Carbohydrate Research 345 (2010) 810–816

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Carbohydrate Research

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Acetylation and characterization of spruce (Picea abies) galactoglucomannans

Chunlin Xu a,c,*, Ann-Sofie Leppänen a, Patrik Eklund b, Peter Holmlund b, Rainer Sjöholm b,Kenneth Sundberg c, Stefan Willför a

a Process Chemistry Centre, Laboratory of Wood and Paper Chemistry, Åbo Akademi University, Turku/Åbo Fi-20500, Finlandb Laboratory of Organic Chemistry, Åbo Akademi University, Turku/Åbo Fi-20500, Finlandc Water & Paper Treatment Segment, Ciba (BASF), Raisionkaari 55, PO 250, FI-21201 Raisio, Finland

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 September 2009Received in revised form 22 December 2009Accepted 11 January 2010Available online 18 January 2010

Keywords:AcetylationGalactoglucomannansSEC-MALLS/RIStructural featuresO-Acetyl migrationThermal stability

0008-6215/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.carres.2010.01.007

* Corresponding author. Tel.: +46 (0)8 55378599; fE-mail addresses: cxu@abo.fi, chunlin.xu@abo.fi (C

Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species andcan be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coatingpolymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under differentconditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, asa classic method, it can be potentially transferred to the industrial scale. The effects of the amount of cat-alyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated.A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylatedGGMs were determined by SEC-MALLS/RI, 1H and 13C NMR and FTIR spectroscopy. NMR studies also indi-cated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. Thethermal stability of the products was investigated by DSC-TGA.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

To improve sustainability in forest and forest-based industries,the concept of the biorefinery, which deals with biomass-based en-ergy, materials, and chemicals, has recently attracted a lot of atten-tion.1,2 A majority of the chemicals and materials used today aresynthetic products of oil or natural gas. These products haveencountered problems of a shortage of supply and of being detri-mental to the environment. More effort has therefore been focusedon natural alternatives. The main chemicals, which are derivedfrom lignocellulosic biomass and which are in competition withpetrochemicals, have 6–12 carbon atoms and multiple functionalgroups.3 Ethanol can be produced through natural fermentationof starch and sugars from wheat or corn, straw, sugarcane and su-gar beet, and wood.4–8 Biodiesel is a fuel that can be made fromvegetable oils or animal fats.9 So far, the utilisation of wood-de-rived chemicals, mainly extractives and non-starch polysaccha-rides, has only been sparingly investigated.10–14 Water-solublepolysaccharides, mainly galactoglucomannans, have been studiedas hydrocolloids.15–20

Acetylated galactoglucomannans (GGMs), which are the mainsoftwood hemicelluloses, are partially water soluble, and are pre-dominant in most industrially important softwood species usedfor pulping and papermaking.21,22 GGM of high purity can, for

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ax: +46 (0)8 55378468.. Xu).

example, be recovered from process waters in mechanical pulpmills using spruce, where 5–10% of the cell wall GGM is dis-solved.15 Spruce GGM consists of a linear backbone of randomlydistributed (1?4)-linked b-D-mannopyranosyl and (1?4)-linkedb-D-glucopyranosyl units, with (1?6)-linked a-D-galactopyranosylunits attached as single side units to the mannosyl units.23–25 Thesugar unit ratio has been suggested to be 3.5–4.5:1:0.5–1 (Man:-Glc:Gal) for water-soluble GGM from Norway spruce.21–25 Thehydroxyl groups at C-2 and C-3 in the mannose units are partiallyacetylated. Spruce GGM can be produced on an industrial scale andutilized as, for example, bioactive polymers, hydrocolloids,papermaking chemicals, or coating polymers.15,26 The possibleapplications of spruce GGM in novel natural materials and hydro-colloids, such as barriers in food packaging, bioactive oligosaccha-rides, dietary fibers, and health-promoting agents, have beenreviewed.27 To explore the applications of native GGM, its physico-chemical properties have been thoroughly studied.17–20 GGM-based hydrogels and films have been investigated as well.28,29

Spruce GGM as an emulsifier in beverages has recently been stud-ied.30 Naturally acetylated and deacetylated spruce GGMs havebeen reported to be potential biological-response modifiers andtherapeutic agents.31

Acetylation is known to improve some properties of polysac-charides. For example, it may enhance the dimensional stabilityand durability of wood.32 Acetylation of mannans or other polysac-charides can, for example, be used to control their solubility, waterabsorbency, hydrophobicity, and physical properties.33–36 The

C. Xu et al. / Carbohydrate Research 345 (2010) 810–816 811

degree of acetylation of polysaccharides, such as chitosan, has aninfluence not only on their physicochemical properties, but alsoon their biological activity.37,38 Acetylated mannans can potentiallybe used in papermaking and paper coating, in packaging to givebarrier properties, or in food products and pharmaceutical formu-lations as additives with immuno-potentiating and antioxidantproperties.27

In this work we have studied different reaction conditions foradditional acetylation of spruce GGM using acetic acid in the pres-ence of pyridine as a catalyst. The molar mass distribution of theacetylated GGMs was determined. The structural features of theacetylated GGMs were characterized by NMR and FTIR spectros-copy. Their thermal stability was furthermore determined by athermal analyzer.

2. Results and discussion

2.1. Preparation and characterization of spruce GGM

In preparation of spruce GGM from TMP water, fibres, fines, andother larger particles were removed by filtration. Small-moleculesubstances, such as inorganic salts and oligomeric sugars were re-moved by ultrafiltration. Lignin and other hydrophobic substanceswere removed by passing through an XAD-7 column. The sugarcomposition of the recovered spruce GGM is shown in Table 1.GGM accounts for 87 mol % of the total sugars. The molar massof GGM is 55 kDa using DMSO–H2O as an eluent instead of aqueousNaNO3 solution, which was used in previous studies,17–20 with con-sideration of the insolubility of some highly acetylated spruceGGM.

2.2. Acetylation of spruce GGM

Acetylation of spruce GGM was carried out in acetic anhydridewith pyridine as catalyst, which is a heterogeneous reaction due tothe insolubility of GGM. The reaction conditions and results areshown in Table 2. Native GGM is coded as AC00. Acetylated GGMsare coded as AC01 to AC34.

2.2.1. CatalystPyridine was used as a catalyst by adding a different amount

from 1 to 3 mL. The DS value increased slightly with an increasein the amount of pyridine as seen by comparing AC04, AC05, andAC13 (Table 2). Acetylation of carbohydrates with various hetero-geneous and homogeneous methods has been carried out usingdifferent acetylating agents such as acetic anhydride, acetyl chlo-ride, and ketene with acidic and basic catalysts.39–41 Among thosecatalysts, for example, zinc chloride could cause a significant deg-radation compared to pyridine.42 It has been stated that noremarkable degradation was observed in the acetylation of starch,glucuronoxylan, and konjac glucomannan when pyridine was used

Table 1Characteristics of spruce GGM

Sugar composition (mol %) GGM

Mannose 50Glucose 23Galactose 14Arabinose 4.0Xylose 1.7Rhamnose 1.2Glucuronic acid 2.2Galacturonic acid 4.0others <1GGM 87DS (DA) 0.32Average molar mass (kDa) 55

as catalyst.35,43,44 Recently, ionic liquids have been reported to actas solvents for homogenous acetylation, which is either catalystfree or uses iodine as a catalyst.45 Ionic liquids, also called greensolvents, have been stated to be recyclable and environmentallycompatible. However, they are still far from being feasible forapplication in actual production. Iodine is also used as a catalystfor a solvent-free acetylation.46 Titration by a large amount ofsaturated sodium hyposulfite is required to transform iodine toiodide. Therefore, in the current study, pyridine as a conventionalcatalyst was utilized.

2.2.2. Acetic anhydrideDifferent amounts of acetic anhydride were applied in different

trials. It did not show any trend of variation of DS depending on theapplied amount. It might be ascribed to an excess amount of aceticanhydride, which is much larger than the actual consumption.

2.2.3. TimeThe DS values of acetylated GGM after 1, 2, or 3 h (AC06, AC07,

and AC13) are shown Table 2. DS after 2 h increased to 0.76 from0.51 at 1 h; however, it dropped to 0.53 after 3 h. It has been re-ported that a longer acetylation time would considerably increasethe DS.47–49 But in those studies, the reaction time was shorterthan 1 h. Gröndahl et al. found that the DS would reach a maxi-mum value without any further increase after 100 min in the pres-ence of a catalyst in a certain amount.44 Therefore, in this study,the DS reached a value which did not increase after a prolongedreaction time, but in fact, it decreased instead.

2.2.4. TemperatureA variation of temperature from 40 to 60 �C, showed that the DS

was slightly higher at a higher temperature by comparing AC01and AC13 (Table 2). Furthermore, refluxing increased the DS con-siderably, from 0.82 at 60 �C to 2.61 by comparing AC16 and AC22.

2.2.5. Pretreatment and ice bath during reaction terminationPretreatment of the sample by dissolving GGM in 50 vol % acetic

acid was applied in the preparation of AC31 and AC33. The pre-treated GGM resulted in a higher DS after acetylation. Due to thepretreatment, GGM was better dispersed in acetic anhydride,which caused a more efficient acetylation. An ice bath was appliedfor the reaction mixtures right after acetylation, before water addi-tion, for terminating the reactions from AC16 to AC34. The ice-bathtreatment apparently increased the DS, as shown by comparison ofAC13 and AC16. Water addition, which is applied for terminatingthe reaction, may cause acid hydrolysis of GGM in the presenceof the acetic acid generated, thus resulting in further degradationthat was suppressed by the ice-bath treatment.

Selected reactions were repeated three times. Among those, inTable 2, AC34 is a duplicate of AC22. These results imply a goodrepeatability of the reaction.

In conclusion, temperature, time, and the amount of catalystand acetic anhydride are the main factors to affect the final DS val-ues. Higher temperature can accelerate the acetylation. Further-more, fully acetylated GGM may be produced in the refluxingreaction. In addition, pretreatment of GGM before acetylationand treatment to terminate the reaction are also critical for acety-lation. This provides knowledge that can be used to design a pro-cess for a GGM product matching a certain DS value.

2.3. Mw distribution

Both MALLS and RI detectors were used to analyze the elutedfractions. There was no exclusion peak detected. The MALLS detec-tor was less sensitive to the lower Mw fractions. Therefore, the RIchromatograms of the samples are reported here (Fig. 1).

Table 2Conditions and results of acetylation of spruce GGM

Code Aceticanhydride(mL)

Pyridine(mL)

Temperature(�C)

Time(h)

DS_titrationc DS_NMRd

AC00 0.32 0.24AC01 50 3 40 2 0.53 0.55AC03 50 3 80 2 0.64AC04 50 1 60 2 0.48AC05 50 2 60 2 0.54AC06 50 3 60 1 0.51AC07 50 3 60 3 0.53AC08 70 3 60 2 0.53 0.55AC09 30 3 60 2 0.57AC13 50 3 60 2 0.76AC16a 50 3 60 2 0.82AC22a 50 3 Reflux 2 2.61AC31a,b 50 3 60 2 1.09AC32a 50 3 60 2 0.90 1.10AC33a,b 50 3 Reflux 2 2.79 3.10AC34a 50 3 Reflux 2 2.65 2.70

a Ice bath.b Pretreatment.c DS_titration for degree of substitution by titration.d DS_NMR for degree of substitution by NMR spectroscopy.

4000 3000 2000 1000 0

AC34

% T

Wavenumbers (cm-1)

AC00

Figure 2. FTIR spectra of unmodified and acetylated GGMs.

812 C. Xu et al. / Carbohydrate Research 345 (2010) 810–816

The starting sample, unmodified spruce GGM (DS 0.32), showedtwo major peaks which correspond to 54.6 and 16.5 kDa, respec-tively, as determined by a MALLS detector (see Supplementarydata, Fig. S1 and Table S1). With an increase in DS, the higher Mw

peak with a shorter elution time gradually shrank, while the lowerMw peak with a longer elution time became larger. When spruceGGM was almost fully acetylated (DS 2.79), only the low Mw frac-tion was eluted. It might be ascribed to degradation of GGM mol-ecules. Another hypothesis is that the high Mw fraction mightstand for aggregates of a few molecules rather than a single largemolecule. Therefore, DMSO dissolved more highly acetylatedGGM slightly better. A study of acetylation of konjac glucomannanshowed a decrease in Mw after acetylation.42 However, Ren et al.proposed an increase in molar mass of wheat-straw hemicellulosesafter acetylation based on a theoretical assumption, although nomeasurement on Mw was reported.48

2.4. FTIR spectra

The IR spectra of unmodified (AC00) and acetylated (AC34)GGMs are shown in Figure 2. After acetylation to a DS of about2.65, the hydroxyl stretching band around 3500 cm�1 became con-siderably smaller.55 A band around 2950 cm�1, representing thesymmetric C–H vibration decreased too.56 At the same time, thecarbonyl stretching band around 1752 cm�1, the carboxyl stretch-

0

0.2

0.4

0.6

0.8

1

1.2

10 15 20Time,

Rel

ativ

e sc

ale

Figure 1. HPLC–SEC/RI chromatograms of un

ing band around 1238 cm�1, and the C–CH3 stretching bandaround 1372 cm�1 became tremendously larger.55 A band around1050 cm�1, assigned to C–O and C–O–C stretching, as well asC–OH bending, became sharper after acetylation.48 All these pro-vide evidence for significant acetylation of the starting sample.In addition, a band at around 900 cm�1 is ascribed to the b-gluco-sidic linkages between the sugar.57

As shown in Figure 3, GGMs AC13, AC31, and AC34 have a DSvalue of 0.76, 1.09, and 2.65, respectively. Both AC31 and AC34were immersed in a water bath when the reaction was terminated.The reaction for AC34 was heated to reflux compared to 60 �C forthe AC13 and AC31 reactions. The hydroxyl-stretching band ataround 3500 cm�1 clearly decreases in the order of increasing DSvalues or more gently terminating reaction condition and harsherheating treatment during the reaction. This confirmed the acetyla-tion of GGM and its variation of DS values. Although IR analysishere is not quantitative, the extent of change in correspondingpeaks was in good agreement with the titration and NMR results.

2.5. NMR spectra

In order to characterize the structural features and to determinethe DS of the acetylated GGMs, the samples were investigated byquantitative 13C NMR. The acetylated GGM samples were dissolvedin DMSO-d6 (approx. 100 mg mL�1) and run at 151 MHz (600 MHz)at 50 �C. A 15-s pulse delay (D1) and an inverse-gated decouplingpulse sequence were used for quantitative measurements. Onaverage, approximately 20,000 scans were accumulated for anacceptable signal-to-noise ratio.

For the determination of the DS values, the carbonyl carbonsignals between �168.5 and �170.5 ppm, as well as the methyl

25 30 35 40 minutes

DS 0.32UnmodifiedGGMDS 0.53

DS 0.76

DS 2.79

modified and acetylated spruce GGMs.

C. Xu et al. / Carbohydrate Research 345 (2010) 810–816 813

(acetyl group) signals between �19.5 and �21 ppm were carefullyintegrated. By comparison of the integral values of the above-men-tioned signals with those of the C-1 (anomeric) carbon signals(�93.5–101.5 ppm), the DS was readily calculated.

The 13C NMR spectra of AC00, AC01, and AC33 are shown in Fig-ure 4 with DA values of 0.24, 0.55, and 3.10, respectively. The val-ues are in agreement with those which were determined by thetitration method (Table 2). By comparison of the acetylated sam-ples with the native sample, it was clearly shown that the signalsat �60 ppm in the native sample, ascribed to the free C-6 carbonatoms (non-bonded) of the galactose, glucose, and mannose unitswere shifted to �63 ppm upon acetylation (Fig. 4).23,24 In additionto higher intensity, the amount of different carbonyl carbons wasclearly detected. In the almost fully acetylated samples, an upfieldshift of the anomeric carbons from �101 to �97.5 ppm was ob-served, which indicated that the positions 2 and 3 in the sugar moi-eties were acetylated.

2.6. Migration of an O-acetyl substituent

In addition to characterizing acetylated GGMs, 13C NMR spec-troscopy was conducted on a native GGM after treatment in a90 �C water bath overnight (12 h). The partial 13C NMR spectra dis-playing the ring carbon signals of native GGM, treated GGM, andfully acetylated GGM are shown in Figure 5. After treatment, a sig-nal at �63 ppm, from an acetyl-substituted O-6, appeared, whichwas not found in the native GGM, but found in the fully acetylatedGGM. This indicates a possible migration of the acetyl groups fromO-2 or O-3 to O-6. In addition, the intensity of the C-1 signal at�104 ppm decreased by the same amount as the signal at�63 ppm increased. In a previous study where GGM was preparedby hot-water extraction of spruce wood and TMP, both sprucewood and spruce TMP gave the same signal (�63 ppm) after treat-ment at 90 �C for 12 h.23 It has been accepted that the acetyl groupis positioned at O-2 or O-3 of mannose in the glucomannan back-bone of GGM. Yaku et al. and Tanaka et al. reported evidence of thepresence of a 6-O-acetyl group in pine acetylglucomannan basedon periodate degradation.49,50 However, Tanaka’s group statedlater that the 6-O-acetyl group was not found in the original acet-ylglucomannan.51 To the authors’ best knowledge, there are no re-ports of O-6 substitution by acetyl groups of mannose in nativeGGM, although acetyl as well as other acyl groups may migrate un-der the proper spatial conditions and in the presence of eitherbases or acids or in neutral conditions.52–54 To understand themechanism of migration, that is, whether the acetyl groups mi-grate from O-2 or O-3 directly or stepwise to O-6 within the same

4000 3000 2000 1000 0

AC31

AC34

% T

Wavenumbers (cm-1)

AC13

Figure 3. FTIR spectra of acetylated GGMs with different DA values AC13 (withoutpretreatment and reflux), AC34 (without pretreatment but reflux), and AC31 (withpretreatment and reflux).

sugar unit or from a different unit, a systematic study on acetylgroup migration is underway in our research groups.

2.7. Thermal analysis

The effect of acetylation on the thermal behavior of spruce GGMwas also studied using DSC-TGA in the temperature range fromroom temperature to 600 �C at a rate of 10 �C per minute. As de-picted by the TG curves in Figure 6, with an increase in tempera-ture, there was a slight decrease in mass at 210 �C for nativeGGM (AC00) and at 270 �C for acetylated GGM (AC34). After thesamples were heated further, a significant loss of mass occurredafter 250 �C for native GGM (AC00) and after 340 �C for modifiedGGM (AC34). At 50% weight loss, the decomposition temperaturesof native GGM (AC00) and acetylated GGM (AC34) are 309 and380 �C, respectively. Figure 7 shows that the decomposition tem-perature of GGMs increased with an increase in DS value; there-fore, the thermal stability increased as well. A thermal study onnative and acetylated wheat-straw hemicelluloses (xylans)showed the same properties.48 In that study, the higher thermalstability of the more highly acetylated xylans was hypothesizedto be due to a possible increase in molar mass of hemicelluloseafter acetylation. However, in our study, the molar mass of GGMsbefore and after acetylations was determined to be lowered afteracetylation. It indicates that acetylation might contribute to ther-mal stability more than a change in Mw.

Figure 6 also shows DTA curves of GGMs, which implies that thetransitions of the GGM polymers are affected by acetylation. Asmall exothermic peak, which represents the release of heat, wasobserved at 340 �C for native GGM (AC00). In comparison acety-lated GGM (AC34) showed a peak at 400 �C, which confirmed thehigher thermal stability of GGM with a higher DS value.

In summary, the classic acetylation method using pyridine as acatalyst and acetic anhydride as a reaction agent in the presence ofacetic acid can be controlled by reaction conditions, for example,temperature, time, dosage of pyridine, and acetic anhydride. Thereis potential to enlarge the scale for further development in produc-tion of acetylated GGMs. Acetylation may cause a decrease in Mw,but an increase in thermal stability.

3. Experimental

3.1. Isolation, purification, and characterization of the nativespruce GGM

At a Finnish mill producing TMP of Norway spruce (Picea abies),GGM from process water was concentrated and purified using dif-ferent filtration and ultrafiltration techniques.10,17 The resultingconcentrate was spray-dried. The dried sample was further dis-solved in distilled water and then purified by passing the solutionthrough a column of XAD-7 resin, followed by dialysis using amembrane with a 12–14 kDa cut-off. Afterward, the GGM solutionwas concentrated by vacuum evaporation in a water bath at 50 �C.EtOH was added to the concentrate to a 9:1 EtOH–H2O ratio. GGMwas allowed to precipitate overnight in a cold room, and the prod-uct was filtered and dried in a vacuum desiccator at 40 �C.

The total sugar composition and amount of GGM were analysedby acid methanolysis in 2 M HCl–CH3OH for 3 h at 100 �C, followedby silylation and GC analysis of the silylated sugar monomersaccording to the method of Willför et al.58

3.2. Acetylation of spruce GGM

GGM (10 g) was placed in a round-bottomed flask equippedwith a magnetic stirrer. Ac2O (50 mL) was added to the flask and

Figure 4. Quantitative mode 13C NMR spectra of native spruce GGM (AC00), and modified GGMs (AC01, AC33) at 50 �C.

Figure 5. Quantitative mode 13C NMR spectra of native spruce GGM, GGM treated at 90 �C overnight ((12 h), and highly acetylated GGM (AC33). Measurement temperaturewas 50 �C.

814 C. Xu et al. / Carbohydrate Research 345 (2010) 810–816

0 100 200 300 400 500 600

0

20

40

60

80

100

-1

0

1

2

3TG

DTA

, tem

pera

ture

diff

eren

ce (º

C)

TG, w

eigh

t (%

)

Temperature (ºC)

DTA

AC00AC34

Figure 6. Thermograms of unmodified GGM and acetylated GGMs.

y = 28.224x + 304.96R2 = 0.98

250

300

350

400

450

0 0.5 1 1.5 2 2.5 3DS

Dec

omp.

Tem

p. (º

C)

at 5

0% w

eigh

t los

s

Figure 7. Decomposition temperature at 50% weight loss unmodified and acety-lated GGMs.

C. Xu et al. / Carbohydrate Research 345 (2010) 810–816 815

mixed thoroughly with the GGM sample under slow stirring for30 min. The predetermined amount of pyridine used as catalystwas then added. The reaction was controlled at the required tem-perature for different times. Afterward, deionized water (50 mL)was added. For selected trials, the flask was immersed in an icebath. The solution was mixed for 10 min. The precipitate was ob-tained by adding EtOH (100 mL) followed by centrifugation. EtOH(60–80%, 100 mL) was added, and the solution was mixed for30 min and centrifuged. This was repeated twice. 100 mL of EtOHwas added, and then the mixture was filtered or centrifuged. Thefinal product was obtained by drying in a vacuum desiccator at40 �C.

A pretreatment was applied in selected trials in which the GGMwas mixed with 20 mL of 50% HOAc for 30 min and then dried at60 �C for 30 min before acetylation.

3.3. Determination of degree of substitution (DS) by titration

The degree of substitution (DS), as also named degree of acety-lation (DA) in some previous studies, was determined by titrationaccording to Laignel et al.59 A dried sample of acetylated GGMwas placed in a 100 mL flask and 75% EtOH (10 mL) was added.The dispersion was stirred at 50 �C for 30 min, then cooled to roomtemperature and 0.5 M NaOH (8 mL) was added. The mixture wasslowly stirred for 72 h, and the excess alkali was then titrated with0.5 M HCl (phenolphthalein end point). The dispersion was al-lowed to stand for 1–2 h, and the trace excess of alkali was titratedagain. A blank was titrated for comparison. The following formula

was used for calculation of % acetyl, as moles of acetyl per 100 mo-les of hexose.

% acetyl ¼ ðVa � VbÞ � NHCl �Macetyl

ms� 100

where Va stands for volume of HCl acid consumed for the blank inliters, Vb the volume of HCl acid consumed for the sample, NHCl

the molarity of the HCl acid, Macetyl 43 g mol�1, and ms the weightof the sample in grams.

The DS was calculated as

DS ¼ 162�% acetylðMacetyl � 100Þ � ðMacetyl � 1Þ �% acetyl

3.4. Characterization of native and acetylated spruce GGM

3.4.1. HPSECAverage molar mass, Mw, was determined by size-exclusion

chromatography (SEC) in on-line combination with a multi-anglelaser-light-scattering (MALLS) instrument (miniDAWN, WyattTechnology, Santa Barbara, USA) and with a refractive index (RI)detector (Shimadzu Corporation, Japan). A Jordi gel GBR mixedbed 10 � 250 mm column (Alltech, Deerfield, USA) with a guardcolumn was used. DMSO–H2O (85:15) solution, after being filteredthrough a 0.1 lm Anodisc 47 membrane filter, was used as the elu-tion solvent at a flow rate of 0.5 mL min�1. The samples were fil-tered through a 0.22-lm nylon syringe filter before injection. Theinjection volume was 300 lL. A dn/dc value of 0.060 mL g�1 wasused.60,61 Astra software (Wyatt Technology, Santa Barbara, USA)was applied to analyse the data.

3.4.2. FTIR spectroscopyFTIR spectra were recorded on an FTIR spectrophotometer (Per-

kin–Elmer FTIR Spectrum 1000) using a KBr disc containing 100–120 mg of dried KBr and about 1–5 mg sample. The spectra wereobtained in the frequency range of 4000–400 cm�1 at a resolutionof 2 cm�1 in the transmittance mode.

3.4.3. 1H NMR and 13C NMR spectroscopyThe dried native and acetylated GGMs were dissolved in DMSO-

d6 and TMS was used as the internal standard. The spectra were re-corded with a Brucker AV 600 instrument at 50 �C. For quantitative13C measurements, an inverse-gated decoupling pulse sequenceand a pulse delay (D1) of 15 s were used.

3.4.4. DSC-TGAThe thermal stability of GGM and acetylated GGMs was investi-

gated by DSC-TGA (differential scanning calorimetry—ThermalGravimetric Analysis) (Q600, Ta Instruments). The samples weredried before thermal analysis. The heating rate in the experimentswas 10 �C min�1 up to 600 �C. A platinum cup was used in thesetests. The weight of the sample and the DTA-signals (the differencebetween the sample and reference temperature) were recorded.

Acknowledgements

Docent Andrey Pranovich is thanked for his advice and con-structive suggestions. Financial aid was gratefully received fromthe Academy of Finland. This work is part of the activities at theÅbo Akademi Process Chemistry Centre within the Finnish Centreof Excellence Programme (2000–2011) by the Academy of Finland,and also part of the activities within the EPNOE network (EuropeanPolysaccharide Network of Excellence). Part of the work was donewithin the EU project WaCheUp (EU STREP 013896).

816 C. Xu et al. / Carbohydrate Research 345 (2010) 810–816

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.carres.2010.01.007.

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