Determination of Cellulose and Apparent Hemicellulose

7/29/2019 Determination of Cellulose and Apparent Hemicellulose

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2997Reprinted from ANALYTICAL BIOCHEMISTRY. Volume 43. ::\umber 2. October 1971

Copyright 19i1 by Academic Press. Inc. Printed in U. S. A.,INALYTlCAL BIOCHI,;MISTRY 43, 539-546 (l9i1)

Purchased by Agricultural Research Service, U.S. Dept . of Agriculture, for official use

Determination of Cellulose and Apparent HemicelluloseIn Plant Tissue by Gas-Liquid Chromatography

,J. H. SLONEKER,'Vorthem Regional Research Laboratory,' Peoria, lliinoi.$ 6160'f

Received February 24. 19i1Quantitative determination of cellulose and hemicellulose in biological

materials is tedious and time-consuming by established methods. For example, the determination of cell wall constituents and fiber content offeeds and forages is based on gravimetric procedures ( l ,2) . These methodsrequire that a rather large sample be repeatedly extracted with a seriesof solvents 'which successively remove lipids, proteins, lignin, etc. Eachdesired fraction is collected by some means, dried, and weighed. Accuracyof these methods depends upon the efficiency '\vith which extraction removes unwanted material from the appropriate fractions.

Cellulose is much easier to determine than other cell wall polysaccharides. A simple extraction procedure is available that solubilizes essentiallY all the protein, lignin, lipid, and hemicellulose, leaving the cellulosefibers intact (3). Combined with a colorimetric assay for hexose, thismethod can measure the cellulose content of bacterial cultures (4).

Likewise, pentosans have been measured colorimetrically in biologicalmaterials (5). However, this method, like the one for cellulose, lacks inspecificity and is subject to error because color produced by noncarbohydrate material accompanying the polysaccharides interferes.GLC2 is a simple quantitative method for measuring simultaneouslyup to eight aldoses as their alditol acetate derivatives (6). This methodis effective for the five aldoses found in wood pulp (7) and the cell wallpolysaccharide constituents of suspension cultures of sycamore cells (8).Although it requires a hydrolysis step, GLC has advantages over gravimetric and colorimetric procedures because individual aldoses aremeasured directly and because variations in the aldose content of differentsamples are readily visualized.

Our objective was to develop a procedure using GLC to determine cel-, This is a laboratory' of the ::\orthern Marketing and ::\utrition Research Division,

Agricultural Research Service, U. S. Department of Agriculture., Abbreviations: GLe. gas-liquid chromatography; R. response factor.

539


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.540 .r. H. SLO::\EKERlulose and apparent hemicellulose in foods. fced. and feedlot \vastes bymeasuring the neutral carbohydrates within these materials. In themethod described here, total neutra l carbohydra te content is measuredin one sample and cellulose is isolated by an extraction procedure andmeasured in a like sample. The content of hemicellulose, based on theneutral aldoses. can be readily established by difference. The content ofstarch may be closely approximated abo from the difference between thetotal D-glucose and that derived from cellulose. In addition our methodcan be med for materials containing up to 75% protein.

METHODSJlateriuls. Alditol acetates were separated OIl a 7 ft X in. stainless-

steel column li.d. 0.085 in.) packed with 370 ECNSS-}\I coated on 100120 mesh Gas-Chrom Q (Applied Science Laboratories, State College,Pa.) ." The column was conditioned overnight at 220 and operated at190-19;'). Carrier gas flow rate was maintained at 40 ml/min. The c11l'0-matographic instrument was an F&M model 810 equipped with a model3370A electronic integrator (Hewlett-Packard, Avondale, Pa.).

Standard sugars obtained commercially were used without further purification. Two internal stanclarcb were : i-inositoL which was added immecliately after the sulfuric acid hydrolysis step, and 2-deoxY-D-glucose,which was added immediately after the samples were autoclaved becauseof its relative lability in acid. The former standard may be used only ifthe material to be tested is free of the polyalcohol. Standard sugar mixtures were run with each set of determinations to minimize error due tounavoidable minor variations during autoclaving that cause small variations in R values.Determination oj Total Aldose Content . Samples were prepared bygrinding the material fine enough to pass a 60- or SO-mesh screen. Moisture values were determined by drying each sample (1.50-300 mg) in atared weighing bottle overnight under vacuum and over phosphoruspentoxide at room temperature followed lly heating for 4 hI' at 100.To measure total neutral carbohydrate, the samples (20-30 mg) were

carefully \H-ighed into 1.5 X 12,5 mm Teflon-lined screw-capped testtubes. Sulfuric aeid (0.3 ml of 7270 coneentrationJ was added aeeuratelyto each tube and the tube contents were heated to 30 for 1 hr. To disperse the material better in sulfurie aeid, the contents were stirred withboth a small glass rod and a test tube mixer, after which the glass rod

"The mention of firm names or trade products does not imply that they areendorsed or recolllmended by the Department of Agriculture o\' er other firms orsimilar products not me!ltioned,


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GLC OF POLYSACCHARIDES Ii\" PL\ i\ "T TISSI-E 541\vas broken, leaving the bottom half in the test tube. After 1 hI' thesamples ,vere completely dispersed except for those high in lignin.Distilled water (8.4 ml or 7.4 ml plus 1 ml of water containing 5 mg ofl:-inositol) was added to each sample to dilute the sulfuric acid to 1N.The diluted samples were placed in a preheated autoclave and were hydrolyzed for 1 hI' at 120. After the hydrolyzates cooled. the tubes ,verecentrifuged before they were opened to add 2-deoxY-D-glucose standard(5 mg in 1 ml of waterl. After the contents in each tube had beenthoroughly mixed, two 2 ml portions of hydrolyzate were transferred toindividual 12 ml conical centrifuge tubes where the sulfuric acid wasneutralized with lead carbonate. Lead salts were removed by centrifugation, and aldoses in the supernatant were reduced to alditols in 1 hI' atroom temperature upon the addition of sodium borohydride (10-20 mg in0.5 mI). Excess borohydride was destroyed by adding acetic acid ( ldropwise until the effervescence of hydrogen ceased.

Each reduced sample was passed over a column composed of a disposable Pasteur pipet (5 mm i.d. X 145 mm length) containing 1 ml ofAG-50X4 resin (200-400 mesh) in the H-;- form (Bio-Rad Laboratories,Richmond, Calif.) and was collected in a 15 X 125 mm Teflon-linedscrew-capped test tube. Each column was washed with 1 ml of water thatwas pooled with the appropriate reduced sample. Contents of each tubewere evaporated to dryness on a test tube evaporator (Buchler Instruments, Inc., Fort Lee, N . .T.) adapted to handle the screw-capped testtubes. Borate ions produced during reduction were removed as trimethylborate by repeatedly (3X) dissolving the contents of each tube inmethanol (1 ml) and removing alcohol by evaporation. The residualalditols in each tube were acetylated for 16 hI' at 100 with pyridineacetic anhydride (0.2 ml of a freshly prepared 1: 1 mixture). The acetylated alditols were injected directly onto the GLC column.Percentage of individual aldoses in a given material was calculated fromthe relation:

(mg s)(area a)(C) (100)aldose % = - = : ' - " - ; = ' - - ' - - - ' - ~ - - - ' - ' - - ' ; - ' - - ~(R)(area s)(mg unknown)where s and a represent the internal standard and aldose, respectively. Crepresents the conversion factor for aldose to polysaccharide: 0.88 forpentoses and 0.90 for hexoses. R represents a response fador, which wasdetermined for each aldose using a known aldose mixture, and ,vas obtained from the relation:

R = (mg s) (area a). (mg a) (area s)


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542 .T. H. tiLOXEKER/)etenuiuatiou of Cellulose. (':pllulo"p wa" i"olatpd for GLC :muly"i"

by an extraction procedure (3,41 which was modified slightly to improyerecovPry of the product. Finely ground "amplps (30-150 mg dependingon the cellulo"e content) were weighed into ];) X 125 mm TpfJon-linedscre\v-capped test tubes. Acetic-nitric acid rpagent i3 ml of a mixturecontaining 150 ml of 80% acetic acid and 15 ml of concentrated nitricacid) was added slowly to each tube while the contents were being mixed.Each tube was tightly capped and heated for 0.5 hI' at 100, after whiehthe tubes were cooled and centrifuged. The supernatant was remoyed witha disposable Pasteur pipet and discarded. The fibrous precipitate waswashed twice with thp acetic-nitric acid reagent (3 ml) and twice withacetone 2 mil . .:\Iost of the residual acetone remaining after the lastwash was remoyed by eyaporation. The cellulosic residue was analyzedas described for the tota l sugars.

Determination of Starch. To obtain a more accurate determination ofstarch by GLC, the materials to be hydrolyzed were first extracted fourtimes with 80% methanol (3 ml each) to remoye free glucose and glucosecontaining oligosaccharides. The quantity of starch was then equal to thetotal glucose recovPrecl (calculated as C"H",O,:;) minus that for cellulose.

RESl'LTS A.'\D DISCl'SSIONThe two-stage hydrolysis technique using 72% and 1N sulfuric acid

(9) is superior to other methods of hydrolysis, especially for materialsthat are insoluble in water or dissolve with difficulty. Even so, such mate-rials as whole corn, one of the more difficult materials to hydrolyze, mustbe finely ground and must he stirred vigorously to achieye complete dissolution in 12% sulfuric aciel. Also, materials high in lignin will not dissol\'e completely in nrc sulfuric acid because lignin remains insolubleeyen at this acid concentration.A threefold concentration of protein in the form of boyine serum al

bumin i Pentex, Inc., Kankakee, II1. I has no detectable effect on the recO\'ery of the fiye aldoses commonly found in plant tissues (Table 11. Thishydrolysis technique should therefore permit the accurate analysis of car-bohydrate in materials containing up to 75% protein. The higher concentrations of protein did interfere with neutralization and borohydride reduction of the hydrolyzates by causing excessiYe foaming. Howeyer, noproblems are encountered during acetylation and GLC because the AG-50resin column remoyes most of the free amino acids and peptides formedduring the partial hydrolysis of the protein.

Gnder the hydrolysis conditions employed, degradation of the aldosesis less than 5% except for D-xylose, for which it is approximately 20%.Howeyer. these figures may yary from one determination to anothl'1' be-


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.Oli ~ , O . O : l ,;.0:1 O.o:l '1.!l0 0 .05 4 .SU 0.0,;10 2S Ii '1.!l2 01,0.01\ 5 .(H lUll 'l .!ll (Ull 4 sli 0.01i -I.K) O.OIi 5.02 ,1,0.11 '>.11 ,1,1J.01i 5.01 0.0" 5 .0 1i 0.21 4.!H 0.1215 :G tl 5 .04 , ~ I L O t ; 5 .HJ0 .01 5.07 O . I I 4.!I!I 0 . 0 7 5.05 (Ulli ';.O!l ,I ,O.H " .2 1 O.O!l ;,.12 ::i: 0.02 5. () .! 0.02 5.10,j ,0.o:!2[, ',IJ.O 4.S!I ,I,O ..(H ';.Oli O.(H ';.O!l 01,0.14 1.!lS 01, O. H 1.!l2 o. 1;; 4.77 01,0.11 I. !I:! 0.12 1.!lU 01,0.02 4 . S,; 0.02 'I. 7!1 lUlla5 M";,4 .1. SS O.OlJ 5.05 O.O!l .1.!J.1 01,0.10 'I.SS 01,0.07 -I.SI 0.05 4.!l0 0.01 5.07 0.02 -1.\lU O.o:! 4 . !l0 (Ull -I.S:I O.o:!t,O tW.t 'l.S7 O. O!I ' ;. O! l 0 .l J: l , ;. OS 0.10 I.!I!I O.OS Ul l (Ull 4.7U 01,0.0.; I.!lS 0.07 I.!ln O.D:! I.SS , ~ O . O ; , 4.S0 , ~ O . 127:, 7t,.O 4.!lS ~ , o . m ! '; .2,; (1. OS 5.2S ~ , 0 . 1 l \ 5.12 ~ , 0 . 1 : 1 5.00 0 .07 ' 1 . 7 : l , ~ 0 . 0 1 1.!lS ol,O.(ll ".01 0 .07 I.SU O.IH ,1.75,1,0.01

Av.: 4. !I:l 5.10 5.0r, 1.HfJ 1.02 .j .sn ;'.on ;'.01 4. \I:! I.SS!;oj

'"t-


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544 J . H. SLONEKERcame of small differenees in acid strength and temperature of hydrolysis.Inaccurate addition of the small volume of 727'10 sulfuric acid to thesamples will produce a rather large error in acid strength after dilution.This source of error can be minimized if automatic repeating dispensersare used when 7 2 ? ~ acid and diluent are added. Effects of variable auto-clave temperatures can be eliminated by running aldose standards alongwith each set of analyses.Avicel, a microcrystalline cellulose obtained by selective removal of

the amorphous regions in native cellulose by controlled acid hydrolysis(American Viscose Division, FMC Corporation, Newark, Del.), was usedto establish the efficiency of the extraction procedure for the recovery ofcellulose (Table 21. Average yield of carbohydrate was in excess of 99%while that of the unextracted sample averaged 101 %. Slightly lower yieldsfor the extracted samples can be attributed to a small loss of celluloseduring removal of the extraction reagent and wash solvent. Use of thefresh reagent to wash the extracted cellulose instead of water as reportedpreviously (3,41 minimized this loss because in ivater the extracted cellulose swelled and became difficult to pack during centrifugation. Theach'antage of the GLC method over the gravimetric and colorimetricmethods is readily apparent from Avicel data (Table 2). D-Xylose andD-mannose in respective yields of approximately 0.6 and 1.0% are in the

TABLE 2Determination of Neutral Sugars and Cellulose in Selected Materials"

Carbohydrate content,

Sample2-DeoxY-D-glucose, internal stanclard

L-Arabino;;e D-Xylo;;e D-Mannose D-Galactose D-Glucose Total

15.3 0.6 28.1 0.9

Avice!"Avicel\Vhole com'"\Vhole corn",i\Vhole corn'Cornpericarpb.,Cornperi carp'

SucroseFructose

4 0.1.f) 0.1

0.6 0.10.6 0.1Trace

2.2 0.22.2 0.20.4 0.1

1.0 0.11.0 0.1TraceTrace


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GLC OF POLYSACCHARIDES I::\ PLA:: \T TISSUE 545crystalline cellulosic material. Furthermore, these t\\'o sugars are not removed by the extraction procedure and would be undetected with othermethods of analysis.

VVhole corn. chosen for its relatively low content of cellulose and hemicellulose, \\'as analyzed three ways: for cellulose, for total carbohydrate,and for total carbohydrate less the extractable mono- and oligosaccharides(Table 2). As with Avicel, the cellulose fraction from whole corn contained a small amount of D-xylose. In the whole corn samples good agreement is shown for L-arabinose and D-xylose, the major constituents ofthe hemicellulose fraction, even after one group of samples was extractedwith 80% methanol. However, methanol extraction did change the yieldof D-glucose, as would be expected because free D-glucose and D-glucosecontaining oligosaccharides are removed by the aqueous methanol. Thequantity of starch in this sample of corn can be calculated by subtractingthe value for cellulose from that for total D-glucose in the methanolextracted samples. The value, 70.4%, is in excellent agreement with that,70.8%, obtained on this sample of corn by two different extractionprocedures (10).

The quantity of cellulose and hemicellulose in corn pericarp is approximately tenfold greater than for whole corn (Table 2). Besides, most ofthe D-mannose and D-galactose in corn is concentrated in the pericarp.Like the other cellulose determinations, the cellulose isolated from pericarp contains a small quantity of D-xylose in some form. Apparent starchcontent of the pericarp fraction is low compared to whole corn.Sucrose and D-fructose were examined to determine the fate of D-fruc

tose during hydrolysis and reduction. Potential ly, D-fructose can be reduced to either D-mannitol or D-glucitol and could cause error in thedetermination of D-mannose and D-glucose. The results indicate that ap-proximately 95% of the D-fructose is destroyed during acid hydrolysisand its contribution to the error in the determination of the two hexosesis negligible in the samples chosen (Table 2). However, in materials containing large quantities of sucrose or the fructose polymer, inulin, thiserror may become significant.

This method is now being used routinely to measure the aldose contentof a wide variety of biological materials such as feed, feed fractions, andfeedlot wastes in order to follow the digestibility of cellulose andhemicelluloses.

SUM1VIARYA GLC method is described by which the neutral aldoses and cellulose

can be measured in whole and digested plant tissue. Apparent hemicellulose is measured by difference. Accuracy of the method is unaffected byprotein in concentrations up to 75%.


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546 ,}. H. SLONEKER

ACKNOWLEDGMENTThe technical assis tance of Mr. Joseph F. Mitton, who carried out the sucrose

and D-fructose analyses, is gratefully acknowledged.REFERENCES

1. VAN SOEST, P. J. , J. Anim. Sci. 23, 838 (1965).2. COLBURN. M. 'Y., EVANS, J. L., AND R.nlAGE. C. H., J. Dairy Sci. 51, 1450 (1968).3. CRAMPTON, E. W., AND MAYNARD, L. A., J. NutI'. 15, 385 (1938).4. UPDEGRAFF, D. M., Anal. Biochern. 32, 420 (1969).5. "Annual Book of ASTM Standards ," American Society fo r Testing and Materials.

Philadelphia, Pa. , Part 15, 1970. ASTM Designation D1787, p. 595.6. S.\WARDEKER, J. S., SLONEKER, J. H., AND JEANES, A., Anal. Chern. 37, 1602 (1965).7. CROWELL, E. P. , AND BURNETT, B. B., Anal. Chern. 39, 121 (1967).8. ALBERSHEll\I, P. , NEVIN, D. J. , ENGLISH, P. D., AND KARR, A. Carbohyd. Res. 5,

340 (1967).9. SAEMAN. J. F., MOORE, ,Yo E., MITCHELL, R. L .. AND MILLETT, M. A .. Tappi 37,

336 (1954).10. GARCIA. ,Yo J. , AND WOLF, M. J. , Cereal Chem, in preparation.

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Determination of Cellulose and Apparent Hemicellulose