'I °
AGRlC' TtJT ROW
0
U. S . Department of Agriculture Forest Servic e
FOREST PRODUCTS LABORATOR Y
L
In cooperation with the University of Wisconsi n
MADISON, WISCONSI N
HOLOCELLULOSE, TOTAL CARBOHYDRATE FRACTIO N
OF EXTRACTIVE-FREE MAPLE WOO D
Its Isolation and Properties
By GEORGE J. RITTER
Chemis t
and
E . F . KURTH
Junior Chemist
Published in
INDUSTRIAL & ENGINEERING CHEMISTR Y
Vol . 25, page 1250, November, 1933
cantiossear maims MP
Z4TT-IWE=F'
' k Mx4
Its Isolation and Prop-+
By
Geoff J, Ritter, Chemis t
and E . . F . Furth, Junior Chemist
The total carbohydrate portion of e.ta4'ti?e free maple wood, here termed p°holocel,u ' .©4 i s
isolated in solid form by a rapid meti
•40qta oped at the Forest Products Laboratory for
routine work .
The total acetyl group's, the total ogVkl§m
dioxide-forming material, and a part of to
m
methoxyl groups of the wood are in the n .olocellulose,
.
Research workers .have long had the desire t o
develop rapid methods for isolating, in a solid fraction ,
the entire carbohydrates in wood which has been extracte d
with alcohol-benzene solution a,id with hot water . Such a
wood fraction would afford a convenient means for studying
the nature and the relationship of the carbohydrate components and their substituent groups, acetyl, carboxyl, an d
methoxyl . Some of these components and substituent group s
are partially or wholly lost in the isolation of Cromsl and
Bevan cellulose which constitutes only about 80 percent o f
the carbohydrates .
-The authors propose the word "holocellulose" as preferabl e
to Skelettgabsstanzft, maintaining that the Mtt•ea ter m
does not de scri .e t e. material correctly either fxlpm the
physical or chemical point of view . The wo-"ioc .cellulose" as yet has not been formally or generally wepted
by workers in this field .
a, ;rt.y at the
?Presented before the Division of Cellulose
85th meeting of the American Chemical Society, Washington ,
D . C ., March 26-31, 1 933 .
R1052
!'
Chlorine dicide in a soluti . of p-idine i n
water has 1*4wiused by .chmidt (12) in isolating a carbo h
drate fr *
4B;j;gAsAsd Skelet t
nzen which constitut e
praotioaft las
carbohydra
extractive-free wood, ,
S.ci .idt' a teesergE'a
quires *If
i.y one month fo r
t
mi
:;
and 4~ tOgIne ore objectionable fo r
111
.tork ig AGA.
the west Products Laborator y
the
#!e , ~. d method . It was foun d
t
atec~
teatmeii*. of the extractive-fre e
chlOWL%t and Bohol-pyY
• ution removed i n
v-4
$~ lame*
eexcMpVO small '
Jigs ;
tom►
tentage wao
minutee with
$
~
ochlorite, 200 P60due remainin g
tkMo1bi I
pce
iiii the
ate fraction .
1R Sae pY
t
sttudy
' * emicaf iaracter comp gethose o
t~
.
tine .
iparisons Via. shor t
ne toy
400sirable . Since the
▪ ' .ial is composed and cellulose, it ha s
b
tom, "'aloe
o le or entire cellusmme
lo
^I
tR1
0t
Material
Maple sawdust (0--80 mesh) t ile was extracted wit i ..
alecaleaAenzene solon and with hod Water was used .
The alt
prepared by dilut 4
l-pyridine so ton
*PC 15 o it -of C . P . pyridine with
i&h : 95 percent ethy l
ala0hol % farm 100 cc . of soluti a
- calcium hypooil.orite solution was prepared
eil r
commercial
chlorinated
lime
with
distilled
wate
r
g
arm
ting off the supernatant clear solution . The result a cooled to below 10° C. and made just aci d
'At
to Utica* befove use .
F
Quantative Prcedure for I lO
ting I olocellulos e
fromExtractive-free MapleSawdust
Since repeated tlternat•9 chlorine and alcohtl -
tc treatments leave - 1 to 2
ffeoat o-f lignin in th e
the the stage in the
o,
it
is
necessary
to
Uete
Le
pros hre at wlttgk the calcium hypvchl.o. ite treatment should
be e ► .ied for 'Ot final elignifi
ioi treatment . That
1
J
stage can be approximated by following the loss in weight o f
wood samples whose lignin content has been previously deter mined . Determining the loss in weight, however, require s
drying the sample which renders some of the holocellulos e
soluble . In the quantitative isolation of holocellulose i t
is, therefore, advantageous to weigh triplicate samples o f
sawdust whose lignin content has been previously determine d
by the 72 percent sulfuric acid method (10), using one sampl e
as a control in following the lignin removal and the othe r
two for check determinations .
The detailed procedure used at the Forest Product s
Laboratory for isolating holocellulose is as follows :
e. e
Weigh approximately 1 .7 grams of air-dry extracte d
sawdust in an alundum crucible, moisten the material wit h
distilled water, and remove the excess moisture by suction .
Transfer the sawdust to a 250 cc . beaker and treat the material with chlorine gas in a chlorinating chamber for 3 to 14minutes ; remove from the chamber and add a few cubic centimeters of alcohol-pyridine solution to the contents in th e
crucible to neutralize the acid formed during chlorination ;
transfer to the alundum crucible and remove the excess solution by suction . Transfer the crucible with its contents t o
a Soxhlet apparatus and extract with alcohol-pyridine solution for 2 .5 hours ; remove from the extractor and wash wit h
cold distilled water . Repeat the chlorination and extractiv e
treatments three times or until the loss in weight of th e
control is within 1 to 2 percent of the lignin conten t
previously determined .
To remove the remaining lignin, transfer the residu e
to a 400 cc . beaker and add 300 cc . of the cold dilute solution of calcium hypochlorite, which has been made just neutra l
to litmus with acetic acid . The bleach then has a pH o f
approximately 7 .0 as determined by the glass electrode .
Maintain a temperature of 10° C . and stir the mixture occasionally during a period of approximately 30 minutes . When
the residue is bleached to a uniform white, filter it, was h
well first with cold distilled water and then with alcohol s
Dry in a vacuum oven and weigh t.
Physical Properties of Holocellulos e
In color, holocellulose resembles well-bleache d
Cross and Bevan cellulose ; in physical structure it mor e
nearly resembles wood sawdust in that it tends to retain
iR .
the geer:al . structure ,f
K;moo .
cleavage -between the atructur
zits is we,
.siderab].y, as is., fiiwt by a ram tgg , percent-4ge o f
single detached fib$rs .
It is , •o, s. ble that retention -o-f
the wood structure in the c•arb:O'de ,trial is due largely tea. me +hani'mal 1adnting bittmawn: the fibers and ray
cells which is reenforc:ed by ecoaffkell0A_tiny flakes of
re residual cementing nrateri.al, perhaps: .
.ewe
t pe,
fore a more conckl ive explanation Of ttt .0iria.cure i t
offered, further chemical-and micros co is research will
be r equiar ed .
Chem. .4
Oomposa.tion , of Holocellulos e
In. order to characterize the holoce,l.lulose, th e
following de-terminations were glade ; Lignin was determine d
by the 72 percent 'sulfuric acid method (10), carbon dioxid e
by the 12 percent hydrochloric acid method (' ;, methoxy l
by the Zeisel Method (1), acetyl by the uc1u jele---sulfoni o
acid method (4), and pentosans by Tollen : e method (1) .
Extractions with 1 .0 percent sulfuric acid and 2 .0 percen t
sodium sulfite were made by treating 1 gran of the material with 50 cc . of the solution at boiling to p&r ..t 4_ 6 f
the solutions for 45 minutes .
For coir._1 arative purposes the same an rt eaal
ts wex
cr e h of the following nine Aeteria .s :
ta1'pt
ri aIcbk -benzene an d
(1)
Maple sawdust extraeter
hot water .
(2)
Material 1 treated with .
V.
alcohol-pyridine solution .
chlorine and
{iwz u - l roxehlorite:.
(3) . Material 2 treated with o
solution (holoce . .lulose) .
1
' 4I Material I treated with chlorine dioxtd -in
water-pyridine -a-elution (, kelettsub•stanzen) .
.
R1052
:s with 1 percen t
(5)
Material 3 treated 45 rai 'boiling
i L.f-rzric acid ..
(6)
Material 4- treated 45 rtes will, ; percent ,
boitlxr- -glfuric acid
(7)'
Material 3 treated 45 mli tee with 2 percen t
boiling ;odium sulfite .
-4-
S
(8)
Cross and Bevan cellulose from material 1 .
(9)
Lignin from material 1 .
The holocellulose and Skelettsubstanzen were
in orde r
treated with the dilute acid and dilute sulfite
to determine what proportion of the polyuronides, the remaining methoxyls, and the acetyls was removed by suc h
treatments . The compositions of the residues (material s
5, 6, and 7) were compared with that of Cross and Beva n
cellulose . In order to determine the distribution of th e
methoxyl in the wood, it was necessary to determine th e
methoxyl content of the lignin .
Table 1 gives the actual composition of each o f
the nine materials ; table 2 gives the composition of eac h
material calculated on the basis of the extractive-fre e
ea-oast in order to indicate what proportion of extractivefc- wood components and substituent groups remain in th e
diiierent residual materials .
Results and Discussio n
Yields and actual chemical composition of th e
materials prepared in this study are listed in table 1 .
•
Material 2 which was prepared from material 1
represents the cellulosic product which was obtained by
repeated alternate treatments with chlorine and alcohol pyridine . Except for lignin content its yield and chemica l
composition correspond closely with that of holocellulos e
which was prepared from material 2 by means of the calciu m
hypochlorite treatment . This indicates that very littl e
substance other than lignin was removed by the hypochlorit e
treatment . It may be noted that in both yield and chemica l
composition holocellulose corresponds closely with th e
Skelettsubstanzen which was isolated by the chlorin e
dioxide procedure . Because of the removal of the ligni n
and the retention of practically all of the other component s
in isolating the holocellulose, it is richer than material 1
and Cross and Bevan cellulose in carbon dioxide-forming
components, pentosans, methoxyl, and acetyl groups .
Further, some of each of these components and groups ar e
still retained in the residue remaining after holocellulos e
is hydrolyzed with boiling dilute acid and alkaline solutions as shown by the composition of materials 5 and 7 .
Moreover, the chemical composition of material 5 is similar
R1052
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to that of material 7 which was prepared from Skelettsubstanzen by means of 1 .0 percent acid hydrolysis . From the
carbon dioxide yields of material 8 it is shown that polyuronides are still present in Cross and Bevan cellulose .
This finding is contrary to the limited distribution o f
polyuronides as suggested by Hawley and Norman
(0 .
The slight difference between the theoretical an d
the experimental yields of holocellulose can best be show n
by calculating the analytical results on the basis of th e
extractive-free wood as was done in table 2 .
If the lignin value is subtracted from the extractive-free wood value, there appears to be a theoretica l
caroohydrate fraction of 77 .2 percent (100 - 22 .8 = 77,2 )
as compared to 76 .2 actually isolated, indica-zng a los s
of ' .0 percent . The data indicate that 0 .6 p e rcent o f
per, ; osans in the wood are missing in the holoee.llulose ,
which would increase the yield of holocellulose to
6.8
pe-cent . If the 0 .6 percent of pentosans is added to th e
holocellulose and the lignin (0 .6 + 76 .2 + 22 .8 = 99 .6) ,
there is still a discrepancy of 0 .40 percent for the analysers of the extractive-free wood . Most of this discrepanc y
is clue to loss of methoxyl . In the isolated lignin and th e
holocellulose is found 5 .75 percent of methoxyl (4,8 + 0,9 5
= 5„75), leaviL 0,35 percent unaccounted for (6 .1 - 5 .75
= 0 .35) . From the data it is impossible to say whethe r
the loss in methoxyl occurred in the lignin or the holocellulose, but in either case it should be added to th e
lignin and the corrected holocellulose values, making a
total of 99 .95 percent (99 .6 + 0 .35 = 99 .95) . Aside fro m
a small percentage of pentosans, the holocellulose, there fore, represents practically the total carbohydrates o f
the extractive-free wood .
In addition to yields, table 2 also shows the pro portion of the total components and substituent groups tha t
remain after the ch e lical treatments employed for preparin g
the various materials .
Methoxyls .--Methoxyl to the extent of 0 .95 percent ,
which is 1546 percent of the total (0 .95 : 6 .1 x 100 = 15 .6 )
is present in the holocellulose . This finding disprove s
the conception that all of the methoxyl in wood is associate d
with the lignin and confirms published results (7, 11) .
Even after the holocellulose is hydrolyzed wit h
boiling 1 .0 percent sulfuric acid for 45 minutes, the remaining material 5 still retains 4 .8 percent of the tota l
R1052
-6-
to
O
RIO 52
O
methoxyl (0 .29 : 6 .1 x 100 = 4 .8) . In a like manner, a s
shown by material 7, the holocellulose hydrolyzed with boiling 2 .0 percent sodium sulfite solution for 45 minutes als o
retains 4 .0 percent of the methoxyl in the wood . And material S, Cross and Bevan cellulose, likewise retains 7 . 0
percent of the methoxyl (0 .43
6 .1 x 100 = 7 .0) .
•
Carbon dioxide-forming material .--The total materials that liberate carbon dioxide in the extractive-fre e
wood are present in the holocellulose . While they ar e
attacked by acid and alkaline hydrolysis, nevertheless mor e
than 25 percent of them withstand those treatments, as i s
shown by the carbon dioxide in materials 5 and 7 . Further ,
approximately 43 .0 percent of those materials are retaine d
in the maple Cross and Bevan cellulose . This percentag e
of carbon dioxide liberated from the maple Cross and Beva n
cellulose is higher than that found in catalpa Cross an d
Bevan cellulose; (9), indicating that the polyuronide conten t
of Cross and Bevan cellulose from different woods may vary .
Acetyls .--Acetyl groups present in the wood with stand the treatments for isolating holocellulose an d
Skelettsubstanzen and they can be recovered quantitativel y
in those two residual products . Those acetyl groups ar e
partially removed by mild acid and alkaline hydrolysis an d
also by the treatments employed for isolating Cross an d
Bevan cellulose as indicated by materials 5, 7, and 8, respectively . The presence of acetyls in Cross and Bevan
cellulose confirms results by another investigator (3) .
The finding of those groups in holocellulose, in material s
5 and 7, and in Cross and Bevan cellulose therefore disprove s
another conception -- namely, that the acetyls are presen t
in the lignin .
Pentosans .--Holocellulose contains 96 .9 percent o f
the pentosans of the extractive-free wood . When holocellulose was hydrolyzed with boiling 1 .0 percent sulfuric aci d
and 2 .0 percent sodium sulfite solution, respectively, i t
sustained a decided loss in pentosans as shown by the composition of materials 5 and 7 in which the pentosans conten t
is lower than that in the Cross and Bevan cellulose .
Summation of Individual Losses Due to Chemical Treatment s
•
After subjecting wood fractions to chemical treatments, difficulties are generally experienced when attempt s
are made to account for the total losses by summarizing th e
R1052
-7-
individual losses, As already shown, such was not the cas e
in preparing materials 3 (holocellulose) and 4 (Skelettsubstanzen) from material . I . This scheme of analysis was teste d
still further 'concerning 'its quantitative aspects . The test
was made''by determining and summarizing the . individua l
losses sustained by material 1 in the preparation of Cros s
and Bevan cellulose, by the holocellulose in the preparatio n
of materials 5 and 7, and by Skelettsubstanzen in the preparation of material 6 .
The treatments employed and the tota l
losses suffered by the three materials are recorded i n
columns B and C of table 3 . To make a summary of the individual losses required the calculation of the carbo n
dioxide (column D, table 3) in terms of uronic acid anhydride ,
its mother substance . That is done according to the following formula,. IJronic acid anhydride = carbon dioxide x 4 .
During the pentosan determination, uronic acid anhydride s
liberate 16 .6 percent of their weight asfurfural (6) which ,
in terms of pentosans, is equivalent to 22 .8 percent of the
uronic acid, anhydride . Consequently,_ a correction facto r
equal to 22 .8 percent of the uronic acid anhydride valu e
must be subtracted from the pentosan value as determined b y
the method used . This has been done in column E, table 3 .
r
Losses in methoxyl and ash .sustained in the isolation of Cross and Bevan cellulose require further explanatio n
because some of each of those losses is recovered in th e
lignin . Thus, when Cross and Bevan cellulose was isolate d
from the wood, 5 .67 percent of methoxyl was lost (6 .1 - 0 .4 3
= 5 .67) . Of this amount 4 .8 percent was recovered in th e
isolated ligni n i leaving 'a net loss of 0 .87 percent (5,67 - 4 . 8
= 0 .87), line 4, column
.During the same procedure 0 .24
percent (0+30
0406 = 0424) of ash wa 's lost and of tha t
amount 009 percent was recovered in the lignin, leaving a
net loss of 0 .15 percent as shown in line 4, column H . Th e
individual losses are summarized in column J . Column K point s
out the close agreement between the sum of the individua l
losses and the totalioss in weight, column C .
In table 3, then, it appears that the carbohydrate s
removed by the treatments in column B consist of uronic acid s
and pentosans, perhaps in the form of polyuronides . Wit h
these materials methoxyl and acetyl groups are also removed .
The nature of the chemical combination between those substituent groups and the polyuronide components is unknown .
Before that combination is discovered, it will be necessar y
to isolate and identify polyuronide components to whic h
methoxyls and acetyls are attached . As a first step i n
that direction the products removed by mil d
hydrolytic treatments of the maple holocellulose are bein g
R1052
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identified . Whether results similar to those reported i n
this paper will be obtained when other woods are treate d
according to the procedure employed for maple will be ascer tained later .
t
Literature Cite d
(1)
Bray, M . W ., Paper Trade J ., 57, 59-65 (1928) .
(2) Dickson, A ., Otterson H ., and Link, K ., J . Am . Chem .
Soc ., 52, 775 (1930) .
(3) Dore, W . H ., J . Ind . Eng . Chem ., 12, 475 (1920) .
(4) Freudenberg, K ., Ann ., 433, 230-7 (1923 .
(5)
Hawley, L . F ., and Norman, A . F . Ind . Eng . Chem . 24 ,
1190 (1932) .
(6) Norman, A . F ., Biochem . J .
(7)
22, 524 (1929).
O'Dwyer, M . H ., Ibid ., 22, 381 (1928) .
(S) Ritter, G . J ., Ind . Eng . Chem . 16, 947 (1924) .
(9) Ritter, G . J ., and Fleck, L . C ., Ibid ., 20, 371 (1925) .
(10) Ritter, G . J ., Seborg, R 02
. .
Ibid., Anal . Ed ., ,
anj• Mitchell, R . L . ,
(11) Schmidt, E ., Meinel, K ., and Jandebeur, W. ,
Cellulosechem ., 12, 129-39 ( 1 93 2 ) .
i
(12) Schmidt, E ., Tang, Y . O ., and Jandeb,ar, W ., Ibid . ,
12, 201 (1931) .
•
R1052
-9-