Characterization of the Molar Masses of Hemicelluloses from Wood

Biomacromolecules 2001, 2, 894-905

894

Characterization of the Molar Masses of Hemicelluloses from Wood and Pulps Employing Size Exclusion Chromatography and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Anna Jacobs and Olof Dahlman* Swedish Pulp and Paper Research Institute (STFI), P.O. Box 5604, S-11486 Stockholm, Sweden Received March 8, 2001; Revised Manuscript Received May 8, 2001

The molar mass parameters for arabino-4-O-methylglucuronoxylans, arabinohexenuronoxylans, 4-Omethylglucuronoxylans, hexenuronoxylans, and galactoglucomannans extracted from wood and pulps have been determined. To characterize different types of hemicelluloses, delignified wood (spruce, pine, larch, aspen, and birch) and chemical pulps (unbleached and totally chlorine-free bleached) were extracted with dimethyl sulfoxide (DMSO) or alkaline aqueous solutions. Size exclusion chromatography (SEC) with offline matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) were employed to characterize the molar masses. The hemicellulose extracts were separated by SEC into fractions each containing components with a narrow range of molar masses and the average molar mass of each fraction subsequently determined by MALDI-MS. The molar mass parameters for the hemicelluloses were then calculated on the basis of the SEC distribution curves and MALDI-MS spectra. As expected, in most cases the hemicelluloses extracted from wood (holocellulose) exhibited higher molar masses than did the corresponding hemicelluloses from chemical pulps. The molar mass parameters for hemicelluloses isolated from pulps derived from cooking samples of the same batch of softwood chips decreased in the following order: ASAM pulp > MSSAQ pulp > kraft pulp. The lowest molar masses were demonstrated by the glucuronoxylans extracted from pulps obtained by cooking with acidic sulfite. The xylans from bleached kraft pulp were characterized by molar masses that were only slightly lower than those of the corresponding xylans from unbleached pulp. The xylans extracted into DMSO exhibited somewhat lower molar masses than did the corresponding xylans extracted into alkaline aqueous solutions. In all cases the range of molar masses demonstrated by the hemicelluloses investigated was found to be rather narrow, i.e., the polydispersity index Mw/Mn was found to be approximately 1.1-1.4. Introduction Hemicelluloses are polysaccharides that occur together with cellulose in most plant tissues. In woody plants, hemicelluloses constitute approximately one-fourth to onethird of the total organic material present.1,2 The hemicelluloses can be isolated from the original or delignified wood (holocellulose) by extraction with alkaline aqueous solutions or organic solvents, such as dimethyl sulfoxide (DMSO).1,3-6 Only a few hemicelluloses (e.g., the arabinogalactan in larch wood) can be extracted from wood using water alone.2,7 However, partly depolymerized hemicelluloses can be extracted from steam-treated wood by water.8,9 The hemicelluloses are heteroglycans containing several different types of sugar components. In softwood species such as spruce and pine, the major hemicelluloses present are O-acetyl-galactoglucomannan and arabino-(4-O-methylglucurono)xylan, whereas the predominant hemicellulose present in hardwoods such as birch and aspen is an O-acetyl* To whom correspondence may be addressed. Phone: +46-8-6767120. Fax: +46-8-108340. E-mail: [email protected].

(4-O-methylglucurono)xylan (structures 1, 2, and 3 in Figure 1).1,2 Although less abundant than cellulose, the hemicelluloses are among the most abundant naturally occurring polysaccharides. Thus, these polymers represent a large and renewable source of raw material for industrial processes. In recent years, interest in utilizing hemicelluloses as raw material for novel technological applications, e.g., involving cationic biopolymers,10 hydrogels,11 long-chain alkyl ester derivatives,6,12 and thermoplastic xylan derivatives,13 has been growing. In connection with chemical pulping and bleaching, the hemicelluloses present in the wood fibers may be chemically modified and, to various extents, depolymerized.14-16 For example, hexenuronic acid (i.e., 4-deoxy-L-threo-hex-4enopyranosyluronic acid (HexA)) residues are produced from 4-O-methyl-D-glucuronic acid residues (4OMeGlcA) in the xylan under the conditions involved in alkaline pulping processes, e.g., the kraft cooking process.17,18 Thus, arabinohexenuronoxylans and hexenuronoxylans (structures 4 and 5, respectively, in Figure 1) have been extracted from alkaline pulps derived from softwood and hardwood, respectively.19

10.1021/bm010050b CCC: $20.00 © 2001 American Chemical Society Published on Web 06/14/2001

Molar Masses of Hemicelluloses from Wood

Biomacromolecules, Vol. 2, No. 3, 2001 895

Figure 1. Chemical structural formulas for O-acetyl-galactoglucomannan (1), arabino-4-O-methyl-glucuronoxylan (2), O-acetyl-4-O-methylglucuronoxylan (3), arabinohexenuronoxylan (4), and hexenuronoxylan (5).

Hemicelluloses influence the properties of paper made from wood fibers significantly.20,21 The abundance and distribution of these polymers in the fiber wall as well as their chemical constitutions and molecular sizes explain, to a certain extent, the often quite large variations in the properties of paper made from fibers obtained by different

processes.16,22-24 The solution properties, adsorption behavior, and polymer strengths of the hemicelluloses are influenced to various degrees by their carbohydrate compositions and molar mass parameters (e.g., the degree of polymerization).24-27 A variety of different analytical approaches have been employed previously to determine the molar mass (MM) or

896

Biomacromolecules, Vol. 2, No. 3, 2001

the degree of polymerization (DP) of wood hemicelluloses26,28-32 Different procedures result in different types of MM or DP values. The number-average molar mass (Mn) is measured by osmometry26,29,30 or can be calculated from the ratio between the number of reducing end groups and the total number of sugar residues present in the hemicellulose.32,33 The weight-average molar mass (Mw) value can be obtained by characterizing light-scattering or sedimentation equilibrium.26,31 Size-exclusion chromatography (SEC), which separates molecules on basis of their size (i.e., hydrodynamic volume), has proven to be very useful in connection with analysis of the molar mass parameters for polysaccharides.34 In early studies,7,35-37 SEC was employed to determine the apparent molar mass and the molar mass distribution for certain wood hemicelluloses. An appropriate collection of calibration substances is required when SEC is utilized to determine molar mass. More recent studies have utilized hemicellulose fractions, characterized by viscometry, for calibration.38-40 Recently, we have described the application of SEC with off-line matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-MS) to the analysis of hemicelluloses isolated from wood and pulps.41,42 MALDIMS is a relatively new technique, which, among other applications, has been employed to determine the molar mass of different types of polysaccharides.43-53 The major advantages associated with the use of MALDI-MS include a high level of sensitivity, applicability to molecules exhibiting a wide range of masses, little or no fragmentation of the molecules being analyzed, and the rapidity with which results can be obtained. Interpretation of data is facilitated by the fact that almost only single-charged ions are generated. This fact also implies that the mass-to-charge ratios of the ions formed in connection with MALDI analysis are directly related to the absolute molar masses of the molecules present. It has been reported that in the case of mixtures of polymers exhibiting a wide range of molar masses, MALDI-MS may yield somewhat inaccurate values for the average molar mass, due to discrimination of signal from the larger polymers.54-59 However, this problem can be minimized or eliminated simply by separating the polymer mixture into fractions each containing components within a relatively narrow size range by SEC prior to MALDI-MS analysis (so-called SEC/ MALDI-MS).45,52,60,61 Although the major hemicellulose components of common pulpwoods have been characterized previously by several investigators,1,2,28 only a few investigations on hemicelluloses derived from modern pulping processes have been described.19,27 Therefore, the aim of the present study was to investigate the molecular properties of hemicelluloses present in pulps originating from modern cooking and totally chlorine-free (TCF) bleaching processes as well as to compare these properties with those of the corresponding hemicelluloses obtained from wood. The isolation, carbohydrate compositions, and molar mass parameters (as characterized by SEC/MALDI-MS) of hemicelluloses extracted from different species of soft- and hardwood and from different types of chemical pulps are reported here.

Jacobs and Dahlman

Experimental Section Reagents. All reagents employed were of analytical grade. Water used for the preparation of reagent and buffer solutions was purified utilizing a Millipore Milli-Q Plus apparatus (Millipore, Bedford, MA). In connection with the carbohydrate analyses, commercially available solutions of cellulases and hemicellulases (Celluclast 1.5 L, Novozym 188, and Novozym 431 obtained from Novo Nordisk A/S, Bagsvaerd, Denmark), 4-aminobenzoic acid ethyl ester (ABEE), and sodium cyanoborohydride (Sigma-Aldrich Chemical Co.; St. Louis, MO) were utilized. These commercial enzyme solutions were purified on a Pharmacia PD10 gel filtration column and mixed in the volume ratio of 1:2:2 prior to use. The oligosaccharides β-(1-4)-D-xylotriose, β-(1-4)-D-xylotetraose, β-(1-4)-D-xylopentaose, and β-(14)-D-xylohexaose were procured from Megazyme (Sydney, Australia). The matrix employed in the MALDI-MS analyses, i.e., 2,5-dihydroxybenzoic acid (DHB), and the Nafion substrate (a 5 wt % solution in a mixture of lower aliphatic alcohols and water) were procured from Sigma-Aldrich Chemical Co. Wood and Pulp Samples. The samples of birch, aspen, spruce, pine, and larch wood were collected from different locations in Sweden. Following air-drying, these wood samples were ground into fine powders (40 mesh) in a Wiley laboratory mill. These powders were first extracted with acetone at room temperature in order to remove lipophilic components and subsequently delignified with an aqueous solution of sodium chlorite and acetic acid, according to the procedure described by Ahlgren and Goring,62 to yield holocelluloses. The thermomechanical pulp (TMP) examined was obtained from a Swedish mill. This pulp was also extracted with acetone and delignified with chlorite in our laboratory in the same manner to produce TMP holocellulose. The origins and carbohydrate compositions of these holocellulose preparations (used as the raw material for extraction of hemicelluloses) are documented in Table 1, along with the origins and carbohydrate compositions of the pulps investigated. The unbleached softwood and hardwood chemical pulps listed in Table 1 were obtained from Swedish kraft and sulfite pulp mills. The samples of unbleached and bleached hardwood kraft pulps were collected at different points along the production line in a mill that performed continuous kraft cooking and TCF bleaching. The TCFbleached softwood kraft pulps originated from two different mills carrying out continuous cooking or batch cooking. The unbleached sulfite pulps were obtained from mills employing magnesium- or sodium-based cooking processes. The oxygenbleached softwood ASAM, MSSAQ, and kraft laboratory pulps (Table 1) were gifts from the Department of Pulp and Paper Technology at the Royal Institute of Technology in Stockholm. The properties of these pulps, produced on a laboratory scale using the ASAM (alkaline sulfite anthraquinone methanol), MSSAQ (mini sulfide sulfite anthraquinone) or modified kraft cooking processes, have been described elsewhere by Teder and Sjo¨stro¨m.63 Extraction of the Wood Samples. Air-dried holocellulose (10 g) was extracted with 300 mL of 24% aqueous potassium hydroxide at room temperature for 24 h under an inert



Table 1. Carbohydrate Compositions of the Wood and Pulps Used for Hemicellulose Isolationa sample/cooking process

delignification/ bleaching

carbohydrate content (% of dry mass)

Xyl

Glc

relative carbohydrate composition (mass %) GlcA Man Ara Gal HexA 4OMeGlcA

birch aspen

75 89

Hardwood Holocellulose 34.1 57.6 3.1 23.3 70.4 3.6

spruce pine larch TMPb

90 87 94 100

Softwood Holocellulose 9.1 75.9 9.1 9.3 75.5 9.3 6.3 80.0 10.4 8.1 80.2 9.1

GalA

0.3 0.0

0.5 0.3

4.4 2.4

e e

e e

1.4 2.0 1.1 e

1.7 1.3 0.6 0.6

2.7 2.4 1.4 1.8

0.1 0.1 0.2 0.2

0.3 e e e

kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) Mg-based sulfite

none oxygen peroxide ozone none

96 94 95 97 95

Hardwood Pulp 26.1 72.1 23.8 71.7 23.9 71.8 24.8 74.2 12.9 83.7

e 2.7 2.6 e 2.7

e e e e 0.1

e 0.2 0.1 e e

1.3 1.2 1.2 0.6 e

0.4 0.5 0.4 0.3 0.5

e e e e e

e e e e e

kraft (batch) kraft (batch) kraft (continuous) kraft (continuous) kraft (lab.) ASAMc (lab.) MSSAQd (lab.) Mg-based sulfite Na-based sulfite (paper grade) Na-based sulfite (dissolving grade)

oxygen peroxide oxygen peracetic acid oxygen oxygen oxygen none none

85 87 89 93 94 87 82 93 93

Softwood Pulp 8.8 83.2 8.7 82.5 8.4 81.3 9.1 83.6 7.6 85.6 8.0 84.0 8.5 83.1 6.1 85.3 5.5 81.8

6.5 7.0 8.6 6.7 5.6 5.3 6.1 8.1 11.9

0.7 1.1 0.6 0.7 0.5 0.9 0.9 0.0 0.0

0.2 0.1 0.2 0.3 0.2 0.3 0.3 0.1 0.1

0.4 0.4 0.7 0.4 0.5 0.5 0.8 0.0 0.0

0.1 0.1 0.2 0.1 0.0 0.9 0.3 0.4 0.8

e e e e e e e e e

e e e e e e e e e

none

94

3.0

0.0

0.0

0.0

0.1

e

e

1.9

95.0

a Key: Xyl ) xylose, Glc ) glucose, Man ) mannose, Ara ) arabinose, Gal ) galactose, HexA ) hexenuronic acid, 4OMeGlcA ) 4-O-methylglucuronic acid, GlcA ) glucuronic acid and GalA ) galacturonic acid. b Thermomechanical pulp. c Cooking with alkaline sulfite anthraquinone methanol. d Cooking with mini sulfide sulfite anthraquinone. e Not detectable.

(nitrogen) atmosphere. After removal of the solid cellulose residue by filtration, the alkaline extract was poured into 1200 mL of ethanol containing 30% acetic acid in order to precipitate the hemicellulose present. The supernatant was removed by centrifugation, and the precipitate then washed and dried. In the case of hardwood extracts, the hemicelluloses (glucuronoxylan) thus obtained were sufficiently pure for the present investigation. For the softwood hemicelluloses, however, a second purification step was required. The crude softwood hemicellulose preparation was dissolved in 5% potassium hydroxide and a saturated aqueous solution of barium hydroxide subsequently added in order to precipitate the galactoglucomannan as a fine white powder. After filtration, the remaining clear solution was poured into ethanol containing 30% acetic acid in order to precipitate the arabinoglucuronoxylan. Birch holocellulose (10 g) was also extracted with 300 mL of 5% aqueous potassium hydroxide solution in the manner described above. In addition, a glucuronoxylan was isolated directly from the sample of acetone-extracted birch wood (without chlorite delignification) by extraction with 24% aqueous potassium hydroxide. Extraction of the Chemical Pulps. Air-dried and acetoneextracted pulp (10 g) was first extracted with 300 mL of DMSO for 20 h at room temperature. After filtration, the filtrate was lyophilized in order to remove the DMSO, leaving the hemicellulose as a colorless solid material. The DMSO-extracted pulp was further extracted with 300 mL

of 5% aqueous potassium hydroxide at room temperature for 24 h under an inert (nitrogen) atmosphere. The undissolved residue was removed by filtration and, after adjusting pH of the solution to 4.5 by addition of acetic acid, the hemicellulose was precipitated by the addition of 1200 mL of ethanol. This precipitate was collected by centrifugation, washed, and, finally, air-dried at room temperature. Extraction of the Chemical Pulps on a Microscale. For extraction on a microscale, 100 mg of pulp was packed into a 1-mL syringe equipped with a glass wool plug at the needle end. Approximately 1 mL of 17% aqueous sodium hydroxide (NaOH) was then sucked into the syringe, and the extraction was performed at room temperature for 3 h. Thereafter, the hemicellulose-containing extract was quickly pushed out of the syringe and an aliquot of this extract diluted with the aqueous sodium hydroxide/acetate solution (containing 0.2 M sodium hydroxide and 0.1 M sodium acetate) employed for elution in connection with the SEC. Subsequently, this solution was filtered through a syringe filter with 0.45 µm pores (Millipore) and then immediately injected onto the SEC column. The remainder of the hemicellulose extract was neutralized with acetic acid and subjected to carbohydrate analysis. Carbohydrate Analysis. Carbohydrate analysis was performed employing enzymatic hydrolysis and capillary electrophoresis (CZE), as described previously.64 In brief, the purified enzyme mixture (0.5 mL) and sodium acetate buffer (0.5 mL, pH 4.0) were added to the sample (10 mg). The resulting suspension was stirred for 30 h at 40 °C to achieve

898


complete enzymatic hydrolysis. The hydrolysate thus obtained was supplemented with 0.040 mL of a ribose solution (10 mg/mL), used as an internal standard for quantification and an aliquot (200 µL) removed for derivatization with ABEE and subsequent CZE analysis. Size Exclusion Chromatography. The SEC system consisted of three columns containing Ultrahydrogel 120, 250, and 500 (Waters Assoc. USA) linked in series with each other and with an instrument for measuring the refractive index of the eluate (Waters). Two different mobile phases were tested, i.e., (i) 0.05 M ammonium acetate (pH 9) and (ii) a strongly alkaline sodium hydroxide/acetate solution (0.2 M hydroxide and 0.1 M acetate, pH 13). The hemicelluloses were dissolved in the eluting solution to obtain final concentrations of 0.2-1% (w/v). All solutions were filtered through syringe filters with 0.45 µm pores (Millipore) prior to injection into the SEC column system. Samples (100 µL) were injected into the SEC system, and 0.13-mL fractions containing components with a narrow range of molar masses were collected from the outlet of the refractometer. The signal from the refractometer was processed utilizing the PL Caliber SEC software and interface (Polymer Laboratories Ltd., U.K.) on a standard PC computer. The molar mass parameters, i.e., the number-average molar mass (Mn), weight-average molar mass (Mw), and polydispersity index (Mw/Mn) of the entire hemicellulose extract were calculated on the basis the SEC profile, together with the MALDI-MS analyses, using the PL Caliber software. MALDI-MS Analysis. MALDI-MS analyses were performed with a Hewlett-Packard G2025 A MALDI-TOF mass spectrometer equipped with a linear detector and using 1-5 µJ energy pulses of the UV (337 nm) laser beam. Data were collected utilizing an extraction voltage of 30 kV and a laser power slightly greater than the minimum required for the generation of analyte ions. Both positive- and negative-ion spectra, representing the sums of 20-60 laser shots, were recorded. The mesa surface of the MALDI probes employed was coated with a Nafion perfluorosulfonated ionomer or nitrocellulose film.53 For the MALDI-MS analysis, the hemicellulose was dissolved in the ammonium acetate or sodium hydroxide/sodium acetate buffer to obtain a concentration of 0.3% (w/v). The matrix solution (2% (w/v), 2,5dihydroxybenzoic acid dissolved in 1:3 (v/v) methanol/water) was then mixed with this hemicellulose solution (or with an SEC fraction) at a ratio of 1:4 (v/v). The volume of each SEC fraction was reduced by about 80% to approximately 25 µL by flushing with a gentle flow of nitrogen prior to mixing with the matrix solution. SEC fractions containing the sodium hydroxide/acetate eluent were also pretreated with a cation-exchange resin (Amberlite IR-120, NH4+ form) prior to concentration. A 0.5-µL aliquot of the resulting solution of the hemicellulose in the DHB matrix was deposited onto the tip of the MALDI probe, dried under a vacuum, and subsequently analyzed by MALDI-MS. Results and Discussion Isolation and Compositions of the Hemicelluloses. The wood hemicelluloses investigated here were isolated by

Jacobs and Dahlman

extraction of delignified birch, aspen, spruce, pine, or larch wood using aqueous potassium hydroxide. Delignification was performed not only to remove the lignin but also to thereby render the hemicellulose more accessible to extraction. Especially in the case of softwoods, delignification is required in order to achieve efficient extraction of the hemicellulose.65 In Table 2, the quantities and compositions of the hemicelluloses obtained are presented. The quantities given were calculated as the difference in the hemicellulose content of the wood samples before and after extraction. As can be seen from this table, all of the xylan and most of the galactoglucomannan could be extracted with 24% aqueous potassium hydroxide. With the less concentrated 5% potassium hydroxide solution, only 15% of the xylan originally present in birch holocellulose could be extracted. The yields of hemicellulose obtained here are similar to those reported in earlier studies.1,4,8,29,65,66 Although chlorite delignification is considered to be a mild and selective procedure, a certain limited amount of degradation of hemicellulose may occur during such treatment.15,62 It is known that hardwood xylans can be extracted from wood utilizing potassium hydroxide solutions without prior delignification.1,2,29 Therefore, for purposes of comparison, birch wood was also extracted directly (i.e., without delignification) with 24% aqueous potassium hydroxide. In agreement with earlier studies,1,29 this procedure yielded a slightly lower quantity of the xylan than that which could be extracted from delignified birch wood (Table 2). Table 2 also documents the carbohydrate compositions of the hemicelluloses extracted. Carbohydrate analysis was performed employing a new procedure developed recently in our laboratory.64 This procedure involves initial hydrolysis of the polysaccharides using a mixture of cellulase and hemicellulase. The reducing saccharides present in the hydrolysate thus obtained are subsequently analyzed by capillary zone electrophoresis. All neutral sugars and uronic acids present as structural elements in wood hemicelluloses can be quantitated in this manner, as can the content of hexenuronic acid in xylans extracted from alkaline pulps. The hemicelluloses extracted from the hardwoods (birch and aspen) were essentially pure glucuronoxylan with a purity of 95-98% as determined by carbohydrate analysis. The molar ratio of 4-O-methylglucuronic acid to xylose in these xylans varied within a range from 4:100 to 12:100, which is quite typical for xylans extracted from hardwoods.1,2 For example, a xylan extracted from trembling aspen by Koshijima et al.67 using 24% potassium hydroxide contained one uronic acid per nine xylose residues. In the present study, the highest relative amount of 4OMeGlcA/Xyl was observed in the xylan extracted with 5% potassium hydroxide. This ratio is comparable to the 1:10-11 ratio of uronic acid/Xyl described by Han and Swan68 in a xylan extracted from delignified (with peracetic acid) birch wood with 7% aqueous potassium hydroxide. The hemicelluloses extracted from delignified softwood (spruce, pine, and larch wood) and TMP with 24% aqueous potassium hydroxide were all found to be mixtures containing predominantly arabinoglucuronoxylan and galactoglucoman-



Table 2. Yields and Compositions of Hemicelluloses Extracted from Wood and Mechanical Pulpa % of hemicellulose extracted

sample

origin

isolation procedureb

HW-1 HW-2 HW-3 HW-4

birch birch birchc aspen

A B B B

15 98 82 98

SW-1

spruce

B

98 82

SW-1a SW-1b SW-2

spruce spruce pine

C D B

SW-2a SW-2b SW-3

pine pine larch

C D B

SW-3a SW-3b STMP-1

larch larch TMPd

C D A

98 55

98 58

e

carbohydrate composition of the hemicellulose hemicellulose (purity)

sugar residues

molar ratio

Hardwood glucuronoxylan (96%) glucuronoxylan (98%) glucuronoxylan (97%) glucuronoxylan (95%)

4OMeGlcA:Xyl 4OMeGlcA:Xyl 4OMeGlcA:Xyl 4OMeGlcA:Xyl

12:100 5:100 4:100 9:100

Softwood arabinoglucuronoxylan (64%) galactoglucomannan (35%) arabinoglucuronoxylan (97%) galactoglucomannan (90%) arabinoglucuronoxylan (71%) galactoglucomannan (29%) arabinoglucuronoxylan (87%) galactoglucomannan (92%) arabinoglucuronoxylan (56%) galactoglucomannan (44%) arabinoglucuronoxylan (91%) galactoglucomannan (95%) arabinoglucuronoxylan (88%)

Ara:4OMeGlcA:Xyl Gal:Glc:Man Ara:4OMeGlcA:Xyl Gal:Glc:Man Ara:4OMeGlcA:Xyl Gal:Glc:Man Ara:4OM-GlcA:Xyl Gal:Glc:Man Ara:4OMeGlcA:Xyl Gal:Glc:Man Ara:4OMeGlcA:Xyl Gal:Glc:Man Ara:4OMeGlcA:Xyl

9:11:100 21:29:100 6:13:100 16:24:100 14:10:100 24:35:100 10:16:100 9:22:100 11:12:100 11:43:100 10:12:100 8:26:100 11:12:100

a Key: Ara ) arabinose, Xyl ) xylose, Glc ) glucose, Man ) mannose, Gal ) galactose, and 4OMeGlcA ) 4-O-methylglucuronic acid. b Key: A ) 5% aqueous KOH; B ) 24% aqueous KOH; C ) 24% aqueous KOH, soluble after addition of BaOH; D ) 24% aqueous KOH, precipitated after addition of BaOH. c Nondelignified wood. d Thermomechanical pulp. e Not analyzed.

nan (Table 2). These crude hemicelluloses could be further purified by treatment with barium hydroxide as described by Banerjee and Timell69 (preparations C and D in Table 2). This procedure resulted in the separation of almost pure galactoglucomannan (SW-1b, SW-2b, and SW-3b) and arabinoglucuronoxylan (SW-1a, SW-2a, and SW-3a). The relative contents of arabinose, 4-O-methylglucuronic acid, and xylose (i.e., the molar ratio Ara:4OMeGlcA:Xyl) in these xylans and of galactose, glucose, and mannose (Gal:Glc: Man) in the mannans are also documented in Table 2 and are comparable to corresponding values reported earlier by Timell.1 For example, in earlier studies arabinoglucuronoxylans with Ara:4OMeGlcA:Xyl ratios of 14:17:100 and 11:14:100 were isolated from spruce70 and pine wood,69 respectively. Here, a number of different hemicelluloses were isolated from unbleached kraft and sulfite pulps as well as from TCFbleached kraft pulps using sequential extractions, as described previously.19 The first extraction involved the use of dimethyl sulfoxide (DMSO), and in the subsequent extraction 5% aqueous potassium hydroxide was employed. In the case of unbleached and TCF-bleached hardwood kraft pulps, DMSO extracted approximately 20% of the total hemicellulose originally present while the corresponding value for potassium hydroxide was 35-56% (Table 3, HK-1 to HK-8). In a similar manner, DMSO extracted 24-34% and potassium hydroxide approximately 10% of the xylan originally present in the softwood kraft pulps listed in Table 3. In the case of the sulfite pulps, larger amounts of the hemicellulose available could be extracted with DMSO (HS-1 and SS-1 to SS-3 in Table 3). Indeed, it has been reported previously that the extraction of hemicellulose from sulfite pulps by DMSO is more effective than extraction from the corresponding kraft pulps.71 The yields of pulp hemicelluloses seen in Table 3 are comparable to those obtained

previously14 employing sequential extraction of kraft and sulfite hard- and softwood pulps. In the present study, hemicelluloses were also extracted from oxygen-bleached pulps obtained by cooking the same softwood raw material, but using different procedures, i.e., the ASAM,72 MSSAQ,73 or modified kraft process74 (SA-1, SM-1, and SK-7 in Table 3). In these cases a microscale procedure similar to that described by Eeremeeva and Bykova39 was employed. This procedure involves extraction of a small amount of pulp (packed in a syringe) with 17% sodium hydroxide. The extracts thus obtained were analyzed directly by SEC, without prior precipitation or purification of the hemicelluloses. This microscale procedure resulted in the extraction of almost all of the hemicellulose from the oxygen-bleached pulps. The carbohydrate compositions of the hemicelluloses extracted in these manners from chemical pulps are shown in Table 3. Analytical procedures capable of quantitating the hexenuronic acid residues in xylans have been developed only quite recently,64,75-78 so that a direct comparison of our findings with older data in the literature is not possible. Sequential extraction of the hardwood kraft pulps with DMSO and 5% aqueous potassium hydroxide yielded pure (>96%) hexenuronoxylans (HK-1 to HK-8 in Table 3). The sugar compositions of these xylans indicated that most of the 4OMeGlcA residues originally present in hardwood xylans (e.g., xylans HW-1 to HW-4 in Table 2) were removed or converted into hexenuronic acid residues during the process of alkaline kraft pulping. The molar ratio of hexenuronic acid, 4-O-methylglucuronic acid, and xylose (HexA:4OMeGlcA:Xyl) calculated for these xylans varied from 1.8:1.2:100 to 4.2:1.0:100 (Table 3). The hexenuronoxylans extracted with DMSO contained slightly higher quantities of HexA residues than did the corresponding hexenuronoxylans extracted with 5% potassium hydroxide.

900


Jacobs and Dahlman

Table 3. Yields and Compositions of Hemicelluloses Extracted from Unbleached and TCF-Bleached Chemical Pulpsa

sample

cooking procedure

procedure calcd bleaching extractionb yield (%)c

predominant hemicellulose present (purity)

carbohydrate composition of the hemicellulose sugar residues molar ratio

HK-1 HK-2 HK-3 HK-4 HK-5 HK-6 HK-7 HK-8 HS-1

kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) Mg-based sulfite

none none oxygen oxygen peroxide peroxide ozone ozone none

E A E A E A E A E

22 40 22 37 22 56 21 31 38

Hardwood Pulp hexenuronoxylan (98%) hexenuronoxylan (99%) hexenuronoxylan (98%) hexenuronoxylan (99%) hexenuronoxylan (99%) hexenuronoxylan (100%) hexenuronoxylan (99%) hexenuronoxylan (98%) glucuronoxylan (96%)

HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl HexA:4OMeGlcA:Xyl 4OMeGlcA:Xyl

4.2:1.0:100 4.1:0.9:100 4.2:1.1:100 4.0:1.2:100 4.2:1.2:100 3.6:1.3:100 2.8:1.5:100 1.8:1.2:100 2.3:100

SK-1 SK-2 SK-3 SK-4 SK-5 SK-6

kraft (continuous) kraft (continuous) kraft (continuous) kraft (continuous) kraft (batch) kraft (batch)

E A E A E E

24 8 27 12 25 34

Softwood Pulp arabinohexenuronoxylan (98%) arabinohexenuronoxylan (93%) arabinohexenuronoxylan (98%) arabinohexenuronoxylan (94%) arabinohexenuronoxylan (94%) arabinohexenuronoxylan (95%)

Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl

7.7:3.6:0.3:100 7.7:4.3:0.5:100 7.1:2.3:0.4:100 7.2:2.9:0.7:100 8.2:5.8:0.8:100 8.2:3.4:1.0:100

SK-7 SA-1 SM-1 SS-1 SS-2

oxygen oxygen peroxide peroxide oxygen peracetic acid oxygen oxygen oxygen none none

kraft (lab.) ASAMe (lab.) MSSAQf (lab.) Mg-based sulfite Na-based sulfite (paper grade) Na-based sulfite none (dissolving grade)

F F F E E

d d d 39 60

arabinohexenuronoxylan (57%) arabinohexenuronoxylan (64%) arabinohexenuronoxylan (61%) flucuronoxylan (74%) glucuronoxylan (90%)

Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:HexA:4OMeGlcA:Xyl Ara:4OMeGlcA:Xyl 4OMeGlcA:Xyl

4.5:5.2:0.0:100 10.1:5.4:4.1:100 7.1:7.5:1.4:100 0.2:4.7:100 14.2:100

E

54

glucuronoxylan (71%)

4OMeGlcA:Xyl

6.3:100

SS-3

a Key: Ara ) arabinose, Xyl ) xylose, HexA ) hexenuronic acid, and 4OMeGlcA ) 4-O-methylglucuronic acid. b Key: A ) 5% aqueous KOH. E ) DMSO. F ) 17% aqueous NaOH on a microscale. c % of the total available hemicellulose. d Not analyzed. e Cooking with alkaline sulfite anthraquinone methanol. f Cooking with mini sulfide sulfite anthraquinone.

The lowest HexA:Xyl ratio observed was exhibited by the hexenuronoxylan extracted from ozone-bleached pulp with potassium hydroxide (HK-8, Table 3). We have previously shown that the unsaturated HexA residues are oxidized and subsequently removed from the xylan during bleaching of pulps using ozone or peracetic acid.16 In contrast, bleaching with oxygen or alkaline hydrogen peroxide had no apparent effect on the compositions of xylans subsequently extracted from these pulps (HK-3 to HK-6). The hemicellulose extracted with DMSO from the hardwood sulfite pulp (HS-1) was found to be a partially acetylated glucuronoxylan (degree of acetylation approximately 0.3) demonstrating a low content of 4OMeGlcA residues, which is consistent with an earlier report.71 In the case of the softwood kraft pulps examined here, sequential extraction using DMSO and 5% potassium hydroxide yielded essentially pure xylans (SK-1 to SK-6 in Table 3). As found for the hardwood kraft xylans discussed above, the sugar compositions of these softwood kraft xylans demonstrated that virtually all of the 4OMeGlcA residues originally present in the native softwood xylans were removed or converted into HexA residues during the process of kraft pulping. The hemicelluloses extracted on a microscale from the oxygen-bleached softwood pulps (SK-7, SA-1, and SM-1) were all primarily arabinohexenuronoxylans, but their sugar compositions differed greatly. The uronic acid content (i.e., the sum of the contents of HexA and 4OMeGlcA residues) for the xylans obtained after employing the ASAM and

MSSAQ pulping procedures (SA-1 and SM-1) were higher than the corresponding value for the kraft xylan (SK-7). Furthermore the molar ratio of 4OMeGlcA and HexA residues in the xylan extracted from the ASAM pulp was higher than those for the xylans from the kraft or MSSAQ pulps, indicating that the ASAM cooking process affects the 4OMeGlcA residues somewhat less than the other two cooking processes. Further examination of the data in Table 3 reveals that the molar ratio of arabinose and xylose (Ara: Xyl) in the kraft pulp xylan is lower than those for the xylans from softwood ASAM and MSSAQ pulps, indicating some loss of arabinose residues during kraft pulping. As expected, the hemicelluloses extracted with DMSO from the softwood sulfite pulps were primarily glucuronoxylans containing different amounts of 4OMeGlcA residues, with very few or no arabinose residues (SS-1 to SS-3 in Table 3). Development of the SEC/MALDI-MS Procedure. The MALDI-MS spectrum (obtained in the negative-ion mode) of the glucuronoxylan extracted from the paper-grade softwood sulfite pulp (SS-2) using DMSO is depicted in Figure 2. This glucuronoxylan was dissolved in the ammonium acetate buffer, and this solution then mixed with the DHB matrix and subjected to MALDI analysis using the Nafioncoated probe.53 This spectrum contains a broad distribution of well-resolved peaks, with the maximum intensity occurring at approximately 3300 mass units. These peaks originate from different glucuronoxylan oligomers with varying sugar compositions. The composition of each oligomer can be


Figure 2. MALDI-MS spectrum (negative-ion mode) of the glucuronoxylan extracted with DMSO from the softwood paper grade pulp obtained by sodium-based sulfite cooking. The mass peaks correspond to xylooligosaccharide chains of increasing lengths containing an odd (O) or even (×) number of 4-O-methylglucuronic acid residues.

Figure 3. MALDI-MS spectra (negative ion-mode) of five fractions collected sequentially (from top to bottom) during SEC of the glucuronoxylan SS-1 extracted from softwood sulfite pulp with DMSO.

derived from the mass associated with its MALDI-MS signal (i.e., from the m/z ratio), since xylose and 4OMeGlcA residues have different molar masses (i.e., 132 and 190, respectively). This MALDI-MS spectrum is evidently composed of two major, partially overlapping series of peaks, in which the distance between adjacent peaks in each series corresponds to 132 mass units (which is the molar mass of a single xylose residue). Thus, these peaks originate from two distinct series of glucuronoxylan oligomers containing different numbers of 4OMeGlcA residues, as we have described earlier.41 The first series contains oligosaccharides with an odd number of 4OMeGlcA residues linked to the xylooligomer backbone (denoted as O in Figure 2; i.e., 4OMeGlcA2n-1Xylx, where n ) 1 or 2 and x ) 10-25). In contrast, the second series contains oligosaccharides containing an even number of 4OMeGlcA moieties as substituents on the backbone (denoted × in Figure 2; i.e., 4OMeGlcA2nXylx, where n ) 1 or 2 and x ) 10-25). The softwood sulfite xylan (SS-2) extracted with DMSO was fractionated using three SEC columns (Ultrahydrogel 120, 250, and 500) linked in series and ammonium acetate (pH 9) as the eluent. The MALDI-MS spectra of five fractions collected sequentially are illustrated in Figure 3. As can be seen, these spectra exhibited signals distributed between m/z values 1500 and approximately m/z ∼ 10 000. Each individual fraction demonstrated a rather symmetric distribution of signals in the MALDI spectrum, but separate


Figure 4. SEC chromatogram and MALDI-MS spectra (negativeion mode) of three sequential fractions (A, B, and C; shaded) obtained with the arabinoglucuronoxylan (SW-1a) extracted from spruce with alkali.

peaks corresponding to individual oligosaccharides with molar masses of greater than approximately 6000 could not be resolved due to the limitations of the MALDI-MS instrument employed. The fractions isolated had peakaverage molar mass (Mp) values of 2800, 3200, 4500, 6000, and 7500 (as determined from the MALDI-MS spectra in Figure 3). In contrast, the MALDI spectrum of this sulfite xylan taken prior to fractionation by SEC (depicted in Figure 2) only contained peaks with an m/z value of less than approximately 6000. Evidently this analysis must have involved discrimination against signals originating from the high molar mass region of the spectrum. Several studies have demonstrated that MALDI analyses may result in inaccurate values for the average molar mass of a mixture of polymers with a wide range of sizes, due to discrimination against signals from the larger polymers.41,54-59 However, as shown here, separation by SEC into fractions containing a narrow size range of polymers prior to MALDIMS analysis (SEC/MALDI-MS) offers a solution to this problem. With this approach, MALDI-MS provides accurate molecular mass values for the fractions obtained.56,61,79 In the present investigation, two different aqueous solutions were employed as eluents in connection with SEC, i.e., an ammonium acetate solution with a pH of 9 and a more alkaline sodium hydroxide/acetate solution of pH 13. The xylans extracted from the softwood holocelluloses and sulfite pulps could be separated well using the ammonium acetate buffer and yielded MALDI-MS spectra of good quality. Figure 4 illustrates fractionation of the spruce arabinoglucuronoxylan SW-1a by SEC and the subsequent MALDIMS analysis of three of the fractions obtained. These fractions gave rise to relatively symmetric distributions of MALDI signals up to a m/z value of at least 35 000. However, in contrast to the spectra shown in Figure 3, no peaks originating from individual polysaccharide constituents could be resolved in this case. Elution from the SEC system with ammonium acetate was, however, found to be unsuitable for certain of the hemicelluloses studied. The galactoglucomannans and the hardwood xylans aggregated to various extents in this eluent, thus making the SEC procedure unreliable. In contrast, good separation of all hemicelluloses on the basis of size was achieved with the more alkaline sodium hydroxide/acetate solution. Figure 5 depicts the two chromatograms obtained

902


Figure 5. SEC chromatograms obtained with the aspen glucuronoxylan (HW-4) employing elution with ammonium acetate (A) or sodium hydroxide/acetate (B). Chromatogram A exhibits peaks originating from xylan aggregates (with an elution volume of approximately 15 mL), as well as from the nonaggregated xylan (elution volume approximately 19 mL). In chromatogram B, only a single peak, corresponding to the nonaggregated aspen glucuronoxylan (elution volume approximately 19 mL) is seen. The DMSO peak, used as a flow marker, is also indicated in these chromatograms.

upon elution of the aspen glucuronoxylan HW-4 through the SEC column system employing the ammonium acetate (A) and sodium hydroxide/acetate (B) solutions. Chromatogram A exhibits peaks originating from xylan aggregates (with an elution volume approximately 15 mL), as well as from the nonaggregated xylan (with an elution volume approximately 19 mL). In chromatogram B only a single peak corresponding to the nonaggregated aspen glucuronoxylan (at an elution volume of approximately 19 mL) is seen. Pretreatment by passage through a cation-exchange resin (Amberlite IR-120, NH4+ form) prior to MALDI-MS analysis was necessary in the case of SEC fractions obtained using the sodium hydroxide/acetate eluent. MALDI analysis is known to be disturbed by the presence of metal ions in the sample, especially if they are in high abundance. With respect to acidic polysaccharides such as the xylans, such disturbance may become severe, since sodium (or potassium) ions bind extensively to the uronic acid groups, thereby producing multiple peaks. Such multiplicity can be the major source of peak broadening in connection with MALDI analysis of acidic polysaccharides.53 Cation exchange was therefore employed to remove most of the sodium ions from the hemicellulose fractions. Despite this pretreatment and the use of MALDI probes coated with Nafion film, MALDI-MS spectra of somewhat lower quality were obtained with the hemicellulose fractions eluted with sodium hydroxide/acetate solution. These MALDI-MS spectra were nonetheless of sufficiently high quality to provide reliable values for the peak-average molar mass (Mp). MALDI analysis was performed on a number of different hemicelluloses following SEC fractionation using both eluent solutions. Five to ten of the SEC fractions obtained with each hemicellulose were characterized by MALDI-MS in order to obtain a sufficient number of data. Subsequently, the logarithms of the Mp values determined by MALDI-MS were plotted as function of the corresponding retention volume ratios (K) obtained from the SEC separation (Figure 6). These K values represent the ratio between the elution volume for a given hemicellulose and the elution volume for DMSO (used as the flow marker), according to the approach suggested by Malfait et al.80 Calculation of the K value in

Jacobs and Dahlman

Figure 6. Relationship between log Mp (determined by MALDI-MS) and the relative retention volume (K) obtained with SEC for different hemicelluloses eluted with ammonium acetate or sodium hydroxide/ acetate. K ) the elution volume of the hemicelluloses/the elution volume of DMSO. The linear relationships and regression coefficients (obtained by least-squares linear regression analysis) are also shown.

this manner compensates for variations in the elution volume of a hemicellulose in connection with different SEC separations, which arise primarily from variations in flow rate. To extend the mass range of these calibration curves, data points for β-(1-4)-D-xylotetraose, β-(1-4)-D-xylopentaose, and β-(1-4)-D-xylohexaose were also plotted. Least-squares linear regression analysis yielded regression coefficients of 0.987 and 0.968 for elution with sodium hydroxide/acetate and ammonium acetate, respectively. These regression coefficients are relatively high considering the fact that they are also influenced by the uncertainty of the SEC procedure. The two regression lines depicted in Figure 6 exhibit virtually the same slope, but with different intercepts. This pattern indicates that separation on the SEC column system with the two eluent systems employed is slightly different. However, this difference is small and of the same magnitude as the standard deviation associated with the SEC procedure (i.e., approximately 5%). Determination of Molar Masses by SEC/MALDI. The number and weight average molar mass values (Mn and Mw), together with the polydispersity indices (Mw/Mn) for the hemicelluloses extracted are documented in Table 4. These data were all collected using SEC/MALDI analysis with the sodium hydroxide/acetate as eluent. As can be seen from this table, the arabinoglucuronoxylans extracted from the spruce and pine holocelluloses demonstrate somewhat higher average molar masses (Mn and Mw) than do the glucuronoxylans extracted from the birch and aspen holocelluloses. The molar mass parameters for the galactoglucomannans are similar to those of the softwood xylans, except for the slightly higher polydispersity index (Mw/Mn) observed for the former. Table 4 reveals that the polydispersity values of all of the hemicelluloses analyzed was low (i.e., Mw/Mn values of approximately 1.1-1.4). Comparison of the average molar mass values for the glucuronoxylans HW-2 and HW-3 in Table 4 indicates that the delignification procedure reduced the molar masses of



Table 4. Molar Mass Parameters for the Hemicelluloses Extracted from Wood and Pulps sample

origin

bleaching procedure

extraction procedurea

Mn

Mw

Mw/Mn

Hardwood HW-1 HW-2 HW-3 HW-4 Softwood SW-1 SW-1a SW-1b SW-2 SW-2a SW-2b SW-3 SW-3a SW-3b STMP-1

birch holocellulose birch holocellulose birch wood meal aspen holocellulose

A B B B

15 000 11 400 14 000 15 600

16 900 13 700 16 500 17 100

1.11 1.19 1.13 1.09

spruce holocellulose spruce holocellulose spruce holocellulose pine holocellulose pine holocellulose pine holocellulose larch holocellulose larch holocellulose larch holocellulose TMPb holocellulose

B C D B C D B C D A

14 600 16 100 14 700 15 700 17 200 16 600 14 000 14 400 15 500 15 100

18 400 19 200 20 200 19 900 20 800 21 400 17 400 17 300 19 100 17 300

1.22 1.16 1.33 1.24 1.18 1.26 1.23 1.18 1.22 1.15

HK-1 HK-2 HK-3 HK-4 HK-5 HK-6 HK-7 HK-8 HS-1

kraft (continous) kraft (continous) kraft (continous) kraft (continous) kraft (continous) kraft (continous) kraft (continous) kraft (continous) Mg-based sulfite

Hardwood Pulp none none oxygen oxygen peroxide peroxide ozone ozone none

E A E A E A E A E

9 900 11 500 8 800 12 100 9 000 11 900 9 200 10 000 4 900

11 600 13 400 10 400 13 300 10 600 13 200 11 000 11 600 6 000

1.18 1.17 1.19 1.10 1.18 1.11 1.20 1.17 1.23

SK-1 SK-2 SK-3 SK-4 SK-5 SK-6 SK-7 SA-1 SM-1 SS-1 SS-2 SS-3

kraft (continous) kraft (continous) kraft (continous) kraft (continous) kraft (batch) kraft (batch) kraft (lab.) ASAMc (lab.) MSSAQd (lab.) Mg-based sulfite Na-based sulfite (paper grade) Na-based sulfite (dissolving grade)

Softwood Pulp oxygen oxygen peroxide peroxide oxygen peracetic acid oxygen oxygen oxygen none none none

E A E A E E F F F E E E

9 000 10 100 9 600 11 600 12 000 12 300 11 600 13 800 12 100 5 000 6 100 2 700

11 900 12 900 12 100 13 100 14 700 14 400 16 500 19 300 17 000 6 200 7 000 3 300

1.32 1.27 1.25 1.13 1.19 1.17 1.34 1.31 1.36 1.23 1.15 1.20

a Key: A ) 5% aqueous KOH. B ) 24% aqueous KOH. C ) 24% aqueous KOH, soluble after addition of BaOH. D ) 24% aqueous KOH, precipitated after addition of BaOH. E ) DMSO. B ) 17% aqueous NaOH, microscale extraction. b Thermomechanical pulp. c Cooking with alkaline sulfite anthraquinone methanol. d Cooking with mini sulfide sulfite anthraquinone.

these polysaccharides by 10-15%. On the other hand, the glucuronoxylan extracted from delignified birch with 5% aqueous potassium hydroxide (HW-1) exhibited higher average molar masses (Mn and Mw) than did the same polysaccharide extracted from both nondelignified and delignified birch using 24% aqueous potassium hydroxide (Table 4). Apparently, both the solvent used for extraction and pretreatment of the wood influence the composition and molar mass of the xylan extracted. Appropriate care is therefore necessary when comparing the molar mass parameters presented in Table 4 with literature data on wood hemicelluloses. The molar mass parameters for the birch glucuronoxylans documented in Table 4 correspond to degrees of polymerization (DP) (calculated as the average number of xylose units per polysaccharide molecule) of DPn ) 84-108 and DPw ) 101-122. These values are somewhat lower than those previously observed for birch xylans using osmometry26 or sedimentation.26 However, the values determined here do agree very well with more recent values obtained for birch

xylans utilizing SEC and viscometry measurements.38,39,81 It is not unlikely that the strong tendency for hardwood glucuronoxylans to form larger aggregates, even in aqueous alkaline solutions (as discussed in connection with Figure 5), may have caused earlier osmometric or sedimentation studies to somewhat overestimate the molar mass parameters. The molar mass parameters for the spruce, pine, and larch arabinoglucuronoxylans (SW-1a, SW-2a, and SW-3a) observed here correspond to degree of polymerization values of DPn ) 89-120 and DPw ) 107-145. These values are similar to those reported for native xylans extracted from spruce30 and from pine.82 In a similar manner, the molar mass parameters for the galactoglucomannans shown in Table 4 (SW-1b to SW-3b) correspond to DPn ) 90-102 and DPw ) 118-132. A galactoglucomannan with a similar degree of polymerization has been isolated from Nordic pine previously.83 Furthermore, the molar mass parameters for the galactoglucomannans investigated here are also consistent with those reported by Eeremeeva et al.39 for spruce glucomannan.

904


Table 4 also contains the molar mass parameters for the softwood and hardwood pulps examined. Xylans extracted from chemical pulps exhibited somewhat lower molar mass parameters than did xylans extracted from chlorite-delignified woods. This difference is as expected, since the pulping of wood modifies the structure of the hemicelluloses and causes partial depolymerization of the polysaccharide chains. The data in Table 4 show that the molar mass parameters of the hemicelluloses extracted were affected to different extents by the various cooking processes employed. Thus, the molar masses of the hemicelluloses isolated from pulps derived from the same batch of softwood chips decreased in the order ASAM pulp > MSSAQ pulp > kraft pulp. Although extraction with alkaline solutions is widely used for isolating hemicelluloses, this procedure suffers from the drawbacks that it may cause a certain amount of degradation of the polysaccharides (so-called alkaline peeling) and that the acetyl groups originally present in certain hemicelluloses are removed. Therefore, in the present study the hardwood kraft pulps and certain softwood pulps were subjected to sequential extraction with both DMSO and alkali. The molar mass parameters for the hardwood xylans sequentially extracted in this manner from the kraft pulps varied somewhat, depending on the solvent used for extraction. As seen in Table 4, extraction with DMSO yielded hexenuronoxylans with lower molar masses (corresponding to DPn ) 58-66) than did corresponding extraction with an alkaline solution (DPn ) 69-80). Hexenuronoxylans extracted from hardwood pulps collected from the same production line in a kraft pulp mill employing TCF bleaching (denoted HK-1 to HK-8 in Table 4) exhibited molar masses which were almost the same as those of the corresponding xylans extracted from the unbleached pulp, indicating that the bleaching processes exert only a minor effect on the molar mass parameters. Consistent with this finding are the relatively small (∼5%) differences in the molar masses observed for xylans extracted from softwood pulps bleached with oxygen, alkaline peroxide, or peracetic acid (SK-1 to SK-6). The glucuronoxylan extracted from the sulfite pulps with DMSO exhibited the lowest molar masses determined here by the SEC/MALDI procedure (Table 4). Again, this was as expected, since acidic sulfite cooking depolymerizes xylans more extensively than kraft cooking. The molar mass parameters of the xylans from the paper-grade pulps obtained using magnesium- or sodium-based sulfite cooking corresponded to degrees of polymerization values (DPn) ranging from 33 to 43. (The xylan SS-1 was deacetylated by the strong alkaline solution employed for SEC elution.) Extraction of the dissolving grade sulfite pulp, which had been subjected to prolonged sulfite cooking, with DMSO yielded glucuronoxylan with a DPn of 20, i.e., a value considerably lower than the corresponding values for the xylans from the softwood kraft pulps. The low DP values demonstrated by sulfite pulp xylans in comparison to the corresponding values for kraft pulps xylans measured here are in agreement with the earlier analyses (viscometry) of xylans extracted from chlorine-bleached birch sulfite and kraft pulps.14

Jacobs and Dahlman

Conclusions The present investigation shows that the molar mass parameters of hemicelluloses isolated from wood and pulps can be determined conveniently (without calibration standards) on an absolute molar mass scale by employing size exclusion chromatography and off-line MALDI-MS. The molar mass parameters of the birch and aspen glucuronoxylans determined here were found to be rather similar to those of the spruce and pine arabinoglucuronoxylans. This finding contradicts some older studies that have reported much higher degree of polymerization values for the glucuronxylan in hardwoods. The strong tendency for hardwood glucuronoxylan to form aggregates in aqueous solutions might have caused earlier investigators to somewhat overestimate molar mass parameters for this hemicellulose. The results demonstrate that the molar mass parameters of hemicelluloses extracted from chemical pulps are strongly dependent on the nature of the cooking process utilized for pulping and decreases in the following order: ASAM pulp > MSSAQ pulp > kraft pulp> sulfite pulp. In contrast, chemical pulps that had been subjected to totally chlorinefree bleaching yielded xylans with degrees of polymerization similar to those of the corresponding xylans extracted from the unbleached pulps. This indicates that such bleaching does not cause any significant depolymerization of the hemicelluloses remaining in the pulp. The carbohydrate analyses (performed employing enzymatic hydrolysis and subsequent capillary electrophoresis) revealed that both the pulping and bleaching processes in many cases altered the compositions of the hemicelluloses quite extensively. This is especially true for the 4-Omethylglucuronic acid residues (present in the xylans), which are converted to unsaturated hexenuronic acid residues during alkaline pulping and subsequently oxidized and split off during TCF bleaching involving ozone or peracetic acid treatments. In summary, the present report presents a comprehensive characterization of the molar mass parameters and carbohydrate composition of the major hemicelluloses in soft- and hardwoods, as well as for the corresponding hemicelluloses in unbleached and TCF-bleached pulps produced from wood. Acknowledgment. The financial support of the Jacob Wallenberg Research Foundation is gratefully acknowledged. The help provided by Ms. Karin Pihlsgård and Mr. Roland Mo¨rck with pulp extractions is also gratefully acknowledged. References and Notes (1) Timell, T. E. Wood Sci. Technol. 1967, 1, 45-70. (2) Shimizu, K. In Wood and Cellulosic Chemistry; Hon, D. N.-S., Shiraishi, N., Eds.; Marcel Dekker: New York, 1991; pp 177-214. (3) Ha¨gglund, E.; Lindberg, B.; McPherson, J. Acta Chem. Scand. 1956, 10, 1160-1164. (4) Lindberg, B.; Meier, H. SVen. Papperstidn. 1957, 60, 785-790. (5) Glasser, W. G.; Kaar, W. E.; Jain, R. K.; Sealey, J. E. Cellulose 2000, 7, 299-317. (6) Sun, R. C.; Fang, J. M.; Tomkinson, J.; Geng, Z. C.; Liu, J. C. Carbohydr. Polym. 2001, 44, 29-39. (7) Simson, B. W. SVen. Papperstidn. 1968, 71, 699-710. (8) Sun, R. C.; Tomkinson, J. Int. J. Polym. Anal. Charact. 1999, 5, 181-193.

Molar Masses of Hemicelluloses from Wood (9) Teleman, A.; Lundquist, J.; Tjerneld, F.; Stålbrand, H.; Dahlman, O. Carbohydr. Res. 2000, 329, 807-815. (10) Ebringerova´, A.; Hroma´dova´, Z.; Kacura´kova´, M.; Antal, M. Carbohydr. Polym. 1994, 24, 301-308. (11) Gabrielii, I.; Gatenholm, P.; Glasser, W. G.; Jain, R. K.; Kenne, L. Carbohydr. Polym. 2000, 43, 367-374. (12) Sun, R.; Fang, J. M.; Tomkinson, J.; Hill, C. A. S. J. Wood Chem. Technol. 1999, 19, 287-306. (13) Jain, R. K.; Sjo¨stedt, M.; Glasser, W. G. Cellulose 2000, 7, 319336. (14) Sjo¨stro¨m, E.; Enstro¨m, B. Tappi 1967, 50, 32-36. (15) Hansson, J. Å.; Hartler, N. SVen. Papperstidn. 1968, 71, 358-365. (16) Bergnor-Gidnert, E.; Tomani, P. E.; Dahlman, O. Nord. Pulp Pap. Res. J. 1998, 13, 310-316. (17) Johansson, M. H.; Samuelson, O. Carbohydr. Res. 1977, 54, 295299. (18) Teleman, A.; Harjunpa¨a¨, V.; Tenkanen, M.; Buchert, J.; Hausalo, T.; Drakenberg, T.; Vuorinen, T. Carbohydr. Res. 1995, 272, 5571. (19) Dahlman, O.; Mo¨rck, R.; Larsson, P. T.; Lindquist, A.; Rydlund, A. Proceedings of the 9th International Symposium on Wood and Pulping Chemistry, Montreal, Canada, June 9-12, 1997; pp M11-M1-4. (20) Rydholm, S. A. Pulping Processes; Interscience Publishers: New York, 1965; pp 1152-1166. (21) Young, R. A. Cellulose 1994, 1, 107-130. (22) Fernandez, E. O.; Young, R. A. Cellulose 1996, 3, 21-44. (23) Kettunen, J.; Laine, J. E.; Yrja¨la¨, I.; Virkola, N.-E. Pap. Puu 1982, 64, 205-211. (24) Pettersson, S. E.; Rydholm, S. A. SVen. Papperstidn. 1961, 64, 4-17. (25) Hartler, N.; Lund, A. SVen. Papperstidn. 1962, 65, 951-955. (26) LeBel, R. G.; Goring, D. A. I.; Timell, T. E. J. Polym. Sci., Part C: Polym. Symp. 1963, 2, 9-28. (27) Bykova, T.; Eeremeeva, T.; Klevinska, V.; Treimanis, A. Holzforschung 1998, 52, 475-480. (28) Fengel, D.; Wegener, G. In Wood: Chemistry, Ultrastructure, Reactions; Walter de Gruyter: Berlin, 1989; pp 106-131. (29) Glaudemans, C. P. J.; Timell, T. E. SVen. Papperstidn. 1958, 61, 1-9. (30) Zinbo, M.; Timell, T. E. SVen. Papperstidn. 1967, 70, 695-701. (31) Wikstro¨m, R. SVen. Papperstidn. 1968, 71, 399-404. (32) Sturgeon, R. J. Carbohydr. Res. 1973, 30, 175-178. (33) Timell, T. E. SVen. Papperstidn. 1960, 63, 668-671. (34) Churms, S. C. J. Chromatogr. A 1996, 720, 151-166. (35) Kringstad, K.; Ellefsen, O ¨ . Das Papier 1964, 10, 583-591. (36) Eriksson, K.-E.; Pettersson, B. A.; Steenberg, B. SVen. Papperstidn. 1968, 71, 695-698. (37) Ettling, B. V.; Adams, M. F. Tappi 1968, 51, 116-118. (38) Eeremeeva, T. E.; Khinonerova, O. E. Cellul. Chem. Technol. 1990, 24, 439-444. (39) Eeremeeva, T. E.; Bykova, T. O. J. Chromatogr. 1993, 639, 159164. (40) Bikova, T.; Klevinska, V.; Treimanis, A. Holzforschung 2000, 54, 66-70. (41) Lindquist, A.; Dahlman, O., Proceedings of the 5th European Workshop on Lignocellulosics and Pulp, Aveiro, Portugal, Aug 31Sept 2, 1998; pp 483-486. Chem Abstr. 1999, 130, 5014. (42) Jacobs, A.; Dahlman, O., Proceedings of the 10th International Symposium on Wood and Pulping Chemistry, Yokohama, Japan, June 7-10, 1999; pp 44-47. (43) Chitzhov, A. O.; Dell, A.; Morris, H. R.; Reason, A. J.; Haslam, S. M.; McDowell, R. A.; Chizhov, O. S.; Usov, A. I. Carbohydr. Res. 1998, 310, 203-210. (44) Dahlman, O.; Rydlund, A.; Lindquist, A. In The European Conference on Pulp and Paper Research. The Present and the Future; Arabatzis, A., Eriksson, L., Seoane, I., Eds.; the European Commision: Brussels, 1997; pp 231-237.

Biomacromolecules, Vol. 2, No. 3, 2001 905 (45) Garrozzo, D.; Impallomeni, G.; Spina, E.; Sturiale, L.; Zanetti, F. Rapid Commun. Mass Spectrom. 1995, 9, 937-941. (46) Garrozzo, D.; Spina, E.; Cozzolino, R.; Cesutti, P.; Fett, W. F. Carbohydr. Res. 2000, 323, 139-146. (47) Hao, C.; Ma, X.; Fang, S.; Liu, Z.; Liu, S.; Song, F.; Liu, J. Rapid Commun. Mass Spectrom. 1998, 12, 345-348. (48) Harvey, D. J. Mass Spectrom. ReV. 1999, 18, 349-451. (49) Karas, M.; Bahr, U.; Ingendoh, A.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F. Anal. Chim. Acta 1990, 241, 175-185. (50) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991, 63, 1463-1466. (51) Stahl, B.; Linos, A.; Karas, M.; Hillenkamp, F.; Steup, M. Anal. Biochem. 1997, 246, 195-204. (52) Yeung, B.; Marecak, D.. J. Chromatogr., A 1999, 852, 573-581. (53) Jacobs, A.; Dahlman, O. Anal. Chem. 2001, 73, 405-410. (54) Axelsson, J.; Scivener, E.; Haddleton, D. M.; Derrick, P. J. Macromolecules 1996, 29, 8875-8882. (55) Byrd, H. C. M.; McEwen, C. N. Anal. Chem. 2000, 72, 4568-4576. (56) Jackson, C.; Larsen, B.; McEwen, C. Anal. Chem. 1996, 68, 13031308. (57) McEwen, C. N.; Jackson, C.; Larsen, B. S. Int. J. Mass Spectrom. Ion Processes 1997, 160, 387-394. (58) Pasch, H.; Rode, K. J. Chromatogr. A 1995, 699, 21-29. (59) Zhu, H.; Yalcin, T.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 275281. (60) Hanton, S. D.; Liu, X. M. Anal. Chem. 2000, 72, 4505-4554. (61) Jacobs, A.; Dahlman, O. Nord. Pulp Pap. Res. J. 2000, 15, 120127. (62) Ahlgren, P. A.; Goring, D. A. I. Can. J. Chem. 1971, 49, 12721275. (63) Teder, A.; Sjo¨stro¨m, K. J. Pulp Pap. Sci. 1996, 22, J296-J300. (64) Dahlman, O.; Jacobs, A.; Liljenberg, A.; Olsson, A. I. J. Chromatogr., A 2000, 891, 157-174. (65) Timell, T. E. AdV. Carbohydr. Chem. 1965, 20, 409-483. (66) Timell, T. E. AdV. Carbohydr. Chem. 1964, 19, 247-302. (67) Koshijima, T.; Timell, T. E.; Zinbo, M. J. Polym. Sci., Part C: Polym. Symp. 1965, 11, 265-279. (68) Han, M.; Swan, B. SVen. Papperstidn. 1968, 71, 552-557. (69) Banerjee, S. K.; Timell, T. E. Tappi 1960, 43, 846-857. (70) Zinbo, M.; Timell, T. E. SVen. Papperstidn. 1967, 70, 597-606. (71) O ¨ hrn, O. E.; Croon, I. SVen. Papperstidn. 1960, 63, 601-605. (72) Patt, R.; Kordsachia, O. Das Papier 1986, 40, V1-V8. (73) Dahlbom, J.; Olm, L.; Teder, A. Tappi J. 1990, 73, 257-261. (74) Norden, S.; Teder, A. Tappi J. 1979, 62, 49-51. (75) Hausalo, T., Proceedings of the 8th International Symposium on Wood and Pulping Chemistry, Helsinki, Finland, June 6-9, 1995; pp 131-136. (76) Dahlman, O.; Rydlund, A.; Lindquist, A., Proceedings of the 9th International Symposium on Wood and Pulping Chemistry, Montreal, Canada, June 9-12, 1997; pp L5-1-L5-4. (77) Gellerstedt, G.; Li, J. Carbohydr. Res. 1996, 294, 41-51. (78) Tenkanen, M.; Gellerstedt, G.; Vuorinen, T.; Teleman, A.; Perttula, M.; Li, J.; Buchert, J. J. Pulp Pap. Sci. 1999, 25, 306-311. (79) Lou, X.; van Dongen, J. L. J.; Meijer, E. W. J. Chromatogr., A 2000, 896, 19-30. (80) Malfait, T.; Slootmaekers, D.; Van Cauwelaert, F. J. Appl. Polym. Sci. 1990, 39, 571-581. (81) Ebringerova´, A.; Eeremeeva, T. E.; Khinoverova, O. E. Carbohydr. Polym. 1991, 15, 255-263. (82) Harwood: V. D. SVen. Papperstidn. 1972, 75, 207-212. (83) Kenne, L.; Rosell, K.-G.; Svensson, S. Carbohydr. Res. 1975, 44, 69-76.

BM010050B

Characterization of the Molar Masses of Hemicelluloses from Wood

Recommend Documents