CELLULOSE CHEMISTRY AND TECHNOLOGY

A THREE-STAGE KLASON METHOD FOR MORE ACCURATE

DETERMINATIONS OF HARDWOOD LIGNIN CONTENT

DANIEL J. NICHOLSON,* AARON T. LEAVITT** and RAYMOND C. FRANCIS***

*
Department of Paper and Bioprocess Engineering (PBE), SUNY College of Environmental Science and
Forestry (SUNY ESF), 1 Forestry Drive, Syracuse, NY 13210, USA
**
Andritz Pulp and Paper, Andritz Inc., 1115 Northmeadow Parkway, Roswell, GA 30076, USA
***
Scientific Consultant, Syracuse, NY 13210, USA (on leave from PBE Department, SUNY ESF)

Received July 20, 2013

A three-stage Klason, or 72 wt% sulfuric acid, method is described for hardwoods. As compared to the traditional two-
stage method, the primary hydrolysis (PH) stage was modified to improve mixing of the wood meal into the 72%
H2SO4. The PH stage was followed by two 1 h secondary hydrolysis (SH) stages. The 72% H2SO4 slurry was first
diluted to 40 wt% H2SO4 and heated at 80 °C then diluted to 3 wt% H2SO4 and refluxed. When equivalent PH stages
were used, convincing evidence was obtained showing that the hydrolytic intensity in the cleavage of lignin-
carbohydrate complexes (LCC) was much higher for the modified SH stages, as compared to the traditional refluxing
of a 3% H2SO4 slurry for 4 h. The accuracy in lignin content determination should be equal or superior for the three-
stage method, which takes approximately 2 h less time. Results are presented for 4 hardwoods from 3 different genera.

Keywords: hardwoods, lignin content, sulfuric acid hydrolysis, acid insoluble lignin, acid soluble lignin

INRODUCTION
Research on the sulfuric acid method for lignin and boiled under reflux for 4 h in a secondary
content determination began in the early 1900’s. hydrolysis (SH) stage.3
By 1923, Peter Klason realized that optimization Ritter et al. observed no significant difference
of the primary hydrolysis (PH) stage with in Klason or acid insoluble lignin (KL or AIL)
concentrated H2SO4 was critical.1 He concluded yield, when the PH stage was performed for 1-3 h
that 61-68 wt% H2SO4 at a low temperature was at 20 °C.3 For a 2 h treatment, Ritter et al.
close to optimum and used his method to obtain observed only a slight increase in KL or AIL in
an acid insoluble lignin (AIL) content of 28% for going from 20 °C to 25 °C or 30 °C (21.6% vs.
European spruce wood. AIL values in the range of 21.8% vs. 22.0%). The standard sulfuric acid or
28% for European spruce is now widely Klason method now calls for ~1.0 g of wood meal
accepted.2 The 72% sulfuric acid method was (oven-dried or OD basis) in 15 ml of 72% H2SO4
later published by Ritter et al. in 1932.3 This with 2 h of hydrolysis at 20 °C in the PH stage.
method is still the most commonly used method This is followed by dilution to 3% H2SO4 and
for lignin content determination of woody reflux at ~100 °C for 4 h.4,5 This method is
species4,5 and modifications to the conditions used referred to as the traditional method in this
by Ritter et al.3 have been minor. The AIL by the publication, since there have not been any
method of Ritter et al.3 is now called Klason significant changes to it since 1967.4,5 In the
lignin. Extensive data were presented on the laboratory of the present authors, the PH standard
effect of PH treatment time in 72% H2SO4 at is 1.0 g of air-dried wood meal (of known
different temperatures.3 The hardwood used was moisture content) in 15 ml of 72% H2SO4 for 2 h
sugar maple (Acer saccharum) and the at 25 °C and with a mixing protocol that will be
consistency of the reaction slurry was ~1.0 g of described later.6
air-dried wood meal in 12.5 ml of 72% H2SO4. Whether or not the standard or traditional
After PH the slurry was diluted to 3 wt% H2SO4 method consistently cleaves all carbohydrates

Cellulose Chem. Technol., 48 (1-2), 53-59 (2014)

DANIEL J. NICHOLSON et al.

from the KL or AIL has been an issue of on-going first attempt with 1.0 g of a poplar wood meal in
concern. An example would be the 1984 results of 16 ml of 72% H2SO4, no adjustment was made for
Matsumoto et al.7 Those authors used 1.0 g of the increased time requirement that would be
wood meal in 20 ml of 72% H2SO4 and ran their needed for the mixing of 1.0 g instead of 0.5 g of
PH stage for 4 h at 20 °C. Their PH stage was wood meal in 16 ml of 72% H2SO4. The true
followed by dilution of the slurry to 3% H2SO4 glucan and xylan content of the sample was
and refluxing for 2 h in a SH stage. Matsumoto et 51.0% and 17.5%, respectively and the
al. found that the KL or AIL from a softwood summative analysis was 99.7%.6 However, in
contained 0.75% neutral sugars (% on KL or their initial trial only one of nine samples gave
AIL), while the value was 0.95% for a hardwood glucan and xylan yields >49% and >17%,
KL or AIL.7 Furthermore, they commented that respectively. Those authors later isolated the
the actual sugar contents on the KL or AIL were problem to inadequate mixing in the PH stage.6
likely to be higher than the values above.7 When 1.0 g of wood meal is being used for either
While performing research on carbohydrate lignin or sugar monomers quantification, nine
compositional analyses of hardwoods, Bose et al. runs at a time is not recommended.
obtained data indicating the possibility of a The results of Bose et al.6 would suggest that
significant retention of carbohydrates on the AIL if mixing is inadequate, incomplete hydrolysis of
if mixing in the PH stage was inadequate.6 The lignin-carbohydrate (L-C) linkages, is possible
PH stage used in that research6 will be described when 1.0 g of wood meal is hydrolyzed in 15 ml
shortly and was very similar to that of the of 72% H2SO4 in the PH stage of the Klason
standard Klason method.4,5 Most quantification of method. There is no standard related to adequate
carbohydrate monomers from biomass use a mixing in the PH stage of the traditional Klason
hydrolysis protocol close to the one first reported method.4,5 The objective of this research was to
in 1954 by Saeman et al.8 That method calls for investigate a three stage hydrolytic procedure for
~0.5 g or biomass meal to be PH treated in 3 ml KL or AIL to see if a more extensive cleavage of
of 72% H2SO4 for 1 h at 30 °C, the slurry is then L-C linkages could be obtained. After the PH
diluted to 4% H2SO4 and autoclaved for an stage, the sample would be diluted to 40% H2SO4
additional hour at 121 °C.8-11 Bose et al. tried to and hydrolyzed at 80 °C for 1 h (SH-1) then
improve on xylose yield from the method of further diluted to 3% H2SO4 and refluxed for 1 h
Saeman et al.,8 by increasing the acid hydrolytic at ~100 °C (SH-2).
intensity in a modified two stage hydrolysis.6 The research program was greatly influenced
Their idea was to perform the SH stage in 40% by KL or AIL values reported for extractive-free
H2SO4 at 80 °C instead of 4% H2SO4 at 121 °C. sugar maple, a hardwood species commonly used
The thinking of Bose et al. was aided by the fact by the present research group.13-16 Ritter et al.
that they had access to a method and equipment obtained seven close to optimum KL or AIL
for sugar monomer analysis by 1H NMR.6 A high values in the range of 21.5% to 22.0% for this
H2SO4 concentration is helpful to the 1H NMR species.3 Later, a value of 22% was reported by
method, because it shifts the H2O peak out of the the US Forest Service.17 The present research
carbohydrate region in the NMR spectrum.12 group has analyzed many samples of this species
Saeman et al. used only 3 ml of 72% H2SO4 in PH by the standard or traditional method, but always
because of the large dilution factor in going from with good mixing in the PH stage. At least five
72% to 4% H2SO4 in the SH stage.8 Since their samples were harvested from different sites in
dilution would be from 72% to only 40% H2SO4, Central New York and one sample from Central
Bose et al. could afford to use 0.5 g of wood meal Pennsylvania. The KL or AIL values all fell in a
in 16 ml of 72% H2SO4 for 2 h at 25 °C in the PH narrow range of 21.5% to 22.5%. However, others
stage.6 Due to both stages being performed at have reported KL or AIL values >24% on an
<100 °C, this method could handle more samples extractive-free basis for North American grown
simultaneously.6 After the PH stage, those authors sugar maple.18,19 As previously discussed, the
simply added 21 ml of H2O to the PH slurry, hydrolytic procedures for KL or AIL and the
sealed the centrifuge tube, shook it vigorously and generation of sugar monomers are fairly similar.
placed it in an 80 °C water bath. During their Kaar et al.10,11 used the Saeman method8 to
initial data collection with 0.5 g of wood meal in analyze seven hardwoods and three softwoods.
16 ml of 72% H2SO4, nine samples were Their summative analyses varied from 98.3% to
hydrolyzed simultaneously.6 However, in their 99.6% for the ten samples. One interesting aspect

54
 Lignin

of the research of Kaar et al. was that they only ~10 minutes. The slurry was mixed after 15
estimated their AIL by filtering the slurry after minutes of reaction and every 15 minutes thereafter.
one hour at 121 °C SH hydrolysis.10,11 On the This PH protocol was used for both methods.
other hand, the modified method of Bose et al.6 In the traditional method, there were no changes to
the standard 4 h SH stage.4,5 In the three-stage method,
for sugar monomers along with the traditional
19.6 g of water was added to the PH slurry that started
Klason + acid soluble lignin (ASL) methods were with 15 ml of 72% H2SO4 (density of 1.63 g/cm3). The
used to determine the contents of lignin and centrifuge tube now containing a 40 wt% H2SO4
carbohydrates in three temperate hardwoods,6 a solution was vigorously shaken before being heated in
Brazilian eucalyptus,20 three Mediterranean an 80 °C thermostat controlled water bath for 60
eucalypti,21 a tropical hardwood,22 two sugar cane minutes (SH-1). The sample was shaken every 15 min.
bagasse samples20 and a bamboo sample.20 The That solution was then diluted to 3 wt% H2SO4 and
summative analyses varied from 98.5-101.5% for refluxed for 1 h (SH-2). A few experiments were
all the samples.6,20-22 Coincidentally, sugar maple performed where the PH stage was shortened to 15
was the only overlapping species between the minutes and with frequent mixing (every 1-2 min).
This was followed by the conventional 4 h SH or SH-1
research of Kaar et al.10,11 and the Bose et al.
+ SH-2 treatment from above.
method. Kaar et al. obtained lignin and xylan
contents of 28.9% (Klason + ASL) and 16.1% on Acid soluble lignin (ASL) determination
an extractive-free basis, respectively.11 In The lignin remaining soluble in the 3% H2SO4
contrast, Bose et al. obtained corresponding solution after filtration of the KL or AIL was estimated
values of 25.0% and 18.6%, respectively.6 It by the UV spectroscopic method described in Tappi
appeared to the present group of researchers that Method UM 250.24 There was no change to the
SH stages in 3% H2SO4 for 4 h at ~100 °C4,5 or in traditional filtration process.4,5 A standard dilution
4% H2SO4 for 1 h at 121 °C8-11 may not always factor of 20 and lignin absorptivity of 110 l/g.cm at
offer sufficient cleavage of L-C linkages in 203 nm were used.
hardwoods, thus resulting in an artificially high
Elemental composition of Klason lignins
value for lignin content and an artificially low The analyses were performed by Galbraith
value for xylan content. This was the primary Laboratories, Inc., Knoxville, Tennessee, USA.
motivation for research on SH in 40% H2SO4 at
80 °C. This SH condition was previous shown to RESULTS AND DISCUSSION
be effective at cleaving almost all of the L-C Preliminary research with W 79
linkages in both hardwoods and non-woods.6,20-22 The research was initiated with poplar W 79
with a reported total lignin content (Klason +
EXPERIMENTAL ASL) of 27.3%.23 In order to get some idea of the
Wood meal samples importance of the PH stage on the cleavage of
Starting with debarked wood chips, wood meals
lignin-carbohydrate complexes (LCC), the PH
(30 mesh) were prepared from paper birch (Betula
papyrifera Marsh), sugar maple, aspen (Populus time in the traditional method was shortened to 15
tremuloides) and a poplar identified as W 79. The min. Very high duplicate values of both AIL and
birch, maple and aspen were harvested in Central New ASL were obtained for W 79 (Table 1). The
York, while W 79 was harvested from a commercial duplicate values were 32.7% and 35.3% for AIL
plantation in Wallula, WA and is described and 3.7% and 3.6% for ASL. These AIL values
elsewhere.23 All of the wood meals were soxhlet appeared unexpectedly high because both wood
extracted by a 2:1 (v/v) mixture of toluene/ethanol. meal samples were completely liquefied 5-10 min
into the PH stage. It was assumed that a 4 h reflux
Traditional and modified Klason determinations in 3 wt% H2SO4 would have cleaved most of the
The PH stage was performed with 1.00 g of air-
LCC linkages. The next experiment was to
dried wood meal in 15 ml of 72% H2SO4 in a 50 ml
polypropylene centrifuge tube with a maximum confirm that poplar W 79 did indeed have a lignin
useable temperature of 121 °C. The reaction content of only ~27% when a 120 min PH was
temperature was 25 °C and the wood meal was used. This was confirmed and duplicate total
thoroughly mixed into the 72% H2SO4 at the lignin values of 27.0% and 26.7% were obtained
beginning. A glass rod was used and it was not by the traditional method (Table 1). The KL or
removed from the centrifuge tube until the end of the AIL values were 24.0% and 24.1% for PH = 120
PH stage. The use of the centrifuge tube with a conical min. The experiment with PH = 15 min was
bottom allowed for the sample to be converted to a repeated a few days later and the duplicate values
liquid with only a small amount of fine particles after

55
DANIEL J. NICHOLSON et al.

were 32.8% and 34.2% for AIL and 4.3% and between PH = 15 min and 120 min was 9.7% on
4.0% for ASL. The four AIL data points for PH = wood (33.8%-24.1%). Kaar et al. analyzed all
15 min gave an average of 33.8%. This value seven of their hardwood species for protein and
clearly indicated that significant amounts of non- ash content and the total of the two was <1.0% on
lignin components were attached to the AIL and wood in all cases. Therefore, approximately 9.0%
almost all of these components had to be of on wood of the non-lignin component attached to
carbohydrate origin. The difference in AIL values the AIL had to be of carbohydrate origin.

Table 1
Lignin content of extractive-free W 79 poplar by traditional and modified methods

PH time T or M1 Klason lignin2 ASL2 Total2

(min)
15 T 32.7 (35.3)3 3.7 (3.6)3 36.4 (38.9)3
15 T4 32.8 (34.2) 4.3 (4.0) 37.1 (38.2)
120 T 24.1 (24.0) 2.9 (2.7) 27.0 (26.7)
15 M 27.2 (26.5) 2.8 (2.6) 30.0 (29.1)
15 M4 28.3 (28.0) 2.6 (2.3) 30.9 (30.3)
120 M 24.7 (24.2) 2.4 (2.5) 27.1 (26.7)
1
Traditional or modified; 2 wt% based on wood meal (OD basis); 3 Duplicate result in
parentheses; 4 Performed at a later date compared to run above

0.3

0.25

3-Stage Klason
0.2
Absorbance.

3-Stage Klason

0.15 Traditional Klason

Traditional Klason
0.1

0.05

0
185 205 225 245 265 285 305
Wavelength, nm

Figure 1: UV spectra of filtrate from traditional and modified Klason determinations performed on W 79 poplar
(duplicates for each method)

The modified or three stage method (PH = 120 UV spectra for the four diluted Klason filtrate
min) was then attempted and duplicate total lignin (duplicates for both traditional and modified
contents of 27.1% and 26.7% were obtained. methods) are shown in Figure 1. The trend of
These values were nearly identical to the two slightly higher KL or AIL and lower ASL for the
stage method. However, the KL or AIL values modified method was consistently observed for
were slightly higher for the modified method, all four wood species (W 79, sugar maple, birch
while the ASL values were slightly lower. This and aspen).
may be due to a higher rate of lignin-lignin
condensation25 in the three stage method and Determinations using the modified method and
particularly in SH-1 that utilized 40% H2SO4 at 80 PH = 15 minutes were then performed. The
°C. The duplicate values of KL or AIL were duplicate values for AIL were 27.2% and 26.5%.
24.7% and 24.2% for the modified method, as The experiments were repeated at a later date and
compared to 24.1% and 24.0% for the traditional duplicate AIL values of 28.3% and 28.0% were
method. The corresponding numbers for ASL obtained. The average AIL value for the four runs
were 2.4% and 2.5% for the modified method and was 27.5%, which is much lower than the 33.8%
2.9% and 2.7% for the traditional method. The value obtained when PH = 15 min was used in the

56
 Lignin

traditional method. The PH = 15 min results associated with filtering a 40 wt% H2SO4 slurry is
prove that the hydrolytic intensity was higher likely to be more significant than filtering a 3
when the two SH stages of the modified method wt% H2SO4 slurry. Since SH-1 was a straight-
were compared to the 4 h SH stage of the forward 1 h hydrolysis, it was concluded that an
traditional method. While the traditional method additional 1 h reflux was preferable to filtering a
gave an AIL with 9.7% on wood of non-lignin 40 wt% H2SO4 slurry. Even with this additional
components (33.8%-24.1%), the value was only SH-2 treatment, the modified method is still ~2 h
3.4% for the modified method (27.5%-24.1%). shorter than the traditional method.
Furthermore, when PH = 120 min plus the present
40 wt% H2SO4 SH-1 treatment were used to Other wood species
produce carbohydrate monomers, not much The traditional and modified methods (PH
furfural or hydroxymethylfurfural was generated.6 =120 min in both) were then compared using
If these compounds were to be generated at sugar maple. The results (KL + ASL) for the
significant yields from xylose and glucose, modified method were 22.4% + 2.5% and 22.5%
respectively, they could possibly condense with + 2.5% (Table 2). For the traditional method, one
lignin26 and result in an artificially high KL or sample gave 22.0% + 3.1%, but the other sample
AIL content. was lost due to experimental error. The traditional
Based on the earlier results showing almost method was repeated at a later date and duplicate
complete hydrolysis of carbohydrates from lignin results of 22.0% + 2.9% and 22.3% + 2.8% were
and their depolymerization to monomers,6,20-22 it obtained (Table 2). Therefore, total lignin
is highly likely that the SH-2 hydrolysis in the contents of 24.9% and 25.0% were obtained from
modified method is not needed for accurate the modified method, while values of 25.1%,
determinations of lignin content. However, the 24.9%, and 25.1% were obtained by the
standard method is based on filtration of KL or traditional method. It should be noted that for the
AIL from a 3 wt% H2SO4 slurry after a reflux traditional method, KL or AIL values in the range
treatment. Filtering the KL or AIL from a 40 wt% of 22.0%-22.3% were obtained (Table 2) and this
H2SO4 slurry would introduce an additional is very similar to the 21.5%-22.0% range reported
variable to be investigated. Also, the safety risk by Ritter et al.3

Table 2
Lignin content of extractive-free maple, birch and aspen by traditional and modified methods

PH time T or M1 Klason lignin2 ASL2 Total2

(min)
Sugar maple
120 T 22.0 3.1 25.1
120 T 22.0 (22.3)3,4 2.9 (2.8) 24.9 (25.1)
120 M 22.4 (22.5) 2.5 (2.6) 24.9 (25.1)
Paper birch
120 T 18.5 (18.7) 3.7 (3.7) 22.2 (22.4)
120 M 19.2 (18.9)) 3.3 (3.3) 22.5 (22.2)
Aspen
120 T 18.0 (18.3) 2.3 (2.3) 20.3 (20.6)
120 M 18.5 (18.3) 1.9 (1.9) 20.4 (20.2)
1
Traditional or modified; 2 wt% based on wood meal (OD basis); 3 Duplicate results in
parentheses; 4 Performed at a later date compared to run above

Table 3
Comparison of elemental composition of KL or AIL between modified and traditional method

Wood sample %C %H %O %S
Poplar W79 60.5 (60.6)1 5.6 (5.7) 31.7 (31.9) <0.2 (<0.2 )
Sugar maple 58.7 (59.0) 5.5 (5.6) 33.2 (33.1) <0.2 (<0.2 )
Paper birch 60.4 (60.4) 5.5 (5.7) 32.0 (32.2) <0.2 (<0.2 )
1
Traditional KL or AIL followed by modified KL or AIL in parentheses

57
DANIEL J. NICHOLSON et al.

The traditional method was then compared to effectiveness of the PH stage was deliberately
the modified method for paper birch and aspen. lowered by its treatment time, being decreased
The duplicates for birch by the traditional method from 120 min to 15 min, it was observed that the
averaged a total lignin of 22.3%, while the SH-1 + SH-2 treatments of the modified method
average for the modified method was 22.4% afforded an AIL value of 27.5% for a poplar,
(Table 2). The corresponding values for the aspen while the corresponding value for the single 4 h
were 20.5% by the traditional method and 20.3% SH stage of the traditional method was 33.8%. In
by the modified method (Table 2). each case, the poplar sample was analyzed in
duplicate on separate days, thus each average
Preliminary results regarding the acid soluble above was for 4 different runs. The minimum AIL
lignin (ASL) value (PH = 120 min) for the poplar was 24.1%.
The modified method gave consistently lower The 33.8% and 27.5% AIL values for the
ASL values (~0.4% on wood) than the traditional and modified methods when PH = 15
conventional method. These lower values appear min was used clearly indicated that significant
reasonable in light of the fact that the modified amounts of non-lignin components were attached
method gave a slightly higher KL or AIL. to the AIL (9.7% for the traditional method and
However, some preliminary research was 3.4% for the modified method). Kaar et al.
performed to get an indication as to whether or analyzed all seven of their hardwood species for
not the ASL from the modified method could protein and ash content and the total of the two
have had a significantly different absorptivity was <1.0% on wood in all cases. Therefore,
value, as compared to the ASL from the almost all of these non-lignin components had to
traditional method. If there were to be a be of carbohydrate origin. The difference between
significant difference in ASL, then one would 33.8% AIL for the traditional method and 27.5%
expect some significant differences in the KL or for the modified method is a clear demonstration
AIL as well. When elemental analyses were that the hydrolytic intensity in LCC cleavage was
performed on KL or AIL from three of the wood much higher for the modified method.
species, there were no significant differences in C,
H, O, or S content between the traditional and ACKNOWLEDGMENTS: The authors are most
modified methods (Table 3). grateful for the financial support from NSF
Interestingly, the ranges of C, H, and O Award CHE 1105726 and Andritz, Inc. Thanks
content in Table 3, i.e. 58.7%-60.6% for C, 5.5%- are also due to Mr. Adebukola Abiola and Ms.
5.7% for H and 31.7%-33.2% for O are very Candace Guilford, undergraduate interns, who
similar to values reported by Bjorkman and performed some repeat experiments with the
Person27 for milled wood lignin (MWL) from modified method and confirmed earlier results.
three other hardwoods. Those values were 58.8%-
60.4% for C, 6.1%-6.5% for H and 33.0%-34.0% REFERENCES
1
for O. The decrease in H and O contents for the P. Klason, Svensk Papperstidn., 26, 319 (1923).
2
KL or AIL, as compared to MWL, is due in part D. Fengel and G. Wegener, “Wood
(most likely) to the loss of OH groups from α- Chemistry, Ultrastructure, Reactions”,
carbon atoms in the lignin side chain, as a result Walter deGruyter, Berlin, 1979, p. 56.
3
G. J. Ritter, R. M. Seborg, R. L. Mitchell, Ind. Eng.
of acid-catalyzed lignin condensation (C-C bond
Chem. Anal. Ed., 4, 202 (1932).
formation) reactions during H2SO4 treatments.25 4
B. L. Browning, “Methods of Wood Chemistry”,
However, the losses in H and O were not major Interscience Publishers, New York, Vol. 2, 1967, p.
and KL or AIL may be less condensed than 785.
previously thought.28 5
Tappi Standards, Acid-insoluble lignin in wood and
pulp, Tappi Method T 222 om-06, Tappi Press,
CONCLUSION Atlanta, GA, 2006.
6
The Klason, or 72 wt% sulfuric acid, method S. K. Bose, V. A. Barber, E. F. Alves, D. J. Kiemle,
for the determination of lignin content is very A. J. Stipanovic, R. C. Francis, Carbohyd. Polym., 78,
accurate and reproducible if performed by 396 (2009).
7
Y. Matsumoto, A. Ishizu, J. Nakano, J. Wood Chem.
experienced analysts. However, data in the
Technol., 4, 321 (1984).
literature suggest that the KL or AIL can contain 8
J. S. Saeman, W. E. Moore, R. L. Mitchell, M. A.
significant amounts of carbohydrate residues if Millet, Tappi J., 37, 336 (1954).
mixing is inadequate in the PH stage. When the

58
 Lignin

9 18
Tappi Standards, Carbohydrate composition of T. B. Vinzant, C. I. Ehrman, W. S. Adney, S. R.
extractive-free wood and wood pulp by gas-liquid Thomas, M. E. Himmel, Appl. Biochem. Biotechnol.,
chromatography, Tappi Method T 249 cm-85, Tappi 62, 99 (1997).
19
Press, Atlanta GA, 1985. P. Watson, A. Hussein, S. Reath, W. Gee, J. Hatton,
10
W. E. Kaar, L. C. Cool, M. M. Merriman, D. L. J. Drummond, Tappi J., 2 (6), 26 (2003).
20
Brink, J. Wood Chem. Technol., 11, 447 (1991). E. F. Alves, S. K. Bose, R. C. Francis, J. L.
11
W. E. Kaar and D. L. Brink, J. Wood Chem. Colodette, M. Iakovlev, A. Van Heiningen, Carbohyd.
Technol., 11, 479 (1991). Polym., 82, 1097 (2010).
12 21
D. J. Kiemle, A. J. Stipanovic, K. E. Mayo, ACS M. El Moussaouiti, B. Barcha, E. F. Alves, R. C.
Symp. Series, 864, 122 (2004) Francis, BioResources, 7, 1558 (2012).
13 22
R. C. Francis, T. S. Bolton, N. D. Biswas, M. Misbahuddin, U. Roy, R. C. Francis,
Abdoulmoumine, N. Lavrykova, S. K. Bose, S. K. Bose, Bioresource Technol., 102, 1284 (2011).
23
Bioresource Technol., 99, 8453 (2008). S. K. Bose, R. C. Francis, M. Govender, T. Bush, A.
14
S. K. Bose, S. Omori, D. Kanungo, N.-H. Spark, Bioresource Technol., 100, 1628 (2009).
24
Shin, R. C. Francis, J. Wood Chem. Technol., Tappi Standards, Acid-soluble lignin in wood and
29, 214 (2009). pulp, Tappi Method UM 250. Tappi Press, Atlanta GA
15
D. J. Nicholson, G. V. Duarte, E. F. Alves, D. J. (1985)
25
Kiemle, R. C. Francis, J. Wood Chem. Technol., 32, J. Gierer, Wood Sci. Technol., 19, 289 (1985).
26
238 (2012). H. Koch, J. Pein, Polym. Bull., 13, 525 (1985).
16 27
R. C. Francis, U.S. Patent 8,303,767 (Nov. 6, A. Bjorkman, B. Person, Svensk Papperstidn., 60,
2012). 158 (1957).
17 28
R. C. Pettersen, Adv. Chem. Series (ACS), 207, 57 S. Yasuda and K. Ota, Holzforschung, 41, 59 (1987).
(1984).

59