2997

Reprinted 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 Hemicellulose
In Plant Tissue by Gas-Liquid Chromatography
,J. H. SLONEKER
,'Vorthem Regional Research Laboratory,' Peoria, lliinoi.$ 6160'f
Received February 24. 19i1
Quantitative determination of cellulose and hemicellulose in biological
materials is tedious and time-consuming by established methods. For ex-
ample, the determination of cell wall constituents and fiber content of
feeds and forages is based on gravimetric procedures (l ,2) . These methods
require that a rather large sample be repeatedly extracted with a series
of solvents 'which successively remove lipids, proteins, lignin, etc. Each
desired fraction is collected by some means, dried, and weighed. Accuracy
of these methods depends upon the efficiency '\vith which extraction re-
moves unwanted material from the appropriate fractions.
Cellulose is much easier to determine than other cell wall polysaccha-
rides. A simple extraction procedure is available that solubilizes essen-
tiallY all the protein, lignin, lipid, and hemicellulose, leaving the cellulose
fibers intact (3). Combined with a colorimetric assay for hexose, this
method can measure the cellulose content of bacterial cultures (4).
Likewise, pentosans have been measured colorimetrically in biological
materials (5). However, this method, like the one for cellulose, lacks in
specificity and is subject to error because color produced by noncarbohy-
drate material accompanying the polysaccharides interferes.
GLC2 is a simple quantitative method for measuring simultaneously
up to eight aldoses as their alditol acetate derivatives (6). This method
is effective for the five aldoses found in wood pulp (7) and the cell wall
polysaccharide constituents of suspension cultures of sycamore cells (8).
Although it requires a hydrolysis step, GLC has advantages over gravi-
metric and colorimetric procedures because individual aldoses are
measured directly and because variations in the aldose content of different
samples 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
.540 .r. H. SLO::\EKER
lulose and apparent hemicellulose in foods. fced. and feedlot \vastes by
measuring the neutral carbohydrates within these materials. In the
method described here, total neutral carbohydrate content is measured
in one sample and cellulose is isolated by an extraction procedure and
measured in a like sample. The content of hemicellulose, based on the
neutral aldoses. can be readily established by difference. The content of
starch may be closely approximated abo from the difference between the
total D-glucose and that derived from cellulose. In addition our method
can be med for materials containing up to 75% protein.
METHODS
Jlateriuls. 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 100-
120 mesh Gas-Chrom Q (Applied Science Laboratories, State College,
Pa.) ." The column was conditioned overnight at 220 and operated at
190-19;'). Carrier gas flow rate was maintained at 40 ml/min. The c11l'0-
matographic instrument was an F&M model 810 equipped with a model
3370A electronic integrator (Hewlett-Packard, Avondale, Pa.).
Standard sugars obtained commercially were used without further puri-
fication. Two internal stanclarcb were : i-inositoL which was added imme-
cliately after the sulfuric acid hydrolysis step, and 2-deoxY-D-glucose,
which was added immediately after the samples were autoclaved because
of its relative lability in acid. The former standard may be used only if
the material to be tested is free of the polyalcohol. Standard sugar mix-
tures were run with each set of determinations to minimize error due to
unavoidable minor variations during autoclaving that cause small varia-
tions in R values.
Determination oj Total Aldose Content. Samples were prepared by
grinding the material fine enough to pass a 60- or SO-mesh screen. Mois-
ture values were determined by drying each sample (1.50-300 mg) in a
tared weighing bottle overnight under vacuum and over phosphorus
pentoxide 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 test
tubes. Sulfuric aeid (0.3 ml of 7270 coneentrationJ was added aeeurately
to each tube and the tube contents were heated to 30 for 1 hr. To dis-
perse the material better in sulfurie aeid, the contents were stirred with
both 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 are
endorsed or recolllmended by the Department of Agriculture o\'er other firms or
similar products not me!ltioned,
GLC OF POLYSACCHARIDES Ii\" PL\i\"T TISSI-E 541
\vas broken, leaving the bottom half in the test tube. After 1 hI' the
samples ,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 of
l:-inositol) was added to each sample to dilute the sulfuric acid to 1 N.
The diluted samples were placed in a preheated autoclave and were hy-
drolyzed for 1 hI' at 120. After the hydrolyzates cooled. the tubes ,vere
centrifuged 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 been
thoroughly mixed, two 2 ml portions of hydrolyzate were transferred to
individual 12 ml conical centrifuge tubes where the sulfuric acid was
neutralized with lead carbonate. Lead salts were removed by centrifuga-
tion, and aldoses in the supernatant were reduced to alditols in 1 hI' at
room temperature upon the addition of sodium borohydride (10-20 mg in
0.5 mI). Excess borohydride was destroyed by adding acetic acid (l
dropwise until the effervescence of hydrogen ceased.
Each reduced sample was passed over a column composed of a dis-
posable Pasteur pipet (5 mm i.d. X 145 mm length) containing 1 ml of
AG-50X4 resin (200-400 mesh) in the H-;- form (Bio-Rad Laboratories,
Richmond, Calif.) and was collected in a 15 X 125 mm Teflon-lined
screw-capped test tube. Each column was washed with 1 ml of water that
was pooled with the appropriate reduced sample. Contents of each tube
were evaporated to dryness on a test tube evaporator (Buchler Instru-
ments, Inc., Fort Lee, N. .T.) adapted to handle the screw-capped test
tubes. Borate ions produced during reduction were removed as trimethyl
borate by repeatedly (3X) dissolving the contents of each tube in
methanol (1 ml) and removing alcohol by evaporation. The residual
alditols in each tube were acetylated for 16 hI' at 100 with pyridine-
acetic anhydride (0.2 ml of a freshly prepared 1: 1 mixture). The acety-
lated alditols were injected directly onto the GLC column.
Percentage of individual aldoses in a given material was calculated from
the relation:
(mg s)(area a)(C) (100)
aldose % = - = : ' - " - ; = ' - - ' - - - ' - ~ - - - ' - ' - - ' ; - ' - - ~
(R)(area s)(mg unknown)
where s and a represent the internal standard and aldose, respectively. C
represents the conversion factor for aldose to polysaccharide: 0.88 for
pentoses and 0.90 for hexoses. R represents a response fador, which was
determined for each aldose using a known aldose mixture, and ,vas ob-
tained from the relation:
R = (mg s) (area a)
. (mg a) (area s)
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 improye
recovPry of the product. Finely ground "amplps (30-150 mg depending
on the cellulo"e content) were weighed into ];) X 125 mm TpfJon-lined
scre\v-capped test tubes. Acetic-nitric acid rpagent i 3 ml of a mixture
containing 150 ml of 80% acetic acid and 15 ml of concentrated nitric
acid) 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 whieh
the tubes were cooled and centrifuged. The supernatant was remoyed with
a disposable Pasteur pipet and discarded. The fibrous precipitate was
washed twice with thp acetic-nitric acid reagent (3 ml) and twice with
acetone 2 mil . .:\Iost of the residual acetone remaining after the last
wash was remoyed by eyaporation. The cellulosic residue was analyzed
as described for the total sugars.
Determination of Starch. To obtain a more accurate determination of
starch by GLC, the materials to be hydrolyzed were first extracted four
times with 80% methanol (3 ml each) to remoye free glucose and glucose-
containing oligosaccharides. The quantity of starch was then equal to the
total glucose recovPrecl (calculated as C"H",O,:;) minus that for cellulose.
RESl'LTS A.'\D DISCl'SSION
The two-stage hydrolysis technique using 72% and 1 N sulfuric acid
(9) is superior to other methods of hydrolysis, especially for materials
that are insoluble in water or dissolve with difficulty. Even so, such mate-
rials as whole corn, one of the more difficult materials to hydrolyze, must
be finely ground and must he stirred vigorously to achieye complete dis-
solution in 12% sulfuric aciel. Also, materials high in lignin will not dis-
sol\'e completely in nrc sulfuric acid because lignin remains insoluble
eyen 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 re-
cO\'ery of the fiye aldoses commonly found in plant tissues (Table 11. This
hydrolysis technique should therefore permit the accurate analysis of car-
bohydrate in materials containing up to 75% protein. The higher concen-
trations of protein did interfere with neutralization and borohydride re-
duction of the hydrolyzates by causing excessiYe foaming. Howeyer, no
problems are encountered during acetylation and GLC because the AG-50
resin column remoyes most of the free amino acids and peptides formed
during the partial hydrolysis of the protein.
Gnder the hydrolysis conditions employed, degradation of the aldoses
is 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-
<I Eadl value is the average of analyses of 11 single hydrolyza teo
J, HtH"ine sefllm nlhumill wns weiglwd into tube 25 mg tot.al of t.he five aldoses thai had been in solution and eVllpomtetl t.o
2.5 9.1 L!l1 0.02 5.07 Il.OO ,->.00 O.O:! l.m; O.O', 1.9,; O.O:! -I.!I:! ,I,O.OS 5.0!l 0.10 5.02 1.!lS 1.!17 O.OS
" .0
16 7 '1.-nS O.K} ,; . O!l 5.IHi (J.OI -1.!l2 O.O:l I.S!I ,1,0.0:1 4 .\15 '>.Oli ,;.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" 5.01i 0.21 4.!H 0.12
15 :G tl 5 . 04 5.HJ0.01 5.07 O.II 4.!I!I 0.07 5.05 (Ulli ';.O!l ,I,O.H ".21 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 lUll
a5 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.lJ:l ,;.OS 0.10 I.!I!I O.OS Ull (Ull 4.7U 01,0.0.; I.!lS 0.07 I.!ln O.D:! I.SS 4.S0 12
7:, 7t,.O 4.!lS ';.2,; (1. OS 5.2S 5.12 5.00 0.07 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
</():HS.t) 10l.!I lOO.!1 un.! !lSA H7.7 101.0 100.0 HS.H B7.ti
TABLE 1
Determination of Neutral Sugars in the Presenee of Protein"
standard
c:
t-<
()
o
":l
>-
!;oj
'"
o
t-<
.-:
if'"

()
()
:r:

::>:l
H
C
M
u:
'" t-'

Z
>-l
>-l
W
"f.
r:
M

mg added
G.on, mg

mg added
5.01, Illg
n-:\IHIlllose
mg added
;;,01, mg
i-Inositol Ht.andllnl
I,-Xylose
Illg added
,;.00, Illg
1.-ArniJillOHl!
Ill!.!; added
".OJ, lilt!:
J)-C:lll(:OHU
mg addull
5.00, mg

tng added
5.01, Jill!;

added
tl.Ol. mg

JIlg added
:>.00, mg:
l .. -Aral)inose
mg add.ed
.5.01, IIlg
Protein
eont.ent"
mg (It)
:.)1
..0..
W
544 J. H. SLONEKER
came of small differenees in acid strength and temperature of hydrolysis.
Inaccurate addition of the small volume of 727'10 sulfuric acid to the
samples will produce a rather large error in acid strength after dilution.
This source of error can be minimized if automatic repeating dispensers
are used when 7 2 ? ~ acid and diluent are added. Effects of variable auto-
clave temperatures can be eliminated by running aldose standards along
with 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 used
to establish the efficiency of the extraction procedure for the recovery of
cellulose (Table 21. Average yield of carbohydrate was in excess of 99%
while that of the unextracted sample averaged 101 %. Slightly lower yields
for the extracted samples can be attributed to a small loss of cellulose
during removal of the extraction reagent and wash solvent. Use of the
fresh reagent to wash the extracted cellulose instead of water as reported
previously (3,41 minimized this loss because in ivater the extracted cel-
lulose swelled and became difficult to pack during centrifugation. The
ach'antage of the GLC method over the gravimetric and colorimetric
methods is readily apparent from Avicel data (Table 2). D-Xylose and
D-mannose in respective yields of approximately 0.6 and 1.0% are in the
TABLE 2
Determination of Neutral Sugars and Cellulose in Selected Materials"
Carbohydrate content, ' ; ~
Sample
2-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'
Corn
pericarpb.,
Corn
peri carp'
Sucrose
Fructose
4 0.1
.f) 0.1
0.6 0.1
0.6 0.1
Trace
2.2 0.2
2.2 0.2
0.4 0.1
1.0 0.1
1.0 0.1
Trace
Trace
<1
0.5 0 1
1.1O.1
Trace
Trace
3.6 0.2
97.7 2.2
99.6 1.3
1.5 0.1
71. 9 1. 7
77.1 1.1
18.4 0.5
2:3.5 0.5
53.0 1. 0
1.8 0.1
99.3
101.2
1.5
75.5
808
18.8
71 ;j
.-., -
;).). u
2.9
a Each value is the average of duplicate analyses from two hydrolyzates.
b Extraeted with acetie-nitric acid reagent to recover cellulose.
. Variety, Pioneer :3:306: harve"ted, fall 1968.
d Extracted with SOc; met hanoi to remove low Illoleeular weight oligosaceharides.
, Extractecl 48 hr with 95S'; ethanol.
GLC OF POLYSACCHARIDES I::\ PLA::\T TISSUE 545
crystalline cellulosic material. Furthermore, these t\\'o sugars are not re-
moved by the extraction procedure and would be undetected with other
methods of analysis.
VVhole corn. chosen for its relatively low content of cellulose and hemi-
cellulose, \\'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 con-
tained a small amount of D-xylose. In the whole corn samples good agree-
ment is shown for L-arabinose and D-xylose, the major constituents of
the hemicellulose fraction, even after one group of samples was extracted
with 80% methanol. However, methanol extraction did change the yield
of D-glucose, as would be expected because free D-glucose and D-glucose-
containing oligosaccharides are removed by the aqueous methanol. The
quantity of starch in this sample of corn can be calculated by subtracting
the value for cellulose from that for total D-glucose in the methanol-
extracted samples. The value, 70.4%, is in excellent agreement with that,
70.8%, obtained on this sample of corn by two different extraction
procedures (10).
The quantity of cellulose and hemicellulose in corn pericarp is approxi-
mately tenfold greater than for whole corn (Table 2). Besides, most of
the D-mannose and D-galactose in corn is concentrated in the peri carp.
Like the other cellulose determinations, the cellulose isolated from peri-
carp contains a small quantity of D-xylose in some form. Apparent starch
content of the peri carp 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. Potentially, D-fructose can be re-
duced to either D-mannitol or D-glucitol and could cause error in the
determination of D-mannose and D-glucose. The results indicate that ap-
proximately 95% of the D-fructose is destroyed during acid hydrolysis
and its contribution to the error in the determination of the two hexoses
is negligible in the samples chosen (Table 2). However, in materials con-
taining large quantities of sucrose or the fructose polymer, inulin, this
error may become significant.
This method is now being used routinely to measure the aldose content
of a wide variety of biological materials such as feed, feed fractions, and
feedlot wastes in order to follow the digestibility of cellulose and
hemicelluloses.
SUM1VIARY
A GLC method is described by which the neutral aldoses and cellulose
can be measured in whole and digested plant tissue. Apparent hemicel-
lulose is measured by difference. Accuracy of the method is unaffected by
protein in concentrations up to 75%.
546 ,}. H. SLONEKER
ACKNOWLEDGMENT
The technical assistance 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 for 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.
GPO 807-780