Toward a Better Understanding of the Lignin Isolation Process
ANDERSON GUERRA,†ILARI FILPPONEN,†LUCIAN A. LUCIA,†CARL SAQUING,†
STEPHANIE BAUMBERGER,‡AND DIMITRIS S. ARGYROPOULOS*,†
Department of Forest Biomaterials Science and Engineering, College of Natural Resources,
North Carolina State University, Raleigh, North Carolina 27695-8005, and UMR Chimie Biologique,
INRA/INA PG, 78850 Thiverval-Grignon, France
The recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) coupled with
the novel combination of derivatization followed by reductive cleavage (DFRC) and quantitative31P
NMR spectroscopy were used to better understand the lignin isolation process from wood. The EMAL
protocol is shown to offer access at lignin samples that are more representative of the overall lignin
present in milled wood. The combination of DFRC/31P NMR provided a detailed picture on the effects
of the isolation conditions on the lignin structure. More specifically, we have used vibratory and ball
milling as the two methods of wood pulverization and have compared their effects on the lignin
structures and molecular weights. Vibratory-milling conditions cause substantial lignin depolymeri-
zation. Lignin depolymerization occurs via the cleavage of uncondensed ?-aryl ether linkages, while
condensed ?-aryl ethers and dibenzodioxocins were found to be resistant to such mechanical action.
Condensation and side chain oxidations were induced mechanochemically under vibratory-milling
conditions as evidenced by the increased amounts of condensed phenolic hydroxyl and carboxylic
acid groups. Alternatively, the mild mechanical treatment offered by ball milling was found not to
affect the isolated lignin macromolecular structure. However, the overall lignin yields were found to
be compromised when the mechanical action was less intense, necessitating longer milling times
under ball-milling conditions. As compared to other lignin preparations isolated from the same batch
of milled wood, the yield of EMAL was about four times greater than the corresponding milled wood
lignin (MWL) and about two times greater as compared to cellulolytic enzyme lignin (CEL). Molecular
weight distribution analyses also pointed out that the EMAL protocol allows the isolation of lignin
fractions that are not accessed by any other lignin isolation procedures.
ball milling; lignin
Enzymatic mild acidolysis lignin; MWL; CEL; DFRC;31P NMR; spruce; vibratory milling;
Lignin is a heterogeneous and highly cross-linked macro-
molecule that represents the second most abundant natural
polymeric material on earth (1). Despite extensive investigations,
the complex and irregular structure of lignin is not completely
understood (2). It is known that the bulk of lignin in wood
consists of nonphenolic aryl-glycerol-?-O-aryl ether units. Other
units, such as phenylcoumaran (?-5), resinol (?-?), and diben-
zodioxocins (5-5/?-O-4, R-O-4) are also present within the lignin
macromolecule (1, 3). Furthermore, lignin is covalently linked
to carbohydrates (4, 5) forming a lignin-carbohydrate network
made up of benzyl-ether (4, 6), benzyl-ester (4, 7, 8), and
phenyl-glycoside (9-11) bonds.
One of the most important problems in elucidating lignin
structure has been the isolation of the total lignin from wood in
a chemically unaltered form (12-14). Despite many efforts, the
isolation of a highly representative and totally unaltered native
lignin is still a challenge (14, 15). A major advance toward this
objective was made when Bjo ¨rkman (16, 17) developed a lignin
isolation procedure based on the extraction of extensive ball-
milled wood by neutral solvents at room temperature. This
procedure has been widely used to isolate lignin from different
species (15). However, it offers lignin that may not be fully
representative of the total lignin present in milled wood (18,
19). The moderate yields usually achieved in this procedure can
be increased by up to 50% if one extends the milling time (20).
However, a milled wood lignin (MWL) less representative of
the native lignin is obtained (14, 21).
Another important contribution to the isolation of lignin was
introduced by Pew and Weyna (22). They treated ball-milled
* To whom correspondence should be addressed. Tel: 919-515-7708.
Fax: 919-515-6302. E-mail: email@example.com.
†North Carolina State University.
‡UMR Chimie Biologique.
J. Agric. Food Chem. 2006, 54, 5939−5947
10.1021/jf060722v CCC: $33.50© 2006 American Chemical Society
Published on Web 07/19/2006
wood with cellulolytic enzymes and obtained an insoluble
residue containing almost all of the lignin present in spruce and
aspen woods. Nevertheless, the residue had as much as 12% of
carbohydrates and no further characterization was attempted.
Chang et al. (12) also treated milled wood with enzymes, but
used an enzymatic preparation with greater cellulolytic and
hemicellulolytic activities than the enzyme used by Pew. In
addition, they extracted the insoluble residue obtained after the
enzymatic hydrolysis successively with 96% and 50% aqueous
dioxane. The combination of these two fractions offered higher
yields than MWL (12). However, the lignin fraction soluble in
50% aqueous dioxane contained twice as many carbohydrates
as MWL or cellulolytic enzyme lignin (CEL96). Recently,
Holtman et al. (23) have also reported some subtle structural
differences between MWL and CEL.
Wu and Argyropoulos (15) have proposed a novel lignin
isolation procedure composed of an initial mild enzymatic
hydrolysis of milled wood, followed by a mild acid hydrolysis
stage. In this procedure, the initial cellulolytic action removes
most of the carbohydrates while the mild acidolysis is designed
to cleave the remaining lignin-carbohydrate bonds, liberating
lignin in high yield and purity. Despite the significant improve-
ments in yield and purity offered by this method, few efforts
have been made to further advance the understanding on the
effect of each step on the structure of the resulting enzymatic
mild acidolysis lignin (EMAL). In a similar procedure aimed
at residual kraft lignins, Argyropoulos et al. (24) have optimized
the enzyme charge to minimize lignin contamination by proteins.
Furthermore, Wu and Argyropoulos (15) have reported the
effects of the acid concentration on the structure of such lignins.
Nevertheless, no attempts have been made to unravel and
address the effects of the milling conditions on the yield, purity,
and structure of EMAL.
In another front, many efforts have been made to elucidate
the effects of mechanical action on the structure of lignin.
However, the conclusions reached have been limited to the
characterization of only 25-30% of the overall lignin actually
present within the original wood (13, 14). Furthermore, these
studies have been restricted to the characterization of the
uncondensed moieties of the lignin. Few efforts have been made
to evaluate the effect of milling conditions on the condensed
lignin moieties (25). Consequently, a study on the effect of
milling on the structure of EMALs, which is known to provide
lignin in high yield and purity, by using a characterization
method designed to quantify condensed and uncondensed
structures is warranted.
A novel approach for the quantification of different lignin
structures using the combination of derivatization followed by
reductive cleavage (DFRC) and quantitative31P NMR was
recently described (26). Because quantitative31P NMR deter-
mines the amounts of the various hydroxyl groups, such spectra
“before DFRC” provide quantitative information about the
aliphatic hydroxyls, carboxylic groups, and condensed and
uncondensed units bearing phenolic hydroxyl groups within
lignin. Such hydroxyl groups are revealed and quantified by
31P NMR after phosphitylating lignin with 2-chloro-4,4,5,5-
tetramethyl-1,3,2-dioxaphospholane (26). Unfortunately, quan-
etherified or carbon-carbon-linked bonding pattern of lignin.
However, when the aryl ether linkages are selectively cleaved
by DFRC, the corresponding phenolic hydroxyls released can
be quantified by31P NMR. In this way, the31P NMR spectra
“after DFRC” offer detailed information about condensed and
31P NMR cannot offer any information about the
uncondensed units connected through ?-aryl ether linkages (26)
as well as dibenzodioxocins.
Overall, therefore, this study applies the recently described
procedure to isolate EMAL and then uses the combination of
DFRC with quantitative31P NMR spectroscopy (DFRC/31P
NMR) to better understand the lignin isolation process from
Norway spruce wood. An assessment on the effects of the
isolation procedure on structures never reported before (diben-
zodioxocins and ?-aryl ether linkages connected to condensed
units) is made. The detailed structural analyses of the EMALs
obtained are also compared to MWL and CEL isolated and
purified from the identical batch of milled wood.
MATERIALS AND METHODS
Isolation of EMALs, MWL and CEL. Unbleached Norway spruce
thermomechanical pulp (TMP) was sampled in a Swedish mill. The
TMP was of approximately 38% consistency and 85 mL of Canadian
Standard Freeness prepared by one-stage refinement and a subsequent
reject refinement (about 20%) stage. The pulp was sampled at the press
stage after the refined and refined reject pulps had been combined.
This pulp currently represents a standard sample, which is the subject
of Cost Action E 41 entitled “Analytical Tools with Applications for
Wood and Pulping Chemistry” operated by the European Union. The
pulp was ground to pass a 20 mesh screen in a Wiley mill and Soxhlet
extracted with acetone for 48 h. The resulting Wiley-milled wood
powder was air-dried and stored in a desiccator under vacuum. Rotary
ball milling was performed in a 5.5 L porcelain jar in the presence of
474 porcelain balls (9.4 mm in diameter), which occupied 18% of the
active jar volume. One hundred grams of extractive-free wood powder
was loaded into the jar, creating a porcelain ball/wood weight ratio of
16.6. The milling process was conducted at room temperature for up
to 25 days with a rotation speed of 60 rpm. Vibratory milling was
performed in a 0.6 L jar loaded with 10 g of extractive-free wood
powder and 2.44 kg of stainless steel balls (6.1 mm in diameter), which
occupied 32% of the active jar volume, for periods ranging from 2 to
EMALs were isolated from samples (10 g) of vibratory- or rotary
ball-milled wood according to the procedure described by Wu and
Argyropoulos (15). The ground wood meal was treated with cellulase
(Iogen, Canada; filter paper activity, 130 FPU mL-1) in a previously
optimized (24) ratio of 40 FPU/g wood. The enzymatic hydrolyses were
carried out at 40 °C for 48 h using 50 mM citrate buffer (pH 4.5) at
5% consistency in an orbital water bath shaker. The insoluble material
remained after the enzymatic hydrolysis was collected by centrifugation
(2000g), washed twice with acidified deionized water (pH 2), and
freeze-dried. The crude lignin obtained was further submitted to a mild
acid hydrolysis using an azeotrope (bp, 86 °C) of aqueous dioxane
(dioxane/water 85:15, v/v, containing 0.01 mol L-1HCl) under an argon
atmosphere. The resulting suspension was centrifuged (2000g), and the
supernatant was carefully withdrawn, neutralized with sodium bicarbon-
ate, and finally added dropwise to 1 L of acidified deionized water
(pH 2). The precipitated lignin was allowed to equilibrate with the
aqueous phase overnight, and it was then recovered by centrifugation,
washed (2×) with deionized water, and freeze-dried.
Caution. The acidolysis residue after centrifugation should be
carefully decanted and discarded. Efforts to wash it, so as to increase
the lignin yields, may cause serious carbohydrate contamination in the
MWLs and CELs were isolated from the extractive-free wood and
milled in the vibratory mill for 48 h, according to the methods described
by Bjo ¨rkman (16, 17) and Chang et al. (12), respectively. Both
preparations were purified as described elsewhere (14, 16, 17).
Determination of Lignin Content. Klason lignin (acid insoluble)
and acid soluble lignin contents of wood meal, EMALs, MWLs, and
CELs were measured according to the method reported by Yeh et al.
DFRC Procedure. DFRC was performed as described by Lu and
Ralph (28). The precise amounts of the lignin and precautions due to
the ensuing NMR steps were nearly identical to those reported by
J. Agric. Food Chem., Vol. 54, No. 16, 2006Guerra et al.
Tohmura and Argyropoulos (26). More specifically, 25 mL of acetyl
bromide in acetic acid (8:92, v/v) was added to about 100 mg of a
lignin sample (EMAL, MWL, or CEL) in a 50 mL round-bottom flask.
The flask was sealed and placed in a water bath set at 50 °C for 2 h
with magnetic stirring. The solvent was rapidly evaporated in a rotary
evaporator connected to a high vacuum pump and a cold trap. The
residue was dissolved in an acidic solvent (dioxane/acetic acid/water,
5:4:1, v/v), zinc dust (500 mg) was added, and the mixture was stirred
at room temperature for 30 min. The zinc dust was filtered off, and
the filtrate was quantitatively transferred into another 50 mL round-
bottom flask. The solvent was evaporated, and the DFRC products were
stored at -10 °C for subsequent quantitative31P NMR.
Thioacidolysis. Thioacidolysis was performed on 5 mg of isolated
lignins in 10 mL of reagent according to a published method (29). The
reagent was prepared by introducing 2.5 mL of BF3etherate (Aldrich)
and 10 mL of ethane thiol EtSH (Aldrich) into a 100 mL flask and
adjusting the final volume to 100 mL with dioxane (pestipur grade).
The reagent and 1 mL of a solution of gas chromatography (GC) internal
standard (nonadecane C19, 0.50 mg mL-1in CH2Cl2) were added to
the lignin sample in a glass tube closed with a Teflon-lined screw cap.
Thioacidolysis was performed at 100 °C (oil bath) for 4 h. The cooled
reaction mixture was diluted with 30 mL of water, and the pH was
adjusted to between 3 and 4.0 (aqueous 0.4 M NaHCO3) before
extraction with 3 × 30 mL CH2Cl2. The combined organic extracts
were then dried over Na2SO4 and then evaporated under reduced
pressure at 40 °C. The final residue was redissolved in approximately
1 mL of CH2Cl2before silylation and GC-MS analyses according to
the method of Lapierre et al. (30).
Acetobromination Derivatization Procedure. Acetobromination
was used as the derivatization method of choice for all samples prior
to size exclusion measurements. Approximately 2.5 mL of a mixture
composed of 8 parts of acetyl bromide and 92 parts (v/v) of glacial
acetic acid was added to about 10 mg of a lignin sample (EMAL, MWL,
or CEL) in a 15 mL round-bottom flask. The flask was sealed and
placed in a water bath set at 50 °C for 2 h with continuous magnetic
stirring. The solvent was rapidly evaporated in a rotary evaporator
connected to a high vacuum pump and a cold trap. The residue was
immediately dissolved in THF (5 mL) and subjected to size exclusion
analysis as described below. One precaution, however, that should be
mentioned is the fact that the installed bromine on the lignin is a good
leaving group. As such, the acetobrominated samples need to be rapidly
evaporated from excess solvent using a rotary evaporator in good
working order (preferably equipped with a vacuum pump connected
to a cold trap). In addition, once the sample is derivatized, it should be
immediately diluted with the required amount of THF and never be
allowed to dry. THF solutions of acetobrominated lignins should be
stored in a refrigerator until further use.
SEC. SEC of EMAL, MWL, and CEL samples were performed on
a size exclusion chromatographic system (Waters system) equipped
with UV set at 280 nm and refractive index detectors. The analyses
were carried out at 40 °C using THF as an eluent at a flow rate of 0.44
mL min-1. One hundred and twenty microliters of the sample dissolved
in THF (2 mg mL-1) was injected into a HR5E and HR1 (Waters)
system of columns connected in series. The HR5E column’s specifica-
tion allow for molecular weights up to 4 × 106g mol-1to be reliably
detected. The SEC system was calibrated with polystyrene standards
in the molecular weight range of 890-1.86 × 106g mol-1, and
Millenium 32 SEC software (Waters) was used for data processing.
Quantitative31P NMR. Quantitative31P NMR spectra of all lignin
preparations were obtained using published procedures (31-33).
Approximately 40 mg of dry lignin was transferred into a sample vial,
dissolved in 400 µL of pyridine and deuterated chloroform (1.6:1, v/v),
and left at room temperature overnight with continuous stirring.
N-Hydroxynaphthalimide (200 µL, 11.4 mg mL-1) and chromium(III)
acetylacetonate (50 µL, 11.4 mg mL-1) were used as an internal
standard and relaxation reagent, respectively. Finally, 100 µL of
phosphitylating reagent I (2-chloro-1,3,2-dioxaphospholane) or reagent
II (2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane) was added and
the mixture was transferred into a 5 mm o.d. NMR tube. The spectra
were acquired using a Bruker 300 MHz spectrometer equipped with a
Quad probe dedicated to31P,13C,19F, and1H acquisition. Dried material
after DFRC was dissolved in 800 µL of pyridine and deuterated
chloroform (1.6:1, v/v), and an aliquot of 400 µL was transferred to a
vial. Internal standards and relaxation reagents were added, and the
mixture was analyzed as described before.
RESULTS AND DISCUSSION
The demonstrated association between lignin and carbohy-
drates greatly influences the amount and structure of CELs and
MWLs that can be extracted from wood (13, 15, 16, 23). In
general, the longer the milling times are, the higher the yield,
but a MWL less representative of the native lignin has been
obtained (13, 20). To improve yields while minimizing damage
on the lignin structure, the extent of mechanical action during
milling must be reduced (21). Because a mild acid hydrolysis
can liberate lignin from lignin-carbohydrate complexes (known
to preclude lignin isolation in high yields), it may facilitate the
isolation of less-modified lignin from milled wood (15).
Consequently, the recently developed procedure aimed at
isolating lignin with the combination of enzymatic and mild
acidolysis presents a real opportunity in this respect since it
can be combined with low-severity milling. In this paper,
therefore, the effects of different milling severities on the various
steps involved in the isolation of EMAL were evaluated.
Vibratory and rotary ball milling were used in order to better
understand the effects of severe and mild milling on the yield,
purity, and structure of the obtained EMALs.
Effect of Milling on the Efficiency of Enzymatic Digestion.
It was initially determined that the efficiency of enzymatic
digestion for vibratory- and ball-milled wood varied similarly
for both methods of pulverization and was a function of milling
time (Figure 1). No weight loss was observed when Wiley-
milled wood was directly treated with cellulases. Powdered
wood before milling consists mostly of large particles with the
basic morphological features of the wood unchanged (34). Such
preparations are known to be impenetrable to cellulolytic
enzymes (35). After milling, the accessibility of milled wood
to cellulase increased, as shown by the wood weight loss
observed during the enzymatic treatment (Figure 1). The longer
the milling times are, the higher the wood weight loss up to 48
h of vibratory or 10 days of ball milling, when the weight loss
reached a maximum of 70%. Longer milling times did not lead
to greater weight loss (Figure 1). Progressive mechanical
treatments are known to lead to the formation of increasing
amounts of disordered cell wall material, which is rapidly
digested by cellulases (34). In this way, the more the charac-
teristic structure of wood breaks down, the more effective the
enzyme becomes in attacking the disordered cell wall material.
Besides cellulose and hemicelluloses, minor amounts of lignin
Figure 1. Weight loss during the enzymatic hydrolysis step as a function
of vibratory (9) and ball milling (4).
Lignin Isolation from WoodJ. Agric. Food Chem., Vol. 54, No. 16, 2006
also became soluble during the enzymatic treatment. However,
no further attempts were made to recover such soluble lignin
Effect of Milling on Lignin Yield and Purity. The effect
of different milling severities on the yield and purity of the
resulting EMALs was extensively examined, and the main data
are shown in Figure 2. As anticipated, progressive mechanical
treatments are seen to facilitate the disruption of the wood cell
wall structure allowing for more lignin to be extracted. As a
result, the longer the milling time is, the higher the EMAL yield,
regardless of the type of milling used (Figure 2A). However,
the combination of enzymatic hydrolysis and mild acid hy-
drolysis permitted the extraction of high yields of lignin even
under the considerably milder rotary ball-milling conditions.
More specifically, after 1 day of ball milling, the EMAL yield
(w/w, based on the amount of Klason lignin of the starting wood
and the isolated lignin) was 10% and it increased 5-fold,
reaching about 50% after 25 days of milling. Prolonged ball
milling beyond this time was found to offer more lignin, but
the material liberated was not extensively examined. As a point
of comparison, it is worth mentioning here that only 18.1% of
MWL was obtained after 6 weeks of porcelain ball milling (14).
Moreover, the data show (Figure 2A) that the more severe
mechanical action applied onto the material by vibratory milling
provided significantly higher yields within shorter periods of
time (75% after 72 h). Vibratory milling for longer than 72 h
had negligible effects on the yield indicating that the maximum
EMAL yield obtained from Norway spruce is about 75%. As
compared to the MWL and CEL isolated from the same batch
of Norway spruce, milled for 48 h (vibratory milling), the yield
of EMAL was 3.9 and 1.9 times greater than the corresponding
MWL (11.6%) and CEL (23.4%), respectively. These data
corroborate previous findings stating that the concerted effect
of cellulolytic action and mild acid hydrolysis offers significant
yield improvements over the traditional procedures for isolating
lignin from wood (15, 36).
Lignin samples isolated from wood still contain associated
carbohydrates and other nonlignin contaminants, regardless of
the isolation and purification procedures applied (12, 14, 15).
Although always present, the amounts of such nonlignin
materials may be affected by different factors, i.e, isolation
procedure and wood species. As such, the purity of a lignin
preparation, which is based on the sum of Klason and UV
soluble lignin contents, represents the total amount of lignin
after removal of such contaminants through a severe hydrolysis
with 72% (w/w) H2SO4. During our work, progressive vibratory
or ball milling was found not to have a significant effect on the
purity of the lignin isolated as evidenced by the data of Figure
2B. The purity of these samples was nearly constant up to 48
h of vibratory milling or 25 days of ball milling. However, a
sample isolated after 96 h of vibratory milling reached 90%
purity. This increase may suggest that during extended vibratory
milling some lignin-carbohydrate linkages may be cleaved. For
example, Staccioli et al. (37) reported that the amount of ester
groups formed by polyoses and lignin decreases due to extensive
mechanical treatments performed. Besides esters groups, it is
speculated that other types of lignin-carbohydrate interactions
may also be cleaved, assisting the isolation of more pure EMAL.
The purities of MWL (85.8%) and CEL (81%) isolated from
the same batch of milled Norway spruce and purified according
to Bjo ¨rkman (17) were similar to the values reported for MWL
and CEL from different wood species (12, 14, 15).
Effect of Milling on Molecular Weight of the Lignin.
Native lignin samples are usually sparingly soluble in solvents
commonly used for SEC, with the EMAL samples studied here
being of no exception, regardless of whether they were
acetylated in pyridine or phosphitylated with 2-chloro-4,4,5,5-
tetramethyl-1,3,2-dioxaphospholane. Under these circumstances,
the following derivatization scheme for lignin that involved its
reactive solubilization in acetyl bromide was established. The
system has been extensively examined and offered as an
effective alternative to sparingly soluble lignins or lignins
requiring mixed polar solvents in the presence of inorganic salts.
By dissolving a lignin sample in neat acetyl bromide diluted
with glacial acetic acid (8:92, v/v), the primary alcoholic and
the phenolic hydroxyl groups are acetylated, while the benzylic
R-hydroxyls are displaced by bromide (28). Similarly, benzyl
aryl ethers are quantitatively cleaved to yield aryl acetates and
acetylated R-bromide products (28). The concerted effect of
acetylation when coupled with the polarity induced by the
selective R-bromination caused every lignin sample examined
in this work to become highly soluble in THF, allowing rapid
SEC analyses. In an effort to ensure that the aforementioned
acetobromination represents a feasible alternative derivatization
technique, this procedure was compared with the traditional
acetylation derivatization procedure using an organosolv (Al-
drich) lignin. This lignin was selected for the comparison since
it was completely soluble in THF after acetylation with acetic
anhydride/pyridine. The SEC chromatograms displayed in
Figure 3A show only minor differences in the UV response
regarding both absorption and, most importantly, elution profiles.
Overall, such data are supportive of the viability of using
acetobromination as a derivatization technique since it represents
a facile and rapid alternative to the complete solubilization of
sparingly soluble lignin samples.
Figure 3B shows a typical set of SEC chromatograms of
various lignins isolated from the same batch of Norway spruce
after acetobromination. The size exclusion chromatograms of
the EMAL samples displayed a highly polydisperse behavior
as far as their molecular weight distributions are concerned. A
high molecular weight fraction (albeit of low abundance) was
apparent. Such polydispersity caused by this higher molecular
weight fraction is not obvious neither in the CEL nor in the
MWL. This is not totally surprising, however, when viewed in
Figure 2. Yields (A) and purities (B) of EMALs as a function of the
vibratory (9) and ball milling (4) times.
J. Agric. Food Chem., Vol. 54, No. 16, 2006 Guerra et al.
the light of the gelation statistics applied to lignification and
delignification (38-40) as well as the recent conclusions of
Lawoko et al. (41), where the molecular weight of lignin in
spruce is described to be rather large. Furthermore, the often
encountered lignin association effects, causing the formation
of the aforementioned high molecular weight fraction, cannot
be ruled out (42).
For the purposes of the present investigation, lignin associa-
tion phenomena are not taken into account and the apparent
molecular weight averages reported in Tables 1 and 2 are on
purpose uncorrected. This is done in order to demonstrate that
the EMAL isolation procedure offers significant opportunities
for the study of these interactions since the MWL and the CEL
procedures do not offer lignin samples with such characteristics
(Figure 3B). Current work in our laboratory attempts to evaluate
the origin and the propensity of various lignin preparations to
associate, and this is the subject of a manuscript in preparation.
To ensure that such association phenomena would not lead
to a misinterpretation of the effects of milling on the molecular
weight distributions, all analyses were carried out on freshly
prepared lignin solutions (analyzed immediately after deriva-
tization) ensuring that the comparisons made from sample to
sample are valid. Moreover, our molecular weight calculation
strategy was based on integration of the whole SEC chromato-
gram, without arbitrarily excluding the high and the low
molecular mass portions of it, as suggested by Baumberger et
The data of Figure 4 and Table 1 show that for the case of
ball-milled wood samples, the apparent molecular weight
averages (Mwand Mn) were nearly constant for up to 10 days
of ball milling. Longer milling times resulted in significantly
greater molecular weight EMAL samples. This is indicative that
as the ball milling time is prolonged the accessibility to higher
molecular weight lignin fragments is increased. This becomes
more apparent when one couples the data of Figure 2A and
Table 1. It is clear that the concerted effect of mild ball milling
followed by the EMAL sequence of enzymatic and mild
acidolytic hydrolyses affords the isolation of significant amounts
of lignin without any apparent damage on the lignin macro-
molecular structure. In this light, one should note that efforts
to isolate MWL in similar yields necessitate extensive milling
(20). Despite the fact that a yield of up to 50% can be achieved
by increased milling times, a MWL that is less representative
of the native lignin is known to be obtained (12, 14, 16, 21).
In contrast to ball milling, the Mwand Mnof EMALs isolated
from vibratory milled wood monotonically decreased as a
function of vibratory milling time (Table 2 and Figure 4). As
anticipated, the severe mechanical action exerted on the
macromolecule during vibratory milling apparently degrades the
lignin macromolecular structure.
For comparative purposes, the size exclusion chromatograms
of MWL and CEL isolated from the same batch of milled wood
after 48 h of vibratory milling are also included in Figure 3B.
While the chromatograms of EMAL and CEL display a bimodal
behavior, the chromatogram of CEL does not contain the small
fraction of the very high molecular weight material. The lack
of such high molecular weight components was even more
acetylation and after acetobromination with acetyl bromide (A) and typical
SEC chromatograms of lignin samples isolated from the same batch of
milled Norway spruce (B).
Gel permeation chromatograms of organosolv lignin after
vibratory (9)- and ball (4)-milled wood.
Weight-average molecular weight of EMAL isolated from
Table 1. Weight-Average Molecular Weight (Mw), Number-Average
Molecular Weight (Mn), and Polydispersity (D) of EMALs Isolated from
Ball-Milled Norway Spruce Wooda
aAggregation phenomena are not taken into account for the calculation of these
Table 2. Weight-Average Molecular Weight (Mw), Number-Average
Molecular Weight (Mn), and Polydispersity (D) of EMALs, MWL, and
CEL Isolated from Vibratory-Milled Norway Spruce Wooda
aAggregation phenomena are not taken into account for the calculation of these
averages.bIsolated from the same batch of milled Norway spruce wood.
Lignin Isolation from Wood J. Agric. Food Chem., Vol. 54, No. 16, 2006
pronounced in the chromatogram of MWL. The absence of the
high molecular weight fractions made the weight-average
molecular weights of both MWL and CEL significantly lower
than the corresponding EMAL. More specifically, the Mwof
EMAL isolated after 48 h of vibratory milling was over 78000
g mol-1, while the weight-average molecular weights, deter-
mined for MWL and CEL, were 23500 and 53800 g mol-1,
respectively (Table 2). Overall, these data show that the
combination of mild enzymatic and mild acidolyses allows the
isolation of lignin fractions that are not accessible by neither
of the alternative lignin isolation procedures.
Effect of Milling on Uncondensed ?-Aryl Ethers and
Other Structures of the Lignin. The various functional groups
that define significant aspects on the structure of lignin were
then determined using quantitative31P NMR spectroscopy for
all EMAL samples isolated from vibratory- and ball-milled
woods. The ?-aryl ether structural content (Figure 5A) was
determined after phosphitylating the CR hydroxyl groups in
these moieties with 2-chloro-1,3,2-dioxaphospholane (31, 44).
The condensed and uncondensed phenolic hydroxyls as well
as the carboxylic acids (Figure 5B-D) were determined by
phosphitylating the lignins with 2-chloro-4,4,5,5-tetramethyl-
1,3,2-dioxaphospholane (32). Quantification was then carried
out via peak integration using N-hydroxynaphthalimide as an
internal standard. Details of signal acquisition, assignment, and
integration can be found elsewhere (31-33).
The data of Figure 5A show that while ball milling did not
affect the ?-O-4 structural content of the resulting EMALs,
vibratory milling resulted in extensive cleavage of these lignin
moieties. This finding is in accord with the work of Fujimoto
et al. (21, 45, 46) and Ikeda et al. (14). A closer inspection of
the data of Figure 5A, coupled with the data of Table 2, shows
that when Norway spruce wood was subjected to vibratory
milling for a period of 96 h, a moderate decrease (13%) in the
?-aryl ether content of the lignin was observed, resulting in
extensive lignin depolymerization (from 95600 to 56500 mol
g-1). For the same period of vibratory milling, the actual
reduction of ?-aryl ether content was 0.2 mmol g-1. This
reduction was found to be accompanied by an increase in
uncondensed phenolic hydroxyls of 0.6 mmol g-1(Figure 5B).
This overproportional increase in phenolic hydroxyl groups
indicates that ?-aryl ether cleavage is not the only reaction taking
place within lignin during vibratory milling. This is not
surprising, since free radicals are known to be formed during
mechanical treatment of lignocellulosic material (47). Such
reactions may be accompanied by a series of autoxidation
reactions, which could eventually result in oxidative fragmenta-
tion of the lignin macromolecule with the concomitant creation
of carboxylic acid groups in the lignin, apparent in vibratory
milling EMALs as shown in Figure 5D. Under ball-milling
conditions, no such oxidation reactions are apparent and the
COOH content of the emerging EMALs was found to be
remarkably constant throughout the milling period extending
into 25 days (Figure 5D). Another side reaction that could also
take place, once free radicals are formed, is that of radical
coupling, causing the creation of condensed carbon-carbon
bonds within the lignin. This is evidenced by the increase of
condensed phenolic hydroxyls apparent during vibratory milling
and totally absent during ball milling (Figure 5C).
Effect of Milling on Other Lignin Structures. Most of the
recent conclusions regarding the bonding patterns of native
lignins have been derived from chemical degradation techniques,
such as thioacidolysis, due to its sensitivity and demonstrated
lack of artifacts (48). In 1997, a new selective ?-aryl ether
cleavage protocol, termed DFRC, was proposed by Lu and
Ralph (28, 49-51). The DFRC method uses a mild depoly-
merizing environment and has the advantage of avoiding the
use of malodorous reagents. While DFRC provides a clean and
selective protocol for ether scission, the primary DFRC mono-
mers detected and quantified by GC are confined to phenyl-
propane units connected to ?-aryl ether bonds on both sides of
the phenyl propanoid units or terminal phenylpropane units
connected to the polymer via a ?-aryl ether bond. Consequently,
by using GC alone for detecting the DFRC monomers, the total
amount of ?-aryl ether linkages cannot be revealed. For example,
if a ?-aryl ether connects two macromolecules or oligomers that
themselves are interlinked via structures other than ?-aryl ethers,
the size of the fragments will preclude them from being detected
Figure 5. ?-O-Aryl ether functional groups (A), uncondensed (B) and condensed (C) phenolic hydroxyl, and carboxylic groups (D) of EMALs as a
function of the vibratory (9) and rotary (4) ball milling time.
J. Agric. Food Chem., Vol. 54, No. 16, 2006Guerra et al.
by GC as DFRC monomers (26, 49-52). The coupling of the
DFRC technique with31P NMR can overcome this limitation
and was recently shown to offer new quantitative information
about ?-aryl ethers linked to condensed and noncondensed
aromatic moieties, including dibenzodioxocins (26). Such
enquiries could thus prove indispensable in further understanding
the effects of milling on the structure of isolated EMALs,
MWLs, and CELs.
However, to ensure that the conclusions were independently
validated, a sample of Norway spruce EMAL, isolated after 25
days of ball milling, was submitted to comparative analyses that
included DFRC/31P NMR and thioacidolysis. The comparison
between the two analytical protocols is shown in Table 3. There
is apparently a good agreement between the two methods in
the total amount of uncondensed ?-O-aryl bonds, demonstrated
for the first time on the same sample of isolated lignin.
Because31P NMR can distinguish condensed from uncon-
densed phenolic hydroxyls, the combination of DFRC with31P
NMR was used to estimate the effects of milling on the amount
of condensed and uncondensed ?-aryl ether structures (Figure
6). Condensed ?-aryl ether bonds refer to structures that connect
two macromolecules or oligomers that themselves are interlinked
via structures other than ?-aryl ethers. By considering the data
of Figure 5A in conjunction with that of Figure 6A, one arrives
at the conclusion that the lignin depolymerization that is taking
place during vibratory milling is due to the cleavage of
uncondensed ?-aryl ether linkages. Interestingly, the condensed
?-aryl ether structures were found to be resistant to the intense
mechanical action caused by vibratory milling and remained
unchanged up to 96 h of vibratory milling (Figure 6B). Figure
6 also shows that condensed and uncondensed ?-aryl ethers were
not affected by ball milling.
Dibenzodioxocins (5-5/?-O-4, R-O-4) can also be determined
using the combination of DFRC with quantitative31PNMR, by
integrating the region from 141.2 to 142.4 ppm, which has been
attributed to the 5-5′-liberated phenols after these moieties open
up during DFRC (26). Figure 6C shows, for the first time, that
the 5-5/?-O-4/R-O-4 ring of dibenzodioxocins is resistant to the
mechanical action of vibratory milling.
The functional group content of EMAL, MWL, and CEL
determined by quantitative31P NMR is shown in Table 4. The
nearly identical amounts of total ?-aryl ether functional groups
of EMAL and CEL indicate no evidence of ?-aryl ether bond
degradation within the lignin during the mild acid hydrolysis
step of the EMAL protocol. MWL is seen to contain slightly
higher amounts of ?-aryl ether linkages (∼3.7/100 C9) than the
EMAL and CEL. However, as shown by its yield and molecular
weight, MWL represents the low molecular weight extractable
lignin fragments soluble in dioxane rather than the overall lignin
The aliphatic hydroxyl group data for EMAL, MWL, and
CEL were found to be 1.2/C9, 1.0/C9, and 0.9/C9, respectively.
These values compare reasonably well with the literature range
(0.8-1.2/C9) (15), considering the uncertainties associated with
carbohydrates contaminants in this determination.
The contents of carboxylic acid groups (2.3-2.9/100 C9)
were also very similar to those reported in the literature for
Table 3. Thioacidolysis and DFRC/31P NMR Data from EMAL Isolated
from Norway Spruce Ball-Milled for 25 Days
G units involved
721 ± 15 (13.5)a
740 ± 10 (13.8)a
H units involved
6 ± 1 (0.1)a
70 ± 15 (1.3)a
727 ± 15 (13.6)a
825 ± 15 (15.3)a
aIn mol % (based on C9 unit molecular weight of 187).
Figure 6. Uncondensed (A) and condensed (B) phenolic hydroxyl groups
(equivalent to the amount of uncondensed and condensed ?-O-aryl ether
linkages, respectively) and dibenzodioxocins units (C) as a function of
the vibratory (9) and rotary (4) ball milling time. Condensed ?-O-aryl
ether linkages are defined as structures that connect two macromolecules
or oligomers that themselves are interlinked via structures other than ?-O-
Table 4. Functional Group Contents,aYields, and Weight-Average
Molecular Weight (Mw) for EMAL, MWL, and CEL Isolated from the
Same Batch of Milled Norway Spruce
(per 100 C9)
(per 100 C9)
(per 100 C9)
total ?-aryl ether
aDetermined by quantitative31P NMR (error ± 1) based on a C9unit molecular
weight of 187 g/mol.bOn the basis of Klason lignin contents of extracted ground
Lignin Isolation from Wood J. Agric. Food Chem., Vol. 54, No. 16, 2006
EMAL and MWL from spruce (15). These similarities provide
further support for the effectiveness of the EMAL protocol in
providing nonoxidized lignin.
The uncondensed phenolic hydroxyl content of EMAL was
found to be somewhat higher than that of MWL and CEL.
Because there is no evidence of liberation of phenolic hydroxyl
groups from ?-aryl ether linkages (Figures 5 and 6 and Table
4), the higher content of such functional groups in EMAL is
supportive of the notion that this lignin preparation is more
representative of the overall lignin present in milled wood. As
shown before, one of the effects of vibratory milling is the
increase in the amount of uncondensed phenolic hydroxyl
groups. Such phenolic groups are seen to increase 3-fold when
compared to the reduction of ?-aryl ether linkages. Because the
yield of EMAL is 3.9 and 1.9 times greater than MWL and
CEL, respectively, the higher phenolic contents in such lignins
are not surprising when one considers the more representative
nature of the EMAL sample.
Conclusions. It can be surmised that the combination of
enzymatic and mild acid hydrolysis offers the possibility to
isolate lignin samples that are more representative of the total
lignin in milled wood. EMAL is released by cleaving lignin-
carbohydrate bonds rather than via the degradation of ether
bonds within lignin. The cleavage of the lignin-carbohydrate
bonds afforded during the mild acidolysis step of the EMAL
protocol allows the isolation of lignin fractions that are not
accessed by any other isolation procedures. Furthermore, the
liberation of lignin from lignin-carbohydrate complexes pro-
vided by the mild acid hydrolysis step offers the possibility of
obtaining high yields using low intensity milling. Intensive
milling protocols offered by vibratory or orbital milling devices
should be considered with caution since they provide higher
lignin yields within relatively short milling intervals at the
expense of the integrity of the lignin macromolecule and
associated condensation and oxidation reactions.
We thank Professor Stefan Willfo ¨r of A ° bo Akademi University
(Turku, Finland) for providing the Cost E41 sample of Norway
spruce. The contributions of Dr. Nestor Soriano and Ana Xavier
are also acknowledged with respect to the size exclusion
chromatographic effort and the Klason and UV analyses,
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Received for review March 14, 2006. Revised manuscript received June
12, 2006. Accepted June 15, 2006. This work became possible by a U.S.
Department of Energy Grant DE-FC36-04GO14308.
Lignin Isolation from Wood J. Agric. Food Chem., Vol. 54, No. 16, 2006