigned to the ring protons of xylan

acetate. The strong signal at

2.0 ppm arising from the methyl pro-

tons (–CH 3 ) conﬁrms the successful acetylation of xylan. Fig. 2 b

presents the DQF-COSY spectrum showing the correlation between

the ring protons of xylan acetate. Based on the spectrum, the

signals at

3.3. Thermogravimetric analysis (TGA)

3.3, 3.7, 3.9, 4.5, 4.7 and 5.0 ppm are assigned to

H-5 a , H-4, H-5 e , H-1, H-2 and H-3 of xylan acetate. The weak

cross peak, which is seen at

One of the factors that affect the thermal properties of a polymer

is its composition. The chemical modiﬁcation of a polymer leads to

a change in its thermal stability. Fig. 4 a shows a comparison of the

thermal properties of xylan and xylan acetate with different DS.

The sudden drop in the curve of xylan during the early stage of

heating is due to the water loss of the sample. At 50% weight loss,

the decomposition temperature of xylan was at 297 ◦ C. In the case

of xylan acetate with a DS of 0.6, 1.1, 1.6 and 2.0, the decomposi-

tion temperatures were at 294, 312, 348 and 355 ◦ C, respectively.

The high thermal stability of xylan acetate is brought about by the

decrease in the number of remaining hydroxyl groups after acety-

lation ( Aburto et al., 1999 ). These hydroxyl groups are oxidized

during heating.

H y = 3.9/3.7 indicates the cor-

relation between the H-5 at the equatorial position and the H-4

at the axial position. A more distinct cross peak is observed at

ıH x /ıH y = 3.3/3.7 showing the correlation between the H-5 at the

axial position and the H-4 at the axial position. Hence, the signals

H x /

3.3 and 3.9 ppm are assigned to H-5 a and H-5 e , respec-

tively.

The

13 C NMR spectrum of xylan acetate

Fig. 2 c. The signal at 20.8 ppm originates from the methyl car-

bons. This further supports the data indicating the acetylation

of xylan. The signals at

is presented

62.4, 70.8, 71.8, 74.5 and 100.1 ppm

are assigned to C-5, C-2, C-3, C-4 and C-1 of xylan acetate. The

N.G.V. Fundador et al. / Carbohydrate Polymers 87 (2012) 170– 176

173

Fig. 1.

1 H (a) and DQF-COSY (b) and 13 C and (c) NMR spectra of xylan extracted from hardwood kraft pulp with 10% NaOH.

Fig. 2. 1 H (a) and DQF-COSY (showing the region of the ring protons) (b) and 13 C (c) NMR spectra of xylan acetate (DS = 2.0).

174

N.G.V. Fundador et al. / Carbohydrate Polymers 87 (2012) 170– 176

Table 1

DS of xylan acetate obtained at different reaction times.

Time (h)

Integral

DS Ac

DS Pr

C O Ac

C O Pr

Total

0.5

0.77

0.51

1.19

1.89

0.4

0.2

0.6

1.4

0.39

0.34

0.94

1.00

0.3

0.6

1.4

1.00

0.81

0.74

0.86

0.6

0.5

1.1

0.9

1.26

1.53

0.29

0.40

0.7

0.9

1.6

0.4

0.60

–

1.0

2.0

Fig. 4 b compares the TG curves of xylan acetate (DS = 1.1)

and xylan acetate propionate (DS Ac = 1.1). At 50% weight loss, the

decomposition temperatures of xylan acetate and xylan acetate

propionate occurred at 312 and 373 ◦ C, respectively. These results

indicate that the perpropionylation of partially substituted xylan

acetate leads to an increase in its thermal stability. The same results

were also observed with the other samples of different DS.

acetate remained partially soluble in CHCl 3 (

58%) even after

complete acetylation (DS = 2.0). In certain case, fully acetylated cel-

lulose is also partially soluble in CHCl 3 ( Shaikh et al., 2009 ) and

the use of mixed solvents provides better solubility ( Bochek &

Kalyuzhnaya, 2002 ). In the case of xylan acetate propionate, the

solubility decreased with increase in the DS Ac . Xylan acetate pro-

pionate samples with DS Ac values of 0.6 and 1.1 were completely

soluble in CHCl 3. The sample with a DS Ac = 1.6 was still partially sol-

uble in CHCl 3 . However, the solubility increased from 48% to 58%.

This shows that the solubility of partially substituted xylan acetate

is improved after perpropionylation.

∼

3.4. Solubility

Altering the molecular structure of xylan by introducing a

hydrophobic acyl group changes its solubility property. In this

study, the solubility behavior of xylan acetate and xylan acetate

propionate having different DS values

3.5. Electrospinning

investi-

gated. Fig. 5 shows the solubility behavior of xylan acetate and

xylan acetate propionate. The solubility of xylan acetate in CHCl 3

increases with an increase in DS. This is due to the increase in

hydrophobicity of the polymer after acetylation. However, xylan

in CHCl 3 was

In this study, xylan acetate nanoﬁbers were formed by electro-

spinning method using HFIP a solvent. Xylan acetate with a DS = 2.0

can be electrospun into nanoﬁbers. Xylan acetates with DS < 2.0

were not soluble in HFIP. Fig. 6 shows the SEM image of the as

Fig. 3. 13 C and expanded HMBC (embedded) NMR spectra of perpropionylated xylan acetate (DS Ac = 0.6, DS Pr = 1.4).

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