UTILIZATION OF BARK WASTE
OREGON STATE UNIVERSITY
PREPARED FOR
ENVIRONMENTAL PROTECTION AGENCY
JULY 1973
PB-221 876
Distributed By:
National Technical Information Service
U." S. DEPARTMENT OF COMMERCE
-------
BIBLIOGRAPHIC DATA
SHEET
EPA-6 70/2-73-005
I. Title and SuBtitl?
UTILIZATION OF BARK WASTE
7. Author(s)
R. A. Currier, M. L, Laver
V. Performing Organization Name and Address
Department of Forest Products
School of Forestry
Oregon State University
Corvallis, Oregon
. .
_ PB-221 876 _
57 lU-fMMI l>:m- "
19 7 3-i s s ui ng date
6.
8. Performing Organization Rept.
No.
10, Project/Task/Work Unit No,
11. fnntrart /Grant No.
R-EP 00276-04
12. Sponsoring Organization Name and Address
U.S. Environmental Protection Agency
National Environmental Res <•.••.: i ch Center
Office of Research & Development
Cincinnati, Ohio 45268
13. Type of Report & Period
Covered
Final
14.
15. Supplementary Notes
16. Abstracts
The prohleo of bark waste that le generated by the forest products industry in s-
the United States haft be coma Increasingly important-* The major overall goal of the
work covered in this report was to utilise physical end cheaical sciences in coor-
dinated studies to promote eeononic uses of bark in order to relieve pollution
created by present methods of disposal. Physical utilization research included:
(1) investigating the preparation of bark pellets from bark in s series of exper-
iments designed to control several variables, such as particle size and configura-
tion, moisture content and addition of chemical agents; (2) determining the compo-
nents responsible for "self-bonding" of bark; and (3) investigating potential pro-
ducts from or applications of "as-is" end modified bark wastes obtained from pro-
duction sources.
Chemical utilization research included: (.1) preparing, for chemical studies,
natural bark, bark that had been ammoniated to contain 4 percent nitrogen,and bark
. that had bean molded into pellets and then broken down into small particles;
and (2) Investigating tha chemical composition of each type of bark prepared.
17, Key Words mad Document Analysis. 17*. Descriptors
Pollution, Waste disposal, *Wastes, *Bark> Wood wastes, Wood products,
*Pelleting, Pellets, Chemical properties, Physical properties, Economics,
Moisture content, Softwoods, *DougIas fir wood, Electric power, Agri-
cultural engineering, Bonding, Carbohydrates
17b. Identifiers/Open-Ended Terms
*Bark waste> *Ammoniscion, n-Haxane-soluble wax, Benzene-soluble wax,
Solid waste disposal, Resource recovery
17c. COSAT! Field/Croup 13-B, 1 1 ~ L
18. Availability Statement
Release to public
I }?. Sorority Class (This
I Hcf-.orc)
UNjCLASSiFn-f?
21. No. o{ Pa«es
FORM NTlS-33 (REV. 3-72)
S«-.-. urity Chi.-:;, 'This |22, Price
_ UNCLASSIFIED
7
USCOMW-DC
-------
REVIEW NOTICE
The Solid Waste Research Laboratory of the National
Environmental Research Center, Cincinnati, U.S. Environmental
Protection Agency, has reviewed this report and approved its
publication. Approval does not signify that the contents
necessarily reflect the views and policies of this laboratory
or of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
The text of this report is reproduced by the National
Environmental Research Center, Cincinnati, in the form re-
ceived from the Grantee; new preliminary pages have been
supplied.
-ii-
-------
. Man and bis - envi conmen t must be p-^c.-rted from the
adverse affects Q.* pesticides, radi'.tlon, noise and oche
forms of . pollutidu , and the .unwise manc.~enent of solid
waste. Efforts to protect tne envi ror:m2n t require a
focus th*t recognizes the inter-play between the com-
ponente'ftof pur 'physical envtron
-------
: Pag«
SUMMAIY 07 PROOtWSTOWARD ORIGINAL* dbatfr . . 1
Physical, UtiiizntioA Research . T'i ". . • i
Chndcal Utilization Resjlarcft - . .v '+> 3
Publicmtiooe . ... .;.'.. ..... V.: 5
Plans for; Further Publication . . V'.v 6
.- •< • '"'.; ' • :' V>- '>'" , . •
.- ' - - - ' •" • /A
'PHYSICAL UtlLIZATION RESKAI^R „' 7
Bark Pelleting ..' . . '; . . .. . . . . . . 7
Bark Molding ....,../. 11
Other Bark Products or Osea- 14
15
CHEMICAL UTILIZAf ION RESEARCH *...... 16
I • , ' j* - »'
Bark Carbohydrates f. .-.* . .^, . , 16
,*••.•• • '
Historical Bevl*^ ...... ^. . 16
»'; -'•- Bxperiaen^al,.. ^;\ •. . . .. . ,:V .. ; 20
' Results and 'Di^cuaalim ...;..,.....;.. 78
Douglas-Fir Wax' .'.>., ^ ./.- ; . . . ............. 118
' •
Historical l^jdr.-.s-y;. :.,'... ,'t .: .......... us
Ejc^ftimeotalY-a-^M^n^Soluble Waoc* ........... 121
Ext>earlaei^i(l.:;'^iBn|MHte^Soluble Wax ............ 128
Rasultv aa/j- DlaCtiiMibn \ . . T-l ........ ...... 135
of-Bark,; ..',f : ...... v. : 145
Historical Re^leVf;'. ..... ^-' 145
Results >an4 Dis'cusalon.\,' .<•...'.. 147
BIBLIOGRAPHY .... .... ,.%>1:.^.^.;. .,....'.' 150
TABLES-. . ;....-. . >*• ..^. -rf,. «.. t=r.- ,«....*y •-.•••••••...•. . . ' 157
. .•'•;•/. . . .- , . . .«*.,*..... ......f 173
-v-
-------
-1-
SU11MARY OF PROGRESS TOWARD ORIGINAL COALS
A recent estimate of the amount of bark waste penerated by the forest
products industry in the entire United States totals some 14 million tons,
oven-dry basis. Other estimates show six million tons ar« available within
the west coast states of California, Oregon and Washington. Approximately
half of the bark in these states now is used in some manner, leaving three
million tons for which disposal problems remain. The major overall goal
of the work covered in this report was to utilize physical and chemical
sciences in coordinated studies to promote economic uses of bark in order
to relieve pollution created by present methods of disposal. We believe
substantial progress has been made toward finding economic uses for bark.
Discussion of progress toward specific goals as originally outlined follows.
Physical Utilization Research
Goal 1. To investigate the preparation of bark pellets from bark in
a seTies of experiments designed to control several variables, such as
particle size and configuration, moisture content, addition of chemical
agents, etc.
Progress in fulfilling Goal 1 has been very good. We have found that
all species of softwood barks investigated will form acceptable pellets.
This includes some eastern and northern species of bark, in addition to
those from the west coast. We have found that Douglas fir bark, in particular,
may be added to other types of forest industry residues (such as sawdust) in
order to make a mixture which will result in formation of pellets. One
significant finding was that pelletizing of bark greatly increases its bulk
density and results in a product capable of being easily conveyed, stored
and spread, compared to bark in natural form.
-------
-2-
There are at least three areas where basic information resulting from
our studies of bark palletizing may be applied directly to future commercial
Venturas utilizing waste bark. These are as follows:
1. Palletizing bark residues to form a fuel for production of electrical
poorer in a centralized plant.
2. Pelletizing of bark mixed with fertilizers or other chemicals for
agricultural applications.
3. Pelletizing bark to increase its bulk density and handling
characteristics for easier transport of this renewable raw material.
Goal 2. One. of the original goals was determination of the components
responsible for "self-bonding" of bark. By a process of selective chemical
extraction of Douglas fir bark followed by pelletlzing (molding), it was
hoped to pinpoint the compounds essential to activation of bonding (cohesion).
With departure of the original Principal Investigator, this portion of the
research could not b® accomplished since we did not have an extractives
chemist on the grant staff. He did learn that Douglas fir bark extracted
by one cheaical solvent sysfcsta did retain its ability to form pellets and
other saoldod itesss. The Forest Research Laboratory now has a separate
project, F858, "Cohesions in Consolidated Bark Products" with the objective
of determining the mschanism responsible for "self-bonding" of bark. We
have cooperated by palletizing bark materials.
Goal 3. To Investigate potential products from or applications of "as-ls"
and modified bark wastes obtained from production sources.
We consider this phase of our physical utilization research efforts to
be the most rewarding since it has the potential of culminating in the
establishment of more than one commercial enterprise resulting directly from
efforts expended in pursuing Goal 3. At this time, there are two products
-------
-3-
on the market in the area of pelletized and extruded bark; both obtained
from us samples and technical information generated during the course of
our studies. In addition, there is at least one other excellent possibility
for the eventual establishment of a large-sized plant centered upon the
chemical-physical utilization of bark wastes.
Chemical Utilization Research
Goal 1. To prepare, for chemical studies, natural bark, bark which
has been ammoniated so as to contain about 4 percent nitrogen, and bark
which has been molded into pellets and then broken down into small particles.
Goal 1 was completed in the 01 year of the research. The samples were
then used for further chemical utilization research.
Goal 2. To investigate the chemical composition of each type of bark
prepared as outlined in Goal 1.
Progress in fulfilling Goal 2 has been very good. The carbohydrate
content of natural inner bark has been found to be about 50-60%. The
carbohydrates have been fractionated into a soluble fraction and an insoluble
"holocellulose" fraction. The ratio of sugars in the hydrolyzate from the
soluble fraction has been determined as follows: glucose, 59.1; arabinose,
11.9; galactose, 3.9; mannose, 3.7; xylose, 1.0; rhamnose, 1.0. Rhamnose
has not been previously reported in Douglas fir bark. The results show
that the soluble fraction is composed mo.stly of a glucan-like polymer.
The insoluble "holocellulose" carbohydrate fraction is very easy to
isolate and comprises some 35-40% of the inner bark. This fraction could
become of commercial interest as a source of carbohydrates. Some suggestions
for uses have been as an animal feed, and in the food industry. Hydrolysis
showed the "holocellulose" to be composed of the following ratio of sugars:
-------
-4-
glucose, 26.5; mannose, 4.1; xylose, 2.7; galactose, 1.0; arabinose, 1.1.
The "holocellulose" has been further fractionated into a xylan, a glucomannan,
a mannan,, and a glucan.
Douglas fir bark contains an n-hexane-soluble fraction and a benzene-
soluble fraction. These are called "waxes." The waxes have been studied
by column chromatography, thin-layer chromatography, gas-liquid chromatography
and gas-liquid chromatography combined with rapid-scan mass spectrometry.
We have found sterols (6-sitosterol and campesterol) in the n-hexane wax as
well as evidence for terpenes. These classes of compounds are in addition
to those reported in the early literature (see text of following report).
The benzene wax contains monocarboxylic fatty acids, dicarboxylic acids and
hydroxy fatty acids in addition to phenolic compounds.
Whole bark was treated with gaseous ammonia to a nitrogen content of
A.08%. Extraction of the bark with organic solvents and hot water removed
1.10% of the nitrogen but did not leach out 2.98% of the nitrogen. This
suggests that the ammoniation of bark may provide a material for soil
amendment which possesses a quick release nitrogen supply followed by a slow-
release nitrogen supply as the bark decomposes.
Bark which had been pelletized and then broken down into small particles
was extracted with the organic solvents, ri-hexane, benzene, ethyl ether,
ethyl alcohol and hot water. These extracts were investigated by ultraviolet
spectroscopy. No significant differences were noted between these extracts
and similar extracts prepared from non-pelletized bark. Therefore, pelletizing
does not appear to grossly alter the chemical make-up of the bark. Thus,
most of the efforts of Goal 2 was directed to studies of the raw bark as
described above.
-------
-5-
Publications
1. Currier, R. A. 1971. What is bark? Physical considerations.
Proceedings of .conference "Converting Bark into Opportunities," Oregon
State University, Corvallis, Oregon, March 8-9, 1971.
2. Currier, R. A. and W. F. Lehmann. 1971. Bark as an Ingredient
in molded items, particleboards, adhesives and other products. Proceedings
of conference "Converting Bark into Opportunities," Oregon State University,
Corvallis, Oregon, March 8-9, 1971.
3. Currier, R. A. 1972. An assessment of current bark utilization
opportunities. 27th Proceedings Northwest Wood Products Clinic, Spokane,
Washington.
4. Laver, M. L. 1971. What is bark? Chemical considerations.
Proceedings of conference "Converting Bark into Opportunities," Oregon
State University, Corvallis, Oregon, March 8-9, 1971.
5. Laver, M. L. 1971. Chemicals from bark. Proceedings of conference
"Converting Bark into Opportunities," Oregon State University, Corvallis,
Oregon, March 8-9, 1971,
6. Fang, H. H-L. 1971. Douglas-fir bark; n-hexane soluble fraction.
Master's thesis, Oregon State University, Corvallis, Oregon.
7. Laver, M. L., H. H-L. Fang and H. Aft. 1971. The n-hexane-soluble
components of Pseudotsuga menziesii bark. Phytochemistry 10(12) :3292.
8. Lai, Y. C. L. 1972. Douglas-fir bark; carbohydrates solubilized
by the acidified sodium chlorite delignification reaction. Master's thesis,
Oregon State University, Corvallis, Oregon.
9. Laver, M. L., J. V. Zerrudo and H. Aft. 1972. Treatment of Douglas-
fir bark with gaseous ammonia. Forest Products Journal 22:82.
-------
-6-
10. Loveland, Patricia M. and M. L. Laver. 1972. Monocarboxylic
and dicarbosylic acids from Pseudotsuga menziesii bark. Phytochemistry
11(1):430.
11. Loveland0 Patricia M. and M. L. Laver. 1972. w-hydroxy fatty
acids and fatty alcohols from Pseudotsuga menziesii bark. Phytochemistry
11 (10):3080.
12. Zerrudo, J. V. 1973. Douglas-fir bark; water-soluble carbohydrates
and alkaline degradation of a xylan. Doctoral thesis. Oregon State University,
CorvalliSs Oregon.
Plans for Further Publications
Results of research covered by the bark pelleting studies will be
presented in a paper to be published in the Forest Products Journal; title
of this report will be "Pelletizing bark residues."
The research area covering bark molding appears to be appropriate for
preparation of a "Technical Note" to be published in the Forest Products
Journal. Plans are not finalized as yet, however.
A paper has been accepted for presentation at the IV Canadian VJood
Chemistry Symposium, Quebec City, Quebec, next July. It will be published
in Carbohydrate Research and cover the alkaline degradation of a xylan.
Manuscripts on "Water-soluble Carbohydrates of Bark," "Holocellulose
Characteristics and Properties," "The Tannins in Douglas fir Bark" and "The
Terpenes in the n_-Hexane Soluble Components of Douglas-fir Bark" are in the
early stages of preparation.
-------
-7-
PHYSICAL UTILIZATION RESEARCH
I. Bark Pelleting
Using a laboratory model pelleting mill (California Pellet Mill Company,
Model CL, Type 3), approximately 200 pelleting trials have been made. Raw
material involved 16 species of bark plus numerous mixtures of species.
In addition, runs were made on mixtures of bark plus woody residues. See
Table 1 for a list of bark species and the mixtures utilized in pelleting
trials.
Variables studied other than raw material mix included particle size,
moisture content, fraction of bark, addition of fertilizers and seeds,
pellet diameter and degree of pellet densification. Most trials consisted
of 10-15 pound lots, but several weighed over 100 pounds. Various types
of pellets produced are depicted in Figure 1; also shown is the hammermilled
Douglas fir bark serving as standard pelletlzing raw material. Pelleting
concentrated on Douglas fir .and western hemlock barks, since both are
available in large quantities as a residue material.
Suitable pellets were formed from all species of bark studied,
although problems were encountered with a few species. Where mixtures of
barks or bark plus woody residues were pelleted, addition of 25% or less
Douglas fir bark to the mix often spelled the difference between success
and failure. Douglas fir bark always pelletized readily under a variety
of conditions.
Pelleting trials were made of selected barks from the northern,
eastern and southern portions of the United States in order to learn if
the techniques we developed could be applied to waste barks from other
regions of the nation having disposal and pollution problems. Results of
our limited tests indicated bark from the species selected could be made
-------
-8-
into well-formed pellets quite readily, although the southern oak gave
trouble since it was about half wood.
A potentially significant application of data collected on bark
pelleting is in transportation of bark wastes. Compared to raw bark in
chunk or comminuted form,, the weight of pelletized bark per unit of
volume occupied is 2.0 to 3.8 times as heavy. Compression factors and
bulk densities for a number of bark species and pellet diameters are
listed in Table 2. In addition, pelletized bark is easily conveyed, stored
and spread compared to bark in natural form.
One of the most promising potential commercial applications for
pelletized bark is to utilize bark as a carrier for fertilizers or other
chemicals used in agriculture. During the last year of this grant,
practically all the pelletizing trials were run on bark-fertilizer mixes.
Small-diameter (1/8" or 3/16") pellets were made using thin dies, since
this combination gave good rates of production and resulted in a product
which could be spread by present standard methods of fertilizer application.
Several drums of bark-fertilizer pellets were made for growing trials
during the 1972 season.
Cooperation has continued with Schroeder Sales Company, Division of
Pacific Kenyon Corporation, Long Beach, California, who have provided
fertilizer and conducted field trials. Results of tests on a golf course
in the Los Angeles area were encouraging, as were trials on home lawns in
the Corvallis area and on the Forest Research Laboratory lawn. Pellets
for lawn use were formulated to yield either 7.5-0-0 or 9-3-6 fertilizer
values. The source of nitrop.cn was urea in prill form. This is a fast-
acting form of nitrogen, but when mixed with bark and pelletized, the
rate of nitrogen release appears to be slowed significantly. Possible
-------
-9-
economic benefits could result since slow release types of fertilizers
are priced considerably higher compared to urea. Another potential source
of slow release nitrogen in bark is the ammoniation process discussed in
this report.
Cooperation also has continued with Bohemia Lumber Company (now
Bohemia, Inc.), Eugene, Oregon. In fact, when this grant concluded on
June 30, 1972, Bohemia, Inc. made a grant to us in order to continue
research on a bark-fertilizer pellet, utilizing Douglas fir bark previously
extracted chemically in their pilot plant. Work planned will involve
production of pellets and conducting field trials on the product. Chances
of constructing a commercial plant to manufacture pelletized bark-fertilizer
appear very promising. A bark-plus-fertilizer pellet offers a means of
complete utilization of the residue from chemically extracted bark and
definitely adds to the feasibility of such an operation.
Several hundred pounds of bark-fertilizer pellets have been given to
George D. Ward & Associates, Portland, Oregon, pollution control consultants,
They have a contract with the Naval Facilities Engineering Command, Seattle,
Washington, for reclaiming and stabilizing blow sand areas in eastern
Oregon. The bark-fertilizer pellets may have the attributes of retaining
moisture and slowly releasing nutrients to trees, shrubs, or grasses
planted on the sand dunes.
Samples of bark pellets have been distributed widely. Included have
been forest products manufacturing concerns, universities, governmental
laboratories, chemical and plastics manufacturers and private individuals.
A partial list of recipients of bark pellet samples may be found in Table 3.
Some limited commercial application of pelleted bark has resulted
aince our research work commenced. It has been learned that the largest
-------
-10-
forest products company in Canada,, MacMillan Bloedel Ltd. has test marketed a
bark-ccmtaining-fertilizer pallet product. The original idea appears to
have come from our exploratory pelleting studies, and we have maintained
contact with their research personnel.
Another company in Oregon has developed a fuel product from bark.
TriWest Products of Springfield, Oregon, makes the pelletized fuel sticks.
A principal of TriWest Products has consulted with us several times
regarding bark pelleting, and has informed us the product idea was initiated
by our research.
Trials on use of 1/2-inch diameter pelletized Douglas fir and western
hemlock bark for fuel have been made by Forest Research Laboratory personnel.
To date, pellets have been tried both as a stove fuel and in fireplaces.
They are somewhat difficult to ignite, but once started, give off excellent
heat. There has been recent discussion regarding the possibilities of
using pallatized bark as the fuel source for a centrally located electric
power generating plant. Instead of disposing of bark wastes through the
present practices of incineration at the sawmill sites, the residues would
be pelletized to improve their bulk density and shipping characteristics.
Transportation to the power generator could be by means similar to the
way coal is handled today. Since bark is a renewable raw material, this
idea may gain more proponents as time passes.
Limited trials have been made of palletizing bark plus seeds such as
i
clover or grass seed. Techniques have been developed whereby the mixture
can be pelletized with some degree of germination resulting. No detailed
study has been made, but this is an area we hope to investigate soon.
Another use of bark in pellet form is being investigated by the U.S.
Forest Service, Forest Products Laboratory, Madison, Wisconsin. Some of
-------
-11-
our 1/4-inch Douglas fir bark pellets have been used as the large aggregate
in cement-bark building blocks. Tests are continuing on the process and
resulting blocks.
II. Bark Molding
Much of the research on molding of bark has consisted of demonstration-
type projects in cooperation with various segments of the forest products,
adhesives, and plastics industry. This method of attacking the problem
was utilized in order to probe the potentials of various end markets for
comminuted Douglas fir and western hemlock barks and to increase awareness
that such material was available from bark residues. A brief description
of the various investigations follows.
A. Planter block from bark
1. Using compression molding, trays, each consisting of six planter
blocks, were prepared from western hemlock bark. In the preparation of
these blocks, the following variables were studied: moisture content of
the bark, binder use, and addition of other materials such as lime, clay,
chemical fertilizer (Vigoro) and fungicides. Greenhouse studies, followed
by field planting, indicated that the bark planter blocks could grow
tomatoes and pansies from seed.
2. Douglas fir and red alder barks were extruded into blocks and
limited growth trials were made utilizing tomatoes. Blocks were formed
with and without added urea-formaldehyde binder; chemical fertilizer
(Vigoro) was added to all barks prior to forming.
B. Bark added to plastics
1. One investigation involved a three-way cooperative study with
industry. We prepared dry corminuted bark, a lumber company (Dant and
-------
-12-
Russell, Inc.5 Portland, Oregon) provided the raw bark and financed
experimental time on molding machines at a plastics producer (Grant and
Roth Plastics, Inc., Hillsboro, Oregon). Several types of extruded,
sheet formed and injection molded products have been produced, with bark
extension of the plastic in the order to 40-60 percent. Types of plastics
extended by bark include polyethylene, cellulose butyrateB cellulose
acetate, polyurethane, polystyrene and poly-vinyl chloride. Photographs
showing some experimental products may be found in Figure 2.
2. The plastics company involved in study (1) above (Grant and Roth
Plastics) sent some of the bark extender to Eastman Chemicals, Inc.,
Kingsport, Tennessees for their evaluation. A laboratory report indicated
promise for a cellulose acetate + plasticizers + bark mix. Cost per pound
of this mix was estimated to be lower than polyvinyl chloride„
3. Large samples (60 Ib) of -20 mesh Douglas fir and western hemlock
bark were shipped to Borden,, Inc., Thermoplastic Division, Leominster,
Massachusetts, where they were evaluated as fillers for extruded rigid
PVC pipe. The resulting laboratory report by Borden, Inc. indicated the
bark contained an excessive amount of volatiles, leading to porous and
lumpy extrusions. On the positive side, addition of bark lowered specific
gravity of the product, resulting in a cost-volume advantage. A follow-up
study now is underway whereby Borden, Inc. has been provided another
sample of Douglas fir bark which has had some potential volatiles removed
by chemical extraction. No results have been received to date.
A. The Hysol Divison of the Dexter Corp., Industry, California, has
formulated a knofchole or other void filling compound from -20 mesh
Douglas fir bark plus epoxy plastic materials. The compound has been
used commercially to fill voids in rough-sawn textured plywood panels.
-------
-13-
We have provided over 100 Ib of comminuted bark for these experimental
formulations, which have resulted in commercial production of products
numbered AE1502 and AE1504.
5. Preliminary runs have been made extruding Venetian blind slats
from polystyrene plus -20 mesh Douglas fir bark in both plain and
extracted form. This experimentation was sponsored by Omega Industries
of Salem,..Oregon. Five different color slats have been run, with
promising results. The extracted bark appeared to be the more suitable
extender.
6. During the last few weeks of the grant period, an excellent
contact was made with B. F. Goodrich Chemical Company, Avon Lake, Ohio.
This company is one of the largest suppliers of plastics molding compounds.
Large samples of both unextracted and extracted Douglas fir bark, and
bast fibers have been shipped for trial runs on soil pipe, conduit and
etc. No results have been received yet.
7, A few furniture drawer guides were made from 50% -20 mesh Douglas
fir bark and 50% general purpose polystyrene at Modcom, Inc., Canby,
Oregon. The management there is willing to conduct more evaluation tests
when some free plastic machine time is available.
C. Other molded products from bark
1. Molded bark products not involving plastics have resulted in
cooperative efforts with two different commercial companies interested
in production of extruded "fuel logs" from bark wastes. A sample lot of
Douglas fir bark was prepared for Glomera-American, Inc., Reedsport, Oregon,
who shipped it to Switzerland where the log forming machines are made.
Trail runs indicated a log containing mostly bark can be formed by this
process. Another company, Oregon Timber Products Company, Albany, Oregon,
-------
-14-
has purchased log extrusion machines from Japan; we cooperated with them
in developing a log containing substantial quantities of Douglas fir and
western hemlock barks. Unfortunately, this company has suffered financial
problems and is not operating at the present time. Photographs depicting
these various logs may be found in Figure 2.
Ill. Other Bark Products or Uses
Samples of bark or bark fractions have been prepared for a number of
other companies and researchers. Some examples follow.
1. Bark particles have been prepared for experimental work on a
trickling filter system for disposal of animal wastes. This is being
done by Dr. Myron Cropsey, Agricultural Engineering Department, Oregon
State University. We have provided all of the 3/4- to 3-inch Douglas fir
bark chunks used in his research. Dr. Cropsey presented a paper covering
his results to date at a meeting of the American Society of Agricultural
Engineers in Portland, Oregon, October 8, 1971. His tests will continue
during the 1972-1973 academic year.
2. Bohemia,, Inc., Eugene, Oregon, is interested in developing a
process for extraction of wax from Douglas fir bark, and has built a
pilot plant. After wax extraction, over 90% of the bark still remains;
we have cooperated with Bohemia, Inc. in determining potential uses for
this spent bark. One effort has been tb turn the residue into an extender
for phenolic plywood glues. Extracted bark was shipped to us and we
ground and screened over 400 Ibs to prepare -100 mesh material. This was
successfully evaluated as an extender for plywood glues, undergoing
standard test procedures prescribed by the American Plywood Association.
Several of the chemical companies manufacturing plywood glues are interested
-------
-15-
in the bark extender. Cooperation is continuing with Bohemia, Inc. in
separating the physical components (cork and fiber) of extracted Douglas
fir bark prior to grinding the remaining portion as an extender. Possibilities
of commercialization of the process appear encouraging.
3. Samples of the -100 mesh extracted bark extender mentioned in
(2) above, as well as -100 mesh unextracted whole bark have been sent to
Colorado State University, where a graduate student is evaluating their
possible use as an extender for binders currently used in manufacture of
particleboard. Preliminary results look promising; additional bark has
been requested. No published report is available as yet.
IV. Miscellaneous
During the period January 1, to June 30, 1972, the physical utilization
research done under this grant was presented orally at four meetings as
follows.
1. Western Builders Short Course, Portland, Oregon. February 11, 1972.
2. Twenty-seventh Annual Northwest Wood Products Clinic meeting
jointly with the Inland Empire Section of the Forest Products Research
Society. Spokane, Washington. April 17-19, 1972.
3. Pacific Northwest Section of the Forest Products Research Society,
Vancouver, B. C., Canada. May 9, 1972.
4. Twenty-sixth Annual Meeting, Forest Products Research Society,
Dallas, Texas. June 19-22, 1972.
In addition, a report for local radio broadcast also was made; this
covered both physical and chemical research sponsored by this grant.
-------
-16-
CHEMICAL UTILIZATION RESEARCH
INTRODUCTION
There are a number of general review articles on bark utilization,
but thrse pertinent ones a,re by Harkin and Rowe (1), the Bark Committee
of the Forest Products Research Society (2) and J. Alfred Hall (3). Hall
cosasents that one cannot w;rite a meaningful "Chemistry of Douglas-fir
Bark" in the present state of our knowledge. This statement demonstrates
the great gaps in the knowledge of the chemistry of bark. The research
herein reported is an effort designed to provide a better understanding
of the chemistry of Douglas-fir bark with the goal of a more complete
and better utilization of 'this abundant natural raw material.
The work has been organized into three areas: the carbohydrates;
the waxes; aad the ammoniation of whole bark. For clarity of reporting,
each of these subjects is presented separately. Each section Includes
an historical review,, an experimental, and a results and discussion.
BARK CARBOHYDRATES
I. Historical Review
Authors have referred to Douglas-fir as Pseudotsuga taxifolia (Poir.)
Britt. and other names. However, the presently preferred botanical name
is Pseudoteuga menziesii (Mirb.) Franco, The names all refer to the same
genus and species. Mention is made of this to avoid confusion about the
exact species investigated.
The present work is concerned with Douglas-fir inner bark. This is a
specific anatomical part of the bark and a brief description of bark
anatotay is included for purposes of definition.
-------
-17-
For a detailed anatomical description of Douglas-fir bark, reference
is made to Grilles (4), Grilles and Smith (5), Chanp. (6) , and Ross and
Krahmer (7). Briefly, however, bark can be considered to consist of
inner bark and outer bark (Figure 4). The inner bark (phloem cells) is
the portion from the vascular cambium to the innermost cork layer. The
outer bark (rhytidome) is'.everything to the outside of the innermost
cork cambium (Figure 4).
The inner bark comes from the vascular cambium, that layer of living
cells between the wood and bark which divide to form wood to the inside
and bark to the outside. The inner bark is composed mainly of sieve
cells, axial and ray parenchyma, and sclereids (Figure 4). Much of the
inner bark is living in the living, tree because many of the parenchyma
and sieve cells remain alive as long as they are components of the inner
bark.
Douglas-fir sclereids are short, sharply pointed, spindle-shaped
fibers of a red brown color (Figure 4). They are often referred to as
bast fibers. They are lignifled cells and develop from axial parenchyma
cells some distance from the vascular cambium. In becoming sclereids,
axial parenchyma cells approximately 0.1 to 0.5 mm in length elongate to
1 to 2 mm by apical intrusive growth, and form thick walls. The sclereids
are commonly straight and somewhat cigar-shaped. Kiefer and Kurth (8)
and Ross and Krahmer (7) describe and illustrate the general appearance
and position of the sclereids in Douglas-fir bark.
The outer bark of Douglas-fir consists of layers of cork in which
growth increments are usually visible, as shown by Ross and Krahmer (7).
Interspersed among the corky layers are areas of phloem tissue that contain
the sclereids and other ce^l types found in the inner bark (Figure 4).
-------
-18-
The cork layers form frorv the cork cambia which are livinp cells that v/ere
once living parenchyma cells of the inner hark. New cork cambia form in
the inner bark and cut away part of the inner bark, which now becomes part
.of the outer bark. The cork cambium produces cork cells to the outside
and a few storage cells to the inside. Cork cells have thin cellulose
walls which are coated with suberin. Suberin is essentially an ester
condensation polymer of hydroxylated, saturated and unsaturated stralp.ht-
chnln fatty acids (9). The cork layers may also contain tannin,
clihydroquercetin and starch. All cells outside the Innermost cork cambium
arc dead because no food supply can pass through this layer of cork cells.
This then results in an outer bark composed of cork cells and dead phloem
cells, which were once inner bark.
The studies on the anatomy of bark have shown it to be a complex
physical material. The chemical composition of bark is equally complex.
It is knowns howevers that the major constituents of bark are the
carbohydrates j, just as they are the major constituents in v;ood (9, 10).
However, little attention has been devoted to the carbohydrates present
In the hark of trees. In contrast, the carbohydrates in the wood of
trees have been extensively studied. Polysacch.ir ides such as pectinlc
acids, galacturonogalactans, arabinopalactans, 4-0-methylp,lucuronoxylans,
arabino-4-O-methylglucuronoxylans, glucomannans , galactop.lucomannans ,
and cellulose have been isolated from the wood of numerous species of
both gymnosperras and arborescent angiosperns and their chemical structures
elucidated (11, 12, 13, 14, 15 p. 447)
One reason for this has undoubtedly been the greater economic
importance of wood as compared with bark. Another is probably to be
found in the occurrence in bark of non-carbohydrate constituents such as
-------
-19-
suberin, tannins, phlobaphenes, and various phenolic compounds, all of
which make the isolation of polysaccharides from bark difficult. Wood
contains none or few of these components.
Segall and Purves (9) discussed these difficulties in a review of
the early literature on the chemistry of bark. The so-called "extractives"
were known to interfere unless they were removed by exhaustive, successive
extractions with alcohol, water or other neutral, chemically inert liquids,
such as diethyl ether or petroleum ether. This early literature showed
that after all of the solvent-soluble materials had been removed by
exhaustive extraction, some 70 to 90% remained as an undissolved residue.
*
After removing the extractive materials it was possible to acid
hydrolyze the remaining residue and investigate some of the monosaccharides
released. It appeared that the polysaccharides in bark were based
predominantly upon glucose, and to a lesser extent on galactose, mannose,
xylose, arabinose and rhamnose. The existence of true cellulose could
not be established at that; time.
Kurth (10) also reviewed the early literature on the chemical
composition of barks. He reported that barks contained hemicelluloses
and "cellulose"; the sugars associated with the hemicelluloses were
glucose, galactose, mannose, arabinose, and xylose. The "cellulose" was
simply that fraction which remained after treating extracted bark with
four successive portions of a mixture of one part nitric acid and four
parts alcohol. No additional evidence that the material was cellulose
was given.
Kiefer and Kurth (8) isolated a holocellulose fraction from the bast
fibers, or sclereids, of Douglas-fir bark. Whole bark was collected,
ground and screened. All fractions larger than 40-mesh were reground and
-------
-20-
rescreened. The fibers were obtained in an almost pure state by stirring
tlio crude fiber fraction in five times its volume of distilled water at
room t etnpernturs?. By vlrtui1 of their high apeclClr gravity, t:l>t- fibers
readily sank to the bottojm of the container, whereas cork and other
impurities remained on tHe surface and were skimmed off.
The overall percentage composition of the bast fibers was determined
and is presented in Table 4. For sake of comparison, analyses of Douglas-
fir wood, taken from the literature (16), are included in the table. The
data indicate that the bajst fibers may be a lignocellulose material with
a composition similar to that of wood. The higher lignin content of the
bast fibers and the lower, methoxyl content of this lignin, however, are
notably dissimilar from those found in wood.
Paper chromatograns of an acid hyilrolyzate of the holocellulose
'showed the presence of glucose, pa lactose, mannose and xylose. The limited
techniques of paper chrortatography at that time did not resolve mannose
and arabinose. Their fermentation procedures for quantitative analyses
indicated an apparent absence of arabinose in Douglas-fir bark. This
paper by Keifer and Kurth (8) and the one by Holmes and Kurth (17) appear
to be the major works published on the carbohydrates in Douglas-fir bark.
These materials seem to have been largely ignored to date.
I1. 'Experimental
A. Collection of Bark Samples
On May 22, 1969, the outer bark was chipped from a standing Douglas-
fir of diameter 18.8 inches at breast height at Black Rock, Oregon. The
inner bark plus cambiup were then carefully stripped from the tree and
Immediately brought to ttye laboratory where the cambium layer was removed.
-------
-21-
Tlie cambium-free inner bark (4832.0 g, moisture content 44.9%, hot air oven
at 110°C) was immersed in 18 liters of 95% ethanol. Water (1207.0 ml) was
later added to adjust tlie solution to ethanol-w.it IT (4:1 v/v) wJth
calculations for the moisture content of the inner hark.
The tree was later cut and by count of the annual rings was 130 years
old. .
B. Sample Preparation
The inner bark, after soaking in the ethanol-water (4:1 v/v) for three
days, was recovered by filtration and washed well with fresh ethanol-water
(4:1 v/v).
The combined filtrate and wash liquors were concentrated on a rotary
evaporator (Buchi, Rotavapor, Switzerland) to less than 2 liters, Water
was added to exactly 2 liters in a volumetric flask and three 10-ml aliquots
of the adjusted solution were removed and the nonvolatile solids determined
by the Celite-vacuum drying method (18). The values obtained were 2.057 g
per 10-ml aliquot which corresponded to a total of 411.4 g of material in
the ethanol-water (4:1 v/lv) extract or 15.4% of the original inner bark on
a dry weight basis.
The extract was tesrjed for monosaccharides by paper chromatography
using the solvent system ethyl acetate-pyridine-vater (8:2:1 v/v/v) . A
trace of glucose was detected by spraying the chromatograms with o-
aminodiphenyl reagent (0.4 g o-aminodiphenyl dissolved in a solution
prepared from 100 ml of jjlacial acetic acid and 20 ml of distilled water)
and heating at 100±2° in an oven for 5 min. (19).
The residue of innei} bark (air-dried) was ground in a Wiley Hill (A.
H. Thomas Company, Philadelphia, PA) and fractionated according to particle
size hy screening with'.screens of an increasing number of meshes per inch
-------
-22-
(The W. S. Tyler Company, Cleveland, Ohio). All fractions were examined
under a microscope. It was observed that those materials retained on the
screens of 20 and 35 meshes per inch were bundles of fibers. These
fractions were reground arid rescreenecl. All materials (1612.8 g) v.'hich
passed through 35, 60, and. 80 mesh per inch screens and were retained on
n 100 mesh per inch screen were used in prcp.irinp. tin- sample for future
investigations. In addition 238.3 p. out of the 390.u p. which passed through
a 100 mesh per inch screert but were retained on a 150 mesh per inch screen
were included. None of the larper material (23.6 g) retained on the 35
mesh per inch screen was used.
C. Benzene-Ethanol Extfraction
A part of the recombincd bark fractions (1500.0 ?., dry weight) was
divided into three portions (618.0 g, 618.0 g, and 264.0 g dry weight).
Each portion was extracted with a solution of benzene-ethanol (2:1 v/v;
1400 ml of benzene and 700 ml of ethanol for the large portions, and
598 ml of benzene and 299 ml of ethanol for the small portion) in a
Soxhlet extractor. Each Extraction was continued for 37.5 hr (a minimum
of 50 solvent exchanges).
The non-volatile solids in the benzene-ethanol extract were determined
by the Celit-vacuum drying method (18); weight, 77.9 g or 4.4% of the
original inner bark on a dry weight basis.
The extract was tested for monosaccharides by paper chromatography
using the solvent system ethyl acetate-pyridine-vater (8:2:1 v/v/v). No
sugars were detected by the o_-aminodinhenyl spray reapent described earlier.
0. Hot-Water Extraction
A' part (1458.3 p, dr^ weight) of the air-dried residue remaining, from
the benzene-ethanol (2:1 v/v) extraction was divided into four portions
-------
-23-
(three of 363.8 g, and one of 366.9 p, dry weight). F,ach portion was stirred
into 3 liters of distilled water at 55° in a 4-liter beaker. The extraction
was continued at 50°-6Q° for 24 hr with intermittent stirring.
The mixture was separated by filtration using a Buchner funnel. The
filtrate was condensed on a rotary evaporator, freeze-dried and the solids
weighed; weight, 202.5 g or 11.1% of the original inner bark on a dry
weight basis.
K. Anmonium-Oxalate Extraction
A part (1458.3 g, dry weight) of the residue (dried in a hot-dry room
at 32° and relative humidity 31% for 4 days) was divided into 4 fractions
each containing 312.0 g. Each fraction was extracted with 3 liters of
0.5% aqueous ammonium oxalate in a 4-liter beaker at 70-80° for 26 hr.
The insolubles were recovered on a Buchner funnel, washed thoroughly
with water and dried in a hot-dry room at 32° for 3 days; weight 1198.0 g
or 66.3% of the original inner bark on a dry weight basis.
The filtrate was concentrated on a rotary evaporator, freeze-dried,
.•mil the sollfls (3.5 g nlr-dry weight) were stored for future reference.
F. Acidified Sod i urn Chlorite Uelign If ication; laolat ion c.' ,a Holocell'ulose
Fraction
An amount (1194.0 g dry weight basis) of the above residues from the
ammonium oxalate extraction was divided into six batches. Five batches
contained 196.8 g each and one contained 210.0 g. Each batch was stirred
into 3.0 liters of distilled water at 75-80° and the temperature was
maintained throughout the reaction. A steady stream, of nitrogen was
bubbled through the mixture to prevent the accumulation of gases and the
mixture was stirred mechanically. Glacial acetic acid (20.0 ml) was added,
followed by sodium chlorite (60.0 g). Fresh glacial acetic acid and sodium
-------
-24-
chlorite were added two more times at one hour intervals. During, the
1
second addition foaming occurred on top of the beaker so 2-5 ml of isoamyl
alcohol was added into the foaming mixture to prevent additional foaming.
At the end of 4 hours, the yellow solids were recoverd by filtfd.feion using
a Biichner funnel with Whatman No. 1 filter paper, and wasrw*Vi*h'distilled
•. ' »-..-.
...^aa, V/ •<•.•• • • •
water. The yellow solids were dialyzed for one week, wfl8nca,;with distilled
water, dried with ethanol and finally dried in the air for two weeks;
weight 797.6 g, dry weight basis or 44.3% of the original- firmer bark. The -;
filtrate was dialyzed for one week, concentrated on a rpjtflty evaporator ,;
and the solids recovered by freeze-dryinp. They were s'ttiFOtft'of1 future
reference (sec section II-H). •-" —••*—>-•• - t" "
The yellow color of the residue from the acidified1'Wdiufiv chlorite
treatment indicated incomplete delipriification. Therefore, the delignification
was repeated on 783.9 g (dry weight basis) of the yellow eoHd.8 (20). The
•'."•'' v
residue was recovered by filtration, dialyzed for one? vefefe'$f_ the Hot-Water-Soluble Solids
(Isolation described in section Il-D) ' . ^'^--:.
's;'V;-'-;V .
1. Elemental Analyses- for Nitrogen, Sulfur, Phosphorus; jidA^tne Halogens
'•\ '^*•/•.;• •- + :••'*•
i . V'' '•• . -l
A small piece of freshly cut sodium was wiped tlioroufenKt ta. remove
* T ^^if^f'tt *. » •
"• •' V'.' '
all traces of kerosene and placed in a small glass teat tuffeX The tuhe was
-------
-23-
gently heated-in a flame until the sodium melted and the vapors rose 1-2 cni
up the walls of the tube. A snail amount of the hot-water-soluble sol ids
was added to the molten sodium and the tube was heated strongly over an open
flame. Heating was continued for 1 to 2 minutes. After the entire end of
the tube was red hot, the tube was plunged into an evaporating dish which
contained about 10.0 ml of distilled water. The hot end of the tube
shattered and the resulting mixture was heated to boiling, the tnsolubles
removed by filtration, and the filtrate recovered for elemental analyses.
An aliquot (2.0-3.0 ml) of the filtrate was added to powdered ferrous
•
sulfate (0.1 g) in a test tube. The solution was heated gently with
shaking until it boiled. Sufficient dilute sulfuric acid was added to
dissolve the iron hydroxides and to p,ive an acid solution. A precipitate
of Prussian blue formed, which indicated the presency of nitrogen. For
purposes of comparison, alanine was fused with sodium, and tested for
nitrogen. A Prussian blue color formed which showed the presence of nitrogen.
A second aliquot (2.0 ml) of the filtrate was acidified with dilute
acetic acid, and a few drops of lead acc'tate were added. No precipitate
formed, indicating that sulfur was not present. As a control for the
analysis of sulfur, cystine was fused with sodium and analyzed exactly as
described above. A yellow precipitate formed which confirmed the presence
of sulfur.
A third aliquot (1.0 ml) of the filtrate was acidified with concentrated
nitric acid (3.0 ml) and boiled for 1 r.inute. The solution was cooled and
an equal volume of ammonium molybdate reapent was added. The solution was
warmed to 40-50° and allowed to stand. No precipitate formed, indicating
that phosphorus was not present. Olucose-1-phosphate was fused with sodium
and tested for phosphorus. A yellov precipitate (avimonium phosphomolybdate)
-------
-26-
formed which Indicated the presence of phosphorus.'ft
A fourth aliquot (2.0 ml) of the filtrate was acidified with dilute
sulfurlc acid, boiled gently to remove any hydrogen cyanide which might
be present, and the solution was treated with a few drops of aqueous
silver nitrate. No precipitate formed indicating that none of the halogens
were present. ;
A small sample of the hot-water-soluble solids was quantitatively
analyzed for nitrogen (Kjeldahl, 3.63%; Pascher and Pascher, 53 Bonn,
Buschstrasse 54, West Germany). ;
2. Test for Tannins and Starch
A small amount of the hot-water-soluble solids was dissolved in
distilled water. An aliquot (1.0 ml) of this solution was treated with
a few drops of a ferric chloride-potassium ferricyanlde solution (21, p. 227)
(1% solutions are mixed prior to use). A blue color developed which
Indicated the presence of phenolics. ......
A second aliquot (5.0 ml) of the solution was created with a few drops
of iodine indicator. A deep blue color developed which Indicated the
presence of. starch.
3. Strong Acid Hydrolysis
A portion of the hot-water soluble solids (0.07 g) was treated with
72% sulfuric acid (0.9 g) and allowed to stand for 45 minutes at room
temperature. Water (20.1 ml) was added slowly with stirring to provide
a final concentration of 3.0% acid. The .solution was refluxed for 5 hours,
Jr
cooled to room temperature, and neutralized to pH 5.0 with saturated >
aqueous barium hydroxide solution. The resulting barium sulfate
precipitate was removed by centrifuge, washed well with water and the
washings were added to the decantate. The combined decantate was
concentrated under vacuum on a rotary evaporator to about 50.0 ml.
-------
-27-
4. Mild Acid Hydrolysis
A portion of the hot-water-soluble solids (0.32 g) was dissolved in
3.0% sulfuric acid (96.0 ml) and the solution refluxed for 5 hours. After
cooling to room temperature, the solution was neutralized to pH 5.0 with
saturated aqueous barium hydroxide solution. The precipitate of barium
sulfate was removed by centrifuge and washed with water. The decantate
plus the washings were concentrated to about 100.0 ml under vacuum on a
rotary evaporator (22) .
5. Qualitative Araino Acid Analysis by Paper Chromatopraphy
The hydrolyzates from the mild acid hydrolysis were subjected to
ascending two-dimensional paper chromatography (23, 24, p. 93) on Vlhatman
No. 1 paper, using water-saturated phenol as a developer in one direction
in an atmosphere of ammonia, and n-butanol-forraic acid-water (20:6:5 v/v/v)
as developer in the second direction. A solution of ninhydrin (1.0 g)
dissolved in n-butanol (500.0 ml) was used as the spray reagent.
6. Carbohydrate Analysis by Paper Chromatography
A portion of the hot-water-soluble solids was dissolved in distilled
water, spotted on Whatman No. 1 filter paper and paper chromatographed as
described below to determine if free sugars were present. No free sugars
were detected.
The hydrolyzates from the strong acid hydrolysis and the mild acid
hydrolysis were applied at intervals of about 1 inch along one edge of a
Whatman No. 1 filter paper, 18 x 22.5 inches, in such amounts as to produce
a colored spot easily detectable with the naked eye in a natural light or
under ultraviolet light. A standard solution cbntaininp about 1.0% each
of the known monosaccharides, glucose, mannose, galactose, arabinose, xylose,
and rhamnose v?as also spotted on the filter papier. The sugars were
-------
-28-
separated by descending development with ethyl acetate-pyridine-vater (8:2:1
v/v/v) (25) as developer. The solvent was allowed to migrate almost to the
bottom of the papers at which time they wets removed from the tank and
air-dried for at least 6 hours. Some of the papers were returned to the
tank and developed as before (repeated up to 3 or 4 tines) in order to
obtain a better separation.
The paper chromatograms were sprayed with <>-aminodiphenyl reagent
(0.4 p £-aminodiphenyl dissolved in 100.0 ml of glacial acetic acid and
20.0 ml of distilled water) and heated at 100i29 in an oven for 5 minutes
(26, 19). The spots were outlined in pencil untfer ultraviolet light. The
rates of movement of the hydrolyzate sugars werfe compared with those of
authentic samples when run simultaneously on the same chromatograms.
The o-aminodiphenyl was purified by recryskallizing the technical
grade material twice from aqueous ethanol. Activated charcoal was used
as a decolorant. The purified crystals were dried at room temperature
under vacuum.
7. Purification of the Hot-Water-Soluble Solids
A portion of the hot-water-soluble solids (3,0-4.0 g) was stirred into
a small amount of distilled water until it was thoroughly wetted. Distilled
water at room temperature was then added slowly with stirring until the
mixture had a concentration of 1.0%. Stirring was continued for at least
2 hours at room temperature. The mixture was ail lowed to stand and was
then cenfcrifuged. The supernatant liquid was recovered. The undissolved
solids were again stirred into water and the whole process repeated 3 more
times. The liquor from the fourth washing was colorless and clear.
The undissolved solids which remained were freeze-dried; yield 29.8%
of the original hot-water-soluble solids. Thesis were labeled "Fraction A."
-------
/
/
-29-
A portion of "Fraction A" was tested for starch (positive) and tannins
(negative). A second portion of "Fraction A" was hydrolyzed by strong acid,
and the hydrolyzate was examined on paper chronatograms. The chromatograms
showed glucose only. A large sample of "Fractiqn A" was later prepared for
additional research purposes.
All of the liquors from the above treatments were combined and
concentrated to about 1.0 liter under vacuum at less than 30° temperature
in a rotary evaporator. Ethanol (95%) was added to provide a final solution
of 70.0% ethanol (1.0 liter each tine). After the fourth washing, the
precipitate was dissolved in water and traces of ethanol were removed under
vacuum in the rotary evaporator. The polysaccharide was then recovered by
freeze-drying and labeled "Fraction B"; yield 25.9% of the original hot-
water-soluble solids. A portion was tested for starch (positive) and
tannins (faintly positive). Another portion was hydrolyzed under mild
acid conditions, and examined by paper chromatography. The chromatoprams
showed the presence of amino acids and sugars.
The mother liquor and washings from "Fraction B" were combined,
concentrated, and freeze-dried. This sample was labeled "Fraction C".
The yield of "Fraction C" (by difference) was 44.3% of the original hot-water-
soluble solids. A portion of this sample was tested for starch (negative)
and tannins (positive).
8. Enzyme Hydrolysis to 'Remove Starch
The enzymes used were two commercial preparations purchased from
Marschall Division, Miles Laboratories, Elkhart, Indiana. The first
enzyme, HT-1000 (27) is a mixture of anylolytic and proteolytic enzymes,
capable of faster and more economical liquifactions of starch than many
other a-amylases. It has been derived from Bacillus sub tiles, and is in
-------
-30-
the form of a white, dry powder. The second enzyme (commercial name,
Diazyme L 30) is an amyloglucosidase (28). It is sold in liquid form.
A part (15.0 g) of "Fraction B" was dissolved in distilled water (P^.l
ml) and the pH was adjusted to 5.5-7.0 with sodium carbonate solution
(1.0 N). HT-1000 (7.5 mg, 0.05% based on sample weight) dissolved in a
small amount of distilled water was added to the sample solution. The
i
mixture was heated to 75° in a wofer bath with continuous agitation and
held at that temperature for 15 minutes. Heating was then continued
until the temperature reached 85-87° and held at this level for 30-40
minutes. At the end of this time, the sample and water bath were cooled
to 60°, and the pH of the sample was adjusted to 3.8-4.2 with hydrochloric
acid (0.1 N). Diazyme (0.09 raJ , equivalent to 80 units/lb starch) was
added directly to the cooled carbohydrate solution. The mixture was
incubated at 60° with stirring for 72 to 96 hours. At the end of the
incubation period, the mixture was transferred to a dialysis bap. and
dialyzed for two days in distilled water, one week in running tap water1
and another day in distilled water. The non-dialyzahle portion was
concentrated in a rotary evaporator at less than 40° and the concentrate
was freeze-dried; yield 9.9% of the original hot-water-soluble solids.
The materials passing out of the dialysis bag during the first two
days were collected, concentrated, and freeze-dried. A portion of the
freeze-dried sample was dissolved in watert spotted on Whatman No. 1 filter
paper and paper chromatographed. The chromatograms showed glucose only.
Another portion of the sample was hydrolyzed under mild acid conditions
and the hydrolyzate was tested by paper chromatography. The chromatograms
showed glucose only.
-------
-31-
9. Enzyme Hydrolysis to Remove Protein
The non-dialyzable, freeze-dried material recovered from the enzyme
hydrolysis to remove starch was dissolved in 1.5 liters of distilled
water. Tris (hydroxymethylamino)-methane was added to the solution and
the pH was adjusted to 8.5 with 0.1 N hydrochlotic acid. Chymotrypsin
(160 mg) and trypsin (150 ing), dissolved in a small amount of water, were
added to the buffered solution and the volume adjusted to 2.0 liters. The
solution was placed in a hot-water bath and heated at a constant temperature
of 30-40° with constant stirring for 12 days. At the end of the incubation
period, the solution was transferred to a dialysis bap. and dialyzed for
12 days against running tap water. The precipitate which had formed
during the reaction was separated from the mother liquor by centrifupation
and washed three times with distilled water. The mother liquor and the
washings were combined, concentrated in a rotary evaporator and freeze-dried
(29). The freeze-dried material had a tan, fluffy appearance. This sample
was labeled "Fraction D"; yield 1.5% of the original hot-water-soluble
solids. "Fraction D" was hydrolyzed with 3.0% sulfuric acid and investigated
by paper chromatography. The chromatograms showed the presence of galactose,
glucose, arabinose and traces of rhamnose, xylose, and mannose.
10. Carbohydrate Analysis by Gas-Liauid-Chromatography
The gas-chromatograph used was a Hewlett-Packard 5751B Research
Chromatograph (Hewlett-Packard Company, Palo Alto, California) equipped
with dual flame ionization detectors. The conditions were: column, 6.5%
ECNSS-M on Gas Chrom 0 100/120 mesh, 6 ft x 1/6 in. O.D. stainless steel;
injection port 200°, detector 235°; column temperature 175° isothermal;
2
helium flov 30 ml/rain; range setting 10 ; attenuation setting 16.
The various samples were hydrolyzed and th£ derivatives prepared for
-------
-32-
injection into the gas chromatogsraph as follows. The polysaccharide sample
(0.32 g) was dissolved in 3.02 sulfuric acid (96.0 ml) and refluxed for 5
hours (mild acid hydrolysis). The solution was cooled and authentic
ayo-inositol (0.1000 g) was added. The hydsrolyzate was then neutralized
to pH 5.0 with a saturated aqueous barium hydroxide solution. The resulting
barium sulfate precipitate was removed by centrifuge. An aliquot (25.0 ml)
of the clear supernatant solution was transferred to a round-bottomed flask
(IGOoO ml). Sodium borohydride (0.08 g) was added to the flask and allowed
to react for 2 hours at room temperature (308 31).
The excess sodium borohydride was decomposed by adding acetic acid
until gas evolution ceased. The solution was concentrated to a sirup in
a rotary evaporator, and methanol (10.0 ml) was added and re-evaporated.
The addition and removal of methanol was repeated five times (32). The
resulting sirup was dried in an oven at 105° for 15 minutes to ensure
complete removal of water.
Acetic anhydride (7.5 ml) and concentrated sulfuric acid (0.5 ml) were
added to the sirup and the solution was heated for 1 hour at 50-60° in a
water bath. After cooling for 5 minutes0 the acetylation mixture was
poured slowly with stirring into about 70.0 ail of ice-water. The mixture
was transferred to a separatory funnel and the alditol acetates were
extracted with three successive amounts of freshly distilled methylene
chloride (25.0 sal5 1590 ml and 10.0 ml). The methylene chloride extract
was concentrated to dryness on a rotary evaporator at 75°. Distilled
water (1.0 ml) was added to the residue and re-evaporated. The alditol
acetates were dissolved in 2.0 ml of freshly distilled methylene chloride
and about 4.0 pi of the solution were injected into the gas chromatograph
(21, 33). The peaks In the resulting spectra wery identified by comparison
-------
-33-
of retention times with authentic known alditol acetates and by peak
enhancement techniques. The areas under the peaks in the resulting
spectra were measured by means of a planimeter.
H. Characterization of the Carbohydrates Solubilized by the Acidified
Sodium Chlorite Delignification Reaction
(Isolation described in section II-F)
1. Ash Determination
The inorganic ash was determined by a modification of the procedure
of Paech and Tracey (3A).
Six samples (0.170 g to 0.180 g dry weight) were weighed into silica
dishes. The samples were saturated with concentrated sulfuric acid (0.2 ml).
The mixtures were stirred thoroughly with glass rods and set aside for 1-1*5
hours. They were placed in a muffle furnace and heated gently until
charring occurred. The temperature was increased to 300° to drive off the
sulfuric acid (about 30 min). The temperature was increased to 500° and
heating continued until ashing was complete (about 5 hours). The ashed
samples were placed in a dessicator to cool and were weighed after 20 min.
The ash content was 13.39±1.57%.
2. Dialysis and Precipitation with Ethanol
In an attempt to separate and purify the carbohydrates in the acidified
sodium chlorite soluble solids, a sample was dialyzed and re-precipitated
(35).
An aliquot (22.2 g, dry weight) was dissolved in distilled water (500
ml) and dialyzed against distilled water. The water was changed each day
for four days, concentrated to about 200 ml under reduced pressure on a
rotary evaporator and lyophilized to yield a brov/n solid; weight 5.6 g or
-------
-34-
22.4% of the starting material. An aliquot of the fraction was subjected
to paper chromatography, under conditions to be described in detail later.
The solution in the dialysis bag was concentrated under reduced
pressure on a rotary evaporator to a volume of 400 ml. The concentrate
was added, with stirring, into 1200 ml of 95% ethanol to provide a 70-71%
etHanoi concentration resulting in the formation of a flocculent white
precipitate. The mixture was allowed to stand at room temperature for
several hours and then centrifuged. The decantate was concentrated and
lyophilized to yield a fluffy white powder; weight 7.4 g or 33.3% of the
starting material.
The white residue was lyophilized to yield a fluffy white powder;
weight 7.7 g or 34.7% of the starting material; ash content 11.88±2.83%.
Dialysis and precipitation did not lower the ash content of the
original material beyond the degree of error of the ash determinations,
and so all further work was done on the original material.
3. Elemental Analysis for Nitrogen, Sulfur, Phosphorus and the Halogens
The procedures used are slight modifications of the Lassaigne's sodium
fusion method (36, p. 1039). A small glass test-tube was supported in a
clamp and a small cube of sodium metal was added. The tube was gently
heated in a flams until the sodium melted and the vapors rose 1-2 cm up
the walls of the tube. A small amount of solid was added directly
onto the molten sodium. The tube was strongly heated over an open flame
until the entire end was red hot. The heating was continued for one or
two minutes and the tube was then plunped into an evaporating dish
containing about 10 ml of distilled water so that the hot tube shattered.
The mixture was heated to boiling, the insolubles removed by filtration,
and the filtrate recovered for elemental analysis.
-------
-35-
An aliquot (2-3 ml) of the filtrate was added to a test-tube containing
about 0.1 g of powdered ferrous sulfate. The mixture was gently heated
with shaking until it boiled. Without cooling, sufficient dilute sulfuric
#
acid was added to dissolve the iron hydroxide and give the solution an
acid reaction. A precipitate of Prussian blue formed, indicating the
presence of nitrogen.
A small aliquot (15.0 mg dry weight) of the acidified sodium chlorite
soluble material was quantitatively analyzed (Pascher and Pascher, 53 Bonn,
Buschstrasse 54, West Germany); nitrogen content, 0.84%.
A second aliquot (2.0 ml) of the filtrate from the sodium fusion
reaction was acidified with dilute acetic acid and a few drops of lead
acetate was added. No reaction resulted, indicating that no sulfur was
present.
A third aliquot (1.0 ml) of the filtrate from the sodium fusion reaction
was acidified with 3.0 ml of concentrated nitric acid and boiled for one :
minute. The solution was cooled and an equal volume of ammonium molybdate
reagent was added. The solution was warmed to 40-50° and allowed to stand j
but no yellow precipitate formed, indicating that no phosphorus was present.
A fourth aliquot (2.0 ml) of the filtrate from the sodium fusion j
reaction was acidified with dilute sulfuric acid and boiled gently until >
it had been reduced to about 1 ml to remove any hydrogen cyanide which
might be present. A few drops of aqueous silver nitrate was added. No
i
precipitate formed, indicating that no halogens were present. >
!
4. Strong Acid Hydrolysis i
An aliquot (177.6 mg dry weight) of the solids solubilized by the
acidified sodium chlorite reaction was dissolved in 77% sulfuric acid
(3.0 g) and allowed to stand for 30 minutes at room temperature. Water
-------
-36-
(56.29 g) was slowly added with stirring to provide a 3.9% sulfurlc acid
solution. The solution was refluxed for 5 hr., cooled to room temperature,
and neutralized to pH 5.0 by tltration with a saturated aqueous barium
hydroxide solution. The resulting precipitate of barium sulfate was
removed by centrifuge and washed well with water. The decantate plus
washings were concentrated on a rotary evaporatory to 50 ml.
5. Mild Acid Hydrolysis
A part (17706 mg dry weight) of the solids solubilized by the acidified
sodium chlorite reaction was dissolved in 3% sulfurlc acid (50 ml) and the
solution was refluxed 5 hr. After cooling to room temperature the solution
was titrated to pH 5.0, with a saturated solution of aqueous barium
hydroxide. The resulting precipitate of barium sulfate was removed by
centrifuge and washed well with water. The decantate plus washings were
concentrated to 25 ml on a rotary evaporator.
6. Qualitative Amino Acid Analysis by Paper Chromatography
The hydrolyzates from the strong acid treatment and the mild acid
treatment were subjected to two dimensional paper chromatography (Whatman
No. 1 paper) using water-saturated phenol (beakers of 0.3% ammonium hydroxide
were placed in the bottom of the developing tank) as developer in one
direction and rv-butanol-formic acid~water (20:6:5 v/v/v) as developer in
the second direction. After air-drying, the papers were sprayed with
ninhydrin spray reagent (0.02% ninhydrin in n-butanol) and heated at
100±5° in an oven for 5 minutes (37, pp. 93-96).
7. Qualitative Carbohydrate Analysis by Paper Chromatography
The hydrolyzates from the strong acid hydrolysis and the mild acid
hydrolysis were subjected to paper chromatography using ethyl acetate-
pyridine-water (8:2:1 v/v/v) as developer. The solvent was allowed to
-------
-37-
mlgrate almost to the bottom of the papers at which time they were removed
from the tank and air-dried. The papers were returned to the tank and
developed as before (repeated 3 tines). The paper chromatograms were
sprayed with o-aminodiphenyl reagent (0.4 g o-aminodiphenyl dissolved in
a solution prepared from 100 ml glacial acetic acid and 20 ml distilled
water) and heated at 10012° in an oven for 5 min (19).
8. Quantitative Carbohydrate Analysis by Gas-Liquid Chromatography
The gas-chromatograph used was a Hewlett-Packard 5751B Research
Chromatograph (Hewlett-Packard Company, Palo Alto, California) equipped
with dual flame lonlzation detectors. The conditions were: column, 6.5%
ECNSS-M on Gas Chrom Q 100/120 mesh, 6 ft. x 1/8 in. O.D. stainless steel;
injection port 200°; detector 230°; column temperature 180° isothermal;
2
helium flow 30 ml/min; range setting 10 , attenuation setting 16.
The solids solublllzed by the acidified sodium chlorite reaction were
hydrolyzed and the derivatives prepared for injection into the gas
chromatograph as follows. A portion of the solids (0.2721 g, dry weight)
was dissolved in 32 sulfuric acid (80 ml) and refluxed for 5 hours. After
cooling, authentic myo-lnosltol (0.1004 g) was added. The solution was
neutralized to pH 5.0 with a saturated aqueous solution of barium hydroxide.
The resultant precipitate of barium sulfate was removed by centrifuge and
the decantate plus washings were concentrated to about 25 ml and transferred
to a 100-ml round-bottomed flask. Sodium borohydride (0.08 g) was added
to the flask and allowed to react for 2 hr. at room temperature (30).
The excess sodium borohydride was decomposed by adding acetic acid
until gas evolution ceased. The solution was concentrated to a sirup on
a rotary evaporator and methanol (10 ml) was added and re-evaporated. The
addition and removal of methanol was repeated five time (32). The resulting
-------
-38-
sirup was dried in an oven at 105° for 15 min to ensure complete removal.
of water.
Acetic anhydride (7.5 ml) and concentrated sulfuric acid (0.5 ml)
were added to the sirup and the solution was heated for 1 hour at. 50-60°
in a water bath. After cooling for 5 min. the acetylation mixture was
poured slowly with stirring into about 70 ml of ice water. The mixture
was transferred to a separatory funnel and the alditol acetates were
extracted with three successive amounts of methylene chloride (25 ml, 15
ml, and 10 ml). The methylene chloride extract was concentrated to dryness
on a rotary evaporator at 75°. Water (1 ml) was added to the residue and
re-evaporated. The alditol acetates were dissolved in 2 ml of methylene
chloride and about 1.0 pi of the solution was injected into the gas
chrotnatograph for quantitative analysis (31) .
The areas under the peaks in the resulting spectrum were measured
with a planimeter.
9. Qualitative Uronic Acid Analysis by Infrared Spectroscopy
A sample (0.5 mg) of dried solids solublllzed by the acidified- sodium
chlorite with potassium bromide (200 mg) and pressed into a pellet. A
spectrum was taken of the pellet over the range of 4000 to 300 cm
(Beckman IR-20A). The residue from the 0.5% ammonium oxalate extraction
was also analyzed, by Infrared spectroscopy.
An infrared spectrum was also obtained in a Nujol mull prepared by
grinding it to a thick paste in Nujol oil (paraffin oil).
10. Qualitative Uronic Acid Analysis by, Color Reactions
Concentrated sulfuric acid (6.0 ml) was added slowly to an aqueous
solution (1.0 ml) of the 3% sulfuric acid hydrolyzate of the solids solubilized
by the acidified sodium chlorite reaction and cooled under tap water. The
-------
-39-
reaction mixture was heated for 20 min in a boiling v/ater bath and cooled.
An aliquot (0.2 ml) of a 0.1% ethanolic solution of carhazole was added
and the test sample allowed to stand for 2 hours at room temperature. The
development of a purple color indicated a uronic acid. The solution was
scanned in the ultraviolet range and showed maximum absorption at 535 nm
indicating the presence of a uronic acid (38, p. 497).
An aliquot (177.6 g dry weight) of the solubilized solids was dissolved
in 3% sulfuric acid (50 ml) and the solution was refluxed for 5 hr. The
hydrolyzate was neutralized by passage through a column of Amerlite IRA-400
anion exchange resin in the -OH form. An amount (1.0 ml) of the neutralized
solution was tested for uronic acids by the carbazole-sulfuric acid test
described above. The results were positive.
A small aliquot of the 0.5% ammonium oxalate residue was also tested
with carbazole-sulfuric acid. The results showed a strong positive test.
Color tests were also run on authentic galacturonic acid (positive test),
oxalic acid and benzoic acid (negative tests).
11. Qualitative Uronic Acid Analysis by Paper Chromatography
The hydrolyzate prepared from the mild acid hydrolysis of the solids
solubilized by the acidified sodium chlorite reaction was subjected to
paper chromatography. Whatman No. 1 filter paper was employed in conjunction
with the following solvent systems:
1. water saturated n-butanol-absolute ethanol-water (10:9:1 v/v/v)
2. water saturated n-butanol-acetone-water (4:5:1 v/v/v)
3. ethyl acetate-pyridine-water (8:2:1 v/v/v).
Authentic galacturonic acid v/as chromatographed simultaneously with
the unknown hydrolyzate.
For the first two solvent systems, the papers were first impregnated
-------
-40-
vlth a phosphate buffer solution prepared by titrating a 0.01 M solution
of disodlum hydrogen phosphate to pH 5.0 with a 0.1 M solution of
phosphoric acid (39).
The chromatograms developed with solvent three were alternately
irrigated and air-dried for a total of 6 times. They were thus in the
'ln!^: 1 :.
solvent atmosphere for a total of about 30 hr. .: ,
; . i .
After development, the paper chromatograms prepared from the first
two solvent systems were sprayed with aniline hydrogen phthalate reagent
(1.66 g of phthalic acid dissolved in 100 ml of water-saturated n-butanol
containing 0.93 g of freshly distilled aniline) (40). The chromatograms
prepared from the third solvent system were sprayed with the o_-aminodiphenyl
reagent. After.spraying, all chromatograms were heated in. an oven at
100±2° for 5 min.
12. Preparation of Methyl Ethers
An aliquot of the solids solubilized by the acidified sodium chlorite
reaction (1.461 g dry weight) was dissolved in 18% aqueous sodium hydroxide
at 0° with stirring followed by the addition of 6.0 g of sodium hydroxide
pellets. Sodium hydroxide (100 ml, 30%) and dimethyl sulfate (50 ml) were
added simultaneously over a period of 3 hours while maintaining the
temperature at 0°. Acetone (150 ml) was added to prevent foaming and the
mixture was stirred for an additional 45 hr at room temperature. The
solution was cooled to 0°, neutralized with 10% sulfuric acid, dialyzed
against water (3 days), concentrated under reduced pressure (200 ml), .
lyophilized, and the entire methylation sequence repeated (41). '
The product was further methylated by the method of Falconer and
Adams (42) by solution in 150 ml of tetrahydrofuran followed by treatment
over a period of 60 hours with seven 10 g portions of crushed sodium
-------
-41-
hydroxide, each followed by a 12 ml portion of dimethyl sulfate. The
solution was stirred vigorously and additional solvent was added as needed
to maintain fluidity. The product was recovered as described above for
the first methylation, and the concentrated dialyzate (600 ml) was
extracted with three 300 ml portions of chloroform. The chloroform layer
was concentrated under reduced pressure, yielding a light brown solid
which was dissolved in acetone and filtered. The concentrated filtrate
(50 ml) was poured into petroleum ether (b.p. 30-60°, 200 ml) and the white
precipitate which formed was recovered by centrifuge (after the mixture
was refrigerated for 6 days) and lyophllized, affording a white, fluffy
powder; yield 80 mg.
13. Acid Hydrolysis of the Methyl Ethers
An aliquot (40 mg) of the methylated material prepared above was
hydrolyzed for 3 hr at 97° with 88% formic acid (10 ml). The formic acid
was removed by evaporation under reduced pressure followed by the addition
and removal of water, then hydrolyzed with 0.5 N sulfuric acid (5 ml for
2.5 hr at 97°). Upon cooling the solution was neutralized with water-
saturated barium hydroxide and the solids were removed by cehtrifugation.
The decantate was concentrated to about 2 ml of sirup. The sirup was
chromatographed on paper using the developer ethyl acetate-pyrldine-water
(8:2:1 v/v/v).
I. Characterization of_ the Holocellulose Fraction
1. Elemental Analysis for Nitrogen, Sulfur, Phosphorus, and the Halogens
To a small test tube (75 x 12 mm), supported in a clamp, was added
0.06 g of sodium metal. The tube was heated in a Bunson Burner flame until
the sodium melted and the vapors rose 1-2 cm up the walls of the tube. The
tube was strongly heated over an open flame until the entire end was red hot
-------
-42-
The tube was then plunged into a small beaker containing 10 ml of distilled
water. The hot tube was shattered. The mixture was heated to ..boiling. The
insolubles were removed by filtration, and-the filtrate recovered for
elemental analysis. . •
A second 10 ml of filtrate from the sodium fusion reaction was similarly
prepared. ^
An aliquot (2.5 ml) of the above filtrate was added:to a test tube
containing about 0.1 g of powdered ferrous sulfate. The mixture-was ;gently
heated with shaking until it boiled. Without cooling, justcenough'dilute
sulfuric acid .was.added to dissolve the iron hydroxide and give the solution
an acid reaction. No.precipitate of Prusslon Blue formed, indicating the
absence of nitrogen. ,,
A known nitrogen containing compound, alanine (0.02 g)., was fused with
sodium aa described above. A.precipitate of Prussian Blue formed upon
reacting the.acidified.sodium .fusion solution with ferrous sulfate:indicating
the presence of nitrogen.
A second aliquot (2.0 ml) of the filtrate'from the sodium fusion reaction
was acidified.with dilute acetic acid. A few drops of lead acetate were
added. No yellow precipitate formed which indicated no sulfur.
A known sulfur-containing compound, cystine, was fused ;wi.th sodium as
above. A yellow precipitate formed in the filtrate by acidifying with
acetic.acid and .adding a few drops of lead acetate. This indicated the
presence of sulfur.
A third aliquot (2.0 ml) of the filtrate from the.sodium fusion reaction
was acidified with 3.0 ml of concentrated nitric acid and boiled 'for one
minute. The solution was cooled and to it was added an equal volume 'of
sjipmonium molyb.date reagent. The solution was warmed to 40-50" .and allowed
-------
-43-
to stand. No yellow precipitate formed which Indicated the absence of
phosphorous.
A known phosphorus containing compound, p.lucose-1-Phosphate was fused
with sodium as described above. A yellow precipitate formed upon reacting
the acidified sodium fusion solution with ammonium molyhdate Indicating the
presence of phosphorus.
A fourth aliquot (2.0 ml) of the filtrate from the sodium fusion
reaction was acidified with dilute nitric acid and added to an excess of
silver nitrate solution. No precipitate formed Indicating the absence of
halogens.
Known sodium chloride (0.02 g) was fused with sodium as described above.
A precipitate formed when the acidified filtrate was added to a silver
nitrate solution, indicating the presence of a halogen. The mother liquor
was decanted and the precipitate was treated with dilute aqueous ammonia
solution. A white precipitate formed which was readily soluble in the ammonia
solution indicating the presence of chlorine.
2. Determination of Lignin
n. Acid-Insoluble Lignin
A portion (0.4566 g, dry weight) of bark holocc-llulosc was placed in
a 500-ml, three-necked round-bottoned flask submerged in a cold-water bath
(18-20°). An awount (10.0 ml) of cold (13-15") 72% siilfuric acid was added
slowly with stirring. The sample was allowed to stand, with frequent
stirring, for 2 hou:t> at 18-20°. The holocellulose became completely
dispersed in the acid. The transparent sirup was diluted to 3.0% sulfuric
acid concentration by slowly adding 376.0 ml of distilled water. The
sample was refluxed for 4 hours. The Insoluble material was recovered by
filtration using a Cooch crucible (fine porosity) which had previously
-------
-44-
been dried and weighed. The residue In the crucible was washed free of
acid with 150.0 ml of hot water and dried In an oven at 105±2° until the
weight became constant (18 hours) (43). The average of three determinations
was 0.0139 g or 3.05% of the holocellulose.
t - •
b. Acid-Soluble Lignin
•-' :
The filtrates from the above Klason lignin determinations were cdmbined
to yield a solution of 1544.0 ml. The solution was analyzed, for the acid-
soluble lignin content by the characteristic lignin absorptions; at 280 nm
and 210 nm (44, 45, p. 259). The instrument used was a Beckmari AGTA TM 111
UV-Visible spectrophotometer. The instrument was standardized by placing
3.0 ml of 3.0% sulfuric acid solution in both the reference and sample cells
(cell widths, 1.0 cm) and scanning over the ultraviolet spectral range from
370 nm to 200 nm. The sample, cell was cleaned, dried and 3.0 ml of the acid
filtrate from the Klason lignin determination was added. The sample was
scanned over the, ultraviolet spectral range from 320 nm to 200-rim. Absorption
peaks were recorded at 280 ran (absorbance 2.60) and at 210 nm (absorbance 3.77)
3. Hydrolysis with 7.7..0% Sulfuric Acid
(
Holocellulpse (913.2 mg, dry weight) was dissolved in 12/.4 g, of 77.0%
sulfuric acid in a 1-liter three-necked, round-bottomed flask, at ice-water
temperature for 1 hour. ' The transparent sirup of dissolved holocellulose
was diluted to 32 sulfuric acid by the dropwise addition of 247.0 ml of
distilled water from a dropping funnel. Small, insoluble particles were
observed In the dilute solution. The dilute solution was refluxed for six
hours. The small, insoluble particles remained. The solution was filtered.
The filtrate wae neutralized to pH 5 with saturated aqueous.barium hydroxide
solution. The resulting precipitate of barium sulfate was removed*by•
centrifuge and washed, well with water. The decantate plus washings were •
*
concentrated on a rotary evaporator to a sirup.
-------
-45-
4. Qualitative Carbohydrate Analysis by Paper Chromatography
The hydrolyzate from the sulfuric acid hydrolysis was separated into
its component oonosaccharides by paper Chromatography. Three drops of the
hydrolyzate sirup was dissolved in 1.0 ml of distilled water. Five
micropipettes of the above dilute hydrolyzate were spotted on Whatman No. 1
chromatographic paper. A solution of a known monosaccharide mixture
containing 1.0% each of glucose, galactose, mannose, arabinose and xylose
were placed along with the unknown hydrolyzate on the front of the
chromatographic paper 3 cm from one end. The chroraatographic sheet was
developed by the descending method in a chromatographic tank of dimensions
61 cm x 67 cm x 82 cm in height, pre-saturated for one day with the developer
ethyl acetate-pyridine-water (8:2:1 v/v/v). The solvent was allowed to
migrate almost to the bottom of the papers (6-8 hr). The papers were
removed from the tank and air dried. They were placed back in the tank
and developed again as above (repeated 3 times). The papers were sprayed
with o-aminodiphenyl reagent (0.4 g o-aminodipher.yl dissolved in a solution
prepared from 100.0 ml of glacial acetic acid and 20.0 ml of distilled
water) and heated at 100±2° in an oven for 5 min to develop the color. The
monosaccharides after separation and reaction with the color reagent were
investigated under ultraviolet light. Hexoses showed a white color and
pentoses showed a red brick color under the ultraviolet light.
5. Isolation of Crystalline Sugar Derivatives
a. Isolation of'Sugars by Preparative Paper Chromatography
A part (6.0 g, dry weight) of bark holocellulose was added to 77.0%
sulfuric acid (90.0 g) in a 5-liter round-bottomed flask which was submerged
in an ice-water bath. The mixture was stirred for 1 hr at the end of which
time it appeared as a transparent sirup. Water (1688.0 ml) was added slowly
-------
-46-
to the sirup to dilute the sulfuric acid to 3% concentration.. The solution
was refluxed for a total of six hours. It was allowed to cool to room
'I
temperature and neutralized to pH 5.0 by the careful addition of aqueous
saturated barium hydroxide solution. The resulting precipitate of barium
sulfate was removed by centrifugalion and washed well with water. The
decantate plus washings were concentrated to about 20.0 ml on a rotary
,j ~ - •.•,•'
evaporator. ...
Aliquots of approximately 0.1 ml each of the concentratedjhydrolyzate
were streaked with a syringe across large pieces of Whatman Njj.. 3 MM
chromatographic paper, 9 cm from one end of the paper. After ,the streaks,
were dry, the papers were developed by the descending method using the
solvent system ethyl acetate-pyridine-water (8:2:1 v/v/v). The solvent
was allowed to migrate almost to the bottom of the papers (about 6-8 hr).
The sheets were removed and dried in the air overnight. The sheets were
developed and air-dried again (repeated 5 more times). A strip 1.0 cm in
width was cut from the center of each sheet along the direction of solvent
migration. The strips were sprayed with o_-aminodiphenyl reagent and -dried
in an oven at 105±3° for 10 rain. The degree of separation was_examined
under ultraviolet light. It required six solvent migrations .to the bottom
of the sheets to adequately separate the monpsaccharides (glucose, galactose,
mannose, arabinpse, and xylpse) in the holocellulpse hydrolyzate,
After assurance that th* sugars were well separated, 1.0 cm strips were
cut from the papers in the direction of solvent flow at 6 cm intervals across
the width of the papers. These strips were sprayed with o-aminodiphenyl
«
indicator as before. After location of the sugars, the strips, were re-fitted
back into the original paper chromatograms and the unsprayed cross^bands pf
each sugar cut out. The sugars were collected from 24 such paper chronatpgrams.
-------
-47-
The paper bands thus collected were placed in 1 liter beakers (a
different beaker for each sugar), and the sugars eluted with 800.0 ml of
distilled water. The eluate of each isolated sugar was concentrated on a
rotary evaporator to a sirup. Methanol was added to the sirup and then
re-evaporated (repeated 3 tines), and final drying was achieved in a
dessicator.
Each isolated sugar sirup was re-chromatographed on paper simultaneously
with a solution of known sugars (glucose, galactose, mannose, arabinose and
xylose; each 1% concentration). In this way each Isolated sugar sirup was
of assured purity before preparation of a crystalline derivative was
attempted.
b. Diethyl Dithloacetal Acetate Derivatives of Galactose and Arabinose
Diethyl dithioacetal acetate derivatives of known monosaccharides were
e synthesized for direct comparison with derivatives of the sugars from the
holocellulose hydrolyzate. The general procedure for each known sugar was
that reported by Wolfrom and Karablnos (46). Galactose is used as a typical
reaction illustration. Galactose (100.0 mg) was dissolved in concentrated
hydrochloric acid (1.0 ml, 12 N) in a 100-ml round-bottomed flask submerged
in an ice-water bath. Ethyl mercaptan (1.0 ml) was added under a well
ventilated hood and the mixture was stirred for one hour. The mixture was
neutralized at ice-bath temperature by the addition of ammonium hydroxide
(13-14 drops, 15 N). During neutralization, white smoke resulted and a
green precipitate was formed. The mixture was concentrated to dryness under
aspirator vacuum on a rotary evaporatory at 40°. Dryness was accomplished
by the addition and re-evaporation of absolute ethanol (repeated 5 times).
The greenish-yellow color disappeared after the second or third evaporation
of ethanol, and a white solid resulted.
-------
-48-
The white solid was acetylated with a mixture (6.0 ml) of acetic anhydride
and anhydrous pyridine (2:1 v/v). The reaction was allowed to continue
overnight at room temperature. The solution was poured into distilled water
(10.0 ml) and extracted twice with chloroform (15.0 ml each time). The
chloroform extract was washed three times with saturated aqueous sodium
bicarbonate (10.0 ml each time). The acid-free chloroform extract was
concentrated on a rotary evaporator to a sirup. Crystallization was attempted
i
by dissolution, of the- sirup in methanol followed by the dropwise addition of
water until a permanent cloudiness resulted. Crystallization.of some of the
derivatives was also attempted from 95% ethanol.
Diethyl dithioacetal acetate derivatives of known mannpse, galactose,
arabinose and xylose were similarly prepared. However, only galactose diethyl
dithioacetal acetate (m.p. 75/.0-77.00; literature value 76.5-77.0°), and
arabinose diethyl dithioacetal acetate (m.p. 78.0-79.0°; literature valfle
79.0-80.0°) crystallized. Therefore, similar derivatives of only the
galactose and arabinose sirups isolated from the holocellulqse were prepared:
galactose diethyl dithioacetal acetate, m.p. 76.5-77.5°, unchanged on
23 5
admixture with, authentic material [a] ' + 10.0° (£ 4.0, chloroform)
arabinose diethyl dithioacetal, m.p. 77.0-78.5°, unchanged on admixture
23.5
with authentic material [ot]_ * - 26.40 (c_ 1.54, chloroform) literature
value -30.0°.
c. Acetate Derivitave of Glucose
An amount (5.4 ml) of acetic anhydride was heated to about 100° and
0.44 g of anhydrous sodium acetate was added. In small portions, 1.0 g of
D-glucose was added with.-stirring over the course of 30 min..
When the addition was. completed, the solution was cooled, to room
temperature, poured into 16. ml of ice-water, and stirred for 30; min. The
-------
-49-
acetylated product was extracted with three 20-ml portions of chloroform.
The extracts were washed with 20 ml of distilled water and concentrated
on a rotary evaporator to a sirup. Glucose pentacetate was crystallized
by dissolution of the sirup in methenol followed by the dropwise addition
of water until a permanent cloudiness resulted. After two recrystallizations
from 95% ethanol, white crystalls of glucose pentaacetate were obtained;
(yield 234.0 mg, m.p. 133.5-135.0; literatuve value 135°) (47).
Glucose sirup (250 mg) isolated from the bark holocellulose hydrolyzate
by preparative paper chromatography was similarily acetylated. White
crystals of glucose pentaacetate were obtained; yield 112 mg, m.p. 133-134°,
23 5
unchanged on admixture with authentic material, [a] " + 3.9° (c 3.4,
L)
chloroform), literature value 3.8°.
d. Di-()-benzylidene Dimethyl Acetal Derivative of D-Xylose
The reagent for preparing di-0-benzylidene dimethyl acetal derivatives
of D-xylose was prepared by dissolving 2 ml of redistilled benzaldehyde in
a mixture of 2.5 N methanolic hydrogen chloride (1.0 ml) and spectroquality
methanol (6.0 ml). An aliquot (2.5 mg) of D-xylose was treated with 5.0 ml
of the reagent at room temperature. After standing for 1 hr, silky needles
appeared in the solution. After standing for 6 hr the solution solidified.
The white crystals were recovered by filtration, washed with 200 ml of ice-
water and dried in a dessicator for 3 days. When recrystallized from
spectroquality methanol, silky needles of di-0-benzylidene-D-xylose dimethyl
acetal were obtained; m.p.210-211.5°, literature value 211-212° (48, p. 88).
Xylose sirup isolated by preparative paper chromatography from bark
holocellulose hydrolyzate was similarily treated with the prepared reagent
and crystalline needles were obtained. After recrystallization from
spectroquality methanol, silky needles of dt-0-benzylidene-D-xylose dimethyl
-------
-50-
acetal were obtained; yield 90.7 nip,, m.p. 211-212.5°, unchanged on admixture
23 5
with authentic material, [al ' - 8.0° (c 1.0, chloroform) literature
1) —
value -7.0° (48, p. 88).
e. Phenylhydrazone Derivative of Mannose
D-raannose (1.0 g) was added to a solution of phenylhydrazine (1.0 ml)
in 95% ethanol (15.0 ml) in a 50-ml round-bottomed flask. The mixture was
warmed for 30 min in a hot-water bath (60°) and then placed in the
refrigerator overnight. The resulting crystals of mannose phenylhydrazone
were removed by filtration and washed successively with a few drops each of
water, ethanol,, and diethyl either; yield 94.5 tng, m.p. 191-192°. The
crystals were again washed with a few drops each of water, ethanol and
diethyl ether; yield 90.5 mg of glistening snow-white crystals, m.p. 194-195°,
[a]~3'5 + 2.5° (c 0.1 pyridine), literature value 199.0-200.0° [u]p6(c 0.1
pyridine) (49, p. 147).
An aliquot (9.0 g air-dried weight, moisture content 8.68%) of
holocellulose was hydrolyzed with 77.0% sulfuric acid as previously
described. One-third of the resulting hydrolyzate was concentrated to
a sirup. The sirup was dissolved in 95% ethanol (45.0 ml) followed by the
addition of phenylhydrazene (3.0 ml) (49, p. 107). The mixture was warmed
for 30 min in a hot-water bath (55°), cooled, and kept in the refrigerator
overnight. The resulting white crystals were recovered by filtration and
washed successively with water, ethanol and diethyl ether; m.p. 160-180°.
These crystals were dissolved in water (30.0 ml) and kept in the refrigerator
overnight. The insoluble white crystals were recovered by filtration and
dried; yield 226.7 mg, m.p. 193.5-194.5, unchanged on admixture with
235
authentic mannose phenylhydrazone; [a] * + 25° (£0.1, pyridine) literature
value +26° (49, p. 147).
-------
-51-
Another aliquot (5.0 g air-dry weight, moisture content 8.68%) of
holocellulose was refluxed with 90% formic acid (50.0 ml) for 1 hr (41).
The residue was removed by centrifugation and filtration. The filtrate
was concentrated to a sirup which was hydrolyzed with 0.5 N sulfuric acid
(50.0 ml) under reflux for 2.5 hr. The acid hydrolyzate was neutralized
to pH 5.0 with a saturated aqueous solution of barium hydroxide and the
resulting precipitate was removed by centrifugation. The remaining
inorganic salts were removed by refluxing the concentrated sirup with
absolute methanol followed by filtration (repeated four times) and removal
of solvent.
The resulting sirup was dissolved in 95% ethanol (15.0 ml) followed by
the addition of phenylhydrazine (1.0 ml) (49, p. 147). The solution was
warmed (55°) for 30 min and then kept in a refrigerator overnight. The
resulting white crystals were recovered by filtration washed successively
with water, ethanol, diethyl ether and dried; yield 132.4 mg; m.p. 194-196°,
12:
'U
23.5
unchanged on admixture with authentic mannose phenylhydrazone; [a]n * + 25C
(c 0.1, pyridine), literature value +26° (49, p. 147).
6. Quantitative Determination of Reducing Sugars by Copper Reduction
a. Standardization of Sodium Thiosulfate Solution
Potassium iodate (1.41387 g) was dissolved in distilled water and the
solution was diluted to 1 liter In a volumetric flask. An aliquot (100.0 ml)
of the solution was removed with a pipette. A 1.8 M potassium iodide
solution (10.0 ml) and a 12 M hydrochloric acid solution (3.0 ml) were
added to the aliquot. This solution was titrated with the sodium thiosulfate
solution to be standardized (about 0.005 M) to a starch end-point. The
exact molarity (0.0058 M) of the sodium thiosulfate solution was calculated
from the average of triplicate samples (50, p. 460; 51, p. 379).
-------
-52-
b. Preparation of Somogyi Copper Reagent
Rochelle salt (40.0 g) (potassium sodium tartrate), disodium hydrogen
phosphate heptahydrate (53.0 g), and 1.0 N sodium hydroxide (100,0 ml) were
dissolved in 500.0 ml of water and 80.0 ml of an aqueous solution containing
8.0 g of cupric sulfate pentahydrate was stirred in. Finally, 180.0 g of
anhydrous sodium sulfate was added and dissolved. The solution was diluted
to 1 liter and allowed to stand for 3 days (52, p. 383-386). Potassium
iodate (37.2 mg) was added to a 100-ml aliquot of the reagent to provide a
concentration of 3.72 mg per liter
c. Determination of Total Reducing Sugars in the Holocellulose Hydrolyzate
Holocellulose (300 mg air-dry weight; moisture content 8.68%) was
dissolved in 77% sulfuric acid (4.50 g) in a 150-ml three-necked round-bottomed
flask in an ice-bath. The mixture was diluted to 3.9% sulfuric acid
concentration by the dropwise addition of water. The solution was heated
to reflux and aliquots (2.0 ml) were withdrawn at 30 min intervals.
Each aliquot was neutralized to pH 2.0 with 3% aqueous sodium hydroxide
solution (1.28 ml). Each neutralized solution was diluted to 25.0 ml in a
volumetric flask. Three aliquots (5.0 ml each) of these solutions were
added with a pipette to test tubes (20 cm x 2.4 cm). Similarly, 5.0 ml of
three standard glucose solutions containing 1.0 mg, 0.75 mg and 0.5 mg per
5 ml of solution were added to identical test tubes. A blank of 5.0 ml of
water was added to another test tube. Somogyi reagent (5.0 ml) was pipetted
into each of these test tubes under a stream of nitrogen gas. The solutions
were heated in a boiling water bath for 10 min and cooled in a water bath
for 10 min. Each solution was oxidized by adding 2.5% aqueous potassium
iodide (0.5 ml) and 2 N sulfuric acid (1.5 ml) and each was allowed to stand
for 5 min with occasional shaking. The solutions were titrated to a starch
-------
-53-
end point with standarized sodium-thiosulfate (0.0058 M) within 30 min. The
amount of copper reagent reduced by the reducing sugars was obtained by
subtraction of the titer of the sample from the tlter of the blank. The
amount of reducing sugar in the test tubes of the unknown hydrolyzate was
calculated by direct comparison with the known amount of glucose in the
standard solution (22).
The initial sample of holocellulose was refluxed a total of 10 hr and
then heating was discontinued while the aliquots which had been withdrawn
were analyzed. The titration results showed that monosaccharides were still
being released after 10 hr of reflux. Therefore, a second sample of
holocellulose (300 mg) was hydrolyzed by sulfuric acid as before but the
reflux time was extended to 20 hr. Aliquots (2.0 ml) were removed every
two hours and analyzed for reducing sugars with the Somogyi reagent. The
reducing power of the solution ceased to increase after 12 hr of reflux,
indicating that hydrolysis was complete at this time.
7. Quantitative Carbohydrate Analysis by Gas-Liauid Chromatography
a. Preparation of Authentic Aliditol Acetates
Galactitol (0.50 g) was added to 10.0 ml of an acetylating reagent
composed of pyridine-acetic anhydride (1:1 v/v). The mixture was refluxed
for 1 hr and poured into ice-water (40.0 ml). A white precipitate formed
which was recovered on filter paper and washed well with water. The white
solid (galactitol hexaacetate) was recrystallized 3 times from 95% ethanol;
m.p. 171°, literature value 171° (30, 53).
Glucitol (sorbitol), mannitol, arabinitol, and myo-inositol were
similarily acetylated: glucitol hexaacetate, m.p. 99.5°, literature value
99-99.5° (30, 53); mannitol hexaacetate, m.p. 125.5°, literature value 126°
(30, 53); arabinitol pentaacetate, m.p. 76°, literature value 76° (30, 53);
-------
-54-
inositol hexaacetate, m.p. 215-216°, literature value 214-215° (54, p. 85).
Xylitol was acetylated as above but the derivative would not crystallize
from 95% ethanol. The oil of xylitol pentaacetate was poured into ice water
and extracted 3 times with chloroform. The chloroform extract was condensed
to a sirup and dissolved in 95% ethanol, 85% methanol, acetone, diethyl ether,
or a mixture of acetone, diethyl ether and ligroin. The sirup was dried by
the addition and removal of methanol under vacuum (repeated 3 times). The
xylitol pentaacetate then crystallized from acetone and was recrystallized
two times, m.p. 57°, literature value 61-62° (30, 53).
Rhamnose (0.1 g) was dissolved in 100 ml of water in a 100-ml round-
bottomed flask and was reduced with sodium borohydride (0.8 g) for 2 hr at
room temperature. Acetic acid was added to the solution until no gas evolved.
The solution was concentrated to a sirup and dried by the addition and removal
of methanol on a rotary evaporator (repeated 5 times). The sirup was dried
in an oven at 105° for 15 min. The dry sirup was acetylated with an
acetylating reagent of acetic anhydride (75 ml) and concentrated sulfuric
acid (5 ml) for 1 hr at 50-60°. The acetylated mixture was cooled for 5 min
and poured into ice water and extracted with chloroform (300 ml, 300 ml,
200 ml). The chloroform extract was condensed to a sirup and dissolved in
95% ethanol. No crystals formed. The sirup was dried several times by the
addition and removal of methanol on a rotary evaporator. It still did not
crystallize from any solvent tried.
Each of the above crystalline alditol acetates was dissolved in turn
in methylene chloride (2.0% solution) and injected (2 pi) separately into
the gas chromatograph (Hewlett-Packard 5751B Research Chromatograph equipped
with dual flame ionization detectors, Hewlett-Packard Company, Palo Alto,
California). The conditions were: column, 6.5% ECNSS-M on Gas Chrom Q
-------
-55-
100/120 mesh, 6 ft x 1/8 in O.D. stainless steel; injection port 180°;
detector 240°; column temp 180° isothermal; helium flow 30 al/min; range
2
setting 10 , attenuation setting 16. .
In this way the retention time of each alditol acetate was determined.
A mixture of the alditol acetates were dissolved in methylene chloride (2.0%
concentration of each) and an aliquot (2 yl) of the solution was injected
into the gas chromatograph. Resolution of the six compounds was excellent,
and under the chromatographic conditions outlined above the retention time
ot each compound in the mixture was: xylitol pentaacetate 16 min; arabinitol
pentaacetate 12 min; mannitol hexaacetate 28 min; galactitol hexaacetate
33 min; glucitol hexaacetate 38 min; myo-inositol hexaacetate 48 min. These
retention times were used to identify alditol acetates prepared from the
holocellulose hydrolyzate.
b. Determine of Optimum Gas-Chromatographic Conditions
Glucitol hexaacetate (4.63 mg), galactitol hexaacetate (0.25 mg), mannitol
hexaacetate (0.75 mg), arabinitol pentaacetate (0.25 mg), xylitol pentaacetate
(0.50 mg), and myo-inositol hexaacetate (6.25 mg) were dissolved together in
methylene chloride (1.0 ml). Aliquots (2 ul) of the solution were injected
into the gas-chromatograph under various ranges of injection port temperatures
(180-230°), detector temperatures (210-270°), and column temperatures (180-
185°). The areas under the peaks of the resulting spectra were measured
with a planimeter. The percent recovery was calculated by comparing the
peak areas of the other sugars to myo-inositol hexaacetate as 100%. The
best recoveries were realized under the following conditions: injection port
temperature, 180°; detector temperature, 240° and column temperature, 180°.
These were the conditions generally used in this work.
-------
-56-
c. Determination of an "Instrument K Factor"
The response of the gas chromatograph to the alditol acetates was
determined by preparing 5 mixtures of the known alditol acetates in various
ratio concentrations as shovn in Table 5.
Each of the mixtures in Table 5 was dissolved in methylene chloride (1.0
ml) and an aliquot (2 yl) was injected into the gas chromatograph. The
areas under the peaks of the resulting spectra were measured by a planimeter.
The percent recovery was calculated on the basis of myo-inositol hexaacetate
as 100%. The "Instrument K Factor" for each alditol acetate was obtained
as the slope of the line obtained by plotting the ratio of the area of each
alditol acetate to that of the standard (myo-inositol hexaacetate) against
the ratio of the weight of the alditol acetate to that of the standard
(myo-inositol hexaacetate). The resulting "Instrument K Factors" were as
follows: xylitol pentaacetate 0.95; arabinitol pentaacetate 0.96; mannitol
hexaacetate 0.96; galactitol hexaacetate 0.98; glucitol hexaacetate 0.92.
d. Analysis of the Holocellulose Hydrolyzate
An amount (300 mg, air-dry weight, moisture content 8.68%) of holocellulose
was dissolved in 4.5 g of 77.0% sulfuric acid in an ice-bath for one hour
with occasional stirring. After one hour, the translucent sirup was diluted
to 3.9% sulfuric acid by dropwise addition of 84.42 nil of distilled water
from a dropping funnel. The dilute solution was refluxed for 6 hr. The
solution was diluted to 100 ml from which 25 ml of hydrolyzate was removed.
To this' 25.0 ml of hydrolyzate, 25 mg of myo-inositol was added. The
hydrolyzate was neutralized to pH 5.0 with saturated aqueous barium hydroxide
solution, centrifuged, concentrated to 25 ml and transferred to a 100-nl
round-bottomed flask. Sodium borohydride (0.08 g) was added to the flask
and allowed to react for 2 hr at room temperature (30).
-------
-57-
The excess sodium borohydride was decomposed by adding acetic acid until
gas evolution ceased. The solution was concentrated to a sirup on a rotary
evaporator, and methanol (10.0 tng) was added and re-evaporated. The addition
and removal of methanol was repeated eight times (32). The resulting solid
was dried in an oven at 105* for 15 min to ensure complete removal of water.
An aliquot (8.0 ml) of a mixture containing acetic anhydride (7.5 ml)
and concentrated sulfuric acid (0.5 ml) were added to the dry solid and the
mixture was heated for 1 hour at 50-60° in a water bath. After cooling for
5 min the acetylation mixture was poured with stirring into about 70 ml of
ice-water in a 150-ml beaker. Two immiscible layers (water layer on top
and acetate layer on the bottom) formed. These were transferred to a 150-ml
separatory funnel and the alditol acetates were extracted with three
successive amounts of methylene chloride (25 ml, 15 ml, and 10 ml). The
methylene chloride extract was concentrated to dryness on a rotary evaporator
at 75°. Water (1.0 ml) was added to the sirup to remove remaining acetic
anhydride. The resulting alditol acetates were dried in a dessicator. The
alditol acetates (20.0 mg) were dissolved in methylene chloride and diluted
to 1.0 ml in a volumetric flask (1.5%). An aliquot (2.0 ul) of the solution
was injected into the gas chromatograph for quantitative analysis (31).
The areas under the peaks were measured with a planimeter and compared
to the area under the peak of myo-inositol hexaacetate which represented
the area from 25 mg of inositol. The weights of the other sugars were thus
calculated as: glucose, 156.8 mg; galactose, 5.9 mg; mannose, 26.2 mg;
arabinose, 4.6 mg; xylose, 16.5 mg; total 198.6 mg.
A second sample of holocellulose (300 mg) was hydrolyzed as above except
that it was refluxed for 12 hr instead of 6 hr in 3.9% sulfuric acid. The
alditol acetates were prepared and injected Into the gas chromatograph as above.
-------
-58-
The areas under the peaks were measured with a planimete'r and compared
to the area under the peak of mjro-inositol which represented the area from
25 mg of sugar. The weights of the other sugars were thus calculated as:
glucose, 160.4 mg; galactose, 5.8 mg; mannose, 27.9 mg; arabinose, 7.8 mg;
xylose, 21.8 mg; total 223.6 mg. This increase in sugar return after 12 hr
of reflux over the 6 hr of reflux is in agreement with the Somogyi values
for total reducing sugars which had indicated that a minimum reflux time
of 12 hr was required to completely hydrolyze the holocellulose.
J. Fractionation of the Holocellulose into its Component Polysaccharides
1. Impregnation with 2.0% Barium Hydroxide, then Extraction with 10.0%
Aqueous Potassium Hydroxide Solution
Air-dried holocellulose (54.3 g, moisture content 8.68%) from Douglas-fir
inner bark was slurried for 20 min at 25° in 779.0 g of aqueous barium
hydroxide containing 63.4 g of barium hydroxide octahydrate. The consistency
of the slurry was 6.0% based on the holocellulose content and the concentration
of barium hydro::ide in the liquor was 4.4%. At the end of the 20 min period
952.0 g of 18.38% aqueous potassium hydroxide solution was added which lowered
the slurry consistency to 2.81% and the barium hydroxide concentration to 2%.
The potassium hydroxide concentration of the extraction liquor was 10.0%.
Treatment was continued for a second 20 minute period at 25°. Separation
was attempted by filtration through Whatman No. 1 filter paper and a filter
crucible. It was difficult to filter the colloidal solution. The difficulty
was overcome by filtration through milk filters-disks (rapid). The solids
were washed with distilled water, dialyzed for three days, freeze-dried and
weighed; weight 42.3 g or 84.6% of the starting holocellulose. They were
termed "Residue A" (55).
The filtrate plus the washings were acidified with acetic acid to a pH
-------
-59-
of 5.0 and condensed to 500.0 rol on a rotary evaporator. The polysaccharide
solids were precipitated from the filtrate by the addition of 500.0 ml of
methanol. The precipitate was recovered by centrifuge and redispersed in
700 ml of 70% methanol. The re-precipitation was repeated 4 times. Methanol
was removed from the precipitate by the addition of water followed by
evaporation using a ratuvapor. The solids were recovered by freeze-drying;
weight 3.5 g or 7.0% of the original holocellulose. They were labeled
"Hemicellulose A" (55).
2. Extraction with 1.0% Aqueous Sodium Hydroxide Solution
Residue A (41.4 g dry weight) was extracted with a 1% aqueous sodium
hydroxide solution by adding it to 990.0 g of a 9.97% aqueous sodium hydroxide
solution to give a 4.0% slurry consistency based on the content of Residue A.
The mixture was stirred for 20 min at 25°. The solids were recovered by
filtration using milk filter disks. The residue was washed with 250 ml of
1% sodium hydroxide solution followed by 1 liter of distilled water. The
solids were transferred with water to a dialysis bag and the mixture was
dialyzed for 3 days against running water. The solids which remained in the
dialysis bag were recovered by freeze-drying and weighed; weight 39.1 g or
78.2% of the original holocellulose. This residue was labeled "Residue B"
(55).
The filtrate and combined washings were condensed to 1.0 liters and
acidified with acetic acid to pH 5.0. The solids were precipitated by the
addition of 3 liters of methanol, and then recovered by centrifuge. The
precipitate was redispersed in 700 ml of 70% methanol. This redispersion
purification procedure was repeated two more times. The methanol was removed
from the final precipitate on a rotary evaporator. The solids were slurried
in water and the mixture was freeze-dried. The weight of solids recovered
-------
-60-
was 0.84 g or 1.7% of the original holocellulose. These solids were labeled
"Hemicellulose B".
3. Extraction with 15.0% Aqueous Sodium Hydroxide Solution
To "Residue B" (38.6 g dry weight) was added 923 g of a 15.05% aqueous
sodium hydroxide solution to give a 4.0% slurry consistency based on the
content of Residue B and a solution concentration of 15.0% in sodium hydroxide.
After stirring for 20 min at 25°, the insolubles were recovered by filtration
using milk filter disks. The residue was washed with 250 ml of 15.0%
aqueous sodium hydroxide solution and 1.0 liters of distilled water. The
residue was dialyzed for 5 days against running water and freeze-dried;
weight 31 o 3 or 62.6% of the original holocellulose. The residue was labeled
"Residue C" (55).
The filtrate and washings were combined, acidified with acetic acid,
condensed to 1.0 liters and the solids were precipitated by the addition of
3 liters of methanol. The precipitate was redissolved in alkali and
redispersed three times in 700 ml of 70% methanol. The methanol was removed
on a rotary evapofator and the precipitate freeze-dried; weight 1.46 g or
2.9% of the starting holocellulose. These solids were labeled "Hemicellulose
23 5
C". The rotation was taken in 1 N sodium hydroxide solution, [ot] ' - 40°
(c_ 0.25, N sodium hydroxide).
K. Characterization of_ Hemicellulose A; Isolation of a_ Xylan
1. Qualitative Carbohydrate Analysis by Paper Chromatography
Hemicellulose A (262.2 mg dry weight) was dissolved in 4.5 g of 77%
sulfuric acid at ice-bath temperature in a 150.0 ml round-bottomed three-
necked flask. The mixture was stirred until complete, dissolution of
Hemicellulose A was achieved (1.0 hr). The translucent sirup was diluted
to 3.9% sulfuric acid by the addition of water (84.42 ml). The dilute
-------
-61-
solution was refluxed, neutralized, and paper chromatographed as described
above for holocellulose hydrolysis. Paper chromatograms of the hemicellulose
A hydrolyzate showed large amounts of xylose, minor amounts of glucose,
galactose, and arabinose and trace amounts of mannose. Hemicellulose A was
thus considered to be a "xylan".
The isolation procedure outlined in section J-l was repeated and a
large amount of the crude xylan was isolated.
2. Ash Determination of the Crude Xylan
The ash content of the crude xylan was determined according to a
modified TAPPI standard method (56).
Duplicate samples (0.2 g dry weight) were weighed into previously ignited
, a
and weighed porcelain crucibles. The crucibles were placed in a muffle
furnace and heated gently (200°) until charring occurred. The temperature
was increased to 600° and heating continued until ashing was complete. The
ashed samples were placed in a dessicator to cool and were weighed after 1.0
hr. The samples were then reignited and reweighed until constant weight was
obtained. The ash content was 27.9%.
3. Precipitation of Excess Barium and Dialysis of the Crude Xylan
For purposes of calculation, it was assumed that the ash content of the
crude xylan was due entirely to the presence of barium as barium oxide. From
this value, the percent barium (25.0%) present in the sample was calculated.
The large barium residue was due to the use of barium hydroxide in the
isolation of the crude xylan.
A part (5.06 g) of the crude xylan was dissolved in distilled water
(100.0 ml). A slight excess of 2 M sulfurlc acid (5.0 ml) was added to
precipitate all of the barium in the sample. The precipitate was allowed
Lo settle, recovered by filtration and washed several times with hot distilled
-------
-62-
water (51, p. 134). The washings and the filtrate were recotnbined, placed
in a dialysis bag and dialysed against tap water for 5 days. The material
left in the dialysis bag was concentrated on a rotary evaporator and
freeze-dried (yield,69.8%) . A portion of the freeze-dried material was
ignited to determine its ash content (4.4%). This purified, freeze-dried
material was labeled "xylan".
4. Optical Rotation of the Xylan
A part (0.18235 g) of the xylan was dissolved in water (10.0 ml). Ten
different readings were taken on the polarimeter (Rudolf and Sons) at 25°.
The specific rotation was calculated according to the relationship (57, p.
415):
r ,25 IQOct IQOa
la]D '" TC"c~~
25
where [a]n = specific rotation
a = optical rotation in degrees
b = cell length in dm = 1
c = concentration in g/100 ml
The specific rotation was -30.5, U 1.74 g/100 ml, water) (average of 10
readings) .
5. Qualitative Uronlc Acid Analysis by Color Reaction
Concentrated sulfurlc acid (6.0 ml) was added slowly to an aqueous
solution (1.0 ml) of the 3.0% sulfuric acid hydrolyzate of xylan and cooled
under tap water. The reaction mixture was heated for 20 minutes in a boiling
water bath and cooled. An aliquot (0.2 ml) of a 0.1% ethanolic solution of
carbazole was added and the test sample allowed to stand for 2 hr at room
temperature. The development of a purple color indicated that the xylan
contained a uronlc acid. The solution was scanned in the visible region
-------
-63-
and showed maximum absorption at 535 ran indicating the presence of a uronic
acid (38, p. 497). Color tests were also run on authentic galacturonic acid
(positive test), oxalic acid (negative test), and benzoic acid (negative test).
6. Paper Chromatography and Gas-Liquid Chromatography of the Xylan Hydrolyzate
A part (0.30 g) of the dialyzed xylan was hydrolyzed in 3.0% sulfuric
acid (mild acid hydrolysis) for 5 hours, cooled to room temperature,
neutralized to pH 5*0 with saturated aqueous barium hydroxide and centrifuged
to remove the barium sulfate precipitate. An aliquot of the supernatant
liquor was concentrated under vacuum in a rotary evaporator. The concentrated
liquor was spotted on Whatman No. 1 filter paper. A solution of known sugars
was also spotted on the same paper. The paper chromatogram was developed
and sprayed as described previously. The sugars detected were xylose (strong),
arabinose (weak), galactose (trace), glucose (trace), and rhamnose (trace).
Another aliquot (25.0 ml) of the hydrolyzate was reduced with sodium
borohydride and alditol acetates were prepared as described previously.
The alditol acetates were injected into a Hewlett Packard Gas Chromatograph
using the same column and conditions as described previously. The sugars
detected were the same as those reported above for paper chromatography.
The areas under the peaks were determined by planimetry.
7. Reducing End-Group Analysis (Somogyi Method)
A Somogyi determination of the reducing end group was carried out on a
portion of the dialyzed xylan. The alkaline copper reagent (1945) was
prepared aa follows: Rochelle salt (40.0 g), disodiuro hydrogen phosphate
dodecahydrate (71.0 g) (or 53.0 g of the heptahydrate), and 1 N sodium
hydroxide (100.0 ml). A solution containing cupric sulfate pentahydrate
(8.0 g) was added with stirring followed by a solution of potassium iodate
(0.372 g in 100 ml water). This amount of potassium iodate is good for
-------
-64-
determining up to 1.25 rag glucose. Finally, anhydrous sodium sulfate (180.0
g) was dissolved; the solution was diluted to 1.0 liter and allowed to stand
3 days so that the Impurities would settle out. The clear supernatant solution
was further clarified by filtration through a fritted glass filter. The pH
of the solution was about 9.5 as reported in the directions for preparation.
The solution is supposed to be stable for at least one year (52, p. 383).
A sample 8.40 mg of the unhydrolyzed xylan in solution (5.0 ml) was
placed in a 25 x 200 iron test tube. Alkaline copper reagent (5.0 ml) was
added by pipette and mixed thoroughly. The tube was closed with a glass
bulb and placed in a rack. Blanks were prepared with water (5.0 ml).
Standard xylose solutions (1.25 mg, 0.75 mg and 0.25 mg) were also added to
aliquots (5.0 ml) of the alkaline copper reagent. The rack of tubes was
immersed in a vigorously boiling water bath to a depth of about 5.0 cm above
the solution inside the tubes. The solutions were heated for 30 minutes,
removed from the bath and allowed to cool. Care was taken that the tubes
were not agitated during the heating or cooling periods. Triplicate
determinations were carried out in all cases.
Potassium iodide (2.5%, 2.0 ml) was added to each tube without mixing.
From a fast flowing buret, 2 N sulfuric acid (1.5 ml) was run into each tube
with shaking so that the liberated iodine would oxidize all reduced copper.
After 5 minutes, the tubes were reshaken. The excess of liberated iodine
not reduced by cuprous ions was then titrated with sodium thiosulfate
(0.005 N). When the solution was light yellow, two drops of starch indicator
(1.0%) and two drops of phenol red indicator were added, and the titration
continued until the starch-iodine blue disappeared. The difference in the
amount of titer consumed by the blank and the xylan was attributed to the
reducing end group of the polysaccharide.
-------
-65-
8. Viscosity Measurements
A part (0.19156) of the dialyzed xylan was weighed into a stoppered
volumetric flask (25.0 ml) which had been previously swept free of dry air
by a stream of nitrogen. Diethylenediamine copper II ion (cupriethylenediamine)
(General Chemical Division, Allied Chemical, Columbia Road and Park Ave.,
Morristown, NJ or Ecusta Paper Division, Olin, P.O. Box 200, Pisgah Forest,
NC) at 25° was added and the solution was shaken until the xylan was completely
dissolved. The flask was filled to the mark with the solvent and mixed
thoroughly. The filled flask was carefully weighed to determine the density
of the solution in g/25 ml. An aliquot (10.0 ml) of the solution was
transferred to a Cannon-Ubbelhode dilution viscometer previously placed in
a water bath at 25°±0.01° and flushed with nitrogen. After 5 minutes the
solution was drawn into the bulb of the viscometer by applying pressure with
nitrogen. The time for the miniscus to pass between the two calibration
marks was measured to 0.1 seconds. Duplicate measurements were made until
they agreed to within ±0.3% (58, p. 537, 59, 60).
The solution v»as diluted directly in the viscosimeter with a suitable
amount of solvent and mixed by stirring with a stream of nitrogen.
Measurements were taken with each dilution. A total of six concentrations
were measured. The viscosity of the pure solvent was also measured. The
intrinsic viscosity was found to be 0.42 dl/g in diethylenediamine copper II
ion.
The intrinsic viscosity was similarly determined in M aqueous sodium
chloride (0.48 dl/g) and in distilled water (0.84 dl/g).
9. Gel Permeation Chromatography - Determination of Molecular Weight
Distribution
A chromaflex column (Kontes Glass Co., Vineland, NJ) 400 x 1000 mm with
-------
a total bed volume (V ) of 1250.0 ml was used for the determination of the
molecular weight distribution of the xylan.
Dry sephadex H-75 (40-120 \i) v/as stirred into a 4- liter beaker with
distilled water and swollen for 30 hours. During this time the suspension
was decanted four times to remove the fine particles. The suspension was
well stirred and was transferred through a funnel into the vertically
mounted column which had previously been filled with water. After a layer
of a few centimeters had formed, the capillary outlet at the bottom of the
tube was opened to release a slow stream of water. When all of the gel had
settled, the eluant was allowed to flow through the bed overnight to complete
the stabilization of the column (40) . The void volume (V ) of 369 ml was
o
determined by use of Blue Dextran 2000 (V^ 2,000,000).
A part (500 mg) of the dialyzed xylan was dissolved in water in a 50.0
ml volumetric flask. After the xylan had completely dissolved the solution
was diluted to the 50 ml mark and mixed thoroughly. The xylan solution
(20.0 ml) was added dropwise to the top of the column with great care and
when it had disappeared below the surface a small quantity of water was
applied to wash the surface. Then more water was added to a height of 2-3
era to start the elution. The elution was carried out under a low hydrostatic
pressure at a flow rate of 0.5-0.7 ml/min. The concentration of the eluted
fractions (10,0 ml each) vas estimated by the phenol-sulfuric acid method
to be described later.
10. Acidity of the Xylan
Three samples (49.90 mg, 81,76 mg, 133.43 mg) of the xylan were each
dissolved in separate aliquots (10.0 ml each) of distilled water. An
aliquot (10.0 ml) of aqueous sodium hydroxide solution (0.0526 N) was added
to each of the three samples. Each solution was back titrated to a methyl
-------
-67-
red end point with hydrochloric acid solution (0.0406 N). Three blank
solutions were similarly titrated. The differences between the titers of
the blanks and the titers of the xylan samples in the order of the weights
given above were: 0.15 ml, 0.25 ml, 0.35 ml.
11. Analysis of Periodate Consumed
A portion (0.26959 g) of the dialyzed xylan was placed in a volumetric
flask (50.0 ml), dissolved in water (10.0 ml), after which, 0.5 M sodium
periodate solution (25.0 ml) was added and finally water was added to bring
the volume up to the 50.0 ml mark. The solution was mixed thoroughly. A
blank having the same periodate concentration was also prepared. Both
reaction mixtures were incubated in a constant temperature bath at 5° in
the dark (61). The time at which the periodate was added was taken as time
zero. Allquots (1.0 ml) of each reaction mixture were withdrawn at timed
intervals and added to 250.0 ml iodine flasks containing 1 N sulfuric acid
(5.0 ml) and 20.0% potassium iodide solution (5.0 ml). The liberated iodine
was titrated with 0.1 N sodium thiosulfate solution until only a pale yellow
color remained. Starch indicator (1.0 ml, 1% solution) was added and the
titration continued until the blue color disappeared.
12. Analysis of Formic Acid Released
When the consumption of periodate had stopped, aliquots (5.0 ml) of
each reaction mixture were added to Florence flasks (150.0 ml) containing
ethylene glycol (1.0 ml). The mixture was allowed to stand at room temperature
for 5 minutes with occasional swirling until the unreacted periodate was
consumed. The solution was titrated with 0.05 N sodium hydroxide solution
using methyl red as the indicator.
13. Complete hydrolysis of the Polyalcohol
The oxidized xylan from the periodate reaction was placed in a dialysis
-------
-68-
bag and dialyzed against running tap water for two days. At the end of
this period, it was taken out and concentrated under vacuum in a rotary
evaporator to about 100.0 ml. The concentrate was neutral to litmus
paper.
An excess of sodium borohydride (1.5 g) was added to the concentrate
and the reduction was allowed to proceed at room temperature for 24 hours
with occasional shaking. At the end of the reduction period the reaction
mixture was returned to the dialysis bag and dialyzed apainst running tap
water for 3 additional days (100).
The polyalcohol was hydrolyzed under mild acid hydrolysis conditions as
described previously. The hydrolyzate was spotted on Whatman No. 1 filter
paper and paper chrotnatographed. The sugars in another aliquot (25.0 ml) of
the hydrolyzate were converted to their alditol acetates and analyzed by
gas-liquid chromatography as described previously. The spectrum showed
xylitol pentaacetate and arebinitol pentaacetate and three small unidentified
peaks which came out early. The areas under the peaks were measured with a
planimeter.
14. Degradation of the Xylan in Sodium Hydroxide Solution
A part (4.34 g) of the dialyzed xyIan was placed in a volumetric flask
(25.0 ml), which had previously been evacuated and flushed with nitrogen,
and dissolved in sodium hydroxide solution (1.0 N). After the xylan was
completely dissolved the solution was diluted to the mark with sodium
hydroxide solution and shaken vigorously to attain complete mixing. The time
when the sodium hydroxide was added was recorded.
Class ampules (5.0 ml) were evacuated and filled with nitrogen In a
glove box. Aliquots (1.0 ml each) of the xylan solution were placed in the
ampules. The ampules were taken out of the glove box, sealed, and placed
-------
-69-
in boiling water (62, 63). The ampules were withdrawn from the constant
temperature bath at definite time intervals (30, 60, 90, 120, 180 min and
so on). Each withdrawn ampule was allowed to cool for 1 to 2 min and placed
In ice water to stop the reaction. Normal hydrochloric acid (1.0 ml) was
added to the contents of the ampule to neutralize the sodium hydroxide
reaction mixture.
The degradation was also carried out in sodium hydroxide solutions of
0.001 N, 0.01 N, 0.1 N, 4 N and 6 N concentration.
15. Phenol-Sulfuric Acid Method of Analysis of the Xylan
The analysis for the undegraded xylan was carried out by the phenol-
sulfur ic acid method of Smith and coworkers (64, 65). A Beckman Model DB
spectrophotometer was used for this investigation.
Reagent grade sulfuric acid (95.5%, specific gravity 1.84) conforming
to ACS specifications was used. Phenol (80% by weight) was prepared by
adding glass distilled water (20.0 g) to redistilled reagent grade phenol
(80.0 g). This mixture formed a water-white liquid which was readily
pipetted and has been known to stay the same color after one year of storage
(30). A pale yellow color developed after several weeks but this did not
interfere in the determination since a blank was included.
A fast delivery pipette (5.0 ml) was used to deliver the concentrated
sulfuric acid. The pipette was prepared by cutting a portion off the tip
of a standard 5.0 ml pipette. A high maximum temperature was desired because
it increased the sensitivity of the reagent.
Water (1.7 ml) and 80% phenol (0.05 ml) were added to an aliquot (0.3 ml)
of the neutral xylan solution in a test tube. Concentrated sulfuric acid
(5.0 ml) was added rapidly. The stream of acid was directed against the
liquid surface in order to obtain good mixing. Intense heat was generated
-------
-70-
at this time. The tubes ware allowed to stand for 10 minutes, then shaken.
end placed in a water bath at 25 to 30° for another 10 to 15 rain. The test
tubes were shaken again and samples were transferred to spectrophotometer
cells (1.0 cm light path) and readings taken at 480 nm. The color developed
was stable for several hours. The weight of hydrolyzed pentoses and uronic
acids was expressed as xylose equivalents by using percent transmission in
conjunction with a standard curve prepared from known xylose.
L. Characterization of Hemicellulose Ji; Isolation of a_ Glucomannan
1. Qualitative Carbohydrate Analysis by Paper Chromatography
Hemicellulose B (250.8 mg, dry weight) was dissolved in 4.5 g of 77%
sulfuric aicd for 1.0 hr at ice-bath temperature. The resulting translucent
solution was diluted to 3.9% sulfuric acid by the addition of 84.42 ml of
distilled water. The dilute solution was refluxed, neutralized, and paper
chromatographed as described above for holocellulose hydrolysis. Paper
chromatograms of the Hemicellulose B hydrolyzate showed large amounts of
glucose and mannosep minor amounts of galactose and xylose, and trace amounts
of arabinose.
M. Characterization of Kami eel lulose C_; Isolation of a Mannan
1. Qualitative Carbohydrate Analysis by Paper Chromatography
Hemicellulose C (273.3 mg, dry weight) was hydrolyzed, neutralized, and
paper chromatographed as described above for Hemicelluloses A and B. The
Hemicellulose C hydrolyzate contained large amounts of mannose, small amounts
of glucose and galactose, and trace amounts of arabinose and xylose.
2. Quantitative Carbohydrate Analysis by Gas-Liquid Chromatography
A portion of Hemicellulose C (150 mg, air-dry weight, moisture content
8.9%) was acid hydrolyzed, neutralized, reduced and acetylated as described
previously for the quantitative carbohydrate analysis of the holocellulose
-------
-71-
fraction by gas-liquid chromatography. An aliquot (20 mg) of the resulting
alditol acetate sirup was dissolved in methylene chloride and diluted to the
mark in a 1.0 ml volumetric flask. An aliquot (2 pi) of the solution was
injected into the gas chromatograph under the general conditions previously
established. The areas under the peaks of the resulting spectrum were
measured by a planimeter.
3. Methylation of the Mannan
A portion of Hemicellulose C (1.4 g, air-dry weight, moisture content
8.9%) was dissolved in 182 aqueous sodium hydroxide solution (50.0 ml) at
ice-bath temperature with stirring. The solution was methylated by the
simultaneous addition of 30% aqueous sodium hydroxide solution (100.0 ml)
and dimethyl sulfate (50.0 ml) over a period of 3 hr while maintaining the
solution at ice-bath temperature. Acetone (100.0 ml) was added to prevent
foaming. The mixture was stirred for an additional 45 hr at room temperature.
The solution was cooled to 0° in an ice-bath, neutralized with 10% sulfuric
acid, and dialyzed for 3 days against running tap water. The solution was
concentrated on a rotary evaporator, freeze-dried and the entire methylation
sequence was repeated (41).
The product was further methylated by dissolution in tetrahydrofuran
(150.0 ml) followed by treatment over a period of 60 hr with seven 10.0 g
portions of crushed sodium hydroxide, each followed by a portion (12.0 ml)
of dimethyl sulfate (42). The product was dialyzed against running tap water
for 3 days. The concentrated dialyzate was extracted with three 300.0-ml
portions of chloroform. The chloroform extract was concentrated on a rotary
evaporator, dissolved in acetone and filtered. The concentrated filtrate
(50.0 ml) was poured into petroleum ether (200.0 ml), and the white
precipitate which resulted was recovered and freeze-dried; yield of a white
-------
-72-
powder 40.7 mg. An infrared spectrum (potassium bromide pellet) of a portion
of Che methylated Hemicellulose C shoved no absorption in the regions of
3200 cm or 1750 cm indicating that there were no free hydroxyl groups in
the material. Therefore, methylation was considered complete.
4. Hydrolysis of the Methylated Mannan
The methylated mannan (40.7 mg) was hydrolyzed with 10.0 ml of 90% formic
acid at 97° for 3 hr. The formic acid was removed by evaporation under
reduced pressure followed by the addition and removal of water. The sirup
so obtained was hydrolyzed with 0.5 N sulfuric acid (5.0 ml, for 2.5 hr at
97°). Upon cooling, the solution was neutralized with barium hydroxide, and
the solids were removed by centrifugation. The remaining inorganic salts
were removed by refluxing the concentrated sirup with absolute methanol,
followed by filtration (repeated four times) and removal of solvent (41).
The sirup so obtained was dissolved in a few drops of water and subjected
to paper chromatography using 2-butanone saturated with water as the developer
(67). The chromatogram was allowed to run until the solvent front had moved
32 cm from the origin where the sample was applied (2.5 hr). The developed
chromatogram was allowed to air-dry and then sprayed with aniline phthalate
indicator (1.66 g of phthalic acid dissolved in 100.0 ml of water-saturated
n-butanol containing 0.93 g of freshly distilled aniline) (68, 69). The
papers were allowed to air-dry again for 15 minp heated in an oven at 105°
for 15 min and viewed under ultraviolet light. Three spots were evident
with the following R values: 0.89 (trace), 0.51 (very strong), 0.24 (trace).
(R.. value is the ratio of the distance the spot moved divided by the distance
the solvent front moved, both measured from the point of application of the
material).
A second aliquot of the hydrolyzate of the methylated mannan was subjected
-------
-73-
to paper chromatography using n-butanbl-ethanol-water (5:1:4 v/v/v) as
developer. The solvent front was allowed to migrate 40 cm past the origin
where the spot was applied (19 hr). The papers were sprayed with aniline
phthalate indicator as described above. Seven spots were evident which had
the following R values (R values determined by dividing the distance the
o o
sugars have moved from the starting line by the distance moved by 2, 3, 4,
6-tetra-O-methyl-D-glucopyranose): 0.96 (trace), 0.82 (very strong), 0.64
(trace), 0.52 (trace), 0.38 (trace), 0.28 (trace), 0.21 (trace).
5, Molecular Weight Determination of the Mannan by Viscosity Measurements
An amount (125 mg, dry weight) of the mannan (Hemicellulose C) was
weighed into a 25.0 ml volumetric flask which had been previously weighed
and swept free of air by a stream of nitrogen gas. Diethylenediamine copper
II reagent at 25° was added and the mixture shaken. However, not all of
the mannan dissolved. The mixture was transferred to a 100 ml volumetric
flask and diluted with additional diethylenedlamlne copper II reagent.
However, the mannan still did not completely dissolve.
N. Characterization of Residue C_\ Isolation of a_ Glucan
1. Hydrolysis with 77.0% Sulfuric Acid
A portion (200 mg, air-dry weight, moisture content 11.1%) of the residue
from the 15.0% aqueous sodium hydroxide extraction (Residue C) was hydrolyzed
with 77.0% sulfuric acid according to the procedure previously described.
Paper chromatography of the hydrolyzate showed the presence of a large
amount of glucose, medium amounts of mannose and galactose, and trace
amounts of xylose and arabinose.
2. Four Hour Formic Acid Extraction
A portion (1.0 g, air-dry weight) of Residue C remaining after the 15.0%
aqueous sodium hydroxide extraction was refluxed (97°) with 100.0 ml of 95%
-------
-74-
fonnic acid for k hr. The insoluble residue was recovered by centrifuge,
washed with water and freeze-dried; weight 0.18 g.
A portion (0.15 g) of the above residue was hydrolyzed with 77.0%
sulfuric acid and the hydrolyzate paper chroroatographed as previously
described. The chromatograras showed a large amount of glucose, a small
amount of mannose, and trace amounts of arabinose and xylose. Thus the
formic acid did not completely remove the non-glucose containing polysaccharides,
3. Four Day Formic Acid Extraction
A portion (10.0 g) of Residue C was extracted with 200.0 ml of 95%
formic acid for 4 days in a Soxhlet extractor using a porcelain extraction
thimble. An amount (25.0 ml) of additional formic acid was added each day.
At the end of 4 days the residue was recovered by washing with distilled
water, centrifuged, and freeze-dried; weight 3.6 g.
An amount (150 mg, air-dry weight, moisture content 11.1%) of the above
residue was hydrolyzed with 77.0% sulfuric acid and the hydrolyzate was
paper chromatographed as previously described. The paper chromatogram
showed glucose only.
4. Quantitative Carbohydrate Analysis by Gas-Liquid Chromatography
An amount (150 mg, air-dry weight, moisture content 11.1%) of the
residue from the formic acid extraction was hydrolyzed with 77.0% sulfuric
acid as previously described. The hydrolyzate was reduced with sodium
borohydride „ and the alditols were acetylated and analyzed by gas-liquid
Chromatography as previously described. The areas under the peaks of the
resulting spectra were measured with a planimeter.
5. Acetolysis of the Glucan
An amount (2.0 g air-dry weight, moisture content 11.1%) of the residue
from the formic acid extraction was kneaded into a mixture of acetic anhydride
-------
-75-
(8.0 ml) and concentrated sulfuric acid (0.2 ml), at ice-bath temperature
(3°), until completely wetted. The mixture was placed in an oven at 50°
for 14 days (70). The resulting dark brown sirup was mixed with acetic
acid (10.0 ml). The suspension was stirred with 500.0 ml of cold water
and an additional 250.0 ml of water was added. The mixture was stirred
for 30 min and filtered through a sintered-glass funnel. The residue was
washed free cf acid with water, air-dried, and extracted with boiling
ethanol (95%, 200.0 ml).
The aqueous filtrate from the above filtration was extracted with
chloroform (250.0, 250,0, 100.0 ml). The chloroform extract was washed
with 10.0 ml of a saturated aqueous solution of sodium carbonate and
concentrated on a rotary evaporator to yield a solid. However, the material
resisted crystallization.
Since no crystals resulted from the above acetolysis, a second acetolysis
was attempted simultaneously with the acetolysis of filter paper. The
acetolysis of filter paper (2.0 g) resulted in crystals of cellobiose
octaacetate (1.56 g), m.p. 228-228.5°, literature value 223-224°.
However, still no crystals resulted from the acetolysis of the residue
from the formic acid extraction. Therefore, the reaction was repeated
but the reaction time was shortened from two weeks to one week. Crystals
of cellobiose octaacetate (550 mg) were obtained. The material was
recrystallized three times from 95% ethanol; yield 275 mg, m.p. 224-225°,
23
[a] + 40° (c. 3.0, chloroform); unchanged on admixture with authentic
22
material, literature value 223-224°, [a] +40° (£3.0, chloroform).
6. Molecular Weight Determination of the Glucan by Viscosity Measurements
An amount (100 mg, dry weight) of the residue from the formic acid
extraction was placed into a 25.0 ml stoppered volumetric flask which had
-------
-76-
bsen previously weighed and swept free of air by a stream of nitrogen gas.
Dlethylensdiamine copper II reagent (cupric ethylenediatnine) (General
Cheaical Division, Allied Chemical, Columbia Road and Park Ave., Morristown,
NJ or Ecustic Paper Division,, Olin, P.O. Box 200, Pisgah Forest, NC) at
25° was addad and the solution was shaken until the glucan was completely
dissolved. The flask was filled to the mark with the solvent and mixed
thoroughly. The filled flask was carefully weighed to determine the density
of the solution in g/25 ml. An aliquot (10.0 ml) of the solution was
transferred to a Cannon-Ubbalhode dilution viscometer previously placed in
a water bath at 25±0.01° and flushed with nitrogen. After 5 min the solution
was drawn into the belt of the viscometer by applying pressure with nitrogen.
The pressure was released and the time required for the miniscus to pass
between the two calibration marks was measured to 0.1 seconds. Triplicate
measurements were made until duplicates agreed to within ±0.3% (58, p. 537,
59, 60).
The solution was diluted directly in the viscometer with a suitable
amount of solvent and mixed by stirring with a stream of nitrogen. Measurements
were taken with each dilution. A total of six concentrations were measured.
The viscosity of the pure solvent was also measured. The intrinsic viscosity
was found to be 0.432 dl/g in diethylenediamlne copper II ion.
7. Mathylation of the Glucan
A portion (1,4 g, air-dry weight, moisture content 11.1%) of the residue
from the formic acid extraction was methylated by the procedure previously
outlined for the methylation of Hetnicellulose C; yield 605 mg. An infrared
^spectrum (potassium bromide pellet) of a portion of the methylated material
showed no absorption in the regions of 3200 cm or 1750 cm indicating
that there were no free-hydroxyl groups. Therefore, methylation was
-------
-77-
considered complete.
8. Hydrolysis of the Methylated Glucpn
A.part of the methylated glucan (150 rag, dry weight) was dissolved in
2.25 p of 77.0% sulfuric acid at the ice-bath temperature for 1 hr and then
diluted with 42.2 ml of water to 3.9% sulfuric acid concentration. The
solution was refluxed for 6 hr. There remained considerable insoluble
materials in the solution which were removed by centrifugation. The
decantate was neutralized with saturated aqueous barium hydroxide solution
to pH 5.0 and the precipitate was removed by centrifugation. The resultant
hydrolyzate was condensed to a sirup on a rotary evaporator.
The sirup so obtained was dissolved in a few drops of water and subjected
to paper chromatography using 2-butanone saturated with water as the developer
(67). The sirup from the hydrolysis of the methylated tnannan was chromatographed
on the same sheet of paper. The chromatogram was allowed to run until the
solvent front had moved 32 cm from the origin where the sample was applied
(2.5 hr). The developed chromatogram was allowed to air-dry and then
sprayed with aniline phthalate indicator (1.66 g of phthalic acid dissolved
in 100.0 ml of water-saturated n-butanol containing 0.93 g of freshly
distilled aniline) (68, 69). The papers were allowed to air-dry again for
15 min, heated in an oven at 105° for 15 min and viewed under ultraviolet
light. Four spots were evident from the hydrolyzed glucan with the following
Rf values: 0.82 (trace), 0.56 (very strong), 0.21 (trace), 0.06 (trace).
A second aliquot of the hydrolyzate of the methylated glucan and of the
methylated tnannan were subjected to paper chromatography using ri-butanol-
ethanol-water (5:1:4 v/v/v) as developer. The solvent front was allowed to
migrate 40 cm past the origin where the spot was applied (19 hr). The papers
were sprayed with aniline phthalate indicator as described above. Five spots
-------
-78
were evident from the hydrolyzate of the methylated glucan with the following
R values (Rp values determined by dividing the distance the sugars have
moved from the starting line by the distance moved by 2, 3, A, 6-tetra-O-.
methyl-D-glucopyranose): 1.0 (tr.ace), 0.83 (very strong), 0.67 (trace),
0.41 (trace), and 0.28 (trace).
Ill. Results and Discussion
A sample of inner bark free from outer bark and cambium was desired
because it provided a relatively homogeneous starting material. Outer bark
contains considerable cork material which would interfere with experimental
studies of the carbohydrates. The cambium layer contains proteins as well
as carbohydrates and the two are best separated at the outset.
In the spring of the year from April through June, the outer bark of
Douglas-fir is easily separated from the inner bark by simply chipping it
away. The inner bark for the present work was taken from a standing tree
to reduce contamination from other sources. The cambium layer was then
carefully separated from the inner bark and a relatively homogeneous
sample resulted.
The inner bark after the ethanol-water extraction was carefully ground,
screened and reground to ensure that most of the fibers were separated and
that the surface areas were exposed. Some of the fine material was
included in the fraction to provide a large starting weight to ensure that
sufficient polysaccharides would be isolated for later studies. All of
the fines were not included because preliminary experiments showed that
they plugged the extraction apparatus and filter systems and made laboratory
procedures time-consuming and difficult.
It is desirable, before proceeding with polysaccharide separation, to
-------
-79-
rcmove ao much as possible of the low-molecular weight materials (simple
sugars, organic acids, liplds, waxes, and so on) present in the bark. Some
of these materials arc readily oxidized and would interfere with the
delipnlficatIon reaction.
Figure 5 outlines the scheme followed in the treatment of the inner
bark and the isolation of the different fractions. The yields for each
extraction are shown. None of the extraction procedures shown in this
chart accomplished a clear-cut separation of one type of material from
another, but In general each step in the sequence performed a definite
function.
The ethanol-water (4:1 v/v) extraction served two purposes. It
prevented possible enzyme action which might change the natural materials
from their native state, and it also solubilized the simple sugars which
could be identified to provide a more detailed investigation of the total
carbohydrate fraction in the bark. The ethanol-water (A:1 v/v) soluble
solids were shown to contain the free sugar, glucose, by paper chromatography.
The benzene-ethanol (2:1 v/v) azeotrope removed lipids and viaxes which
would Interfere with later reactions. The extract contained no free sugars
when Investigated by paper chromatography. Having de-fatted the bark it
was possible to carry out a hot water extraction and remove a water-soluble
fraction which amounted to 11.1% of the original bark sample.
Compounds isolated from natural sources often contain one or more of
the elements, nitrogen, sulfur, phosphorus or the halogens. Therefore, it
it> best to qualitatively determine if they arc present.
The sodium fusion test on the water-soluble fraction failed to detect
the presence of sulfur, phosphorus, and the halogens. Nitrogen was shown
to be present as evidenced by the formation of a precipitate of Prussian
-------
-80-
blue with ferrous sulfatc.
A portion of the sample was sent to an analytical laboratory (Pascher
and Pasoher, Mikroanalytisches Lahorntor iuri 53 Bonn, Buschstrasse 54, West
Germany) for analysis of nitrogen by the Kjeldahl method, and was found to
contain 3.632 nitrogen. A part of this nitrogen is in the form of proteins
as discussed later.
A ferric chloride-potassium ferrlcyanide test produced a dark-blue
color. This color (Turnbull's blue) was due to the formation of a 1:1
complex of ferric ion and pnenolic hydroxyl which give the ferrous reaction.
The iodine test, likewise resulted in a blue color characteristic of a
starch-iodine complex.
These strongly positive color tests showed the presence of considerable
amounts of tan.^ns and starch. Kiefer and Kurth (8) indicated the possibility
of phenolic materials in Douglas-fir inner bark and starch has been reported
in the hot-water-soluble fractions from several Inner barks (63). Therefore,
starch was expected to be present in Douglas-fir bark.
A fundamental aspect of polysaccharides is the component monosaccharides
which are linked together to form the polymer chain. .Jften polysaccharides
contain linkages which are resistant to acid cleavage. Thus, to ensure
complete hydrolysis, the hot-water-soluble solids were treated with strong
acid according to the ptocedure of Laver, Root, Shafizadeh and Lowe (22).
A portion of the hot-water-soluble solids would not dissolve in 72.0%
sulfurlc acid. After hydrolysis, the insoluble portion had a dark-red color
and presumably were phenolic acids. This precipitate was not investigated.
The hydrolyzatc was analyzed for monosaccharidcvs as discussed below.
If the polysaccharides under Investigation arc water soluble and if
hydrolysis can be accomplished under mild acid conditions, treatment with
-------
-81-
strong acids as discussed above is not desirable because of deprodative
side reactions. Since the hot-water-extracted solids were water soluble,
a mild ncid hydrolysis (3.0% sulfuric acid) was compared to the 72.0%
suit uric acid hydrolysis. The hydrolyzate was analyzed for amino acids
and tnonosaccharides as discussed belov.
As was the case in the strong acid hydrolysis, the insoluble portion
of the extract had a reddish appearanc . This precipitate was found to
persist later even after purification of the water-soluble solids.
Quantitative analysis showed that the hot-water-soluble solids
contained 3.63% nitrogen. Bark has been known to contain both protein and
non-protein nitrogen, as well as free atnino acids. At least 5 water-soluble
proteins have been detected. The cambium layer of Douglas-fir bark has
also been shown to contain protein (17), and possibly some cambium layer
may have remained with the inner bark during the sairple preparation.
The hydrolyzate from the mild acid hydrolysis (3.02 sulfuric acid) of
the hot.-water-soluble solids was examined for amino acids by two-dimensional
paper chromatography.
The chromatograns showed several purple spots but the spots were not
well resolved. Lai (62) found that 3.0Z sulfuric acid hydrolysis did not
appear to he strong enough to 'lydrolyze the proteins into their component
amino ac.'ds. The strong acid treatment (72.02 sulfuric acid) better
hydrolyzed the proteins into their component amino acids. The evidence in
the present woric shows that proteins were extracted by the hot-water treatment,
Paper chromatography of the original extract failed to detect free
mononaccharidos. Therefore, the carbohydrates present in the hot-water-
soluble fraction are considered to be in the form of polysaccharides.
The mixture of munosaccharide sugars resulting iron acid hydrolysis
-------
-82-
of the hot-water-soluble solids was well resolved by paper chromatography.
The chromatograms showed a very large amount of glucose, a moderate amount
of arabinose, a slight amount of galactose, trace amounts of xylose and
rhamnose and almost no mannose.
The hydrolyzates from both the strong acid hydrolysis and mild acid
hydrolysis contained what appeared to be identical monosaccharides by paper
chromatography. Therefore, strong acid hydrolysis was not necessary, and
to avoid degradation, the 3.0% sulfurii- acid method was used in all
subsequent hydrolyses.
The five major sugars, glucose, mannose, galactose, arabinose and
xylose are those ordinarily found in wood and wood pulp (20). Rhamnose in
Douglas-fir bark was first reported oy Lai (62) in the acidified acid
hydrolyzate of the sodium chlorite-soluble fraction. Its identification
in the acid hydrolyzate of the hot-water-soluble solids supports its
finding in the polysaccharides cf Douglas-fir inner hark.
Figure 6 outlines the scheme followed for the purification of the
polysaccharides extracted by hot water. When the freeze-dried sample was
redissolved in distilled water at room temperature, it was found that 29.8%
of the sample would not dissolve. The insoluble portion (Fraction A) gave
a strong positive reaction to the iodine test shoving the presence of
starch and a negative test to the ferric chloride-potassium fcrrfcyanidc
solution showing the absence of phenolics. Strong acid hydrolysis and
paper chromatography indicated the presence of glucose only. No traces
of other monosacchjrides could be detected. Microscopic examination of
this sample showed some fiber debris and irregularly shaped materials
which were presumably starch granules. Since only glucose was detected
and the most obvious sources of this munosaccharide were starch and
-------
-83-
cellulose, this fraction was not investigated further.
Ethanol was added to the water-soluble portion to provide a solution
of 70.0% In ethanol. A precipitate formed which amounted to 25.9% of the
original water-soluble solids. These precipitated solids were labeled
Fraction B (Figure 6). Those solids which remained soluble in the 70.0%
ethanol solution (44.3% of the original water-soluble solids) were labeled
Fraction C (Figure 6). Fraction C was found to react strongly with ferric
chloride-potassium ferricyanide indicating the presence of phenolics but
gave no reaction with iodine indicating the absence of starch. This
fraction was not studied further. Polysaccharides which may be in this
fraction would be expected to possess a very low molecular weight.
The fraction insoluble in the 70.0% ethanol-water mixture (Fraction B)
gave a positive reaction with iodine indicating the presence of starch. It
also gave a slightly positive reaction with ferric chloride-potassium
ferricyanide, thus indicating that .separation of the polysaccharides
from the phenolics was not complete.
Hydrolysis of Fraction B and two-dimensional paper chromatography of
the hydrolyzate showed the presence of amino acids. Six major spots,
seven light spots, five very light spots and two large smears showed on
the paper chromatogram. The identification of these amino acids was not
investigated further because the main objective of this study was the
carbohydrates. However, the paper chromatogram definitely showed the
presence of protein in the inner bark of Douglas-fir.
Paper chromatography of the monosaccharides showed the same relative
amounts of sugars as found in the whole hot-water-soluble fraction.
Fraction B (Figure 6) was treated with ot-amylase enzymes to remove
starch. At the end of the hydrolysis the reaction mixture was dialyzed
-------
-84-
(Figure 6). The dialyzate showed the presence of glucose only, both before
and after hydrolysis with acid. No other tnonosaccharides were detected.
The enzymea used in this Investigation were u-amylases and therefore
specific for the hydrolysis of the (l->4)-0-a-D-glucopyranosyl-type bond. The
fact that glucose was released proves the presence of an a-D-(l-»-4)-glucan,
undoubtedly the amylose portion of starch. This represents the first time
that starch has been shown by chemical analysis to be present in the inner
bark of Douglas-fir. The starch undoubtedly acts as a food reserve for
the living cells in the inner bark.
The non-dialyzables froin the enzyme hydrolysis showed a blue color
with iodine indicator. Thus the d-amylases did not hydrolyze all of the
starch. This was undoubtedly due to the presence of some amylopectin in
the starch molecule. Amylopectin possesses some cx-D-(l-*6)-glucan branch
chains which are not cleaved by a-amylases. The residue was, therefore,
expected to contain sone residual starch molecules.
The original hot-water-soluble solids were found to have a nitrogen
content of 3.63%. The protein content of natural products is generally
calculated as the product of the nitrogen percentage and the factor 6.25,
which is the usual factor used for conversion of nitrogen content to
protein content. In accordance with this calculation the hot-water-soluble
solids of the inner bark of Douglas-fir contained 21.8% protein. This
appeared to be a very high protein content, but when it was corrected to
the original bark weight (Figure 5) the value was 2.42% protein in the inner
bark of Douglas-fir. Values for bark proteins which have been reported
are 5.0% for the inner bark of birch and an average of 21.6% for the inner
bark of black locust (58, p. 625). Therefore, in comparison, Douglas-fir
bark appears quite low in protein content.
-------
-85-
The quantitative nitrogen content (Kjeldahl) of Fraction D (Figure 6)
was 1.57%. This proved to be a reversal in nitropen content. The original
water-soluble solids contairied 3.63% nitrogen as previously mentioned.
Kxtraction with water at room temperature and precipitation with 70% ethanol
provided Fraction B (Figure 6) which contained 0.28% nitrogen. Apparently
in the re-precipitation of the water-soluble solids, a large amount of the
protein remained in solution with the phenolic compounds. In an effort to
obtain purer polysaccharide samples, and in particular to remove the starch,
Fraction B was treated with starch hydrolyzing enzymes (HT-1000 and Diazyme
T.30) and with protein hydrolyzing enzymes (chymotrypsin and trypsin;
Figure 6). The nitrogen content of the final product (Fraction D) increased
co 1.57%. It would appear from this increase in nitrogen content that not
all of the enzymes were removed by dialysis. Therefore, the net result of
the treatments was to introduce protein impurities into the sample.
Fraction D was hydrolyzed with 3.0% sulfuric acid and paper chromato-
graphed. The paper chromatogram showed a large reduction in the amount
of glucose present, relative to the amount of the other monosaccharides.
f.lucose was still the major monosaccharide present, although the spots for
arabinose and galactose were now more prominent relative to the glucose
spot. A part of the glucose residues may have come from residual starch
that was not destroyed by the enzyme. It was thought, however, that
another treatment with the starch enzyme would not completely remove the
l
starch and that it might even prove detrimental to the other polysaccharides
that were present. Starch enzyme treatment was carried out at a pH of 3.8-
4.2 and at this slightly acidic condition, there was a distinct possibility
that acid hydrolysis might destroy the polysaccharides that were the object
of the purification process.
-------
-86-
Carbohydrates are neither heat resistant nor volatile and so derivatives
must be prepared which will volatilize without degradation in order to
perform gas-liquid chromatographic analysis. There are numerous derivatives
which have been tried but the ones most commonly used today are the "alditol
acetates." In the preparation of the alditol acetates the monosaccharides
are first treated with sodium borohydride to reduce the aldehyde function
to the alcohol function. This has the result of preventing ring isomerization
of the pyranose and furanose forms and so prevents the formation of alpha
and beta forms of the sugars. The end result is only one form, the alditol,
for each of the monosaccharide sugars, rather than three, four or even five
isomers. The acetates are synthesized from the alditols to make a volatile,
heat resistant derivative.
The gas chromatographic resolutions of the alditol acetates prepared
from Fractions B and D (Figure 6) were excellent. The areas under the peaks
of the different sugars were compared to each other to give relative weight
values. The areas under the peaks of the other sugars were compared to the
area of the derivative for galactose which was taken as 1.0. For Fraction
B (Figure 6) the areas under the curves were: galactitol hexaacetate, 1.0;
arabinitol pentaacetate, 1.4; and glucitol hexaacetate, 32.A. Only traces
of rhamnitol pentaacetate (0.1), xylitol pentaacetate and mannitol hexaacetate
were found. For Fraction D (Figure 6) the areas were galactitol hexaacetate,
1.0; arabinitol pentaacetate, 1.3; glucitol hexaacetate, 2.9; and traces of
rhamnitol pentaacetate, xylitol pentaacetate and mannitol hexaacetate.
These relative areas are in agreement with the relative area and intensity
of color developed by the spots on the paper chromatograms of both samples.
The starch enzyme treatment resulted in a decrease of glucitol
lu-xaacetate from 32.4 units to 2.9 units when compared with galactitol
-------
-87-
hexaacetate. The ratio (1.4:1.0) of arabinose to galactose In Fraction B
is the same as in Fraction D (1.3:1.0), allowing for errors in measurements.
The results show that the starch enzyme was effective in reducing the
amount of starch in the sample. The almost constant ratio of arabinose to
galactose in Fraction B and Fraction D confirms what the paper chromatograms
have already shown, that the starch enzymes acted'to hydrolyze the starch
only. All other polysaccharides were left intact by the starch enzyme
treatment. Therefore, it was concluded that the major carbohydrate component
of the hot-water-extract was starch.
The arabinose and galactose monosaccharides released by acid hydrolysis
undoubtedly exist in the inner bark as part of L-arabino- U-galactans which
are known to be present in the wood of conifers. These polysaccharides are
the only major wood glycans that can be isolated in good yield by extraction
of wood with water before delignification. Their ease of extraction and
their useful qualities as gums have brought them into commercial production
marketed as the commercial gum, Stractan.
Since the L-arabino-D-galactans are common to the wood in conifers,
it is considered possible that the arabinose and galactose containing
polysaccharides isolated in the present work are similar. This represents
the first time that possible L-arabino-D-galactan polysaccharides have been
reported In Douglas-fir bark. Because of the large volumes of Douglas-fir
bark which are waste products, it is possible that the bark could become
of interest as a raw-material source for these polymers since they have
aJready been marketed as commercial gums.
Not all of the pectic material of plants is extractable with water.
The insoluble part, protopectin (calcium pectate) was removed from the
water-insoluble residue of Douglas-fir inner bark by extraction with a
-------
-88-
0.5% ammonium oxalate solution (Figure 5). Presumably a cation exchange
occurred in which insoluble calcium oxalate was formed along with soluble
ammonium pectate. The latter was extracted with the filtrate.
Through all of the extractions the bark retained its characteristic
brown color. However, the strong oxidizing conditions of the acidified
sodium chlorite reaction bleached the reaction mixture to a pale yellow
color. Fumes of yellow gases, undoubtedly chlorine and chlorine dioxide,
were visible in the reaction vessel above the mixture. These gases
emphasize the necessity of performing the delignification reaction in a
fume hood and of bubbling nitrogen through the reaction to sweep away
these toxic materials. Fresh sodium chlorite was added to the reaction
at one hour intervals rather than all of it at the beginning to prevent
an excess of gas formation and to give the reaction time to proceed. The
residue from the delignification, termed "holocellulose" was a pale yellow
color.
The filtrate was yellowish in color and was dialyzed to remove
low-molecular weight impurities and the inorganic salts and ions which
resulted from the reaction of the sodium chlorite. The solution which
remained in the dialysis bag after several days was almost colorless and
the solids which were recovered by lyophilization were white.
The delignification reaction has been developed to solubilize the
non-carbohydrate components of plants. Thus the insoluble residue
(holocellulose) remaining is relatively pure carbohydrate material. However,
the reaction does dissolve some carbohydrates and it is these materials
that are part of the subject of this study.
The solubilized solids of the present investigation represented a
complex mixture of carbohydrates, lignin-like compounds, tannins, proteins,
-------
-89-
and so forth. Therefore, as described below, a variety o> approaches were
used to characterize the carbohydrates Ln the mixture.
The ash content was 13.39'].57%, as Hul.fati>. Although a r.onsidvrabl.e
amount of inorganic sodium chlorite had been added to the delignification
reaction, this was a high ash content considering that the fraction had
been dialyzed. Although it would be of interest to know what elements
were present in the ash, no effort was made to determine them in the present
work.
In an effort to lower the ash content and remove more of the low-
molecular weight materials, the acidified sodium chlorite soluble materials
were again dialyzed. More than 22.4% passed through the dialysis membrane.
Paper chromatography of the dialyzate showed a relatively large spot for
glucose, indicating that carbohydrate substances, probably monosaccharides
and short chain oligosaccharides, had passed through the dialysis membrane.
The solution retained in the dialysis bag was made to 70% ethanol, a
method often employed for the purification of polysaccharides (15, p. 449),
and resulted in the precipitation of a white, flocculent material.
However, the sulfated ash content of the precipitate was 11.88+2.83%
indicating that dialysis and precipitation did not lower the ash content
of the original material below the experimental error of the ash
determination. The inorganic materials were either chemically attached
to the polysaccharides, possibly as salts, or were physically wrapped in
the polysaccharides so that diffusion through the dialysis bag was
restricted.
Because of the fact that carbohydrates which might be important to
the present investigation passed through the dialysis bag and might be
lost, and that dialysis and precipitation did nothing to lower the ash
-------
-90-
content, It was concluded that simple purification was not feasible.
Therefore, all additional investigations were done on the original
material.
The original material showed a positive test for nitrogen. However,
quantitative analysis showed only 0.84% nitrogen, an amount not considered
to represent an interfering contaminant and no special efforts were made
to separate the nitrogenous material.
The tests for sulfur, phosphorus and the halogens were negative.
A fundamental aspect of polysaccharides is the component monosaccharides
which are linked together to form the polymer chain. Often polysaccharides
contain linkages which are resistant to acid cleavage. Thus, to ensure
complete hydrolysis the material was treated with strong acid according to
the procedure of Laver, Root Shafizadeh and Lowe (22). The hydrolyzate
was analyzed for amino acids and monosaccharides as described below.
If the polysaccharides under investigation are water soluble and if
hydrolysis can be accomplished under mild acid conditions, treatment with
strong acids as described above is not desirable because of degradative
side reactions. Since the material was water soluble, a mild acid hydrolysis
(3% sulfuric acid) was compared with the 77% sulfuric acid hydrolysis.
The hydrolyzate was analyzed for amino acids and monosaccharides as
described below.
Elemental analysis showed the presence of nitrogen. It could possibly
I
be a result of some residual ammonium oxalate from the ammonium oxalate
extraction or it could be part of protein material. The cambium layer of
Douglas-fir bark has been shown to contain protein and possibly some cambium
layer remained with the inner bark in sample preparation.
The hydrolyzates from the strong acid hydrolysis (77% sulfuric acid)
-------
-91-
and the tnlld acid hydrolysis (3% sulfuric acid) were examined for amino
acids by two-dimensional paper chromatography. It was found that the
second solvent, n^-butanol-formic acid-water (20:6:5 v/v/v) muBt be
prepared just prior to use otherwise gradual esterification occurred and
altered its chromatographic usefulness.
Chromatograms of -the hydrolyzate from the mild acid treatment did
not show amino acids. However, chromatograms of the hydrolyzate from the
strong acid treatment showed ten purple spots. Quantitative analysis showed
only 0.84% nitrogen content indicating that the amount of protein material
was not great. Therefore, no effort was made to identify the amino acids.
The mixture of monosaccharide sugars resulting from acid hydrolysis
was well resolved by paper chromatography. The chromatograms showed that
the hydrolyzates contained a large proportion of glucose, a moderate amount
of arabinose, slight amounts of mannose and galactose and trace amounts of
xylose and rhamnose.
The hydrolyzates from both the strong acid hydrolysis and mild acid
hydrolysis contained what appeared to be identical monosaccharides by
paper chromatography. Therefore, strong acid was not necessary and to
avoid degradation, the 3% sulfuric acid method was used in all subsequent
hydrolysis.
The five major sugars, glucose, mannose, galactose, arabinose and
xylose are those ordinarily found in wood and wood pulp. The trace amount
of rhamnose represents the first time it has been found in Douglas-fir bark
although it has been reported in other barks.
Since the monosaccharides resulting from the hydrolysis of bark are
the same as those found in wood, it is expected that the polysaccharides of
bark are similar to those in wood. If this is so then the major carbohydrate
-------
-92-
component is cellulose, but it also contains some hemicelluloses such as
xylans, glucomannans, glucoRalactomannans and so forth. The isolation of
the cellulose fraction and an evaluation of its properties and its degree
of polymerization will be of future interest.
The gas chromatographic resolution of the alditol acetates prepared
from the acid hydrolyzate was excellent. The myo-inositol peak at the end
of the spectrum resulted from the addition of an accurately measured
amount of myo-inositol as an internal standard. The areas under the peaks
of the other sugars in the spectrum are compared to the area under the
peak of myo-inositol for quantitative results.
The sample taken (moisture-free, ash-free basis) yielded the following
percentages of monosaccharides: glucose, 41.5%; arabinose 8.3%; galactose,
2.7%; mannose, 2.6%; xylose, 0.7%; rhamnose, 0.7%. This total of 56.5% is
less than 100% because the material contains organic substances other than
carbohydrates.
The acidified sodium chlorite delignification reaction is an oxidation
reaction. Therefore, it was anticipated that the primary hydroxyl groups
in position six of the hexose containing polysaccharides would most likely
be oxidized to uronic acids as demonstrated below. Therefore, several
qualitative tests were applied to determine if uronic acids existed. Since
uronic acids are known to be present in natural materials, all of the tests
were run in direct comparison with the residue from the ammonium oxalate
extraction, the material from which the fraction was prepared.
The infrared spectrum of the 0.5% ammonium oxalate insoluble material
showed a strong absorption at 1590 cm , corresponding to absorption by
carboxylate ion. Thus the starting material from which the fraction was
prepared contained uronic acids. The fraction isolated showed an absorption
-------
-93-
band at exactly the same place. However, the treatment with acidified
sodium chlorite, although an oxidation reaction, did not appear to increase
the uronic acid content, at least not as detected by infrared spectroscopy.
The carbazole-sulfurlc acid color reaction is said to be reasonably
specific for hexuronic acids. This was verified by testing monosaccharides
and oxalic acid and benzole acid. These compounds shoved no reaction,
indicating that the simple sugars and carboxylic acid functions other than
those in uronic acids did not interfere with the color reaction.
The 3% sulfuric acid hydrolyzate showed a positive purple color,
indicating the presence of hexuronic acids. The reactions showed a maximum
absorption in the ultraviolet region at 535±1 nm as described for the test.
The color was not particularly strong and it was considered possible
that since the sulfuric acid had been neutralized with barium hydroxide,
some of the uronic acids had formed barium salts and had precipitated
with the barium sulfate. Therefore, a part of the acid hydrolyzate was
neutralized with ion exchange resin but the results were the same.
The pale yellow color of the "holocellulose" from the first delignifi-
cation (Figure 5) reaction indicated incomplete delignification. The
holocellulose was therefore treated a second time with acidified sodium
chlorite solution. A white holocellulose was obtained in a yield of 30.6%
based on the original starting bark sample.
This was a considerable reduction in yield from the 44.3% isolated
after the first acidified sodium chlorite treatment. However, no effort
was made to bleach the material under milder conditions and it is believed
that the second oxidation could be performed under conditions which would
\
provide a white holocellulose with little loss of material.
Organic compounds isolated from natural sources often contain one or
-------
-94-
more of the elements nitrogen, phosphorus, sulfur or the halogens. Therefore,
it is best to qualitatively determine if they are present because purification
techniques are usually required when they are found. However, the tests for
these elements in the holocellulose fraction were negative. Therefore,
plant materials, such as proteins, which contain these elements were not
present in the hdlocellulose fraction.
The separation of the carbohydrate materials from other plant
substances, particularly lignin, is difficult and results in a choice
between two possibilities. Most of the polymeric phenols can be removed
by strong oxidation reactions but in such a reaction the degradation of
the desired carb'ohydrates is extensive. However, when mild oxidative
conditions are used, as in the present work, considerable polyphenolic
substances are not separated from the carbohydrate (holocellulose) fraction.
It is thus necessary to analyze for these polymeric phenols in order
to have a complete knowledge of the holocellulbse fraction. The usual
analysis is the "Klason lignin" determination which involves the hydrolysis
of the carbohydrates with strong (72%) sulfuric acid and weighing the
insoluble solids which remain as "Klason lignin." The holocellulose
isolated from the inner bark of Douglas-fir was found to contain 3.1%
Klason lignin. This is not a particularly high amount of this type of
material and so the holocellulose fraction seemed to be quite free of
highly polymeric lignin-like molecules.
The digestion of a holocellulose material with 72% sulfuric acid
(Klason lignin determination) results in the solubilization of some lignin-
like substances which are thus not measured as "Klason lignin." These
materials are referred to by the term, "acid-soluble lignin" and they must
be determined to obtain a complete analysis of a holocellulose fraction (45).
-------
-95-
These materials are best analyzed by ultraviolet absorption. Lignin-
like compounds strongly absorb energy at 280 nm and this band has been
used for the quantitative determination of acid-soluble lignins. However,
the formation of 5-(hydroxylmethyl)-furfural from hexoses and furfural
from pentoses formed during the refluxing step in the Klason lignin
determination was found to interfere with this determination. Browning
and Bublitz (44) showed that interference by these compounds could be
minimized by determining the absorbance of the filtrate at two wavelengths.
Using absorptivity values for lignin and for carbohydrate degradation
products obtained, respectively, from spruce Brauns lignin and from a
synthetic mixture of glucose, xylose, mannose, and glucuronolactone which
had been subjected to the hydrolysis conditions of the lignin determination,
Browning and Bublitz (44) were able to write the following equations:
A280 = °'68 S + 18 CL
0.15CD+70CL
where A_on and A c were the absorbance values of the lignin filtrate, 0.68
/oU
and 0.15 the absorptivities of carbohydrate degradation products, 18 and 70
the absorptivities of lignin at 280 and 215 nm, respectively, and C and C
D Li
the concentrations in grams/liter of carbohydrate degradation products and
of soluble lignin in the filtrate. Goldschmid (45) showed that by solving
the simultaneous equations, the following expression for the soluble lignin
concentration in the filtrate can be obtained:
4 5T A - A
t.jj A215 280
L 300
In the present work the acid-soluble lignin content of the holocellulose
Isolated from Douglas-fir inner bark was determined by scanning the filtrate
-------
-96-
from the Kalson lignin determination over the wavelength range from 320 to
200 run. Absorption maxima were observed at 280 nra and 210 run. The 280 nm
peak was the usual one observed for lignin compounds and the 210 nra peak was
slightly shifted from the 215 run peak reported by Browning and Bublitz (44) .
However, a shifting of wavelength due to different materials and different
analytical techniques are not uncommon (45).
The absorbances at 280 nm and 210 nm were 2.62 and 3.77 respectively.
These values were used to calculate the acid-soluble lignin as follows:
r *'53 A215 " A280 .. 4.53 x 3.77 - 2.62
C
L 300 300
Using this figure in conjunction with the filtrate volume (1544.0 ml)
and the dry weight of the sample (1.8264 g) the value of 4.07% acid-soluble
lignin in the holocellulose was calculated. Thus the total lignin content
was 3.1% (acid-insoluble) plus 4.1% (acid-soluble) to equal 7.2%.
The amount of acid-soluble lignin thus exceeded the amount of acid-
insoluble lignin as is generally the case with holocellulose material (39).
Therefore, correcting the lignin content for soluble lignin was essential.
Although the absorptivity values for lignin and carbohydrate degradation
products may be somewhat uncertain, as stressed by Browning and Bublitz
(44), this error is small compared with that resulting from not correcting
for soluble lignin at all.
An important step in determining the structures of polysaccharide
materials is to acid hydrolyze them to their monosaccharide components.
For water-insoluble polysaccharides two steps are usually required. The
first is to dissolve the material in a strong acid usually sulfuric, and
second is to dilute the acid with water followed by reflux to hydrolyze
-------
-97-
the glycosidic bonds. It is important to perform the hydrolysis with
complete dissolution of polysaccharides and with minimum decomposition of
sugars.
The holocellulose fraction from Douglas-fir inner bark was found to
completely dissolve In 77.0% sulfuric acid, the concentration used to
dissolve cellulose (22) . It was necessary to add water to the solution
slowly with strong stirring to prevent local heating which might have
caused degradation of the sugars. After dilution to 3.9% sulfuric acid
concentration, the solution was refluxed to bring about hydrolysis.
The acid solution was neutralized to pH 5.0 with aqueous barium
hydroxide, resulting in a heavy precipitate of barium sulfate. This method
of neutralization was preferred because the pH could be controlled easily.
A final pH of about 5.0 was desired because monosaccharide solutions should
not be allowed to become alkaline. The action of alkali on monosaccharides
follows three general courses (22): isomerization, fragmentation, and
internal oxidation and reduction. Such reactions interfere with the
qualitative and quantitative results of monosaccharide analyses and consider-
able care was taken to avoid them.
The identification of the monosaccharides released from polysaccharides
on acid hydrolysis is fundamental to an understanding of these polymers.
Paper chromatography has become the standard way of tentatively identifying
the monosaccharides. Several solvent systems are well established and
operation of the individual sugars can usually be achieved with relative
ease.
Paper chromatograms of the hydrolyzate from the holocellulose fraction
of Douglas-fir inner bark showed the presence of glucose (very strong spot),
mannose (medium spot), xylose (medium spot), galactose (trace spot), and
-------
-98-
arabinose (trace spot) . These sugars moved the same distance as authentic
sugar samples on the same chromatogram.
A trace spot was also evident near the origin. This spot was attributed
to the presence of glucuronic acid.
Qualitative paper chromatography provided an indication of the
monosaccharides present in the holocellulose hydrolyzate. However, it is
possible for two or more sugars to migrate the same distance and so paper
chromatography did not unambiguously identify the monosaccharides. The
chromatography did not show whether the sugars were of the D or the L
configurations and hence did not completely designate the sugars. Therefore,
it was considered necessary to isolate crystalline derivatives of the sugars
to unambiguously prove their presence and their configurations.
To prepare crystalline derivatives it was necessary to first separate
the mixture of monosaccharides into their individual, isolated sirups.
Since large amounts of each sirup were not required, the isolation was
accomplished by preparative paper chromatography as described above. The
only limitation to the procedure was the time required to develop a
sufficent number of paper chromatograms to obtain enough milligrams of
sirup. Each isolated sirup was reacted with reagents which provided
derivatives which crystallized readily. In every case derivatives of
authentic monosaccharides were also crystallized for comparison purposes.
Although the diethyl dithioacetal acetate derivatives of many
monosaccharides have been reported in the literature (46) not all of them
crystallize easily. In the present work only the derivatives from authentic
D-galactose and L-arabinose crystallized easily although diethyl-dithioacetal
acetate sirups of D-glucose, D-xylose and D-mannose were also prepared.
Because only a few milligrams of each monosaccharide sirup was available
-------
-99-
from the holocellulose hydrolyzate, only the sirups from the easily
crystallized galactose and arabinose fractions were reacted to prepare
diethyl dithioacetal acetate materials. In this way crystalline penta-0-
acetyl-D-galactose diethyl dithioacetal and crystalline tetra-0-acetyl-L-
arabinose diethyl dithioacetal were obtained. These materials proved the
presence of D-galactose and L-arabinose residues in the polysaccharides
from Douglas-fir inner bark.
The acetylation of glucose is a common reaction and in the present work
white crystals of penta-0-acetyl-B-D-glucopyranose were readily prepared
from authentic D-glucose. The glucose sirup isolated from the holocellulose
hydrolyzate also yielded penta-()-acetyl-8-D-glucopyranose upon acetylation.
This derivative proved the presence of D-glucose residues in the polysaccharide
from Douglas-fir inner bark.
The acetates and the diethyl dithioacetal acetate derivatives of xylose
do not crystallize easily. However, D-xylose can be easily identified as
its di-0-benzylidene dimethyl acetal and in the present work di-O-benzylidene-
D-xylose dimethyl acetal was readily obtained from authentic D-xylose. The
xylose sirup isolated from the holocellulose hydrolyzate also yielded
di-0-benzylidene-D-xylose dimethyl acetal upon reaction. This derivative
proved the presence of D-xylose residues in the polysaccharides from
Douglas-fir inner bark.
Mannose is a difficult sugar to crystallize and there are also few
derivatives of mannose that crystallize easily. However, D-mannose
phenylhydrazone crystallizes readily and can be used to identify and to
isolate the sugar. The reaction must be carefully done, or a second molecule
of phenylhydrazine will add to the sugar and the phenylosazone will result.
In fact, this is what occurs with the other monosaccharides. Phenylosazones
-------
cannot be used to identify sugars because both Cl and C2 are involved and
sugars which differ only about Cl and C2S such as mannose, glucose, and
fructose, cannot be differentiated. Thus to identify mannose, the reaction
must be stopped after the addition of only one mole of phenylhydrazine to
give mannose phenylhydrazone. Isbell and Frush (49) have reported the
reaction conditions to perform this step.
In the present work, D-mannose phenylhydrazone was readily prepared
from authentic D-mannose. Because D-mannose phenylhydrazone crystallizes
so readily from the reaction system outlined by Isbell and Frush (49), it
can be preferentially crystallized from mixtures of monosaccharides. Thus,
in the present work, a complete hydrolyzate sirup (77.0% sulfuric acid
hydrolysis) of the holocellulose from Douglas-fir bark was reacted with
phenylhydrazine. After standing overnight in the refrigerator, white
crystals of D-mannose phenylhydrazone were obtained. This derivative
proved the presence of D-mannose residues in the polysaccharides from
Douglas-fir bark.
There are numerous modifications of the standard Fehling's copper
reduction test for the presence of reducing sugars. However, one of the
more common methods for the quantitative analysis of reducing sugars is
the Soraogyi (52) procedure. This method is based on the ability of certain
sugars (reducing sugars) to act as reducing agents. The sugar reacts with
Cu in the aqueous alkaline medium to produce cuprous oxide. The cuprous
j_i_
oxide ie oxidized by iodine back to Cu and the excess iodine is titrated
i
with thiosulfate. The reactions are:
RCHO -»- 2 Cu"*"* -»- 5 OH~ -»• RCO~ + Cu-0 + 3 H~0
I0~ + 5 I~ + 6 H* •* 3 I2 + 3 H 0
Cu20 + 2 H"*" + I2 -» 2 Cu""" +21" + H20
-------
-101-
I2 + 2 S203 + 21 + S406
This method has been widely and successfully applied on both milligram
;md microgram quantities, both titrimetrically and colorimetrically. Its
accuracy over a wide range of sugar concentrations, the ease and rapidity
of operation, and its proven reliability place it above other micro-
oxidation methods (52) .
As evidenced from the above equations, thiosulfate anion is used as the
standard tlter to measure the excess iodine concentration. In the present
work the sodium thiosulfate solution was standardized by titration to a
starch end-point of an accurately diluted solution of potassium iodate
which had been prepared from recrystallized and dried potassium iodate
solids.
Several different alkaline copper reagents have been recommended for
use in conjunction with the iodometrlc determination of reduced copper.
The most common ones are Somogyi's 1945 (52) reagent and his 1952 reagent.
Somogyi's 1945 phosphate-buffered reagent possesses an advantage over the
1952 carbonate-buffered reagent, in that amyloses are held in solution in
the 1945 reagent but are precipitated from the 1952 reagent. Therefore,
the 1945 reagent has become the standard procedure and was the method of
choice in the present work. The amount of potassium iodate added to the
reagent determines the amount of iodine released and hence the amount of
sodium thiosulfate titer needed. Therefore, some preliminary titrations
are often required to determine the best' amount of potassium Iodate to add.
The blank should require between 9.0-10.0 ml of 0.005 M sodium thiosulfate
solution. When performed properly the method is very good for determining
the overall reducing power of a solution and the precision is ±0.01 mg for
D-glucose or about ±2%, averaged throughout the range 0.3 to 3.0 mg of glucose.
-------
-102-
When determining the quantitative amounts of monosaccharides released
from a holocellulose fraction on acid hydrolysis, it is important that the
time of refluxing be carefully determined. If the refluxing time is too
short, complete hydrolysis is not realized and the quantitative analysis
will be erroneous because those monosaccharide residues linked by the more
acid-resistant glycosidic bonds will not be determined. If the refluxing
time is too long, acid degradation of the hydrolyzed monosaccharides will
be extensive. Acid reversion to new, complicated polymers may also occur
on extended heating. The importance of the correct reflux time to achieve
maximum monosaccharide yield has been demonstrated by Laver, Root, Shafizadeh
and Lowe (22) in their studies of pulp analyses.
The simpliest way to measure the proper time to reflux the acid solution
is to measure the reducing power of the hydrolysis solution as the reaction
progresses. In the present work this was accomplished by removing aliquots
of the refluxing solution at specific times and determining the reducing «
power of the aliquots by the Somogyi method. Glucose was used as a
convenient standard reducing sugar in which terms the concentration of
monosaccharides was measured. A plot of the reducing power in glucose
equivalents versus the time of reflux showed clearly that 12 hours is the
optimum time to reflux the hydrolysis solution to achieve maximum yield
of monosaccharides.
The reflux time of 12 hours was considerably longer than the 4.5 hours
of reflux time which Laver9 Root, Shafizadeh and Lowe (22) showed to be
necessary for wood pulps. Therefore, the holocellulose fraction from
Douglas-fir inner bark contained glycosidic bonds which were more acid
resistant than those in wood pulps. These acid resistant bonds were
attributed to the mannose containing polysaccharides in the holocellulose.
-------
-103-
Carbohydrates are neither heat resistant nor volatile and so derivatives
must be prepared which will volatilize without degradation in order to
perform gas-liquid chromatographic analyses.
The derivatives most commonly used today are the "alditol acetates."
In the preparation of the alditol acetates the monosaccharides are first
treated with sodium borohydride to reduce the aldehyde function to the
alcohol function. This has the result of preventing ring isomerization to
the pyranose and furanose forms and so prevents the formation of alpha and
beta forms of the sugars. The end result is only one form, the alditol,
for each of the monosaccharide sugars rather than three, four or even five
forms. The acetate esters are synthesized from the alditols to make volatile,
heat resistant derivatives.
Identification of compounds by gas-liquid chromatography is realized
by comparing the time required for the unknown material to pass through the
column with the time required for a sample of the authentic material to pass
through the column. These times are called "retention times." Qualitative
paper chromatography of the hydrolyzate of the Douglas-fir holocellulose
had shown the presence of glucose, mannose, galactose, xylose, and arabinose.
Therefore, authentic crystalline alditol acetates of each of these sugars
were synthesized to positively determine retention times and instrument
response for quantitative analyses. myo-Inositol hexaacetate was also
synthesized to be used as an internal standard. Each of these crystalline
materials was passed through the gas chromatdgraph and the retention time
for each was determined. • The crystalline materials were then mixed and
again passed through the gas chromatograph to ascertain the resolution.
The separation of each alditol acetate was excellent and the retention
times of each was measured at the center of the peak.
-------
-104-
There are nsany parameters which can be varied in gas-liquid chromato-
graphic techniques. The resolution of the peaks will be changed by changes
in the carrier gas flow and changes in the column temperature. Peak heights
and peak areas will also change with changes in injection port and detector
temperatures. Therefore, in the present study several conditions were
systematically changed until the optimum conditions were determined. The
conditions used throughout the rest of the work were: column, 6.5% ECNSS-M
on Gas Chrom Q 100/120 mesh, 6 ft x 1/8 in O.D. stainless steel; injection
port 180°; detector 240°; column temperature 180° isothermal; helium flow
2
30 ml/min; range setting 10 ; attenuation setting 16.
Gas chromatographic detectors respond differently to different compounds,
These response factors must be known to obtain quantitative results. The
recorder is also a possible source of error and the precision obtainable
with standards should be determined. A good way to reduce these sources of
error is to add an accurately weighed amount of an internal standard to the
mixture to be analyzed and compare the peak areas of the compounds to be
measured against the peak area of the internal standard. The internal
standard used in the present work was myjv-inositol hexaacetate.
The response of the gas chromatography to each of the alditol acetates
to be measured was calibrated by analyzing varying, but accurately weighed,
amounts of each crystalline, authentic, alditol acetate with authentic,
crystalline myo-inositol hexaacetate. The calibration factor for each
alditol acetate was obtained by plotting' the ratio of the area of each
alditol acetate to the area of the standard (myo-inositol hexaacetate) as
ordinate against the ratio of the weights of each as the abscissa. Straight
lines passing through the origin resulted for arabinitol pentaacetate,
xylitol pentaacetate, mannitol hexaacetate, galactitol hexaacetate, and
-------
-105-
glucitol hexaacetate. The slope of each of these lines is the "Instrument
K Factor" and represents the overall response of the gas chromatographic
system to each of the alditol acetates calibrated against the internal
standard, myo-inositol hexaacetate. The "Instrument K. Factors" were:
arabinitol pentaacetate 0.96; xylitol pentaacetate 0.95; mannitol hexaacetate
0.96; galactitol hexaacetate 0.98; glucitol hexaacetate 0.92, These
"Instrument K Factors" were used in subsequent calculations.
A sample of the holocellulose from Douglas-fir inner bark was carefully
hydrolyzed to minimize acid degradation. The hydrolyzate so obtained was
reduced by the addition of sodium borohydride and the excess sodium boro-
hydride was decomposed by acetic acid. The solution was concentrated to
a sirup. The sirup was dissolved in methanol and the methanol was
reevaporated. This procedure was repeated a total of eight times.
Albersheim, Nevins, English and Karr (32) showed that the boric acid level
could be reduced to a convenient level by a number of additions and
reevaporations of methanol. The boric acid was presumably converted to
volatile methyl borate which was evaporated from the sirup. The removal
of boric acid is important otherwise the acetylation reaction does not
proceed. Acetylation and the gas-liquid chromatographic analysis was
accomplished as outlined above.
The areas under the peaks were measured with a planimeter and were
found to be (duplicate analyses): arabinitol pentaacetate, 13.2; xylitol
pentaacetate, 39.7; mannitol hexaacetate', 62.0; galactitol hexaacetate,
15.A; glucitol hexaacetate, 386.0; myo-inositol hexaacetate, 232.2.
The following equation was used to determine the individual anhydro-
monosaccharide residue in the holocellulose fraction (31):
C x I x F x 100
% anhydromonosaccharide residue
R x S x H x K
-------
-106-
where
C = chromatographic area of the1 component alditol acetate peak,
R = chromatographic area of the myo-inositol hexaacetate peak,
I = weight of the myo-inositol originally added, in grams,
S = dry weight of the original holocellulose sample, in grams,
F = factor to convert weight monosaccharide to anhydromonosaccharide
residue (0.88 pentose)(0.90 hexose),
H = hydrolysis survival factor (90),
K = "Instrument K Factor".
Using the area under the peak of glucitol hexaacetate as an example the
calculations become:
,. , , , . , 386.0 x 100 mg x 0.90 x 100 ,, ,
L anhydroglucose residue = 232.2 x 273.96 mg x 0.974 x 0.92 = 6l«
In a similar way the percentages of anhydromonosaccharide residues in
the Douglas-fir inner bark holocellulose were calculated (average of two
determinations): anhydroarabinose residues9 2.6; anhydroxylose residues,
6.3; anhydromannose residues, 9.5; anhydrogalactose residues, 2.3;
anhydroglucose residues, 61.1. The total yield of anhydromonosaccharides
was 81.8%. This value is comparable to the values of 7A.9%, 78.0% and
81.9% reported by Borchard and Piper (31) for the total carbohydrate
content of chlorite holopulps and appears to be favorable for materials of
this type.
The value of 81.8% carbohydrates plus the value of 7.2% lignin amounts
for 89.0% of the solids in the holocellulose fraction. This total value is
similar to the values usually reported for the solids content of materials
of this type.
The study of the structure of bark hemicelluloses requires homogeneous
-------
-107-
polytners isolated in high yields with minimal modification. This is usually
accomplished, as in the present work, by delignifying the bark and extracting
the resulting holocellulose with aqueous alkali. Solubility differences
can be exploited for the separation of individual hemicelluloses by judicious
use of cations and concentrations. Thus, relatively dilute alkalis suffice
to dissolve xylans and galactoglucomannans, but higher concentrations are
required for the extraction of glucommannans.
The procedure used in the present work to separate the polysaccharides
which comprise the holocellulose from Douglas-fir inner bark is outlined in
Figure 7. The key feature was selective blocking of the dissolution of
mannose-containing polysaccharides in the first extraction step. This was
accomplished by impregnating the holocellulose with aqeuous barium hydroxide
(55). The impregnated holocellulose was then contacted with 10.0% aqueous
potassium hydroxide which was known to be a good solvent for xylan-rich
polysaccharides. The extract was neutralized and methanol was added to
70.0% concentration. The resulting precipitate was labelled "Hemicellulose
A." Hemicellulose A was recovered in 7.6% yield based on the starting
holocellulose (Figure 7).
Residue A (Figure 7) remaining from the 10.0% potassium hydroxide
extraction was expected to contain most of the mannose-containing hemicelluloses
because of the impregnation with barium hydroxide (55). After removal of
barium ions by dialysis, the residue was extracted with 1.0% aqueous sodium
hydroxide which has been shown to be a gbod solvent for galactoglucomannan
hemicelluloses (55).
The extract was neutralized and methanol was added to 70.0% concentration.
The resulting precipitate was labelled "Hemicellulose B." Hemicellulose B
was recovered in 1.7% yield based on the starting holocellulose (Figure 7).
-------
-108-
The concentration of the sodium hydroxide extraction medium was
increased to 15.0% sodium hydroxide because it has been shown (55) that
polysaccharides rich in mannose residues become soluble in concentrated
alkali solutions. The extract was neutralized and methanol was added to
70.0% concentration. The resulting precipitate was labelled "Hemicellulose
C." Hemicellulose C was recovered in 2,9% yield based on the original
holocellulose (Figure 7).
Residue C (Figure 7) was hydrolyzed by acid. Paper chromatography
showed considerable amounts of mannose and galactose and traces of xylose
and arabinose as well as glucose in the hydrolyzate. It was thought possible
that increasing the sodium hydroxide concentration from 15.0% to 18.0%
would extract more of the mannose- and galactose-containing polysaccharides
and leave a relatively homogeneous glucan as a residue. However, the
residue from the 18.0% sodium hydroxide still appeared to contain the
same ratios of sugars. Therefore, further investigations were concentrated
on the extract and residue from the 15.0% sodium hydroxide extraction
(Figure 7) rather than those from the 18.0% sodium hydroxide extraction.
The minimum hydrolysis time for Hemicellulose A in acid to achieve
the maximum yield of monosaccharide was determined to be 4 hours. A plot
of glucose equivalents (%) versus reflux time in hours indicated complete
hydrolysis after 4 hours and in most cases additional aliquots of the
material were refluxed for 4.5 to 6 hours.
The maximum amount of glucose equivalents released was found to be
59.3%. However, the fraction contained 29.7% ash determined as the oxides.
i I
The major inorganic ion was considered to be Ba because of the previous
impregnation with barium hydroxide. By calculation the percentage of
barium ions in the fraction would be 26.6%. Therefore, the total solids
-------
-109-
accounted for in the fraction was 85.9%. Acid degradation of xylose
containing polysaccharides is known to be extensive. In fact, the
conversion of pentoses to furfural under conditions of acid reflux is the
basis of the TAPPI standard method for the determination of pentosans in
wood and pulp. The relatively low total recovery of materials in the
Hemicellulose A fraction was, therefore, attributed to acid degradation.
A paper chromatogram of the acid hydrolyzate showed a very strong
spot for xylose, weak spots for glucose, galactose, and arabinose and a
trace spot for mannose. There was a slow moving spot which barely migrated
from the origin. This spot was attributed to glucuronic acid.
The trace amount of mannose indicated that the impregnation of the
holocellulose with barium hydroxide worked well in blocking the dissolution
of raannan-containing hemicelluloses. These results are in agreement with
those previously reported (55) for the separation of materials of this type.
The Hemicellulose A fraction was shown to be a "xylan" composed of
the following ratio of sugar residues: xylose, 4.5; arabinose, 1.0;
glucuronic acid, 1.0. The xylan possessed a specific rotation of -30.5.
The intrinsic viscosity of the polysaccharide in molar deithylenediamine
copper II reagent at 25° was 0.42 dl/g which corresponded to a degree of
polymerization of 89. The molecular weight of the xylan as analyzed by
4
end-group analysis was 1.8 x 10 which, in combination with gas-liquid
chromatographic analysis, showed a degree of polymerization of 90.
The xylan was oxidized with periodate anion. It consumed 133.5 moles
of periodate anion and released 22.5 moles of formic acid per mole of xylan.
The oxidized xylan was hydrolyzed and analyzed by gas-liquid chromatography
and showed the following ratio of sugar residues: xylose, 7.0; arabinose
1.0.
-------
-110-
The above data were consistent with a polysaccharide xylan structure
consisting of a backbone of 88 anhydro-D-xylopyranose units hooked to
8-jO-(l-*4) plus a reducing and a non-reducing end group on the backbone.
There were 20 anhydroglucuronic acid side chains and 20 anhydroarabinose
side chains on these 90 units. At least ten of the anhydroarabinose units
were in the form of monoarabinose side chains and up to ten were in the
form of arabinoblose or longer side chains.
The xylan was reacted at 100° with aqueous sodium hydroxide solutions
ranging in concentration from 0.001 N to 3.676 N. The rate of degradation
did not change at alkali concentrations above 0.1 N in sodium hydroxide.
Alkaline degradation of the xylan in 0.1 N aqueous sodium hydroxide
at 100° showed a rate constant for end-group peeling of k.. = k~ = 5.33
-1 -1
hour and a termination rate constant of k, =» 0.66 hour
The peeling reaction stopped when 19.81% of the xylan had been
degraded. This small amount of degradation reflected the relatively high
termination rate constant compared to the peeling rate constant.
Hemicellulose B was hydrolyzed with sulfuric acid and the hydrolyzate
qualitatively analyzed by paper chromatography. The chromatogram showed
strong spots for glucose and tnannose, weak spots for galactose and xylose,
and a trace spot for arabinose.
The fraction still contained some xylose residues indicating that the
extraction of the xylose-containing hemicelluloses was not complete by the
extraction with 10.0% potassium hydroxide'. Beelik, Concac Hamilton and
Partlow (55) also reported xylose residues in similar fractions isolated
from western hemlock holocellulosa and grand fir holocellulose. Hemicellulose
B was considered to be a galactoglucomannan,
Hemicellulose C was hydeolyzed, neutralized, and analyzed by paper
-------
-111-
chromatography by the methods described for the holocelluloae fraction.
The chromatogram showed that the Hemicellulose C fraction was composed of
a large amount of mannose residues, small amounts of glucose and galactose
residues, and trace amounts of arabinose and xylose residues.
The results indicate that all of the xylan hemicelluloses had been
extracted by the 10.0% potassium hydroxide and the 1.0% sodium hydroxide
treatments. This is in agreement with the results reported by Beelik,
Conca, Hamilton and Partlow (55) for hemicelluloses from softwoods.
The gas-liquid chromatographic spectrum of the alditol acetates
prepared from the acid hydrolyzate of Hemicellulose C showed that the major
peak present was from mannitol hexaacetate. The areas under the peaks of
rhamnitol pentaacetate, arabinitol pentaacetate, and xylitol pentaacetate
were too small to be measured. The areas under the remaining peaks as
measured by a disc integrator were (average of triplicate reading):
mannitol hexaacetate, 3407; galactitol hexaacetate, 315; glucitol hexaacetate,
240; myo-inositol hexaacetate, 11617. The percentages of anhydromonosaccharide
residues were calculated similarily to those for the holocellulose fraction
from the formula:
«,.., u-ij jj C x I x F x 100
% anhydromonosaccharide residue =• — ~ —
K x b x n x K.
The results were: anhydromannose residue, 85.7%; anhydrogalactose
residue, 7.0%; anhydroglucose residue, 4.6%; total recovery, 97.3%.
The ratio of anhydrosugars in Hemicellulose C was therefore: anhydro-
glucose, 1.0; anhydrogalactose, 1.5; anhydromannose, 18.6. Therefore, the
great majority of Hemicellulose C is composed of mannose-containing
polysaccharides and is therefore termed a "mannan."
23
The specific rotation of the mannan was [a] - 40° (c_ 0.25, N sodium
hydroxide) . This is close to the rotations of mannans from other plant
-------
-112-
sources and supports the above conclusion that this fraction from Douglas-fir
inner bark is primarily a mannan.
Methylation of a polysaccharide provides information about the positions
of the glycosidic bonds which unite the anhydromonosaccharide residues in
the polymer. The usual procedure is to form methyl ethers on all of the
free hydroxyl groups. These ethers are resistant to the acid hydrolysis
conditions ordinarily applied and so hydrolysis of the completely methylated
polysaccharide frees only the hydroxyl groups involved in the glycosidic
linkages. Location of the positions of the freed hydroxyl groups determines
the point of attachment of the glycosidic bond in the original polymer.
It is important that all of the hydroxyl groups in the original
polysaccharide be methylated. The methylation reaction is usually analyzed
by infrared spectroscopy because the hydroxyl groups absorb readily and
their disappearance as methylation progresses can be followed.
In the present work with the mannan from Douglas-fir inner bark,
raethylation proved difficult. However, after extended reaction time the
infrared spectrum showed no absorption for hydroxyl groups in the usual
regions of 3800 ^ 3300 cm . Therefore, methylation was considered complete.
Formic acid has been shown to be a good medium for the hydrolysis of
mannans and was used in the present work- The initial formic acid treatment
must be followed by an hydrolysis with dilute sulfuric acid to hydrolyze
any formate esters which might have been formed.
Paper chromatography of the hydrolyfeate sirup in two different solvent
systems showed R, and R values consistent with the presence of 2,3,6-tri-
r o
0-methyl-D-mannopyranose (very strong spot), 293p4,6-tetra-0-methyl-p-
mannopyranose (trace spot) and 2.3-di-O-methyl-D-mannopyranose (trace spot).
There were several unidentified spots which are possibly partially methylated
-------
-113-
sugars resulting from an incomplete methylation of the mannan as is
evidenced by the presence of 2,3-di-0_-inethyl-D-mannopyrano8e. They were
present in very trace amounts and could only be detected when the paper
chromatogram was heavily loaded with hydrolyzate sirup. On the other hand,
the spot for 2,3,6-tri-O-methyl-D-mannopyranose was very strong.
The crystallization of the above derivative of 2,3,6-tri-O-methyl-p-
raannopyranose shows that the hydroxyl group on C4 of the mannopyranose
compound was freed on hydrolysis of the methylated mannan. The isolation
of the crystalline derivative supports the paper chromatographic evidence
for the presence of 2,3,6-tri-0_-methyl-D-mannopyranose in the hydrolyzate
of the methylated mannan. Moreover the paper chromatograms show it to be
not only the major spot but essentially the only spot.
Therefore, it is concluded the Hemicellulose C is primarily a mannose
containing polysaccharide and that the anhydroraannose residues are attached
by 3-D_-(l+4)-glycosidic bonds. The B configuration is taken from the negative
optical rotation (-40°).
A paper chromatogram of the acid hydrolyzate of Residue C showed a
strong spot for glucose, medium spots for mannose and galactose, and trace
spots for arabinose and xylose. Residue C therefore appeared to be a
mixture of the hemicelluloses now known to be present in the holocellulose
from Douglas-fir inner bark. While the extractions shown in Figure 7 tended
to dissolve relatively pure hemicelluloses, the final residue was still a
mixture.
Formic acid has been shown to completely remove hemicelluloses from an
anhydroglucose rich material and leave a residue containing only anhydroglucose
residues (70). The degradation of the residue in this procedure can be
quite extensive and relatively short treatment of 4-hour duration was first
-------
-114-
attempted. However, a paper chromatogram of the hydrolyzate of the residue
remaining after the 4-hour extraction showed a weak spot for mannose, and a
trace spot for xylose and arabinose as well as the strong spot for glucose.
Therefore, removal of the hemicelluloses was not complete.
Longer extraction time in the refluxing formic acid was attempted.
The yield of residue was 36.0% and so degradation was quite extensive.
However, a paper chromatogram of the acid hydrolyzate of the residue showed
the presence of glucose only with no contamination from other sugars.
Therefore, the other hemicellulose linkages had been hydrolyzed by the
i
formic acid in preference to those joining the anhydroglucose residues.
Therefore, a glucan was left as the residue.
Since the material under investigation was a glucan, the Somogyi copper
reduction method with glucose as the standard sugar would give a reliable
quantitative analysis of the amount of glucan in the sample. The amount
of reflux time required for total hydrolysis was also determined by removing
aliquots as the reaction progressed and determining the reducing power.
The hydrolysis was complete after 4 hours of reflux and the overall
yield in glucose equivalents was 103.3%. Values of greater than 100%
recovery occur sometimes in this type of procedure due to the accumulative
sources of error in moisture content determinations, dilution measurements
and the error of the Somogyi method. The large yield does show that the
residue remaining after extracting Residue C (Figure 7) for 4 days with formic
acid was a glucan. ,
The. gas-liquid chromatographic spectrum of tha alditol acetate prepared
from the acid hydrolyzate of the glucan showed trace peaks for rhamnitol
pentaacetate, arabinitol pentaacetate, and xylitol pentaacetate. The areas
of these peaks were too small to be measured. Primarily the spectrum shows
-------
-115-
peaks for glucitol hexaacetate and the added myo-inositol hexaacetate. The
areas under these peaks, as measured by a planitneter were (average of three
readings): glucitol hexaacetate, 41; myo-inositol hexaacetate, 165. The
percentage of anhydroglucose residues was calculated similarly to those for
the holocellulose fraction from the formula:
v u j -i a C x I x F x 100
6 anhydroglucose residue = R x s x H x K
The results showed the percent of anhydroglucose residues to be 94.4%.
A definitive method of establishing linkages in a polysaccharide is
by fragmentation analysis. If known dimers can be identified, then the
linkage Joining the two anhydromonosaccharide units is established. In
the present investigation of the glucan, cellobiose octaacetate was isolated
in crystalline form from the glucan. Thus the presence of B-D.-(l-»-4)
glycosidic bonds in the glucan was established. It is interesting to note,
however that the time of acetolysis had to be shortened from two weeks,
which is the usual reaction time for cellulose (70) to one week. No
cellobiose octaacetate was obtained if the acetolysis of the glucan from
Douglas-fir bark was allowed to react for two weeks. This would indicate
that the chain length was short, because if the acetolysis proceeds too
far on a glucan then glucose pentaacetate is produced rather than the dimer.
Thus the acetolysis showed the presence of (3-I>-(l"*^) glucosidic bonds, but
indications were of a short chain.
The calculations of the viscosity data were made as follows:
n - Kpt
nr = n/no
nsp = (T1-V/no = nr " l
H . = reduced viscosity
-------
-116-
where
n = viscosity in centipoise (cp)
n a viscosity in pur® solvent • .
o
H o relative viscosity
[n] a Intrinsic viscosity in deciliters/gran (dl/g)
K ° viscotneter COBStent
p ° density at 25<,0°±0»Q1
t o time in seconds for solution to flow through the viscometer
c a concentration in g/dl
The plot of the reduced viscosities against the concentration showed a
straight line ma determined by linear regression analysis. The value for
the intrinsic viscosity wa© obtained by extrapolating the plots to zero
concentration. This was
-------
-117-
prior to hydrolysis. The Infrared spectra of the methylated glucan showed
the disappearance of the hydroxyl absorption in the 3800 - 3300 cm range
indicating that all of the hydroxyl groups had been methylated.
The methylated glucan was hydrolyzed with sulfuric acid in the same
way that the glucan had been hydrolyzed. Paper chromatography in two
different solvent systems showed R_ and R_ values consistent with the
t u
presence of 2,3,4,6-tetra-O-methyl-D-glucopyranose (trace spot), and
2,3,6-tri-O-methyl-D-glucopyranose (very strong spot). The unidentified
spots are possibly partially methylated sugars resulting from slightly
incomplete methylation of the glucan. These materials were present in only
trace amounts and could be detected only when the chromatograms were heavily
loaded with hydrolyzate sirup. On the other hand the spot for 2,3,6-tri-O-
methyl-D-glucopyranose was very pronounced.
The isolation of crystalline cellobiose octoaacetate from acetolysis
of the glucan proved the presence of B-^-Cl^A) linkages and hence established
the presence of 2,3,6-tri-O-methyl-D-glucopyranose in the hydrolyzate from
the methylated glucan. Thus the identification of 2,3,4,6-tetra-O-methyl-
D-glucopyranose by comparing relative spot movements on paper chromatograms
was placed on firm ground. This material came from the non-reducing end
group of the glucan.
The above data on the glucan is consistent with the presence of a
linear polysaccharide of 65 repeating anhydroglucopyranose units attached
by 8-1)-(1+4) glucosidic bonds plus a reducing and non-reducing end group.
-------
-118-
DOUGLAS-FIR BARK WAX
I. Historical Review
Kurth and Kiefer (71) found that the chemical components of the whole
hark could be partially separated by extraction with various solvents. Table
6 indicates the nature and yields of the Douglas-fir extractives which they
removed by successive extraction with the solvents shown.
Kurth, in a separate publication (72)p reported on the chemical
composition of the "waxes" In Douglas-fir bark. The n-hexane-soluble wax was
saponified by refluxing a mixture of 20 g of the wax, 20 g of potassium
hydroxide and 300 ml of 70% ethanol for three hours. Following this, 100 ml
of water was added, the alcohol was removed by evaporation, and the residue
extracted with n-hexane in a separatory funnel to remove unsaponifiable
compounds. The unsaponifi&ble matter was dried and recrystallized from
acetone. A white crystalline solid was obtained which was identified as
lif.noceryl alcohol, also named tetracosanol-1, a straight-chained, saturated
alcohol, C24H5QOH. The yield was 19.5% of the "wax." The filtrate from
the lignoceryl alcohol crystallization gave a positive Liebermann-Burchard
test for sterols (73, p. 80; 74, p. 100) and a precipitate with digitonin.
After removal of the acetone, the residue was crystallized as white needles
from dilute alcohol. The material was identified as "phytosterols." The
yield was 0.3% of the "wax." "Phytosterols" are now known to be mixtures
of closely related sterols of which the most common one in bark is 8-sitosterol.
The alkaline solution from the separation of the unsaponifiable matter
was acidified with sulfuric acid and extracted with n-hexane in a separatory
funnel. The t^-hexane solution was washed with water, dried over anhydrous
sodium sulfate, and evaporated to dryness. The residue was recrystallized
from acetone and then from ii-hexane. The white crystals were identified as
-------
-119-
lignoceric acid, also named tetracosanoic acid, a straight-chained, saturated
acid, C2,H,gO . The yield was 60.5% of the "wax." The small amount of
yellow solid residue left in the filtrate from this crystallization was
oxidized with cold potassium permanganate to dihydroxystearic acid; m.p.
129-130°. This indicated the presence of oleic acid in the original wax.
A brown resin remained suspended in the aqueous liquor after the
f
n-hexane extraction of the lignoceric acid. The resin was extracted with
diethyl ether. Evaporation of the ether left a brown residue which
crystallized from benzene as fine crystals. Recrystallization from dilute
ethanol gave yellow prisms. These crystals were identified as ferulic acid
(4-hydroxy-3-methoxycinnamic acid). The yield was 22% of the original wax.
The fraction of the bark which was n_-hexane insoluble but benzene soluble
(benzene-soluble "wax") was reported by Kurth and co-workers (75, 76) to
possess a more complicated composition than the n-hexane-soluble "wax."
It had a melting point of 60° to 63°, and Kurth tentatively isolated, after
saponification, a fatty acid mixture in about 25% yield, a dark-colored
phlobaphene in 24% yield, a dark colored diethyl ether-soluble acid fraction
in 26% yield, unsaponifiable matter in 5% yield and glycerol.
The cork fraction of Douglas-fir bark was studied by Hergert and Kurth
(76). The extractives were separated according to their solubilities in
n-hexane, benzene, diethyl ether, ethanol and hot water. The rv-hexane and
benzene fractions were "waxes." The yield of n_-hexane-soluble "wax" was
5.78% and of benzene-soluble "wax" was 1.75% based on an average of nine
trees sampled. The n-hexane-soluble "wax" was found to contain lignoceric
acid, 49.3%; lignoceryl alcohol, 27.5%; ferulic acid, 9.8%; "phytosterol",
0.6%; and n-hexane-insoluble, benzene-soluble acidic material, 8.1%; a
benzene-insoluble phenolic material, 3.6%. The n-hexane-insoluhle but
-------
-120-
benzene-soluble "wax" proved to have a complicated composition. The authors
did not locate the position of the hydroxyl group in the hydroxypalmitic
acid, C..,H_20 , but reported that it was not terminal.
It is interesting to note that dihydroquercetin was the extractive
present in greatest amount from the cork. Although not extracted with
n-hexane or benzene, and not considered a part of the "wax-portion," Douglas-
fir bark represents a major source of this compound. It is easily obtained
from the "wax"-free (benzene extracted) bark or cork by extraction with
diethyl ether. Upon evaporation of the diethyl ether it can be recrystallized
from hot water as long white needles, m.p. 241-248 (71). The quantity of
dihydroquercetin in cork varies from 5% in samples of cork from second-growth
trees to 23% in cork from mature trees (76) . This indicates the desirability
of separating the cork from the other bark components when the extraction of
dihydroquercetin is intended.
In 1967 Kurth reported more recent work on the n-hexane-insoluble,
benzene-soluble "waxes" of Douglas-fir bark and cork (75). In addition to
the compounds previously mentioned, he found an hydroxybehenic acid c?oH,,0~,
and an hydroxyarachidic acid (hydroxyeicosanoic acid), C Jfl,-.0-.
The hydroxybehenic acid was crystalline but gave a range of melting points
depending upon how it was.recrystallized. Kurth comments that this is
characteristic of those hydroxy-fatty acids that form lactones and lactides.
He did not report the position of the hydroxyl group. Likewise, he did not
report the exact position of the hydroxyl' group in the hydroxyarachidic acid.
He also mentions the presence of a dicarboxylic acid of tentative formula
C20H38°4 ^ut ^ic* not reP°rt further characterization.
The chemical composition of the bast fibers of Douglas-fir bark were
also studied by Kiefer and Kurth (77). Table 6 shows the yields of extractives
-------
-121-
resulting from successive extractions with the solvents shown. Thef$ indicated
•that the bast fibers did not contain as much extractive material, particularly
dihydroquercetin and "wax," as did the cork fraction of Douglas-fir^bark.
Further separation of the extractives into their individual components was
not reported.
II. Experimental: a,-Hexane-Soluble Wax
A. Collection of_ Bark Samples
Bark samples used in this study were collected from indigenous sources
of Douglas-fir. They consisted of bark from trees of various apes and
diameters. The specimens were stripped from the trees, sealed in polyethylene
bap,s, and brought to the Forest Research Laboratory, Oregon State University.
B. Sample Preparation and ja.-Hexane Extraction
Bark chips were ground to pass a screen of ten meshes to the inch (The
W. S. Tyler Company, Cleveland, Ohio). Since it was not necessary to dry
the bark before extraction (57) , the ground bark was packed into a Soxhlet
thimble, placed in a Soxhlet extractor and extracted for 48 hours with £-hexane.
The solvent was evaporated under aspirator vacuum on a rotary evaporator
(Buchi, Rotavapor, Switzerland). The residual "wax-like" solid was transferred
to a sample jar with a minimum of n-hexane solvent. The excess solvent was
evaporated by passage of a stream of nitrogen. The ultraviolet absorption
spectrum of the extracted solids was measured in a chloroform-n-hexane (3:1
v/v) solution.
C. Separation of the n-Hexane-Soluble Components
1. Separation by Column Chromatography
Column chromatrography was used as a first step in separating the
n-hexane-soluble materials. A typical separation is described belov?. Silica
Oel G was packed uniformly into a glass column two feet in length and one
-------
-122-
inch in diameter. A portion (2.00 g) of the r^-hexane-soluble fraction was
dissolved in chloroform (15.0 ml) and added to the top of the Silica Gel G
column. Chloroform-n-hexane (3:1 v/v) was used as the developing solvent.
A series of bands were located by use of ultraviolet light.
The fastest moving two bands (blue and light blue bands) were investigated
by H. Aft (personal communication). The present investigation was concerned
with the next two fastest moving bands, the light bluish-green band and the
bright-blue band (designated in combination as the "yellow band"). To
collect these bands the fastest moving zones were first washed from the
column and stored. Becuase of the difficulty of separating the next two
hands, they were washed from the column and collected as a mixture. The
mixture was added to the top of a second Silica Gel G column and developed
with the same solvent system. Then the bright-blue band (fastest moving)
approached the sand in the bottom of the column, fractions of effluent (5.0
ml) were collected. These fractions were tested by thin-layer chromatography.
The fractions were tested in two different solvent systems: diethyl ether-
n-hexane (1:4 v/v) and chloroform-carbon tetrachloride (6:1 v/v). The
chromatograms showed several spots for each fraction, indicating that the
column chromatographic separation was incomplete.
However, as a source of starting meterial for further separations, the
"yellow band" was collected from several Silica Gel G columns.
The Liebermann-Burchard test (59, p. 70; 60, p. 100) was employed to
determine if the material contained unsaturated sterols. A sample of the
"yellow band" from the column chromatographic separation was dissolved in
chloroform. An aliquot (1.0 ml) of the solution was added to a test tube
and dried. Acetic anhydride (2.0 ml) was added followed by the dropwise
addition of concentrated sulfuric acid. A dark green color change indicated
-------
-123-
a positive test.
2. Separation by Thin-Layer Chromatography
The "yellow band" from the column chromatographic separation was
subjected to thin-layer chromatography in several solvent systems in an
effort, to more completely resolve the nixture.
Solutions of the samples to be chromatographed were applied to the
thin-layer plates with capillary tubes in such a way that the surfaces of
Che plates were disturbed as little as possible. The chromatography tanks
used for irrigating the thin-layer plates were saturated with vapors from
the developing solvent at least two hours before the plates were placed in
the tanks.
Aliquots of the "yellow band" in chloroform-n-hexane (3:1 v/v) solution
were added to the thin-layer plates and developed with the following solvent
systems (all hexane solvents were n_-hexane) :
1. Chloroform-carbon tetrachloride (6:1 v/v)
2. Chloroform-carbon tetrachloride (1:1 v/v)
3. Chloroform-carbon tetrachloride (1:4 v/v)
4. Chloroform-hexane (4:1 v/v)
'.' .. 'I
5. Ben^ene-hexane (2:1 v/v)
6. Diethyl ether-hexane (3:1 v/v)
7. Diethyl ether-hexane (1:4 v/v)
8. ;Chloroform-benzene (6:1 v/v)
9. Chloroform-benzene (1:1 v/v)
10. Chloroform-benzene (1:4 v/v)
11. Benzene-methanol-acetic acid (45:8:4 v/v/v)
12. Hexane-diethyl ether-acetic acid (70:30:1 v/v/v)
13. Hexane-diethyl ether-acetic acid (85:15:1 v/v/v)
-------
-124-
14. Chloroform-ethyl acetate-formic acid (5:4:1 v/v/v)
Some thin-layer plates were developed two dimmensionally in the following
solvent systems:
ii-hexane-dlethyl ether-acetic acid (70:30:1 v/v/v) in one direction
followed by chloroform in the second direction; diethyl ether-n_-hexane (1:4
v/v) in one direction followed by chloroform-carbon tetrachloride (6:1 v/v)
in the second direction.
After development, the thin-layer plates were dried at room temperature.
Those plates which were impregnated with silver nitrate were dried in the
dark. The following methods were used to examine the developed plates.
1. Ultraviolet light
2. Ultraviolet light with the plate in ammonia vapors
3. Sprayed with 30% aqueous sulfuric odd, heated for 5 to 10 min
at 105° in an oven and finally viewed under ultraviolet light.
4. Exposure to iodine vapors.
5. Sprayed with bromothymol blue solution (50 mj* bromothymol .blue,
1.25 g boric acid, 8 ml 1 N sodium hydroxide and 112 ml water, or
40 mg bromothymol blue and 100 ml 0.01 N sodium hydroxide)
6. Sprayed with £-nitroaniline solution ((a)£-nitroaniline, 0.3% in
8% (v/v) HC1 (25 ml) and NaN02 (5% w/v)(1.5 ml) mixed immediately
before spraying, (b) Na CO. (20% v/v)). The two solutions were
used successively.
Authentic B-sitosterol (Aldrich Chemical Co., Inc., Milwaukee, V.'isconsin)
was chromatographed by one-dimensional thin-layer chromatography simultaneously
with the "yellow band" in each of the 14 solvent systems listed above. The
spots were located by exposure to iodine vapor. The separation by every
solvent systea showed a compound in the "yellow band" which migrated the
-------
-125-
saire distance as the authentic B-sitosterol.
3. Separation by Gas-Liquid Chromatography
Gas-liquid chromatography was used to test the purity of the samples
separated by column chromatography and thin-layer chromatography and to
collect pure samples. A Hewlett-Packard 5751B Research Chromatograph
(Hewlett-Packard Company, Palo Alto, California) were used. Both instruments
were equipped with flame ionization detectors and used helium as the carrier
gas. Stainless steel columns (1/8 in O.D.) were filled with the packing
material of choice. A glass wool plug was inserted in the end of the
column and the material was packed to a uniform tightness by vibrating the
column with a small vibrating tool. Four different column packings were
used in an attempt to find a column packing which would give good resolution.
Details of the four columns are listed in Table 7.
The columns were put on the instrument for conditioning for 12 to 18
hr at a temperature of 10° above the maximum temperature used for resolution.
Various temperatures, flow rates, sensitivities and chart speeds were tested.
Both temperature programming and isothermal methods were used.
Most of the samples were directly injected into the gas chromatograph
in iv-hexane solution. However, because some of the compounds were thought
to contain hydroxyl groups the trimethylsilyl ethers of several of the
fractions were prepared in an attempt to improve resolution. These
trimethylsilyl ethers were made by reacting dry samples for 5 to 10 min at
room temperature with hexamethyldisilazane and trimethylchlorosilane in
pyridine (2:1:10 v/v/v). The pyridine solution was injected directly into
the gas chromatograph.
The "yellow band" from column chromatography was investigated. Four
different columns were used (Table 7). The experimental conditions are
-------
-126-
shown in Table 8.
Two bands from the thin-layer chromatographic separation [developer,
diethyl ether-n_-hexane (1:4 v/v)] of the "yellow band" were tested for
purity by gas-liquid chromatography. The two bands were readily observed
on the thin-layer plates by ultraviolet light. The faster moving band
showed a yellow-blue color and the slower moving band showed a bright-blue
color. These two bands were separately scraped from the thin-layer plates,
and the organic compounds extracted from the solid Silica Gel G with
r\-hexane. The compounds were separated by gas-liquid chromatography on
the following column packings: SE-32; US W-98; OV-17 (Table 10). Both
silylated and non-sllylated samples were injected under the general
conditions outlined in Table 8.
An authentic sample of 6-sitosterol was subjected to gas-liquid
chromatography using the columns and the conditions shown in Table 8. The
retention times were compared with those for the peaks in the unknown.
A "peak enhancement" study was conducted by adding a small quantity
of authentic 6-sitosterol to the n-hexane solution of the "yellow band"
and injecting the solution into the gas chromatograph under the conditions
outlined in Table 8.
A known sample of stigmasterol (Aldrich Chemical Co., Milwaukee, Wisconsin)
was chromatographed on SE-52 and OV-17 columns under the conditions outlined
in Table 8.
A. Identification by Gas-Liquid Chromatography Combined With Rapid-Scan
Mass Spectrometry
Gas-liquid chromatography in combination with mass spectrometry was
used to separate and identify a range of compounds present in the n-hexane-
soluble fraction of Douglas-fir bark. The following operating conditions
-------
-127-
were used;
Gas-Liquid Chroma t ography
Instrument
Detector
Detector temperature
Injection sample
Injection port temperature
Column
Column temperature
Carrier gas flow rate
Mass Spectrometry
Filament current
Electron voltage
Analyzer pressure
Multiplier voltage
Scanning speed
F & M 810 (Hewlett-Packard)
Hydrogen flame ionization
248°
4 yl of the sllylated mixture
270°
UC W-98
Isothermal at 265°
30 ml/min of helium
70 eV source, 40 MA
20 eV and 70 eV
1 x 10~6 mm Hg
3.00 KV
6.5 seconds from m/e_ 24 to 500
The mass spectrometer used was an Atlas CH-4, Nier-type (nine inch, 60
degree sector) single-focusing instrument. The trimethylsilyl ethers of the
fractions of interest were separated by gas-liquid chromatography and a
portion of the effluent was passed directly into the dual ion source of the
mass spectrometer. The gas-liquid chromatograph was fitted with a 5:1 (EC-1
valve:flame) splitter. The mass spectrometer was equipped with an EC-1
throttle valve which was adjusted to permit approximately 10% of the remaining
column effluent to enter the ionization chamber, the remaining effluent being
vented into the air through a heated tube. The effluent was further split
in the ion source with 50% going to the 70 eV source. The 20 eV source
operated at less than the ionization potential of the carrier gas (helium),
-------
-128-
but above that of organic compounds, and was therefore used as a continuous
total ionization readout without any contribution from ionized helium. The
70 eV source provided the ionization used to obtain the mass spectra. The
spectra were recorded on a Honeywell 1508 oscillograph.
111. Experimental: Benzene-Soluble Wax (78, 79)
A. Bark Collection and Solvent Extraction
Whole bark was stripped from the bottom end of a freshly-felled, 58
year old Douglas-fir at Black Rock, Oregon. The bark was air-dried (moisture
content 9.3%), ground to pass a 1/2-inch screen, and a portion (768.2 g dry
cut) was packed into a stainless-steel mesh basket and extracted in a large
Soxhlet extractor with n-hexane (8.0 liters) for 52 hr. The extract was
filtered while hot through glass wool and the ii-hexane evaporated on a
rotary evaporator at 30° or less leaving a cream-colored, "wax-like" solid;
yield, 30.0 g. This n-hexane "wax" was stored for future investigations.
The bark residue from the n-hexane extraction was air-dried, re-packed
into the stainless-steel mesh basket and extracted with benzene (8.0 liters)
in the Soxhlet extractor for 52 hr. The extract was filtered, and
concentrated as above. The final traces of benzene were removed by freeze-
drying, leaving a light-brown, "wax-like" solid; yield 20.0 g.
B. Saponification and Separation of Acids and Neutrals
An aliquot (10.0 g) of the benzene "wax" was saponified by gently
refluxing for 3.75 hr in 10% NaOH (250.0 ml). The mixture was cooled in
an ice bath and acidified with glacial acetic acid resulting in a
precipitate. The organic compounds were recovered in two ways. In the
first procedure the mixture was extracted with benzene without first
collecting the precipitate. In the second procedure the precipitate was
recovered on a filter, and then only the precipitate was extracted with benzene.
-------
-129-
The benzene solution, containing both acids and neutrals, from both
procedures was extracted in a beaker with 5% NaOH with warming and stirring.
The aqueous layer was recovered and washed several times with fresh benzene.
The benzene solution including washings, containing the neutrals, were
washed first with dilute HOAc, then with water and dried with anhydrous
Na-SO..
24
The aqueous 5% NaOH fraction containing the acids was cooled in an ice
bath, acidified with glacial acetic acid and extracted with benzene. A
little diethyl ether was added to the benzene to break emulsions. The benzene
solution was dried over anhydrous Na~SO and the solvent removed on a rotary
evaporator at 30° or less leaving a tan-colored solid.
C. Preparation of Methyl Esters
The methanol used was reagent grade which had been doubly distilled
through a short Vigreax column and checked by GLC with SE-30 as stationary
phase.
The acids Isolated above were dissolved in MeOH (400.0 ml) containing
5-6% gaseous HC1 by weight. The brown solution was refluxed for 2-4 hr,
cooled under tap water, concentrated to approximately 20 ml on a rotary
evaporator and diluted with water (50-55 ml), resulting in a white precipitate.
The mixture was extracted 3 times with 100 ml portions of doubly distilled
n-hexane. The rv-hexane extract was washed with 1% NaHCO,, with water, and
then dried with anhydrous Na~SO,. The ii-hexane extract was removed on a
rotary evaporator leaving a tan-colored'solid.
D. Thin-Layer Chromatography (TLC)
The methyl esters isolated above were dissolved in benzene, diluted to
25 ml and aliquots were subjected to TLC on layers of pre-washed Silica Gel
G using n-hexane-diethyl ether (70:30 v/v) as solvent and a spray reagent of
-------
-130-
0.2% 2',7'-dichloroFluorescein as indicator. Authentic standard methyl esters
were developed simultaneously with the unknown compounds.
Three major spots or "families" were evident. Spot No. 1 (R 0.74)
migrated essentially the same distance as authentic methyl lignocerate (R 0.76)
indicating that it was composed of non-oxygenated fatty acid methyl esters.
Spot No. 2 (R 0.54) migrated similarily to authentic dimethyloctadecanedioate
(Rf0.54) indicating that it was composed of dicarboxylic fatty acid dimethyl
esters.
Spot No. 3 (Rf0.17) was not readily identified by comparison with known
standards but TLC and gas-liquid chromatographic (GLC) data, as well as
properties of a derivative, leads to the belief that it contains hydroxy-acids.
To isolate the methyl ester "families" for further resolution by gas-
liquid chromatography the methyl ester solution was applied as closely spaced
spots to each of five TLC plates and developed as above. A narrow band on
each plate edge was sprayed with 2' ,7'-dichloro-Fluorescein, and the whole
plate briefly exposed to iodine vapors. As soon as the iodine color disappeared
the spots were scraped from the plates and the organic compounds eluted by
repeated washings with benzene. The eluents were concentrated to 1 ml or
less for GLC.
E. TLC of the Neutral Fraction
The neutral fraction of benzene "wax" was subjected to TLC under the
same condition as described above. No major spots resulted, spot A (R 0.30)
and spot B (R 0.07). Spot B migrated the same distance as authentic 6-
sitosterol. In addition,, the reaction of the following sprays used for the
detection of steroids were noted: 85% phosphoric acid - H^O (1:1 v/v);
Liebermann-Burchard Reagent; Stahl SbCl_-HOAc (1:1 v/v).
F. GLC of the Fatty Acid Methyl Esters
-------
-131-
All GLC was performed on a Hewlett-Packard Model 5751B equipped with
dual flame ionization detectors, a Model 17503A input module, a Model 7127A
strip chart recorder, and a Series 300 DISK Integrator.
Column packings were prepared by coating Gas-Chrom Q (100/120 mesh) with
the desired liquid phase and calculating the loading (w/w) by the filtrate
measurement technique. All columns were made from 1/8 in O.D. stainless
steel tubing. Specific columns used were: (a) 5 ft, 4.75% Sillcone SE-30;
(b) 2.5 ft, 8.25% Apiezon-L; (c) 3 ft, 7.4% Reoplex-400; (d) 6 ft, 11.1%
EGGS-X. Various conditions and temperatures were used as specified in the
results. Carrier gas (helium) flow rates were approximately 30 ml/min.
The methyl esters were injected into the GC in ri-hexane or benzene
solutions. Identification of GLC peaks were made by comparing retention
times of the "unknown" peaks with a standard plot of chain-length vs. log
retention time prepared from authentic standard compounds. Standard compounds
were also added to the unknown samples for "peak enhancement" studies.
Peaks due to unsaturated compounds were located by comparing GLC's of
bromine-treated samples with untreated samples.
GLC of standard mixtures of saturated and unsaturated methyl esters and
of saturated diesters showed that peak areas very closely reflected the
weight composition ratios of each series. In this way the ratios of the
compounds of the two spots from TLC were calculated by comparing peak areas.
G. GLC Combined with Rapid-Scan Mass Spectrometry
Mass spectra (MS) of the compounds'of Spot No. 2 (TLC) were obtained
by tanden GLC-MS. GLC was performed on an F&M 810 (Hewlett-Packard) with
the SE-30 solumn under conditions close to those already described. One-
fifth of the column effluent was fed to the GLC flame ionization detector,
while the remainder was passed to the heated EC-1 inlet of an Atlas MAT CH-4
-------
-132-
Nier type mass spectrometer. The 20eV source provided constant monitoring
of the organic compounds reaching the MS, while the 70eV source provided
ionization for the spectra obtained by rapid magnetic scan.
H. Identification of the Alcohols
Identification of the alcohols of Spot A (section I1I-E) was made by
co-GLC of the free alcohols (4.75% SE-30 and 7% Carbowax 20M) with authentic
compounds, and by co-GLC (SE-30) of the acetates (Ac_0 in pyridine with toluene-
sulfonic acid), methyl ethers (CH I and Ag 0) , and TMSi ethers (HMDS and TMcS
in pyridine) with the respective derivatives made from authentic compounds.
The €„„ and C0, alcohols from benzene wax were collected from a
preparative SE-30 solumn and identity was further verified by IR and m.m.p.
1. 1-Docosanol
1-Docosanol from benzene wax had m.p. 69.5-71°, which was not depressed
by mixture with approximately equal amounts of authentic behenyl alcohol; IR
(KBr), cm"1: 3340 (broad) with shoulder ca 3230 (-OH stretch, H-bonded) , 2910
and 2840 (alkyl), 1470 and 1460 (alkyl) , 1056 (primary -OH), 723 and 712
(-(CH,,) -) , and identical to spectrum of authentic behenyl alcohol. Acetate
i n
IR (KBr), cm'1: 2915 and 2850 (alkyl stretch), 1745 (ester carbonyl) , 1465
(alkyl), 1365 (-CHj) , 1235 (C-O-C for acetate), 1040 (C-O-C) , 715 (-(CH2)n-) ,
and identical to spectrum of acetate prepared from authentic alcohol.
2. 1-Tetracosanol
1-Tetracosanol from benzene wax had m.p. 76.0-76.5°, identical to that
of authentic lignoceryl alcohol; IR (KBr K... cm" : 3330 (broad) with shoulder
~"V- j
C£ 3230 (-OH stretch, H-bonded), 2910 and 2840 (alkyl), 1470 and 1460 (alkyl),
1056 (primary -OH), 722 and 712 (-(CH^-) , and identical to a spectrum of
authentic lignoceryl alcohol.
-------
-133-
I. Identification of the Hydroxy Acids
Hydroxy methyl esters and the methoxy methyl esters were collected
from a preparative SE-30 column for further analysis.
1. Methyl 16-hydroxyhexadecanoate
Methyl 16-hydroxyhexadecanoate from benzene wax had m.p. 54-55° (benzene)
(lit. 54.5-55.5°); IR (KBr), cm'1: 3400 with shoulder c£ 3320 (hydroxyl),
2920 and 2850 (alkyl), 1740 (ester carbonyl), 1470 and 1463 (alkyl), 1435
(-CH , methyl ester), 1362, 1340, series of small bands 1190-1300 (-ClU-
wag and twist), 1160 (methyl ester C-O-C), 1060 and 1045 (C-0), 880 (-CH -COOCH ),
730 and 720 (-(CH_) -rock). (Found: C, 71.5; H, 12.0. Calc. for C.,H,.0,:
i n 17 34 3
C, 71.3; H, 12.0)
Treatment with HI and red P followed by Zn and HC1, and methylation
(MeOH - HC1) gave one product whose retention time (SE-30 and EGSS-X) was
the same as that of authentic methyl hexadecanoate, and the MS of the product
showed the ion fragments expected for methyl hexadecanoate.
Oxidation (CrO- in HOAc) gave a product whose methyl ester had the same
TLC R , the same R (SE-30 and EGSS-X), and the same MS as authentic dimethyl
hexadecanedioate.
— 1
Methyl 16-methoxyhexadecanoate gave IR (KBr)cra : 2995 (weak), 2915, 2855
and 2815 (weak) (alkane), 1740 (ester carbonyl), 1475 (alkyl), 1440, 1380, 8
sharp bands of varying intensity 1345-1203, 1170 (methyl ester C-O-C), 1110
implies ether C-O-C), 940, 880, and 715 ((CH.) ). GLC-MS, m/e: 300 (M+), 285
/ n
(M-CH3), 270 (M-30), 269 (M-31), 268 (M-32), 253 (M-47), 241 (M-59), 236 (M-64),
208 (M-92); also the following fragments (which were common to all homologs):
143, 129, 115, 101, 87, 74 (methyl ester), 55, and 45 (CH OCH +, base peak).
2. Methyl 18-hydroxyoctadecanoate
White crystals from hexane had m.p. 62-63° (lit. 62.5°). TR (KBr) was
-------
-134-
identical to a published spectrum: 3390 cm with shoulder at ca 3330 (-OH),
2910 and 2840 (alkane), 1740 (ester carbonyl), 1475 and 1468 (alkane), 1440
(-CH , methyl ester), 1370 and 1355, 1330. Series of small bands 1180-1310
(-CH2 - wag and twist), 1170 (methyl ester C-O-C), 1055 (C-0), 883 (CH - COOCH-),
730 and 720 (-(CH~) - rock). The MS of the methoxy methyl ester was
i n
identical to a published spectrum of methyl 18-raethoxyoctadecanoate: m/e
328, 313, 298, 297, 296, 281, 269, 264, 236 (assignments as for C 6 homolog);
m/e 143 and lower was identical to that for the C., homolog.
3. Methyl 20-hydroxyeicosanoate
White crystals from hexane had m.p. 69.0-69.5° (lit. 68-68.5°); IR (KBr),
cm"1: ca 3330 (broad, -OH, H-bonded), 2910 and 2840 (alkyl), 1740 (ester
carbonyl), 1475 and 1465 (alkyl), 1438 (-CH , methyl ester), 1385, series of
8 small bands 1310-1185 (-CH- - wag and twist), 1175 (methyl ester C-O-C),
1055 (C-0), 880 (-CH0-COOCH,), 728 and 715 (-(CH.) -).
/ J / n
Reduction of the hydroxyl group gave a product whose methyl ester had
the same R as authentic methyl eicosanoate (SE-30, EGSS-X); the MS contained
ion fragments expected for methyl eicosanoate. Oxidation (CrO., in HOAc)
followed by methylation gave a product whose TLC R , R (SE-30, EGSS-X), and
MS were identical to those of dimethyl eicosandeioate isolated previously
from benzene wax.
The methoxy methyl ester gave a MS similar to that of the lower homologs:
m/e 356, 341, 326, 325, 324, 309, 297, 292, 264 (assignments as for C,£
16
homolog); m/e 143 and lower was identical to lower homologs.
4. Methyl 22-hydroxydocosanoate
White crystals from hexane had m.p. 74.5-75.5° (lit. 73.5-74.5°). IR
(KBr) was similar to that of the lower homologs; there was a series of 9
small bands 1315-1185 cm" (-CH2 - wag and twist).
-------
-135-
Reduction of the hydroxyl group gave a product whose methyl ester had
the same R (SE-30, EGSS-X) as authentic methyl docosanoate; the MS contained
ion fragments expected for methyl docosanoate. Oxidation (CrO. In HOAc)
and methylation gave a product whose TLC R , R (SE-30. EGSS-X) and MS
were identical to those of authentic dimethyl docosanedioate.
The methoxy methyl ester gave a MS similar to those for the lower
homologs: m/e 384, 369, 354, 353, 352, 337, 325, 320, 292; fragmentation
m/e 143 and below was identical to that for lower homologs.
5. Methyl 24-hydroxytetracosanoate
White crystals from hexane had m.p. 80-81° (lit. 80-81°). IR (KBr) was
similar to that of the lower homologs; there was a series of 10 small bands
1310-1180 cm"1 (-CH - wag and twist).
IV. Results and Discussion
Since a reasonably representative sample of Douglas-fir bark was desired,
small amounts were taken from a number of trees in the area of Corvallis,
Oregon. Because of the number of trees sampled and because these samples
were thoroughly mixed into a larger collection, no effort was made to
ascertain the diameter or age of the trees.
The n-hexane extraction closely followed the method of Kurth and Kiefer
(71). These authors reported the n-hexane-soluble material to be a light
colored "wax," whereas other solvents such as benzene extracted dark colored
materials. The light color indicated a'less complex fraction which was more
desirable for an initial study.
Column chromatography on Silica Gel G was used to provide a coarse
separation of the ii-hexane-soluble compounds. A number of distinct bands
were evident as the column developed.
-------
-136-
The following bands labelled "light bluish green" and "bright blue"
(designated in combination as the "yellow band" was eluted from the column
and tested for purity by thin-layer chromatography. The better resolving
power of the thin-layer technique showed that the "yellow band" was a
mixture of several chemical compounds. Some thin-layer plates showed as
many as 11 distinct spots plus a sizable spot which remained at the origin.
The spot at the origin contained an undetermined number of compounds.
A solution of the "yellow band" showed a positive Lieberman-Burchard
test (73, 74) indicative of the presence of sterols.
A quantity of the "yellow band" was accumulated from several columns
and the mixture was further separated by thin-layer and gas-liquid chromato-
graphy.
The extensive thin-layer chromatographic study was conducted to find
out how many compounds were in the "yellow band" and to find a solvent
system which gave good separation which could be used as a method to collect
pure compounds.
The 14 solvent systems employed are widely used for the separation
of lipids as well as for sterols and terpenes. The solvent systems composed
of chloroform-carbon tetrachloride (6:1 v/v), diethyl ether-n-hexane (1:4
v/v), n-hexane-diethyl ether-acetic acid (70:30:1 v/v/v) were the best. Of
these three, chloroform-carbon tetrachloride (6:1 v/v) produced more spots
than the other two, but each spot had considerable tailing. The diethyl
ether-n_~hexane (1:4 v/v) system gave very good separation and although the
chromatograro showed fewer spots than the solvent system of chloroform-carbon
tetrachloride (6:1 v/v) the amount of tailing was reduced. The addition of
acetic acid to the ether and n-hexane solvent systems [n-hexane-diethyl
ether-acetic acid (70:30:1 v/v/v), and ji-nexane-diethyl ether-acetic acid
-------
-137-
(85:15:1 v/v/v)] did not show improvement. Solvent systems with higher
polarity such as benzene-methanol-acetic acid (45:8:4 v/v/v) and chlorofora-
diethyl ether-formic acid (5:4:1 v/v/v) showed poor separation and fewer
spots could be observed than with the less polar solvent systems.
Exposure of the thin-layer plates to iodine vapor proved to be the
best method of detecting the spots. It was the easiest to use and showed
more spots than the other methods tried. Simple exposure to ultraviolet
light was the second best method of spot detection. The combination of
ammonia vapors and ultraviolet light did not show any improvement over the
use of ultraviolet light alone. The other methods, 30% aqueous sulfuric
acid, bromothymol blue spray, and p_-nitroaniline spray showed no improvement
over the iodine vapor method and were more difficult to use.
Four columns (Tables 7 and 8) were used in an attempt to completely
resolve the compounds in the "yellow band" from the column chromatograph.
The Hi-EFF 8 BP column did not give good resolution. Even with
temperature programming, it showed only five peaks. The other three columns
showed no great difference in resolving power. Each of these columns showed
as many as 16 peaks and the UC W-98 column showed 19 peaks.
Separation of the "yellow band" from the column chromatograph by
thin-layer chromatography prior to injection into the gas chromatograph
yielded improved separation. The "yellow-blue band" (high R ) from the
thin-layer plates was shown to contain two compounds by gas-liquid chromato-
graphy.
However, there were no peaks by gas-liquid chromatography which could
be attributed to the "bright-blue band" (low Rf) from the thin-layer plates.
It may be that the "bright-blue band" contained high-boiling compounds
which did not elute under the conditions used.
-------
-138-
The results of gas-liquid chronatop.raphy showed that at least 20
compounds were jammed into the "yellow band" from column chromatography.
Nineteen of these were resolved by the DC W-98 column and there was a
minimum of one in the "bright-blue band" from the thin-layer plates which
dlil not show from the DC W-98 column making a minimum of 20 compounds.
These materials must be similar in chemical nature and structure. This
further increases the difficulty of separation and purification. The
identification of every compound shown by gas-liquid chromatography is
often impossible, mainly because of the time and difficulties involved
and the ninute amounts of material which can be detected.
However, the combination of thin-layer chromatography and gas-liquid
chromatography produced evidence fcr the identity of some of the compounds.
It is concluded from the thin-layer and gas-liquid chromatographic evidence
that the n-hexane-soluble fraction from Douglas-fir bark contains campesterol
and fl-sitosterol.
A portion of the gas effluent from the gas-liquid chromatographic
column was passed directly into the ion-source of the mass spectrometer.
The remaining portion of the gas effluent from the column was passed -.hrough
a flarat ioniz.\tion detector which produced an electrical signal which was
recorded on an X-Y strip recorder. In this way when a peak appeared on
the recorder, a portion of the compounds causing the peak was also in the
nans spectrometer and a rapid scan by the mass spectrometer produced a
fragmentation spectra for that compound. Thus, the gas-liquid chromatograph
separated the mixture into pure compounds and the mass spectrometer yielded
information as to the identity of each pure compound.
An aliquot of the trimethylsilyl ethers of the known campesterol and
K-sitosterol mixture was injected into the gas chromatograph. As the peaks
-------
appeared on the recorder, a scan was made by the mass spectrometer. In this
way standard mass spectra were obtained for known B-sitosterol and campesterol
for reference purposes.
In the same way, the trimethylsilyl ethers of the "yellow band" were
injected into the gas chromatograph. The gas-liquid chromatographic
spectrum was scanned by mass spectrometry. In the mass spectrum of the
trimethylsilyl ether of B-sitosterol, characteristic peaks were found at
m/e 486, 396, 381, 357, 255 and 129, The base peak at m/e 129 s which is
a characteristic fragment for a 5-en-3-ol steroid trimethylsilyl ether was
very obvious. In the fragmentation reaction the triraethylsilyl ether
derivative is split into two parts, one part which is charged and the other
which is neutral. The charge can reside on the m/e 129 fragment or the
m/e M-129 fragment (M is the molecular ion mass). Therefore, both fragments
are recorded, but usually the intensities are different.
The mass spectra were not as conclusive for campesterol as might be
expected from the retention times. However, peaks at m/e 129 and 255, which
are characteristic for sterols, were observed. These spectra indicated
mixtures of terpenes and sterols.
The mass spectral data support the gas-liquid chromatograhic retention
data as to the presence of campesterol. The ratio of campesterol to
B-sitosterol is about 1 to 8 as estimated by areas under the gas-liquid
chromatographic curves.
The identification of campesterol and B-sitosterol in the n-hexane-soluble
(
fraction of Douglas-fir bark has proven very interesting because it leads to
a search for other sterols and the part which they might play in bark. The
I
physiological role of these materials is largely unknown, although Rowe (70)
has suggested that B-sitosterol may have a function in cell-wall permeability.
-------
-140-
The mass spectra also showed peaks at m/c 93 and m/e 121 whicli are very
characteristic of terpenes and it is concluded that this particular fraction
of the bark contains terpene compounds.
The acids recovered from saponified "benzene wax" could be resolved
into three major families by TLC of their methyl esters. Spot 1 (R 0.74)
migrated nearly the same distance as authentic methyl lignocerate (Rf 0.76)
suggesting that it was composed of methyl esters of non-oxygenated fatty
acids. Spot 2 (R 0.54) migrated similarly to authentic dimethyl octadecane-
dioate (R 0.57) suggesting that it was composed of dimethyl esters of
dicarboxylic acids. Spot 3 (Rf 0.17) was not readily identified by standards
in our possession, but further investigation suggests that it contains the
methyl esters of the hydroxy acids observed by Kurth.
TLC of the neutral compounds from saponified "benzene wax" yielded two
major components, spot A (R, 0.30) and spot B (Rf 0.17). The latter spot
reacted positively to steroid-detection reagents and migrated the same
distance as authentic 3-sltosterol. These spots could be the aliphatic
alcohols and "phytosterols" observed by Kurth (72).
CLC resolved the compounds of TLC spot 1 into a number of fatty acid
methyl esters (Table 9), identifications of which were based on studies with
four GLC liquid phases. The ester of greatest abundance was methyl lignocerate,
but substantial amounts of several other esters were present. Several of the
GLC components of spot 1 remain unidentified.
The dimethyl esters identified in' TLC spot 2 are listed, with their
relative abundances, in Table 10. Initial identifications were based on GLC
studies made with three liquid phases: SE-30, Apiezon-L, and Reoplex-400.
Besides the C „ compound reported by Kurth (72) we found substantial amounts
of other dimethyl esters.
-------
-141-
Verification of the identities of the spot 2 dimethyl esters was obtained
by tandem GLC-MS. We examined the high ends of the mass spectra of authentic
dimethyl esters and of the GLC peaks in spot 2 for those m/e ion peaks
characteristic of dimethyl esters of dicarboxylic acids: M, M-31, M-64, M-73,
M-92, and M-105. We positively identified all of these m/e peaks for each of
our authentic compounds except for £„., where the peak believed to be M could
not be accuratley assigned an m/e. For the C , and C__ dimethyl esters of
spot 2, we identified all the above ions; for C~?, all except M; for C ,,
only M-31 and M-73. The spectra of the C g and Clg , dimethyl esters were
of mixtures of the two, since resolution was incomplete at the mass spectro-
meter. However, M, M-31, M-64 and M-73 were identified for the C g _ dimethyl
ester, and M = 340 m/c and (M-CH OH) - 308 m/e were identified, which indicates
the presence of a C10 .. dimethyl ester. The MS taken at various points on
lo'. i
the total-ionization peak showed that the unsaturated diester emerged from
the GLC first. This is consistent with the GLC studies. No obvious signs
of branching were noted in any of the spectra.
Gas chromatopraphic analysis (SE-30) of the TLC spot thought to contain
fatty alcohols showed two major components which were identified as 1-
docosanol and 1-tetracosanol; smaller amounts of 1-hexadecanol, 1-octadecanol
and 1-eicosanol were also present (Table 11). Earlier, Kurth (75) isolated
1-tetracosanol from the neutral fraction of saponified benzene wax.
Identification was established by co-GLC of the free alcohols of benzene
wax with authentic compounds (SE-30 and'Carbowax 20M) and by co-GLC (SE-30)
of the alcohol acetates, methyl ethers, and TMSi ethers with the respective
derivatives prepared from authentic compounds. The IR spectra (KBr) of C~_
and C_, free alcohol and alcohol acetate peaks collected were identical to
those of the authentic compounds. In particular, the -OH stretch bands at
-------
-142-
££ 3330 cm present in spectra of the free alcohols were absent in spectra
of the acetates; in the latter, the ester carbonyl absorption at 1745 cm
appear. Melting points of the C._ and C_, alcohols from benzene wax agreed
with those of authentic compounds, and mixed m.p.'s with respective authentic
compounds showed no depression.
GLC (SE-30) of TLC spot 3, thought to contain methyl esters of hydroxy
acids, showed it to contain a family of six compounds for which a plot of
chain length (arbitrary integer) vs. log R was a straight line. Five of
these compounds were isolated by preparative GLC and identified as the methyl
esters of w-hydroxy acids C.., - C_, (even); not enough of the sixth compound
(C0,?) was present to conveniently collect for positive identification.
Zo
Kurth reported the presence of C , , C__ and C hydroxy acids in benzene
wax, but though he noted the unusual melting characteristics of the free
hydroxy behenic acid, he did not locate the hydroxyl group (76).
Also present in the chromatogram, but unidentified, were two peaks
emerging before w-hydroxy C.,, and a peak (peak "2a") only partially resolved
from w-hydroxy C,0 (Table 12). The Eiethyl ether of peak 2a emerged after
lo
w-hydroxy C,_ on EGSS-X, and contained at least two major components. The
lo
GLC behavior of peak 2a8 as well as the fact that it was liquid at room
temperature, suggest that it may contain unsaturated C,0 w-hydroxy methyl
lo
esters. Kurth claims to have isolated a fraction rich in hydroxy unsaturated
acids from the cork of Douglas fir bark.
Treatment of the mixed w-hydroxy esters from benzene wax with alkali in
a manner similar to saponification of the wax, followed by methylation did
not produce any detectable dicarboxylic esters hence we are confident that
the dicarboxylic acids identified earlier by us are not artifacts of the
saponification procedure. That w-hydroxy acid and dicarboxylic acid families
-------
-143-
of similar composition were found in benzene wax may be of biosynthetic
significance.
Though not as widespread or abundant as the hydroxy acids In general,
u>-hydroxy acids as constituents of plant waxes and extracts have been known
for some time. The inter-esterification of the free hydroxy acids to form
"estolides" occurs readily, especially in acid medium or upon heating above
the melting point, and may partly explain the variable melting point observed
by Kurth (76) for his hydroxy-behenic acid. Murray and Schoenfeld considered
estolide formation upon acidification of a saponification mixture of w-hydroxy
acid salts enough of a problem to avoid isolating the free acids during their
qualitative and quantitative analysis of the co-hydroxy acids of carnauba wax.
Estolide formation could partly explain our inability to totally account for
the weight of benzene wax after isolation of neutrals, methyl esters, and
"phenolic acids"; and it could explain the somewhat unreproduceable relative
abundances of w-hydroxy esters between complete isolations of the esters from
the wax. The relative abundances shown in Table 12 should be considered only
tentative and typical of our Isolation procedure.
Identity of the w-hydroxy acids was established by analyzing individual
hydroxy methyl esters and the corresponding methoxy methyl esters collected
by preparative GLC. The IR spectra (CC1.) contained a weak, sharp band at
3620 cm .indicating a hydroxyl group; this band was absent in the IR
spectra of the corresponding co-hydroxy methyl esters. The IR spectrum (KBr)
of methyl 18-hydroxyoctadecanoate was identical to published spectrum, and
the spectra of the other homologs were similar. The most conspicuous
difference between homologs was the addition of one progression band in the
region between 1180 and 1315 cm for each increase of two methylene units.
The lengths of the hydrocarbon chain of the C..,, C^n and C_9 homologs
-------
-144-
were established by reduction of the hydroxyl group and identification of
the resulting fatty acids (as methyl esters) by GLC (two columns) and by
GLC-MS. Hydroxyl group positions for the C.,, C . and C__ homologs were
established by oxidation of the hydroxyl group and identification of the
resulting dicarboxylic acids (as methyl esters) by GLC (two columns) and
by GLC-MS.
The mass spectra of methyl 18-methoxyoctadecanoate agreed with a
published spectrum, and the spectra for the C.,, C and C00 homologs were
lo zu if.
similar. In all cases, the portions of the spectra m/e 143 and below were
identical. The peak m/e 45, the strongest in the spectra, is mainly due to
the fragment CH_OCH_ and indicates the methoxy is in the terminal (to)
position. The high m/e^ portions of the spectra all shoved significant peaks
assigned as M+, (M-CH3), M-30, M-31, M-32, M-47, M-59, M-64 and M-92.
Finally, the melting points of the C-,-C_, to-hydroxy esters agreed
with values reported in the literature.
-------
-145-
AMMONIATION OF BARK
I. Historical Review (80)
The use of ground bark for mulching, soil conditioning, and ornamental
purposes in home gardens and elsewhere has greatly increased in recent years
(1, 2, 3, 81, 82). A major consideration in these applications is to
ensure that the addition of bark does not lower the nitrogen content of the
soil. Bark contains very little nitrogen and the microorganisms which bring
about its decomposition tend to compete with plant roots for the available
nigrogen (83, 84). According to Bollen (85) the fortification of bark with
nitrogen is vital to the successful use of bark on or in the soil. Treatments
include chemical processing, mechanical mixing with nitrogen fertilizers,
aging, composting and combinations of treatments. The work herein reported
is concerned with fortification by chemical processing and with gaseous
ammonia in particular.
There have been several reports on the subject (3, 83, 84, 85, 86, 87).
Aspitarte (86) and Bollen and Glennie (83) exposed Douglas-fir bark to
anydrous ammonia in a closed screw conveyor system. Bollen and Glennie (84)
also sprayed ground bark with aqueous ammonia, nitric acid, an aqueous solution
of urea, and an aqueous solution of urea and phosphoric acid. Simpler
procedures have involved covering a pile of bark with a polyethylene sheet
and passing gaseous ammonia through it from a perforated pipe or hose (85, 88).
Our objectives in the present study included a determination of the
amount of nitrogen retained by Douglas-fir bark after treatment with gaseous
ammonia under controlled conditions. We also wished to ascertain how readily
this nitrogen was extracted with selected solvents and if any nitrogen
remained after exhaustive extraction. We investigated the extracts to
determine if the ammoniation altered the chemical compounds which are extracted
-------
-146-
by organic solvents and water. Since these compounds are most likely to be
leached into the soils we particularly wanted to determine if any known
harmful products resulted.
II. Experimental (80)
A. Sample Preparation
Samples of bark from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]
were collected near Corvalllss Oregon. They were thoroughly mixed, ground
in a ball mill to a size suitable for research investigations and air dried;
moisture content, 15.8% (oven at 110°C overnight); nitrogen content, 0,46%
(Kealdahl).
B- Bark Extraction
An aliquot (21.04 g) of the bark was successively extracted in a Soxhlet
apparatus (50 exchanges of solvent) with ii-hexane, benzene, diethyl ether,
95% ethanol and hot water. An ultraviolet spectrum of each extract was
obtained over a range of 230 nm to 350 nm. Each extract was concentrated
under vacuum on a rotary evaporator, dried in an oven at 110°C overnight, and
the solids weighed.
C, Treatment with Gaseous Ammonia
An aliquot (233.26 g) of the air-dried bark was loosely packed into a
glass tube to provide a sample 4.6 cm in diameter and 100.0 cm In length.
Typical conditions for ammonia treatment were: tank gauge pressure, 10 psl,
time of gaseous ammonia flowB 2 hr; total time in ammonia atmosphere, 17 hr;
maximum temperature in sample, 68°C; nitrogen content of air-dried bark after
treatment9 4.08%.
An aliquot (25.00 g) of the treated bark after air drying was successively
extracted with n-haxane, benzene, diethyl ether, 95% ethanol and hot water
and the extracts investigated as described above for those from the original
bark. The nitrogen content of the bark residue after extraction was 2.98%.
-------
-147-
III. Results and Discussion (80)
The sample of Douglas-fir bark used in the present treatment contained
0.14% nitrogen. This is similar to the 0.11% and 0.16% reported by Bollen
and Glennie (86). After treatment with gaseous ammonia the nitrogen content
Increased to 4.08% which is in the range of 3.71% and 4.27% obtained by
Bollen and Glennie (83) for bark ammoniated under selected conditions.
This amount of nitrogen has been shown (83, 84) to be more than adequate
for the prevention of nitrogen starvation in soil.
Ammoniation of finely ground bark (passed a screen of 32 meshes to the
inch) resulted in a nitrogen content to 2.34%. The lower nitrogen retention
was attributed to a more solid packing of the fine particles in the treating
tube so that the ammonia gas may not have diffused freely through the sample.
A loose packing of the bark or an increased treating time would be expected
to increase the nitrogen content. This illustrates the flexibility of
ammonlation with gaseous nitrogen. By careful control of treating conditions,
it is possible to tailor the nitrogen content of bark to the desired content.
Bollen and Glennie (83) also demonstrated this control. They obtained
nitrogen contents ranging from 1.68% to 4.27% by changing the treating
conditions.
The amount of solids extracted in each of the organic solvents was not
significantly altered by treatment with gaseous ammonia (Table 13). However,
the hot water extraction of ammoniated bark resulted in an increased solid
t
dissolution of 3.5 times for coarse bark and 3.2 times for finely ground
bark when compared to untreated bark. Ammoniation is known to Increase the
pH of bark (83, 84) which could bring about hydrolysis of some of the
polyphenolic compounds so that they would become water soluble. The data
(Table 13) indicate that there would be a more Immediate release of leachable
-------
-148-
solids Into the soil from ammoniated bark than from untreated bark. However,
there appears to be no evidence of toxic materials from ammoniated bark.
Bollen and Glennie (83, 84) and Bollen and Lu (87) report that tannins, resins,
and other readily extracted compounds from treated and untreated bark have
no toxic effects on plants when added to the soil. The conditions of
extraction used in the present study are considered to be exhaustive and it
appears that the early rate of leaching would not be continuous but would
level off to a slow dissolution of the bark due to microbial action.
Table 13 also shows that grinding the bark to a small screen size has
little or no effect upon the amounts of solids extracted by each solvent.
Therefore, whether the bark is used as chunk-sized decorative bark, or ground
and used as a mulch has little effect upon the immediate dissolution of solids.
However, the immediate leaching of solids should not be confused with microbial
decomposition for in the latter case bark size has a considerable effect.
According to Bollen (85) the finer the bark particles the more rapidly it is
decomposed.
The bark residue from the exhaustive extraction with organic solvents
and hot water analyzed for 2.98% nitrogen. These results support the findings
of Bollen and Glennie (83, 84) that a part of the nitrogen in ammoniated bark
is immediately available but that a substantial part is slowly released only
through normal decomposition. In many soil uses this dual effect is very
desirable and beneficial.
The ultraviolet spectra of the extracts showed that little if any chemical
changes occurred in the extractable materials because of ammoniation (Table
14). Only the major peaks and shoulders are reported, but in general there
were little differences in the spectra. Although the wavelength range
included only the ultraviolet region, it is expected that major changes in
-------
-149-
che leachable compounds' would have been detected. Indications are that
chemical changes which might have occurred took place in those compounds
which were not soluble.
-------
-150-
BIBLIOGRAPHY
1. Harkin, J. M. and J. Wo Rowe. Bark and its possible uses. USDA Forest
Service, Research Not* FP-.-0910, October 1969.
2. Bark Committee of the Forest Products Research Society, Marketing and
selling bark products. Ed. by Jean Mater, R. Martin, D. Williams,
R. Sarles, F. Lamb, and R. Allison, Forest Products Research
Society, Madison, WI. 1969.
3. Hall, J. Alfred. Utilization of Douglas-fir bark. Pacific Northwest
Forest and Range Experiment Station, Forest Service, U. S.
Department of Agriculture, Portland, OR. 1971.
4. Grilles, S. J. Structure and development of the bark of Douglas-fir
Pseudotsuga menziesii (Mlrb.) Franco. Doctoral thesis. Corvallis,
Oregon State University. 1956. 67 numb, leaves.
5. Grilles, S. J. and F. H. Smith. The secondary phloem of Douglas-fir.
Forest Science 5:377-388. 1959.
6. Chang, Y. Bark structure of North American conifers. (U. S. Dept. of
Agriculture, Technology Bulletin 1095). 1954. 86 p.
7. Ross, W. D. and R. L. Krahmer. Some sources of variation in structural
characteristics of Douglas-fir bark. Wood and Fiber 3:35-46. 1971.
8. Kiefer, H. J. and E. F. Kurth. The chemical composition of the bast
fibers of Douglas-fir bark. Tappi 36:14-19. 1953.
9. Segal1, G. H. and C. B. Purves. Chemical composition of wood barks.
Pulp and Paper Magazine of Canada 47:149-161. 1946.
10. Kurth, E. F. The chemical composition of bark. Chemical Reviews
40:33-49. 1947.
11. Aspinall, G. 0. Structural chemistry of the hemicelluloses. In:
Advances in carbohydrate chemistry. Vol. 14. Ed. by M. L. Wolfrom.
New York, Academic Press, Inc. 1959. p. 429-433.
12. Timell, T. E., C. P. Glaudemans and J. K. Gillham. Recent studies on
the polysaccharices of white birch and other hardwoods. Tappi
42:623-634. 1959.
13. Hamilton, J. K. and N. S. Thompson. A comparison of the carbohydrates
of hardwoods and softwoods. Tappi 42:752-760. 1959.
14. Timell, T. E. Wood hemicelluloses. In: Advances in carbohydrate
chemistry. Vol. 19. Ed. by M. L. Wolfrom. New York, Academic
Press, Inc. 1964. p. 247-302.
-------
-151-
15. Whistler, R. L. and E. L. Richards. Hemicelluloses. In: The
Carbohydrates Chemistry and Biochemistry, Vol. IIA, 2nd edition.
Ed. by W. Pigman and D. Horton. New York, Academic Press, Inc.
1970. 469 p.
16. Graham, H. M. and E. F. Kurth. Constituents of extractives from Douglas-
fir. Industrial Engineering Chemistry 41:409-414. 1949.
17. Holmes, G. W. and E. F. Kurth. The chemical composition of the newly
formed inner bark of Douglas-fir. Tappi 44:893-898. 1961.
18. Cleland, J. E. and W. R. Fetzer. Determination of total solids in
glucose syrup. Industrial and Engineering Chemistry. Analytical
Edition 13:858-660. 1941.
19. Timell, T. E., C. P. Glaudemans and A. C. Currie. Spectrophotometric
method for determination of sugars. Analytical Chemistry 28:
1916-1920. 1956.
20. Timell, T. E. Isolation of polysaccharides from the bark of gymnosperms.
Svensk papperstidning 64:651-661. 1961.
21. Browning, B. L. Methods of wood chemistry. Vol. I. New York,
Interscience Publishers. 1967. 384 p.
22. Laver, M. L., D. F. Root, F. Shafizadeh and J. C. Lowe. An improved
method for the analysis of the carbohydrates of wood pulps through
refined conditions of hydrolysis, neutralization, and monosaccharide
separation. Tappi 50:618-622. 1967.
23. Black, R. J., R. LeShange and G. Zweig. Paper chromatography, a
laboratory manual. New York, Academic Press, Inc. 1952. 195 p.
24. Clark, J. M., Jr. Experimental biochemistry. San Francisco, CA, W.
H. Freeman and Co. 1964. 228 p.
25. Hough, L. and J. K. N. Jones. Chromatography on paper. In: Methods
in carbohydrate chemistry. Vol. I. Ed. by R. L. Whistler, M.
L. Wolfrom. New York, Academic Press. 1962. 589 p.
26. Piper, C. V. and L. J. Bernardin. A spectrophotometric method for
the chromatographic analysis of sugars. Tappi 41:16-18. 1958.
27. Miles Laboratories, Inc. HT-1000. 'Product Information. Elkhart,
Indiana. No date.
28. Miles Laboratories, Inc. Diazyme, an amyloglucosidase enzyme for
conversion of starch and/or dextrins to glucose. Technical
Bulletin No. 9-245. Elkhart, Indiana. 1963.
29. Wolfrom, M. L. and D. L. Patin. Carbohydrates of the coffee bean.
IV. An arabinogalactan. Journal of Organic Chemistry 30:4060-
4063. 1965.
-------
-152-
30. Abdel-Ahker, M., J. K, Hamilton and F. Smith. The reduction of sugars
with sodium borohydride. Journal of the American Chemical Society
73:46-91-4692. 1961.
31. Borchardt, L. G. and C. V. Piper. A gas chromatographic method for
carbohydrates as alditol-acetates. Tappi 53:257-260. 1970.
32. Albersheim, P., D. J. Nevins, P. D. English, and A. Karr. A method
for the analysis of sugars in plant cell-wall polysaccharides by
gas-liquid chromatography. Carbohydrate Research 5:300-345. 1967.
33. Sloneker, J. H. Gas-liquid chromatography of alditol acetates. In:
Methods in Carbohydrate Chemistry. VI. General Carbohydrate
Methods. Ed. by R. L. Whistler and J. N. BeMiller. New York,
Academic Press. 1972. 603 p.
34. Paech, K. and M. V. Tracey. Moderne Methoden Per Pflanzen-analyse
(modern methods of plant analysis). 1:468-473. 1956.
35. Wolfrom, M. L. and D. L. Patin. Carbohydrates of the coffee bean.
IV. An arabinogalactan. Journal of Organic Chemistry 30:4060-
4063. 1965.
36. Vogel, I. A. Textbook of practical organic chemistry including
qualitative organic analysis. Third ed., New York, Longmans,
Green and Co. 1956. 1188 p.
37. Clark, John N. Experimental biochemistry. San Francisco, CA, W. H.
Freeman and Co. 1964. 228 p.
38. Pische, S. Color reactions of hexuronic acids. In: Methods of
Carbohydrate Chemistry, Vol 1. Ed. by R. L. Whistler and M. L.
Wolfrom, New York, Academic Press. 1962. 589 p.
39. Jayme, G. and H. Knolle. Paper chromatography of sugar mixtures on
glass-fiber papers. Angewande Chemie 68:243-246. 1956.
40. Partridge, S. M. Aniline hydrogen phthalate as a spraying reagent for
chromatography of sugars. Nature 164:443. 1949.
41. Wolfrom, M. L., M. L. Laver and D. L. Patin. Carbohydrates of the
coffee bean. II. Isolation and characterization of a mannan.
Journal of Organic Chemistry 26:4533-4535. 1961.
42. Falconer, E. L. and G. A. Adams. The aldobiouronic acids of
hemicellulose B of oat hulls. Canadian Journal of Chemistry 34:
338-344. 1956.
43. Acid Insoluble Lignin in Wood Pulp. Tappi Standard T222 M-24.
44. Browning, B. L. and L. 0. Bublitz. The ioslation of holocellulose
from wood. Tappi 36:452-458. 1953.
-------
-153-
45. Goldschmid, Otto. Ultraviolet spectra. In: Lignins, Occurrence,
Formation, Structure and Reactions. Ed. by K. V. Sarkanen and
C. H. Ludwig. New York, Wiley-Interscience. 1971. 916 p.
46. Wolfrom, M. L. and J. V. Karabinos. The identification of aldose
sugars by their mercaptal acetates. Journal of the American
Chemical Society 67:500-501. 1945.
47. Bates, F. J. and Associates. Polarimetry, saccharimetry and the
sugars. National Bureau of Standards Circular C440. 1942. p. 488.
48. Whistler, R. L. and J. N. BeMiller. a-D-xylose. I. Isolation from
corn cobs or xylan. In: Methods in Carbohydrate Cehmistry, Vol.
1. Ed. by R. L. Whistler and M. L. Wolfrom. New York, Academic
Press. 1962. 589 p.
49. Isbell, H. S. and H. L. Frush. d-D-Mannose from ivory-nut meal. In:
Methods in Carbohydrate Chemistry, Vol. 1. Ed. by R. L. Whistler
and M. L. Wolfrom. New York, Academic Press. 1962. 589 p.
•
50. Fales, H. A. and F. Kenny. Inorganic quantitative analysis. New York,
Appleton-Century-Crofts, Inc. 1939. 717 p.
51. Fisher, R. B. Quantitative chemical analysis. Second ed. Philadelphia
and London, W.B., Saunders Co. 1964. 501 p.
52. Hodge, J. E. and B. T. Hofreiter. Determination of reducing sugars
and carbohydrates. In: Methods in Carbohydrate Chemistry, Vol.
I. Ed. by R. L. Whistler and M. L. Wolfrom. New York, Academic
Press. 1962. 589 p.
53. Sawardeker, J. S., J. H. Soneker and A. Jeanes. Quantitative deter-
mination of monoaaccharides as their alditol acetates by gas
liquid chromotography. Analytical Chemistry 12:1602-1603. 1965.
54. Langlois, David L. myo-Inositol from corn steep water or calcium
phytaten. In: Methods in Carbohydrate Chemistry, Vol. II. Ed.
by R. L. Whistler and M. L. Wolfrom. New York, Academic Press.
1963. 572 p.
55. Beelik, A., R. J. Conca, J. K. Hamilton and E. V. Partlow. Selective
extraction of hemicelluloses from softwoods. Tappi 50:78-81.
1967.
56. Technical Association of the Pulp and Paper Industry. Ash in pulp.
Tappi Standards and Suggested Methods. T211 M-58. New York.
1958.
57. Willard, H. H., L. L. Merritt, Jr. and J. A. Dean. Instrumental
methods of analysis. Fourth edition. Princeton, N.J., Van
Nostrand Co., Inc. 1965. 784 p.
-------
-154-
58. Browning. B. L. Methods of wood chemistry, Vol. II. New York,
Interscience Publishers. 1967. p. 384-882.
59. Song, M. J. The structure of a xylan from Tilia americana L. Master's
thesis. Syracuse, N.Y., State University College of Forestry
at Syracuse University. 1970. 97 numb, leaves.
60. Technical Association of the Pulp and Paper Industry. Cupriethylene-
diamine disperse viscosity of pulp. Tappi Standards and
Suggested Methods. T230 SU-66. New York. 1966.
61. Sears, K. D., A. Beelik, R. L. Casebier, R. J. Engen, J. K. Hamilton
and H. L. Hergert. Southern pine prehydrolyzates: characterization
of polysaccharides and lignin fragments. Journal of Polymer Science
Part C:425-443. 1971.
62. Lai, Y. Z. Kinetics of degradation of carbohydrates. Doctoral thesis.
Seattle, University of Washington. 1968. 97 numb, leaves.
63. Lai, Y. Z.' and K. V. Sarkanen. Kinetic study on the alkaline degrada-
of amylose. Journal of Polymer Science, Part C, 28:15-26. 1969.
64. Barnett, A. J. G. and G. A. Tawab. A rapid method for the determination
of lactose in milk and cheese. Journal of Scientific Food
Agriculture 8:437-441. 1957.
65. Dubois, M. , K. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith.
Colorimetric method for determination of sugars and related
substances. Analytical Chemistry 28:350-356. 1956.
66. Samuelson, 0. and A. Wennerblom. Degradation of cellulose by alkali
cooking. I. Formation of carboxyl groups. Svensk Papperstidning
57:827-830.
67. Boggs, L.» L. S. Cuendet, I. Ehrenthal, R. Koch and F. Smith. Separation
and identification of sugars using paper chromatography. Nature
166:520-521. 1950.
68. Hough, L., J. K. N. Jones and W. H. Wadman. Quantitative analysis of
mixtures of sugars by the method of partition chromatography.
Part V. Improved methods for the separation and dectection of
the sugars and their methylated derivatives on the paper chromato-
gram. Journal of the Chemical Society 1702-1706. 1950.
i
69. Partridge» S. M. Aniline hydrogen phthalate as a spraying reagent
for chromatography of sugars. Nature 164:443. 1949.
70. Wolfrom, M. L. and D. L. Patin. Isolation and characterization of
cellulose in the coffee bean. Agriculture and Food Chemistry
12:376-377. 1964.
71. Kurth, E. F. and H. J. Kiefer. Wax from Douglas-fir bark. Tappi
33:183-186. 1950.
-------
-155-
72, Kurth, E. F. The composition of the wax in Douglas-fir bark. Journal
of the American Chemical Society 72:1685-1686. 1950.
73. Fieser, L. F. Experiments in organic chemistry. Third ed. Boston,
D. C. Heath and Co. 1957. 353 p.
a
74. Fieser, L. F. and Mary Fieser. Natural products related to phenantherene.
Third ed. New York, Reinhold Publ. Co. 1949. 704 p.
75. Kurth, E. F. The chemical composition of conifer bark waxes and
corks. Tappi 50:253-257. 1967.
76. Hergert, H. L. and E. F. Kurth. The chemical nature of the cork from
Douglas-fir bark. Tappi 35:59-66. 1952.
77. Kiefer, H. J. and E. F. Kurth. The chemical composition of the bast
fibers of Douglas-fir bark. Tappi 36:14-19. 1953.
78. Loveland, P. M. and M. L. Laver. Monocarboxylic and Dicarboxylic acids
in the benzene extract of Pseudotsuga menziesii bark. Phytochemistry
11:430-432. 1972.
79. Loveland, P. M. and M. L. Laver. u-Hydroxy fatty acids and fatty
alcohols from Pseudotsuga menziesii bark. Phytochemistry 11:
3080-3081. 1972.
80. Laver, M. L., J. V. Zerrudo and Harvey Aft. Treatment of Douglas-fir
bark with gaseous ammonia. Forest Products Journal 22:82-83.
1972.
81. Mater, Jean, ed. Marketing bark agricultural and horticultural o
products. Forest Products Research Society, Madison, WI. 1970.
82. Mater, Jean. Utilization of bark in highway landscaping. Forest
Products Journal 21:17-20. 1971.
L
83. Bollen, W. B. and D. W. Glennie. Sawdust, bark and other wood wastes
for soil conditioning and mulching. Forest Products Journal
11:38-46. 1961.
84. Bollen, W. B. and D. W. Glennie. Fortified bark for mulching and soil
conditioning. Forest Products Journal 13:209-215. 1963.
85. Bollen, W. B. Properties of tree barks in relation to their agricul-
tural utilization. Research Paper PNW-77, U. S. Dept. of Agric.,
For. Serv., Pacific NW For. and Range Exp. Sta., Portland, Oregon.
1969.
86. Aspitarte, T. R. Availability of nitrogen in ammoniated bark used as
a soil amendment. Doctoral thesis, Oregon State University,
Corvallis, Oregon. 1959.
-------
-156-
87. Bollen, W. B. and K. C. Lu, Douglas-fir bark tannin decomposition in
two forest soils. Research Paper PNW-85, U, S. Dept. of Agric.,
For. Serv.j Pacific NW For. and Range Exp. Sta., Portland, Oregon,
1969.
88. Ticknor, R. L. and J. L. Ricard. Oregon State University, North
Willamette Exp. Sta., Aurora, Oregon. Personal communication.
1971.
-------
-157-
Table 1. Raw material for pelleting trials
Pure Barks
1. Douglas fir
2. western hemlock
3. western larch
4. ponderosa pine
5. lodgepole pine
6. sugar pine
7. western white pine
8. white fir
9. noble fir
10. silver fir
11. grand fir
12. western redcedar
13. Sitka spruce
14. Engelmann spruce
15. red alder
Mixtures
1 + 2
2 + 8
1 + 2 + 3 + 4 + 7
Douglas fir bark + shavings
Douglas fir bark + hog fuel
Douglas fir bark + hog fuel + shavings
Douglas fir bark + sawdust
Douglas fir bark + sanderdust
Douglas fir bark + fertilizer
western hemlock bark + fertilizer
chemically extracted Douglas fir bark
western redcedar bark plus woody material
mixed northern hardwood bark from Maine
mixed eastern softwood bark from Maine
mixed eastern softwood bark + sawdust
southern oak bark + woody material from
Tennessee
-------
-158-
Table 2. Bulk densities and compression factors for some pelleted barks,
Bark species
Douglas fir
Douglas fir bast fibers
Douglas fir fines
Western hemlock
Western hemlock
Western hemlock
Ponderosa pine
Ponderosa pine
Lodgepole pine
Lodgepole pine
Silver fir
Silver fir
Silver fir
White fir
White fir
White fir
Grand fir
Grand fir
Red alder
Red alder
Pellet
diameter
(inches)
1/2
1/2
3/16
1/2
1/4
3/16
1/2
1/4
1/4
3/16
1/2
1/4
3/16
1/2
1/4
3/16
1/2
1/4
1/2
3/16
Bulk density
Before
pelleting
14.2
13.0
13.4
14.9
14.9
14.9
12.6
12.6
11.1
11.1
14.0
14.0
14.0
14.7
14.7
14.7
14.9
14.9
22.3
22.3
(Ib/cu ft)
After
pelleting
39.3
49.4
45.7
45.2
43.0
49.6
42.3
38.4
37.6
42.0
45.8
44.9
46.0
38.9
40.3
35.6
37.4
30.4
46.7
46.2
Compres-
sion
factor
2.8
3.8
3.4
3.0
2.9
3.3
3.4
3.0
3.4
3.8
3.8
3.2
3.3
2.6
2.7
2.4
2.5
2.0
2.1
2.1
Mixed eastern softwoods
1/2
15.9
47.5
3.0
-------
-159-
Table 3. Partial list, recipients of bark pellet samples
Company
1. Dant and Russell, Inc.
2. Sigma Plastics
3. Grant & Roth Plastics
4. Colorado State University
5. Small Business Administration
6. California Pellet Mill Company
7. Bohemia, Inc.
8. Fruit Growers' Supply Company
9. Sequoia Forest Products
10. DiGiorgio Corporation
11. MacMillan Bloedel
12. Architectural Short Course Display
13. MEG Company
14. Publishers Paper Company
15. Western Wood Fibre
16. U. S. Plywood-Champion Papers
17. U. S. Forest Service
18. Oregon Timber Products Company
19. Reydco Trading Company
20. University of Maine
21. Borden Company
22. Ellingson Timber Company
23, U.S. Forest Products Laboratory
24. Oregon Lumber Export Company
Location
Portland, Oregon
Portland, Oregon
Hillsboro, Oregon
Ft. Collins, Colorado
Portland, Oregon
Portland, Oregon
Eugene, Oregon
Los Angeles, California
Dinuba, California
San Francisco, California
Vancouver, B. C., Canada
Portland, Oregon
Neodesha, Kansas
Portland, Oregon
Eugene, Oregon
Eugene, Oregon
Washington, D. C.
Albany, Oregon
Eugene, Oregon
Farmington, Maine
Springfield, Oregon
Baker, Oregon
Madison, Wisconsin
Portland, Oregon
-------
-160-
25. Pacific Keynon Corporation
26. Oxford Paper Company
27. National Distillers Products Company
28. Canadian Car (Pacific)
29. H. J. Stoll & Sons
30. W. R. Grace 5. Company
31. Princeton Turf Farms
32. Potlatch Forests, Inc.
33. University of Washington
34. FAO
35. Agricultural Engineering Dept. , OSU
36. Bear River Lumber Company
37. Hercules, Inc.
38. Agricultural Chemistry Dept., OSU
39. B. P. John Furniture Company
40. Crown-Zellerbach Corporation
41. Koppers Company
42. American Forest Products Company
43. Peco, Inc.
44. Head Groundskeeper, OSU
45. NASA
46. Chemistry Dept. Brigham Young Univ.
47. George D. Ward & Associates
48. Weyerhaeuser Company
49. Institute for Wood Biology &
Wood Preservation
50. Collier Carbon & Chemical Company
Los Angeles, California
Rumford, Maine
Memphis, Tennessee
Vancouver, B. C., Canada
Portland, Oregon
Halsey, Oregon
Cranbury, New Jersey
Lewiston, Idaho
Seattle, Washington
Rome, Italy
Corvallis , Oregon
Logan, Utah
San Francisco, California
Corvallis, Oregon
Portland, Oregon
Portland, Oregon
Los Angeles, California
Martell, California
Portland, Oregon
Corvallis, Oregon
Moffett Field, California
Provo, Utah
Portland, Oregon
Longview, Washington
Hamburg, West Germany
Salem, Oregon
-------
-ibi-
51. Unalit S. A.
52. Wood Products Service Company
53. Department of Forestry
54, Ekono
55. Monsanto Company "
56. University of Denver
57. Incentive, AB
58. Agricultural Service Corporation
59. Canadian Forestry Service
St. Usage, France
Memphis, Tennessee
Salem, Oregon
Helsinki, Finland
Eugene, Oregon
Denver, Colorado
Stockholm, Sweden
Salem. Oregon
Victoria, B. C., Canada
-------
-162-
Table 4. Composition of bast fibers and Douglas-fir wood.
Fiberb Wooda
Kther soluble 2.92 1.32
Alcohol soluble 8.65 5.46
Hot water soluble 2.58 2.82
Sum of three extractives 14.15 9.60
Values based on oven-dry extractive-free material
Ash 0.60 0.17
Lignin 44.80 30.15
Holocellulose 54.58b 71.40
I'entosans 8.62 10.11
Methoxyl group 3.89 '4.75
Acetyl group 2.39 0.59
Uronic acid anhydride 4.62 2.80
Methoxyl on lignin 7.16 15.20
Values in percent of oven-dry bark.
Corrected for lignin content.
-------
-163-
Table 5. Mixtures of alditol acetates for determination of an "Instrument
K Factor"
Sugar
Myo-inositol hexaacetate
Glucitol hexaacetate
Galactitol hexaacetate
Mannitol hexaacetate
Acabinitol pentaacetate
Xylitol pentaacetate
Weight In mg
10.0
8.0
0.3
1.4
0.4
1.1
10.0
12.0
6.5
2.1
0.6
1.65
10. 0
16.0
0.6
2.8
0.8
2.2
10.0
20.0
7.5
3.5
1.0
2.75
10.0
24.0
0.9
6.2
1.2
3.3
-------
-164-
Table 6. Extractives content of the bast fibers of Douglas-fir bark.
Solvent
u-Hexane
Itenzene
Diethyl ether
Hot water
Ethanol
Yield %a
1.59
0.95
0.38
9.61
0.96
Nature
Light yellow "wax"
lirown "wax"
Dihydroquercetin
Tannin, carbohydrate, etc.
Phlobaphine
o
Percentages based on oven-dry weight of bast fibers.
-------
Table 7. Columns used in gas-liquid chromatography.
Col.
No.
1
2
3
A
Length
ft.
12
6
6
6
O.D.
in.
1/8
1/8
1/8
1/8
Liquid
coating
SE-52
OV-17
US W-98
Hi-EFF 8 BP
Solid
Concentration support
10 wt.
3 wt.
10 wt.
3 wt.
%
11
/5
%
%
Gas
Gas
Chrom.
Chrom.
W.
Q.
Silicone Gum
Gas
Chrom.
Q-
Mesh
60/80
100/120
80/100
100/120
Instrument
Aerograph 200
Hewlett-Packard
Hewlett-Packard
Hewlett-Packard
Outside diameter
-------
Table 8. Conditions for separation of the "Yellow Band" by gas-liquid chromatography.
Injection
Detector port
Sample temp. °C temp. °C Column
Silylated
Mixture 265 277 SE-52
Mixture 265 277 SE-52
Mixture 270 275 UC W-98
Mixture 255 235 UC W-98
Mixture 278 272 OV-17
Mixture 260 245 OV-17
Mixture 275 270 Hi-Eff 8 BP
Flow
rate Range Attenuation
Column temp. °C ml/min setting setting Instrument
Isothermal 255 60 1
Isothermal 255 60 1
Isothermal 275 28 102
Program 28 10'
148-^300
2°/min
Isothermal 255 30 1Q2
Program 30 10"
170^300
2°/min
7
Program 30 10"
30°/min
4 Aerograph
4 Aerograph
16 Hewlett-Packard
16 Hewlett-Packard
16 Hewlett-Packard
16 Hewlett-Packard
16 Hewlett Packard
-------
-167-
Table 9. Monocarboxylic acids in Douglas-fir bark "benzene wax".
Acid2
I'almLtic
(UnLdent. sat'd.)
(C1Q unsat'd.)
lo
Stearic
(Unident. unsat'd.)
1 1
it
if
Arachidic
ii-Heneicosanoic
Uehenic
n-Tricosanoic
Lignoceric
n-Pentacosanoic
Cerotic
(N-Heptacosanoic)
(Montanic)
Retention time
SK-30, 215°
1 .3
1.6
2.2
2.4
3.1
3.8
4.53
4.53
4.5
6 . 4
8.9
12.1
17.0
23.0
31.7
4
59.3
Percent
relative
abundance
0.9
0.3
3.1
0.4
0.8
trace
0.4
3.3
2.5
tract
22.3
0.7
51.7
0.5
12.7
trace
0.3
99.9
Identified by GLC of methyl esters in TLC spot 1.
2
Named compounds in parentheses, "( )", denote tentative identifications
These peaks were resolved from methyl arachidate at 195°.
4
Observed in a temperature-programmed run on SE-30, and isothermally on
Apiezon-L.
-------
-168-
Table 10. Dicarboxylic acids in Douglas-fir hark "benzene wax".
Percent
relative
Acid abundance
Hexadecanedioic 36.3
Octadec-?-enedioic3 24.8^
Octadecanedioic 14.6
Eicosanedioic 14.3
Docosanedioic 8.7
Tetracosanedioic 1.3
100.0
Identified by GLC of dimethyl esters in TLC spot 2
and verified by tandem GLC-MS.
2
Estimated from SE-30 data.
Location of double bond not established.
4
Includes small, unresolved shoulder.
-------
Table 11. Aliphatic alcohols from benzene wax.
Acid
l-hexadecanol
1-octadecanol
1-eicosanol
1-docosanol
1-tetracosanol
Percent
relative .
abundance
trace
4.0
3.6
45. 0
47.5
100.1
Based on peak areas from GLC of acetate derivatives.
-------
-170-
Table 12. GLC of TLC spot 3 containing methyl esters of oHiydroxy acids.
GLC
peak
(SE-30)
00
0
1
2a
2
3
-
4
5
6
Acid
unident .
unident .
16-hydroxyhexadecanoic
unident .
18-hydroxyoctadecanoic
20-hydroxyeicosanoic
unident.
22-hydroxydocosanoic
24-hydroxytetracosanoic
(26-hydroxyhexaconsanoic)
«tl
(min)
0.8
2.7
9.5
14.1
15.1
21.4
22.9
27.8
33.9
41.1
Percent
relative
abundance
1.7
7.8
25.6
7.1
7.1
18.0
2.0
23.6
5.9
1.3
J
For temp, programed run 180-248°.
?
"Calc. from peak areas, uncorrected for detector response.
Tentative identification.
-------
-171-
Table 13. Solvent extraction yields from bark
Solvent
n-Hexane
Benzene
Diethyl ether
Kthanol
Hot water
Untreated
bark
4.67
2.70
1.81
6.38
2.20
Percent Yield-L
NH -treated
bark2
4.63
2.95
2.21
7.13
7.60
NH -treated
bark
(32 mesh)
4.16
3.92
1.19
6.06
7.00
Percentages based on dry weight.
?
"NH represents gaseous ammonia.
Passed a screen of 32 meshes to the inch.
-------
-172-
Table 14, Ultraviolet absorption of bark extracts.
Extraction solvent Major Shoulder
and bark sample peak nm nm
ri-Hexane
Untreated bark , 264 228
NHj-treated bark „ 264 330
NH3-treated bark (32 mesh) 264 - 229
Benzene
Untreated hark 262
NH3~treated bark 263
NH3-treated bark (32 mesh) 265
Diethyl ether
Untreated bark 254
NH3-treated bark 257
NH3-treated bark (32 mesh) 258
Ethanol
Untreated bark 261
NH3-treated bark 265
NH3-treated bark (32 mesh) 264
Hot water
Untreated bark
NH3-treated bark 249
NH_ represents gaseous ammonia.
2
Passed a screen of 32 meshes to the inch.
-------
Figure 1
1. Douglas fir bark from veneer logs.
2. Through hanonermill containing 1/4-inch screen.
3. Reground to pass 20 mesh screen.
1. Pelleted Sitka spruce bark, 1/2-inch die.
2. Pelleted red alder bark, I/4-inch die.
3. Pelleted Douglas fir bark with fertilizer added, 3/16-inch die,
4. Pelleted Douglas fir bark. 1/lQ-inch die.
NOT REPRODUCIBLE
-173-
-------
Figure 2
A
*
¥#**'•
• wr"
'*
-------
Figure 3
Carbohydrate fraction from Douglas fir inner bark. Left side:
Inner bark starting material, fibrous in nature. Right side:
Carbohydrate (holocellulose) fraction, 40-50% yield, pure
white, still fibrous in nature.
Carbohydrate (holocellulose) fraction from inner bark at a higher
magnification than above. It shows the white, fibrous nature of
the material. --
NOT REPRODUCIBLE
-175-
-------
Figure 4
NOT REPRODUCIBLE
Anatomy of Douglas fir Bark
-176-
-------
Douglas-fir inner bark (100.0 g dry weight)
Extraction with
ethanol-water (A:1 v/v)
r
Kxtract 15.4 g soJids Residue 84.6 g
Extraction with
benzene-ethanol (2:1 v/v)
Extract 4.4 g solids
Residue 80.2 g
Extraction with hot water
Extract 11.1 g solids
Extract 2.8 g solids
Residue 69.1 g
Extract 22.0 g solids
Extraction with 0.5%
aqueous ammonium oxalate
Residue 66.3 g
Acidified sodium
chlorite treatment
Residue 44.3 g
Acidified sodium
chlorite treatment
Extract 14.3 g solids
Residue 30.6 g
(Holocellulose)
Figure 5. Isolation of the holocellulose fraction of Douglas-fir inner
bark, (g/100 g dry-weight basis).
-177-
-------
Hot-water-soluble solids from Douglas-fir inner
bark (11.1 g from 100,0 g of original Inner bark)
Extraction with water
at room temperature
Water solubles 7.8 g
Precipitation with 70%
ethanol then centrifuge
Residue 3,3 g
Fraction A
70% ethanol solubles
4.9 g, "Fraction C"
Residue 2.9 g
"Traction B"
HT-1000 then Diazyme
L 30 then dialysis
Dialyzables
Dialyzables
Non-dialyzables 1.1 g
Chymotrypsin then
trypsin then dialysis
Precipitate Non-dialyzables 0.16 g
"Fraction D"
Figure 6. Purification of the polysaccharides extracted by hot water.
-178-
-------
Hoiocellulose (100.0 g, dry weight)
I'
|
i
Extract
Methanol to 70.0%
Precipitate
Hemicellulose A 7.0 g
20% Ba(OH)2, then KOH to 10.0%
Residue A 84.6
1.0% NaOH
Extract
Methanol to 70.0%
Precipitate
Hemicellulose B 1.7 g
Extract
Methanol to 70.0%
Precipitate
Hemicellulose C 2.9 g
Residue B 78.2 g
15.0% NaOH
Residue C 62.6
Figure 7. Fractionation of the holocellulose from Douglas-fir inner bark
into its component hemicellulosea.
-179-
------- | |