DFRC Method for Lignin Analysis. 1. New Method for β-Aryl Ether Cleavage: Lignin Model Studies

Article abstract of DOI:10.1021/jf970539p

A new method for selective and efficient cleavage of arylglycerol-β-aryl (β-O-4) ether linkages in lignins is described and applied to several lignin β-ether models. The term "DFRC" was coined for derivatization followed by reductive cleavage. Derivatization, accompanied by cell wall solubilization, is accomplished with acetyl bromide (AcBr); reductive cleavage of resulting β-bromo ethers utilizes zinc in acetic acid. Degradation monomers, 4-acetoxycinnamyl acetates, from β-ether cleavage by the DFRC method were identified by NMR, GC-MS, and comparison of GC retention times with authentic compounds. Under the conditions used in this study, the β-ether linkage of all models was cleaved in very high (>92%) yield. The DFRC method produces simpler mixtures of monomers with higher yields than alternative hydrolytic methods. Because of its relative simplicity, mild conditions, and exceptional selectivity, this method should become a powerful analytical method for lignin characterization.

Full text of DOI:10.1021/jf970539p

J. Agric. Food Chem. 1997, 45, 4655
−4660
4655
DF RC Meth od for Lign in An a lysis. 1. New Meth od for â-Ar yl Eth er
Clea va ge: Lign in Mod el Stu d ies
Fachuang Lu and J ohn Ralph*
U.S. Dairy Forage Research Center, USDA-Agricultural Research Service, 1925 Linden Drive West, and
Department of Forestry, University of WisconsinsMadison, Madison, Wisconsin 53706
A new method for selective and efficient cleavage of arylglycerol- -aryl ( -O-4) ether linkages in
lignins is described and applied to several lignin -ether models. The term “DFRC” was coined for
derivatization followed by reductive cleavage. Derivatization, accompanied by cell wall solubilization,
is accomplished with acetyl bromide (AcBr); reductive cleavage of resulting -bromo ethers utilizes
zinc in acetic acid. Degradation monomers, 4-acetoxycinnamyl acetates, from -ether cleavage by
the DFRC method were identified by NMR, GC-MS, and comparison of GC retention times with
authentic compounds. Under the conditions used in this study, the -ether linkage of all models
was cleaved in very high (>92%) yield. The DFRC method produces simpler mixtures of monomers
with higher yields than alternative hydrolytic methods. Because of its relative simplicity, mild
conditions, and exceptional selectivity, this method should become a powerful analytical method
for lignin characterization.
Keyw or d s: Acetyl bromide; lignin; lignin model compound; -aryl ether; thioacidolysis; -bromo-
ether; cleavage; quantitative analysis; gas chromatography; reductive elimination; acetylation;
bromination
INTRODUCTION
Although a great deal of progress has been made in
lignin chemistry, many ambiguities remain regarding
lignin structure and its cross-linking with other cell wall
components (Dence and Lin, 1992; Ralph and Helm,
1993). As a highly abundant, renewable raw material
that is currently underutilized, lignin has attracted
increasing interest in wood chemistry, plant biochem-
istry, and related fields (Fengel and Wegener, 1989;
F igu r e 1. -Ether units, major substructures in lignins.
Chen, 1991).
Unlike other natural polymers such as proteins,
conditions made it difficult to isolate, identify, and
polysaccharides, and nucleic acids, which have interunit
quantitate degradation products. The main degradation
linkages susceptible to enzymic and chemical hydroly-
monomer from dry HI treatment of spruce lignin, for
ses, lignin contains resistant carbon-carbon and diphe-
nyl ether bonds (Morohoshi, 1991; Sakakibara, 1991).
It is a common practice to degrade the polymer to low
molecular weight compounds in order to obtain struc-
tural information. When this strategy is applied to
lignins, however, significant limitations arise such as
the low yield of degradation products, interference from
other contaminants, and side reactions (Lapierre, 1993).
Permanganate oxidation, alkaline-nitrobenzene oxi-
dation, and acidolysis are traditional methods for lignin
characterization, although all have shortcomings. Thio-
acidolysis (Lapierre, 1993; Rolando et al., 1992) was a
major improvement in achieving relatively selective
cleavage reactions to form diagnostic products in good
yields. Recently several iodine-containing reagents
have been used in attempts to degrade lignins under
milder conditions; these include trimethylsilyl iodide
(Makino et al., 1990; Meshitsuka et al., 1987; Shevchen-
ko and Akim, 1995), acetyl iodide (Shevchenko and
Akim, 1995), pivaloyl iodide (Fukagawa et al., 1992),
and dry hydrogen iodide (Shevchenko et al., 1991;
Shevchenko and Akim, 1995). Complicated mixtures of
unstable degradation products formed under those
example, was 1,3-diiodo-1-(4-hydroxy-3-methoxyphen-
yl)propane which was stable for only 1-2 h (Shevchenko
and Akim, 1995). Currently, thioacidolysis remains
probably the most effective diagnostic method for lignin
characterization (Rolando et al., 1992). However thio-
acidolysis is not a simple technique to perform; it
requires optimization in a user’s lab, and utilizes the
malodorous ethane thiol. There remains a need to
develop a more selective, simpler, and more powerful
method for degradation of lignin to provide detailed
information about lignin structure.
During recent research on selective methods for
cleavage of R-ether linkages in lignins, Figure 1 (R )
aryl), it was found that AcBr is an ideal reagent for this
purpose. AcBr treatment of R, -diaryl ether lignin
models in dioxane or acetic acid at room temperature
resulted in complete formation of acetylated R-bromo
derivatives along with selective R-ether cleavage/bro-
mination (Lu and Ralph, 1996b). It is well established
that -bromo ethers or esters can be reductively cleaved
by zinc dust in polar solvents (Rowlands et al., 1952;
Soday and Boord, 1933; Schmitt and Boord, 1932; Kato
et al., 1987), or Cr(II)en complex in dimethylformamide
(DMF) (Kochi et al., 1968; Greene et al., 1987) to form
alkenes. Experiments have shown that the lignin
derivatives from AcBr treatment possess the -bromo
ether skeleton required for the reductive elimination (Lu
* Author to whom correspondence should be ad-
dressed [telephone (608) 264-5407; fax (608) 264-5147;
e-mail jralph@facstaff.wisc.edu].
S0021-8561(97)00539-6 CCC: $14.00
© 1997 American Chemical Society

4656 J. Agric. Food Chem., Vol. 45, No. 12, 1997
Lu and Ralph
F igu r e 2. -Ether models used for the DFRC methods, and some monomers 10-15 derived from dimers 6 and 7.
F igu r e 3. Selective ether cleavage by the DFRC method; monomers 16-18 derive from -aryl ether models and lignins.
methods described in literature (Ralph et al., 1986; Ralph and
Helm, 1991). Hydroxycinnamyl diacetate standards 16-18
(trans-isomers, Figure 3) were prepared by acetylating the
trans-hydroxycinnamyl alcohol parents, synthesized as previ-
ously described (Quideau and Ralph, 1992; Ludley and Ralph,
1996). NMR data for all compounds are given in the NMR
Database of Lignin and Cell Wall Model Compounds (Ralph
et al., 1996). Acetyl bromide, dioxane, acetic acid, and zinc
dust were purchased from Aldrich Chemical Co. and used as
supplied. Commercial analytical reagent grade solvents were
used without further purification.
and Ralph, 1996b). We believed that AcBr treatment
of lignin followed by Zn reductive cleavage would
provide a pathway to selectively cleave R- and -aryl
ethers in lignin, resulting in a new method for lignin
characterization (Figure 3). The new method for cleav-
ing ethers in lignin has been given the acronym DFRC
for derivatization followed by reductive cleavage (and
to reflect the Dairy Forage Research Center where the
method was developed). The protocol for DFRC mono-
mer analysis has been published (Lu and Ralph, 1997a).
The first paper of this series describes a simple and
efficient method (based on AcBr treatment and reduc-
tive ether cleavage) for selective cleavage of ether
linkages in lignins and our evaluations using a range
of representative lignin models. Because of AcBr’s
ability to dissolve lignin and lignin-containing plant
material there is little doubt that this method can be
applied to isolated lignin samples and plant materials.
A following publication will report on those applications.
AcBr stock solution, made by mixing AcBr and acetic acid,
8:92 by volume, was stable for several weeks.
DF RC Meth od . To a 10 mL round bottom flask containing
about 10 mg of lignin model, 2.5 mL of AcBr stock solution
was added. The mixture was kept at room temperature and
stirred gently overnight. After removal of solvent by rotary
evaporation at below 50 °C, the residue was dissolved in 2.5
mL of dioxane/acetic acid/water (5:4:1, v/v/v) solution. Zinc
dust (50 mg) was added to the well-stirred solution. Stirring
was continued for 30 min. After addition of internal standard
(3 mg of teracosane in methylene chloride) the mixture was
quantitatively transferred into a separatory funnel with CH2-
Cl2 (10 mL) and saturated NH4Cl (10 mL). The pH of the
aqueous phase was adjusted to less than 3 by adding 3% HCl,
EXPERIMENTAL PROCEDURES
Ma ter ia ls a n d Rea gen ts. Lignin model compounds 1-9,
shown in Figure 2, were synthesized according to the standard

DFRC Method for Ether Cleavage in Lignins
J. Agric. Food Chem., Vol. 45, No. 12, 1997 4657
F igu r e 4. Mass spectrum of product 17 (coniferyl diacetate) and partial proton NMR spectrum of crude products (17t and 17c)
from dimer 2 by the DFRC method. c ) cis, t ) trans.
Ta ble 1. Ma ss Sp ectr a l a n d GC (RRT a n d RF ) Da ta of Degr a d a tion Mon om er s 10-18
peak
RRT
RF
m/ z (rel intensity)
10
11
12
13
14
15
16c
16t
17c
17t
18c
18t
0.31
0.52
0.48
0.60
0.66
0.78
0.58
0.63
0.69
0.76
0.81
0.88
180(15), 151(100), 135(8), 107(30), 91(24), 77(42)
238(4), 196(56), 167(100), 151(5), 137(7), 122(9), 77(12)
194(11), 165(100), 137(15), 122(22), 107(18), 92(22), 79(83)
252(5), 209(8), 167(100), 139(99), 124(25), 95(20), 77(41)
252(2), 210(36), 181(100), 167(5), 151(12)
310(2), 268(9), 225(15), 183(100), 155(11), 123(15), 95(9)
234(19), 192(100), 149(85), 133(54), 121(44), 107(28), 94(29), 77(24)
1.76
1.76
1.85
1.85
2.06
2.06
264(11), 222(100), 179(37), 163(9), 151(12), 131(27), 119(15), 91(14)
294(8), 252(100), 209(24), 193(10), 181(8), 161(17), 149(13), 133(6)
the solution was vigorously mixed, and the organic layer was
separated. The water phase was then extracted with CH2Cl2
(2 × 5 mL). The combined CH2Cl2 fractions were dried over
MgSO4 and the filtrate was evaporated under reduced
pressure. The residue was acetylated in 1.5 mL dichlo-
romethane containing 0.2 mL acetic anhydride and 0.2 mL
pyridine for 40 min. All volatile components were removed
completely by coevaporation with ethanol under reduced
pressure. The residue was used for NMR, GC, and GC-MS
characterization.
GC Deter m in a tion a n d GC-MS. The degraded mono-
mers from models were dissolved in 1.5 mL of methylene
chloride and 1 L of this solution was used for GC analysis.
The degraded monomers from lignins or models were quan-
titatively determined by GLC (Hewlett Packard 5980, Atlanta,
GA): column 0.20 mm × 30 m SPB-5 (Supelco, Bellefonte, PA);
He carrier gas, 1 mL/min; injector 220 °C, initial column
temperature 160 °C, ramped at 10 °C/min to 300 °C, hold 5
min; flame ionization detector (FID), 300 °C. The amounts of
individual monomers 16-18 were determined using response
factors (RFs) derived from pure monomer standards using
tetracosane as internal standard. Relative retention times
(RRTs) and GC response factors relative to the tetracosane
internal standard are given in Table 1. Electron ionization
(EI)-MS (70 eV) data were collected on a Hewlett Packard
HP5970 mass selective detector attached to the same GC. The
column and flow conditions differed slightly between the GC-
FID and GC-MS setups; thus, absolute retention times differ
between the two.
RESULTS AND DISCUSSION
Because the most frequent interunit linkages in lignin
are arylglycerol- -aryl ethers (Figure 1) (Adler, 1977),
the cleavage of -aryl ethers has been studied exten-
sively (Makino et al., 1990; Meshitsuka et al., 1987;
Fukagawa et al., 1992; Shevchenko et al., 1991; Lapierre
et al., 1985; Lapierre et al., 1983). If these -O-4 ether
linkages were to be cleaved selectively and completely,
the characterization of the degradation monomers (along
with dimers and even trimers) would provide valuable
structural information regarding the initial lignin. It
has long been one of the most important research
targets for lignin chemists to find a mild, selective, and
efficient method for -O-4 ether cleavage (Lundquist,

4658 J. Agric. Food Chem., Vol. 45, No. 12, 1997
Lu and Ralph
1992; Nimz, 1969, 1974; Lapierre et al., 1991). Such
cleavage is a key requirement either for an efficient
degradation of polymeric lignins during chemical pulp-
ing or for analysis of various linkages present in
lignins.
For simplicity many studies on lignin reactions begin
with model compounds. Interpretation of the chemical
behavior of lignin, especially in degradation reactions,
is largely based on results obtained from studies of
appropriate model compounds under analogous condi-
tions. It is essential that any proposed reactions on
representative models yield desired products with high
yields. If this requirement is not met, it is unreasonable
to expect that the significantly more complex plant
matrix will react efficiently. To determine the efficiency
of the AcBr/Zn mediated ether cleavage method, -O-4
aryl ether model compounds shown in Figure 2 were
studied.
Id en tifica tion of Degr a d a tion Mon om er s. As
shown in Figure 3, the DFRC degradation method
requires two key steps: (1) bromination and acetylation
with AcBr, and (2) reductive cleavage with Zn dust.
Final acetylation is used to acetylate free phenolic
groups that are produced following the reduction step
and reduce the number of compounds to be quantitated.
The reaction of lignin or lignin models with AcBr in
acetic acid results in complete dissolution of lignin and
formation of acetylated lignin R-bromo-derivatives which
possess the -bromo ether skeleton. In the next step
the bromo ethers are cleaved by reductive elimination
with Zn dust in aqueous dioxane/acetic acid solution,
forming a pair of 4-hydroxycinnamyl acetate isomers.
Thus -ether dimer 2 was cleaved through the DFRC
procedure resulting in 4-acetoxy-3-methoxycinnamyl
acetates 17, which were identified by NMR and GC-
MS (Figure 4). Monomers derived from DFRC degrada-
tion of various models representing -ether structures
in softwood and hardwood lignins are essentially 4-ac-
etoxycinnamyl acetate 16, 4-acetoxy-3-methoxycinnamyl
acetate 17, and 4-acetoxy-3,5-dimethoxycinnamyl ac-
etate 18. Two trimeric models, 8 and 9, which more
accurately model internal -ether units than do dimeric
models, were subjected to DFRC, forming monomer 17
(from trimer 8) and monomers 17 and 18 (from trimer
9) in 93, 97, and 93% yields, respectively (Figure 5 and
Table 2).
The NMR and mass spectral data of degradation
monomers derived from -ether models by the DFRC
method are listed in Table 1. As shown in Figures 5
and 6, GC chromatograms of the monomers obtained
from the DFRC method are clean and simple. Only one
pair of monomers is formed from the corresponding
uncondensed lignin structural unit.
A further hydrogenation step to reduce the number
of degradation monomer isomers and make quantitation
easier was considered (Lu and Ralph, 1996a). However
competing hydrogenolysis is unavoidable. For example,
hydrogenation of 4-acetoxycinnamyl acetate 18 was
always accompanied by the hydrogenolysis byproduct
propylphenyl acetate 15, which is also derived from
other lignin substructures. Thus hydrogenation does
not actually help quantitative analysis and makes the
procedure less diagnostic and more time consuming.
Distinct advantages of the current DFRC protocol are
that degradation monomers have a C6-C3 skeleton
representing the phenylpropanoid characteristic of lig-
nin, and that ester groups on -positions of lignin side
chains remain fully intact (Lu and Ralph, 1997b,c). To
F igu r e 5. GC-FID chromatograms of DFRC monomers from
(A) dimer 1, (B) dimer 2, (C) dimer 5, (D) trimer 8. c ) cis, t )
trans.
our knowledge, this method is the first one possessing
such features that will be exploited in future work.
The primary degradation products of -O-4 ether
models possessing R-carbonyl groups are propiophe-
nones 12 and 14 and 1-acetoxy-1-phenylacetones 13 and
15, although the mechanisms involved are not under-
stood at present. Those products are well separated in
GC chromatograms from monomers derived from nor-
mal -O-4 ethers. Some minor compounds, 10 and 11,
are also formed as shown in Figure 6. Although reac-
tions are not as clean as for arylglycerol- -aryl ether
structures, it is possible to estimate R-carbonyl groups
present in lignin units connected through -O-4 ether
bonds by using an extended reduction step in the DFRC
method.
Qu a n tita tive Deter m in a tion of th e Degr a d a tion
Mon om er s. We have shown (Lu and Ralph, 1996b)
with lignin models that AcBr in acetic acid at room
temperature is highly selective in its reactions with
lignin, i.e., R-hydroxyls and R-ethers become R-bromo
derivatives and -hydroxyls are acetylated rapidly, and
phenolic hydroxyls are acetylated more slowly. Brom-
inated acetylated derivatives of lignin models (and
lignins) are formed in almost quantitative yields. The
only detectable monomers from Zn reductive cleavage
steps on -ether units are pairs of 4-acetoxycinnamyl
acetates, confirmed by NMR and GC-MS (see Figures
4 and 5). The relative simplicity in the resulting
monomer composition makes quantitative analysis easy

DFRC Method for Ether Cleavage in Lignins
J. Agric. Food Chem., Vol. 45, No. 12, 1997 4659
Ta ble 2. Com p a r ison of Mon om er Yield s fr om Th ioa cid olysis, Acid olysis, a n d DF RC Meth od s
9 (GSS)
1 (PG)
2 (GG)
3 (GS)
4 (SG)
5 (SS)
8 (GGG)
G%
S%
DFRC method
thioacidolysis
acidolysis
93.2
94.5
70/75
69
95.0
93.5
75
96.4
52-57
32
92.6
97.0
93.4
coming publications will examine how the DFRC method
compares with respect to sensitivity to sample type,
temperature, duration of the steps involved, and sample
moisture content.
The high yield of degradation monomers from -ether
models gives the DFRC method the potential to quan-
titate uncondensed lignin structural units and be fur-
ther used in studies on lignin cross-linking chemistry
in cell walls. All reactions involved in the DFRC method
are run at room temperature (up to 50 °C for lignins
and cell wall samples) and released products are rea-
sonably stable, so secondary condensation reactions can
presumably be avoided. This was reflected by high
monomer yields obtained for lignin models subjected to
the DFRC method (Table 2). No apparent differences
were found between monomer yields from p-hydoxyphe-
nyl, guaiacyl, and syringyl models. Therefore, all units
connected by -O-4 ethers in lignin can be cleaved
equally efficiently and the monomer ratio between
p-hydroxyphenyl, guaiacyl, and syringyl derivatives is
diagnostic of the proportions of those units releasable
as monomers.
As for model compounds 6 and 7 possessing R-carbo-
nyl groups, -ether cleavage by the standard DFRC
procedure is less efficient than in normal -ether models
1-5 and 8 and 9, although up to 60% of their ether
linkages were cleaved as shown by GC chromatograms
(Figure 6). Because models 6 and 7 have phenacyl ether
skeletons which can be cleaved almost quantitatively
by Zn in acetic acid (Hendrickson and Kandall, 1970),
the ether linkages in models 6 and 7 could be eventually
cleaved as long as the Zn treatment times are suf-
ficiently long. This idea proved to be correct when
models 6 and 7 were subjected to the DFRC method
with overnight (16 h) Zn treatment. The starting
dimeric compound almost completely disappeared in
GC-MS total ion chromatograms of the corresponding
degradation products (Figure 6). The main products
recovered were 12 or 13 and 14 or 15 plus small
amounts of 10 or 11. Therefore, caution is required
when this method is applied to lignins with high
R-carbonyl contents. Reducing carbonyls to hydroxyl
groups by sodium borohydride (or sodium borodeuteride
to allow mass spectrometric distinction) before the
DFRC procedure, or prolonging the zinc treatment may
be worthy modifications to apply when quantitation of
R-carbonyl groups becomes a crucial consideration.
F igu r e 6. GC-MS total ion chromatograms of DFRC prod-
ucts from R-carbonyl dimers 6 (E, F) and 7 (G, H) using two
Zn treatment times. x ) Impurities, * ) currently unidentified
products.
and accurate. Reproducibility of the DFRC method is
about 3-5%, comparable to other analytical techniques.
Monomer yields of AcBr/Zn degradation of models are
92-97% (Table 2), much higher than those of acidolysis
or thioacidolysis (Lapierre et al., 1985; Grabber et al.,
1996). Extensive nonspecific derivatization of lignin
and numerous side reactions are generally difficult to
avoid in lignin degradation reactions carried out at high
temperatures with reactive acids or Lewis acids. It is
believed that the frequency of condensation reactions
is lower for model compounds than is the case in real
lignin. Even for thioacidolysis, the mildest and most
selective method to date, the best monomer yield from
model compounds is far from quantitative (Lapierre et
al., 1985; Grabber et al., 1996). It is unlikely that, when
applied to real lignins, thioacidolysis can completely
cleave all desired linkages and recover desired mono-
mers quantitatively. Thioacidolysis is also sensitive to
experimental conditions. For example, we recently
found significant quantities of -O-4 ether dimers in our
thioacidolysis runs when the experimental conditions
were not optimized (Ralph and Grabber, 1996). Forth-
CONCLUSION
From this study it has been demonstrated that
-ether linkages in lignin models were efficiently cleaved
through derivatization with AcBr followed by reductive
cleavage, now referred to as the DFRC method. This
method can be applied to isolated lignin and lignins in
situ as will be shown in forthcoming publications. Its
simplicity and cleanliness, and the use of relatively
innocuous reagents, may provide a suitable alternative
to the widely used thioacidolysis procedure for some
analyses.

4660 J. Agric. Food Chem., Vol. 45, No. 12, 1997
Lu and Ralph
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Received for review J une 25, 1997. Revised manuscript
received October 1, 1997. Accepted October 3, 1997.X
This
research was funded in part by a USDA-NRI competitive
grant, #95-03675 (Improved Utilization of Wood and Wood
Fiber section).
Lu, F.; Ralph, J . 9th International Symposium on Wood and
Pulping Chemistry, Montreal, Quebec; Canadian Pulp and
Paper Association: Montreal, 1997c; Vol. 1, p L3.
Ludley, F. H.; Ralph, J . Improved preparation of coniferyl and
sinapyl alcohols. J . Agric. Food Chem. 1996, 44, 2942-2943.
J F970539P
X Abstract published in Advance ACS Abstracts, No-
vember 15, 1997.