Solubility and molecular weight of hemicelluloses fromAlnus incanaandAlnus glutinosa. Effect of tree age

Tatiana Bikova *, Arnis Treimanis

State Institute of Wood Chemistry, 27 Dzerbenes Str., LV 1006 Riga, Latvia

Received 8 October 2001; accepted 28 December 2001

Abstract

The xylems ofAlnus incana Moench. andAlnus glutinosa Gaertn. 8-15-year-old trees were subjected to comparative studies regardingthe molecular weight (MW) of hemicelluloses. Extraction of hemicelluloses according to the sequence 1% NaOH-10% KOH-18% NaOH,followed by direct analysis of each extract using the SEC/UV method, was applied aiming at simultaneous quantitative and MW analysis.Both the tree age and alder species were found to affect the behaviour of hemicelluloses in alkaline solutions as well as their MW. Underequal extraction conditions, the solubility of xylan increased forA. incana and decreased forA. glutinosa with the tree age. Forglucuronoxylan and glucomannan, a cyclic MW oscillation within a certain range throughout the tree growth was found. Both the sinusoid-and impulse-type of MW changes were suggested. The MW of arabinogalactan and xyloglucan decreased to a certain invariant value in thetree age period under investigation. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Alkaline extraction;Alnus glutinosa; Alnus incana; Hemicelluloses; Molecular weight; SEC analysis; Tree age

1. Introduction

The wood cell wall contains up to 30% of hemicellulosesdepending on the wood species. Therefore, whole spectra ofhemicellulose properties should be examined in detail. Thecontent and chemical structure of hemicelluloses as well astheir distribution in the cell wall and wood tissues are thesubject of unremitting intensive investigations. Dudkin et al.[6], Sharkov and Kuibina[20], Katkevich[14], Higuchi[13]have overviewed the state of the art regarding these prob-lems.

Molecular weight (MW) is an important property ofhemicelluloses. The framework of the MW magnitude foran individual polysaccharide appears to be estimated. How-ever, it is so wide (for example, for xylan, from 16 to50 kDa; for mannan, from 5 to 30 kDa; for arabinogalactan,

from 9 to 250 kDa) [5,16,19–21] that more and morequestions arise regarding the actual value, whether it isinvariant along the same tree in different tissues or duringthe tree growth, etc., or the fluctuations are the methodologi-cal reasons only.

Different approaches aiming at studying tree develop-ment have been employed: (a) changes in the compositionthroughout the annual rings[12]; (b) the composition of thecambial tissue in comparison to juvenile and mature wood[15,19]; (c) changes in the composition of polysaccharidesdepending on the tree age[23]; (d) a study on developingthe cell culture[7,13]. A significant effect of wood maturityon the content and distribution of individual polysaccha-rides in the cell wall has been estimated both for an-giosperms and gymnosperms. However, an aspect such asMW of hemicelluloses regarding the tree age was notactually considered. In relation to this question, Katkevichet al. [15] have shown that the xylan fromPinus sylvestrisearlywood and latewood has a similar MWD.

The determination of the MW of hemicelluloses includestwo methodological backgrounds that are equal in strength,i.e. the isolation from the plant material and a method forestimation of MW. The former represents a multi-stageprocedure, which includes the removal of extractives and

Abbreviations: GPC, gel permeation chromatography; HPLC, high-performance liquid chromatography; LCC, lignin-carbohydrate complex;MWD, molecular weight distribution; SEC/UV, size exclusion chromato-graphy with ultraviolet detection; RI, refractive index; UV/VIS, ultravio-let–visible

* Corresponding author. Fax: +371-7550635.E-mail address: koks@edi.lv (T. Bikova).

Plant Physiol. Biochem. 40 (2002) 347–353

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© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 3 7 7 - 3

the isolation of holocellulose followed by alkaline extrac-tion and recuperation from the solution [3,13,19]. However,it is a well-established fact that hemicelluloses undergodegradation during this process. Then, the chemically trans-formed preparation is usually analysed by the GPC methodfor evaluation of MWD. Such a way, with minor modifica-tions, has been applied up to now [11,22,23]. RegardingGPC, the data, when examined, were in fact relative, i.e.according to dextran, polystyrene or pullulan[3,4,8,11,15,22–24]. Hence, the overall technique of theprecise determination of the MW of hemicelluloses shouldbe improved taking into account different aspects.

A direct analysis of alkaline extracts from wood, holo-cellulose and wood pulps utilizing the GPC method hasbeen proposed [2,4,9]. Numerous schemes of the fractionalisolation of hemicelluloses from wood are available fromthe literature [6,16,19–22]. Hence, depending on the plantorigin, the corresponding extraction scheme can be chosen.Such an approach, apart from the minimization of thetransformations of wood cell wall components arising fromtheir isolation, allows simultaneous quantitative and MWDanalyses of hemicelluloses, lignin and the lignin-carbohydrate complex (LCC) [2,4]. Additionally, directinformation about the bonds between the cell wall constitu-ents could be obtained [4].

The goal of our present investigation was to elucidatewhether the MW of hemicelluloses is invariable during treegrowth, utilizing the ‘extraction–direct SEC’ technique. Anapproach of comparative studies of trees of different agewas realized. Fast-growing Alnus incana and Alnus gluti-nosa were chosen for preliminary investigations. The ex-traction–direct SEC technique is under optimization; there-fore, the methodological aspects were also underconsideration.

2. Results and discussion

2.1. Interpretation of SEC data

Fig. 1 illustrates typical chromatograms of the fractionsobtained by step-by-step extraction from the same xylemsample. The chromatograms revealed multi-modal distribu-tion patterns for alkaline extracts reflecting their complexchemical composition. The extracts were directly analysedby SEC, hence, each fraction contained hemicelluloses,lignin and the LCC. Moreover, the first fraction wasenriched in extractives. For both Alnus species, the compo-sition of corresponding fractions was similar. In the case of1% NaOH extracts, the first sub-fraction (MW range 65-25 kDa) contained glucuronic acid, arabinose and galactosein the ratio 1-2:2-5:10; the second one (MW range 15-6kDa), xylose and glucose in the ratio 5-6:10. The polysac-charides extracted here were attributed to arabinogalactanand xyloglucan, respectively [19,20]. In the case of 10%KOH and 18% NaOH extracts, the ratio of glucuronic and

methylglucuronic acids mixture and xylose was 1-2:10,while the ratio of glucose and mannose was 5-7:10 in thefirst and second peaks (Fig. 1), respectively. Therefore, thefirst and second peaks on the RI-chromatogram represent4-O-methyl glucuronoxylan (called xylan) and glucoman-nan (called mannan) [5], correspondingly.

Wong and Jong [24] have shown that, under SEC in basicmedia, a part of hemicelluloses and lignin is eluted in theLCC form and is not simply co-eluted. The objective of adouble-detection SEC mode applied in our study wasmonitoring of LCC in the extracts. In alkaline media, thelignin structure forming sub-units really absorb above280 nm, therefore its distribution along the MW scale wasmonitored by the multi-wave UV/VIS detection mode [1].The combined RI- and UV/VIS-chromatograms (Fig. 1)clearly revealed that, in all fractions, high-MW peaks didnot absorb above 280 nm, i.e. xylan and arabinogalactanwere nearly lignin-free extracted. The second peak (6-16 kDa range), where the absorbance above 290 nm wasobserved, was bonded to lignin. Therefore, it was attributedto the LCC. Hence, mannan and xyloglucan extracted fromthe xylem were LCC-included hemicelluloses. The thirdpeak contained aromatic substances only, which were notconsidered.

Taking into account the fact that each extract representeda mixture of individual polymers, the multi-modal chro-matograms were convoluted into separate Gauss peaks, andthe concentrations as well as MW were calculated for eachof them. Table 1 summarizes the data on the composition

Fig. 1. Combined RI- and UV-chromatograms of fractions obtained bysequential extraction of A. incana xylem from a 12-year-old tree.

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and weight-average molecular weight (Mw) of hemicellulo-ses in the fractions obtained.

2.2. Solubility of hemicelluloses

Table 1 clearly reveals the changes in the chemicalcomposition of the alder xylem depending on the tree age,as well as some distinctions between two alder species. Thecomposition of alder wood has not been systematicallyinvestigated, and the data presented are scant and ratherdifferent [5,6,20]. In the case of A. incana, the content ofxylan ranges from 14 to 24% on wood. For A. glutinosa, theyield of purified xylan from the 10% KOH fraction ofholocellulose has been reported to be 17% [5]. Whencompared, the data on the xylan content obtained by thedirect method were in line with those from the standardtechnique.

The content of arabinogalactan averaged 1.5%, irrespec-tive of the tree age and alder species. The content ofxyloglucan, which was included into LCC, averaged 2%except the xylem of the A. glutinosa mature tree. ForA. incana, the content of xylan in the 10% KOH fractionconsequently increased up to a constant value of 17. 5%,whereas, for A. glutinosa, on the contrary, it decreased from17.4 to 8.3% with the tree age. For xylan additionallydissolved with 18% NaOH, no correlation between thesolubility and the tree age was observed. In the case ofA. glutinosa, a higher content of xylan in the 18% NaOH-soluble fraction was found for the mature tree. The mannanincluded in the LCC, was solubilized from young A. incanatrees with 10% KOH and 18% NaOH nearly in equalportions. Its content did not change in the xylem of youngA. incana trees, while in the xylem of the mature tree, thetotal content of mannan was doubled to be by 87% solublein 10% KOH. The behaviour of the mannan from youngA. glutinosa trees in alkaline solutions was different. It was

more soluble in 10% KOH solution than 18% NaOHsolution. For the mature tree, similarly to A. incana, itscontent was higher than that in young trees, and mannanwas by 88% dissolved in 10% KOH. In our opinion, thexylan and mannan fractions distributed between alkalisolutions originate from different tissues and/or cell walllayers that require further investigation. An insignificantamount (0.3-0.5%) of starch with an MW of 150 kDa wasalso identified in the 1% NaOH extracts of 8-10-year-oldtrees.

The total solubility of hemicelluloses for A. incanagradually increased with the tree age and, vice versa,decreased for A. glutinosa (Table 1). In principle, thetendency observed for A. glutinosa agreed with the findingsfor Eucalyptus globulus [23] and Pinus radiata [12]. Forbirch xylem, the dissolution of hemicelluloses of growingMW under sequential alkaline extraction has been shown[4]. MW data of Table 1 do not agree with results of [4]. Weexamined the correlation between the MW values and thesolubility of hemicelluloses. As shown in Fig. 2, the changesin MW were not the main factor governing the dissolutionof hemicelluloses under the procedure utilized. Cinıte et al.[4] have concluded the importance of the cell wall swelling,the sub-microscopic pore size and the destruction of bonds(mainly ether) for the extraction of hemicelluloses in basicmedia. Taking into account the fact that the techniqueemployed in our study was based on extraction, aspects suchas the degree of sample grinding as well as the time andtemperature of extraction can affect the magnitude of thesolubility of individual substance. However, the aim of thepresent work was not an exhaustive extraction of allhemicelluloses from wood, and the conditions employedwere the same for all samples compared. Hence, thechanges observed in the fractional composition appeared tobe the cumulative effect of the physico-chemical transfor

Table 1Fractional composition and weight-average molecular weight of hemicelluloses from the xylem of A. incana and A. glutinosa

Tree age (years) Component 8 10 12 15 30 - 55

Solvent % (onwood)

M√ w % M√ w % M√ w % M√ w % M√ w

A. glutinosa1% NaOH Arabinogalactan 1.2 54 700 1.3 46 030 1.8 43 550 1.8 33 790 1.5 29 060

Xyloglucan 1.8 11 070 2.1 7 620 2.4 5 370 2.3 5 350 3.7 5 10010% KOH Xylan 17.4 31 920 16.3 28 680 11.8 33 770 11.3 30 330 8.3 27 650

Mannan 2.1 13 600 2.1 16 640 1.8 19 360 1.9 17 600 3.0 11 37018% NaOH Xylan 3.4 34 500 1.9 32 500 1.8 41 010 2.1 35 120 4.6 29 700

Mannan 0.9 18 430 0.8 15 900 1.0 15 470 0.9 15 000 0.4 15 700Total solubility 26.8 24.4 20.4 19.5 20.0A. incana1% NaOH Arabinogalactan 1.2 43 680 1.7 42 290 1.4 40 300 1.7 35 340 1.7 26 250

Xyloglucan 1.9 9 070 2.4 8 870 2.2 7 860 2.6 6 290 2.3 5 00010% KOH Xylan 11.2 34 890 14.9 32 580 17.3 32 230 17.7 33 860 17.5 35 680

Mannan 1.0 10 590 1.0 7 715 1.2 8 420 1.1 9 880 3.5 12 22018% NaOH Xylan 2.6 30 940 2.6 29 640 2.9 29 120 1.9 30 560 2.4 33 740

Mannan 1.1 12 450 1.0 11 440 0.9 11 930 0.9 13 630 0.5 14 850Total solubility 18.9 23.5 25.9 25.9 27.9

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mations in the cell wall structure that occurred during thetree growth.

2.3. MW of hemicelluloses from Alnus

In general, the MW values of xylan and mannan deter-mined in the present work (Table 1) were in accordancewith the values usually indicated for deciduous species[6,16,19–21]. However, Dudkin et al. [5,6] have determinedthe MW of xylan isolated by 10% KOH and DMSO fromA. glutinosa holocellulose to be 16 500 (DP ∼ 110) and17 900 (DP ∼ 120), respectively. For mannan isolated froman 18% NaOH extract of A. glutinosa holocellulose, MWwas 7700-7740 [5]. It is obvious that the standard way ofMW estimation via holocellulose stage [5,6] resulted in anearly twice drop of MW for both polysaccharides. It shouldbe noted that the MW of hemicelluloses in sub-fractionsdecreased by about 30-50% after the isolation from thealkaline solution. Sun et al. [22] also noted the reduction ofthe MW of hemicelluloses and explained the observedphenomenon by degradation in strong alkali. For the tech-nique presented in our investigation, the questions regardingthe alkaline destruction of hemicelluloses, lignin and LCCare very important, and will be considered in a specialpaper. In brief, in all alkaline solutions applied, no visiblechanges in the MW values during the storage at about 24 h(under ambient temperature) were estimated.

Rather distinctive MW values were indicated in theliterature for arabinogalactan. When it was isolated from thecambial tissue of Populus tremuloides, Mn was 200 kDa[19]; when isolated from Larix sibirica bark, Mw was foundto range from 9-10 kDa to 60-70 kDa [6,8]. Apparently, theactual MW values of arabinogalactan depend also on itslocalization in the wood tissue, wood species, etc., thatrequires further elucidation.

For both Alnus species, the xylan and mannan distributedbetween the alkali solutions differ in MW (Table 1). In thecase of A. incana, the MW of xylan dissolved in 10% KOHwas somewhat higher than that additionally extracted with18% NaOH. In the case of A. glutinosa, vice versa, the MWof xylan in the second fraction was somewhat higher. The

xylan of the A. glutinosa xylem was less polydisperse thanthat of the A. incana xylem (Mw/Mn was 1.09-1.16 and1.15-1.25, respectively). At the same time, the MW ofA. incana mannan, which was dissolved in 10% KOH, waspersistently lower than that from the fractions additionallydissolved in 18% NaOH. In all cases, a narrow polydisper-sity of mannan was estimated to be 1.05-1.11.

2.4. MW and tree age

The mean deviations calculated for the Mw values listedin Table 1 to be 20, 27, 10 and 16% for arabinogalactan,xyloglucan, xylan and mannan, respectively, outnumberedthe accuracy of the MW determination by the SEC method(that was 3%). Hence, the variations observed were notrandom and should be examined more closely. It is wellknown that the changes in the Mw magnitude are rathersensitive to the chemical transformations in the elementalring (i.e. oxidation, deacetylation, cleavage of branchesetc.); therefore, the values of both the weight- and number-average degree of polymerization, DPw and DPn, respec-tively, were calculated. Figs. 3-5 visualize the effect of thetree age upon the MW of hemicelluloses. For each polysac-charide under study, the changes in DPw and DPn valueswere similar.

In the case of arabinogalactan, in spite of some distinc-tions between two alder species, MW decreased with thetree age (Fig. 3A). Even a more significant MW drop wasobserved in the case of A. glutinosa. For both Alnus species,DPs were lower in the case of the mature tree xylem.Polydispersity (Mw/Mn) ranged from 1.18 to 1.21 in allcases.

The effect of the tree age upon the MW of xyloglucanwas also observed (Fig. 3B). In general, the MW droppedapproximately twice during the tree growth. At the sametime, the rate of the MW decrease was distinctive forA. incana and A. glutinosa. For the former, after a 10-yearage, MW subsequently decreased, whereas, for the latter,after rather a fast dropping during an 8-12-year growthperiod, MW reached a certain invariant magnitude, whichwas the same for the mature xylem.

Fig. 2. Relationship between the solubility of hemicelluloses in alkaline solutions under sequential extraction and weight-average molecular weight.

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For xylan, Fig. 4 shows another kind of the dynamics ofMW changes as well as some distinctions in MW regardingalder species. Fig. 4 reveals unexpected regular fluctuationsin the MW of xylan with the tree age. For A. incana, the DPvalues smoothly diminished during the 8-12-year periodand, at the further tree growth, DP increased. Higher DPvalues were observed for the 30-year-old tree. In the case of

A. glutinosa, uneven changes of DP with a certain period-icity, i.e. a recurrent great MW increment, followed by agradual decrease, were observed. Apparently, during the treegrowth, cyclic oscillations in the MW of xylan with adistinctive periodicity regarding the wood species occurred.In the case of A. incana, the step of the MW change withinthe 8-15-year period was calculated to be approximately tenand five xylose residues per year for the fractions of xylandissolved in 10% KOH and 18% NaOH, respectively.

For mannan in the case of the A. incana xylem, Fig. 5shows the same dynamics of MW changes as for xylan. Inboth the fractions, DP was the lowest in the case of the10-year-old tree and the highest for the 30-year-old tree. ForA. glutinosa, MW consequently changed through the maxi-mum for the mannan dissolved in 10% KOH, and, viceversa, through the minimum for that dissolved in 18%NaOH. In both the fractions, an extreme was observed forthe 12-year-old tree. For the xylem of young trees, the MWof mannan from A. glutinosa was persistently higher. How-ever, the differences in the magnitude of MW of mannanestimated for young trees between Alnus species werelevelled off for mature trees. A similar phenomenon wasalso observed for xyloglucan.

For xylan and mannan of A. incana and mannan ofA. glutinosa, the MW changes during the tree growth weresimilar to a sinusoid-type curve (Fig. 6A). For A. glutinosaxylan, the MW changes could apparently be attributed to animpulse-type curve (Fig. 6B). Unfortunately, neither the realamplitude of the oscillation nor the initial point of theprocess could be determined from the data obtained owingto the narrow range of tree age under study. At the first sight,MW oscillations for xylans and mannans in the case of

Fig. 3. Effect of the alder tree age on the weight-average and number-average degree of polymerization (DPw and DPn, respectively) of arabino-galactan (A) and xyloglucan (B).

Fig. 4. Effect of the alder tree age on the weight-average and numberaverage degree of polymerization (DPw and DPn, respectively) of mannan.

Fig. 5. Effect of the alder tree age on the weight-average and number-average degree of polymerization (DPw and DPn, respectively) of xylan.

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A. incana in contrast to those of A. glutinosa were synchro-nous (Figs. 4 and 5), although, this synchronous effect mightbe only apparent. From the MW oscillations phenomenon,our hypothesis is as follows. The parameters of the MWoscillation (amplitude, period) for an individual polysaccha-ride in their specific combination are an immanent charac-teristic of a certain wood species, and such a combination isgenetically determinated. This genetic combination of indi-vidual MW oscillations for a single plant object (species)governs the rate of the plant’s maturity. From the standpointof the similarity at evolution, the MW oscillations inrelation to the tree’s growth are a reflection of the similarprocesses on the cellular level. The concentric arrangementof both the cellulose fibrils in wood cell wall lamella [3,10]and the year-rings in a tree is a manifestation of the similarmode for this structure formation at tree evolution.

3. Conclusions

An analysis of the MW of hemicelluloses by an adequatetechnique revealed that the MW of individual polysaccha-rides undergoes transformations during the tree growth. Thephenomenon of periodicity is an immanent property ofliving substances; hence the cyclic oscillations of MWvalues are not unique and appear to be connected with thedeep metabolic transformations during the tree growth. TheMW changes observed regarding the tree age should beassumed to reflect the similar changes that occurred on thecellular level. The similarity in the type of MW changes inline with the distinctive dynamics of changes (i.e. ampli-tude, step and rate of oscillation damp) apparently reflectedthe genetically determinated rate of tree’s maturity amongspecies within the alder family. Thus, the MW of hemicel-luloses should be systematically examined, since the treeage, species, morphology, etc., undoubtedly, have an effecton their magnitude, which, in turn, should be very importantto understand the general laws of plant development.

4. Methods

4.1. Materials

Xylems of 8-, 10-, 12- and 15-year-old trees of Alnusincana Moench. and Alnus glutinosa Gaertn. were subjectedto investigations. Tree sampling and wood characterizationhave been described previously [17]. For comparison pur-poses, the mature xylem of 30- and 55-year-old trees ofA. incana and A. glutinosa, respectively, was used. Thecomposition of mature tree wood has been presented earlier[18].

4.2. Extraction

The sequence for selective extraction of different hemi-celluloses from deciduous trees described in [16] wasutilized. 100 ± 0.05 mg of an oven-dried (50 °C) milledxylem (a fraction of 0.40-0.60 mm) sample was placed intoa centrifuge plastic tube and 2 ml of aqueous 1% NaOHsolution added. After 60 min, the extract was decanted anddirectly subjected to SEC analysis. The xylem residuewashed twice with the same alkaline solution (4 ml), cen-trifuged and decanted was further treated by the sameprocedure with aq. 10% KOH for 2 h, then with aq. 18%NaOH for 1 h. All experiments were carried out at ambienttemperature.

4.3. SEC conditions

Ten microliters of each alkaline extract were analysed bythe double detection SEC technique (a refractometer in linewith an UV/VIS detector) [1]. A cartridge class columnSeparon HEMA 1000 BIO (TESSEK Ltd., Prague, CzechRepublic) was used. Aqueous 2.5 mM NaOH was an eluent.The analysis time at a flow-rate of 0.3 ml·min-1 was 5 min.The concentration of hemicelluloses in the extract wasdetermined from the preliminary obtained calibrationgraphs (peak area vs. calibrant concentration) and recalcu-lated per sample weight. An average MW of hemicelluloseswas determined using calibration with the hemicellulosesfractions that were characterized by viscometry as describedpreviously [9]. Data presented were averaged from threeparallel experiments. For quantitative analysis, the meandeviation was 1.5 and 2.8% for the concentration rangeabove and below 1 mg·ml-1, respectively; for MW determi-nation, it was 3%.

4.4. Calibrant preparation

From 1 and 18% NaOH as well as 10% KOH extracts,the sub-fractions corresponding to each peak on the RI-chromatogram (for each extract, three fractions) were col-lected during the SEC analyses. The solutions were com-bined regarding alder species and the tree age.Hemicelluloses were isolated from the alkaline solution via

Fig. 6. Schematic representation of the MW oscillations for hemicellulosesduring the tree growth. A, xylan and mannan of A. incana as well asmannan of A. glutinosa; B, xylan of A. glutinosa (bold line: periodobserved).

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neutralization with acetic acid, followed by precipitationwith ethanol, then drying by the solvent replacing [20]. Thecomposition of hemicelluloses in the sub-fractions obtainedwas determined via analysis of carbohydrates in the acidhydrolysates by HPLC [18].

References

[1] T. Bikova, V. Klevinska, T. Eremeeva, A. Treimanis, Study onalkali-accessible chromophores from unbleached kraft pulp, Holz-forschung 55 (2001) 554–558.

[2] T. Bikova, A. Treimanis, Direct analysis of molecular-mass andchemical heterogeneity of plant cell wall components, Proc. Work-shop of COST E20 (Wood Fibre Cell Wall Structure), 26-28 April,Uppsala, Sweden, 2001, pp. 20–21.

[3] R.M. Brown, J.W. Herth, W.W. Franke, D. Romanovicz, The role ofthe golgi apparatus in the biosynthesis and secretion of a cellulosicglycoprotein in Pleurochrysis, in: F. Loewus (Ed.), Biogenesis ofPlant Cell Wall Polysaccharides, Academic Press, New York, 1973,pp. 207–258.

[4] V. Cinıte, P. Erins, A. Rebyatnikova, I. Stoldere, Study of birch woodalkali extraction, Khimiya drevesiny (Wood Chemistry) 6 (1975)38–46 (in Russian).

[5] M.S. Dudkin, A.T. Bezusov, N.G. Shantova, Investigation of hemi-celluloses from alder wood, Khimiya drevesiny (Wood Chemistry)14 (1973) 27–30 (in Russian).

[6] M.S. Dudkin, V.S. Gromov, N.A. Vedernikov, R.G. Katkevich,N.K. Cherno, Hemicelluloses, Zinatne, Riga, 1991.

[7] Y. Edashige, T. Ishii, T. Hiroi, T. Fujii, Structural analysis ofpolysaccharides of primary walls from xylem differentiating zonesof Cryptomeria japonica D. Don, Holzforschung 49 (1995)197–202.

[8] T. Eremeeva, T. Bykova, Analysis of larch arabinogalactan by highperformance size-exclusion chromatography, Carbohydr. Polym 18(1992) 217–219.

[9] T. Eremeeva, T. Bykova, High-performance size-exclusion chroma-tography of wood hemicelluloses on a poly(2-hydroxyethylmethacrylate-co-ethylene dimethacrylate) column with sodium hy-droxide solution as eluent, J. Chromatogr. 639 (1993) 159–164.

[10] J. Fahlén, L. Salmén, The lamella structure of the wood fiberwall—radial or concentric, Proc. Workshop of COST E20 (WoodFibre Cell Wall Structure), 26-28 April, Uppsala, Sweden, 2001,pp. 25.

[11] I. Gabrielii, P. Gatenholm, W.G. Glasser, R.K. Jain, L. Kenne,Separation, characterisation and hydrogel-formation of hemicellulo-ses from aspen wood, Carbohydr. Polym. 43 (2000) 367–374.

[12] V.D. Harwood, Variation in carbohydrate analyses in relation towood age in Pinus radiata, Holzforschung 25 (1971) 73–77.

[13] T. Higuchi, Biosynthesis and Biodegradation of Wood Components,Academic press, London, 1985.

[14] R.G. Katkevich, Hemicelluloses, in: V.N. Sergeeva (Ed.), Cell Wallof Wood and its Transformations under Chemical Action, Zinatne,Riga, 1972, pp. 103–127 (in Russian).

[15] R.G. Katkevich, Yu. Yu. Katkevich, A.A. Cinite, D.E. Liepina,Changes in hemicelluloses during pine wood formation, Khimiyadrevesiny (Wood Chemistry) 5 (1977) 3–12 (in Russian).

[16] Z Kin, Hemicellulozy, Chemia I Wykorzystanie, Panstwowe Wy-davnictwo Zolnicze I Lesna, Warszawa, 1980 (in Polish).

[17] V. Klevinska, T. Bikova, Comparison of the properties of black andgrey juvenile alder wood, Holz als Roh- und Werkstoff 57 (1999)246.

[18] V. Klevinska, T. Bykova, A. Treimanis, Differences in the chemicalcomposition and properties of unbleached grey and black alder kraftpulps, Cell. Chem. Technol 34 (2000) 163–172.

[19] B.W. Simson, T.E. Timell, Polysaccharides in cambial tissues ofPopulus tremuloides and Tilia americana, Cell. Chem. Technol. 2(1978) 39–84.

[20] V.I. Sharkov, N.I. Kuibina, Chemistry of Hemicelluloses, Lesnajapromislenost, Moscow, 1972 (in Russian).

[21] E. Sjöström, Wood Chemistry, Fundamentals and Applications,second ed., Academic Press, New York, 1993.

[22] R.C. Sun, J.M. Fang, J. Tomkinson, Z.C. Geng, J.C. Liu, Fractionalisolation, physico-chemical characterization and homogeneous es-terification of hemicelluloses from fast-growing poplar wood, Car-bohydr. Polym. 44 (2001) 29–39.

[23] A.F.A. Wallis, R.H. Wearne, P.J. Wright, Chemical analysis ofpolysaccharides in plantation eucalypt woods and pulps, Appita 49(1996) 258–262.

[24] K.K.Y. Wong, E. de Jong, Size-exclusion chromatography of lignin-and carbohydrate-containing sample using alkaline eluents, J. Chro-matogr. A737 (1996) 193–203.

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