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A facile spectroscopic method for measuring

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Cite this: Green Chem., 2021, 23,

lignin content in lignocellulosic biomass†
Open Access Article. Published on 09 June 2021. Downloaded on 2/22/2022 [Link] AM.

5106
Fachuang Lu, *a,b Chen Wang,a Mingjie Chen, b
Fengxia Yue a
and
b
John Ralph

Although measuring lignin contents is a routine operation for biomass compositional analysis in process
development aiming at efficient utilization of woody biomass, it is still a challenging task requiring many
steps, hazardous reagents, heating, and a significant time. A facile spectroscopic method, our CASA
(Cysteine–Assisted Sulfuric Acid) method, was developed to quantify the lignin content of lignocellulosic
biomass, based on an extraordinary system in which biomass samples are fully dissolved in 72% H2SO4
containing cysteine at 24 °C in 60 min. Using synthetic lignins, the lignin absorptivities were determined
to be 17.25 g−1 L cm−1 for softwood lignin and 11.23 g−1 L cm−1 for hardwood lignin and monocot lignin.
Received 29th April 2021, Seven softwoods, six hardwoods, and six monocots were tested using the CASA method. A high coeffi-
Accepted 8th June 2021
cient of determination (R2 = 0.95) was found between the CASA results and the acid-insoluble lignin con-
DOI: 10.1039/d1gc01507a tents, and an even better R2 (0.98) was obtained when the CASA data were correlated with the total lignin
[Link]/greenchem contents.

Introduction measurement is one of the important routine practices for

biomass compositional analysis in process development and
Lignin is the most abundant aromatic polymer and the second optimization.11,12
most prominent renewable raw material after cellulose.1 In Many methods have been developed and modified over the
plant cells, lignin cross-links to hemicelluloses and cements past for quantitatively measuring the amount of lignin in
cellulose microfibers, enhancing the mechanical strength of certain kinds of plant tissues. The oldest and the most
the plant stem, facilitating the transport of water and nutri- popular method for lignin quantitation, the Klason method,
ents, and providing protection against biological attack.2–4 has been used for more than a century.13–15 During this
Due to the presence of lignin, it is difficult for microorganisms period, other methods have been proposed, and the options
to fully utilize lignocellulosic feedstocks,5,6 although progress for lignin quantitation have expanded from the traditional
has been made in developing technologies aimed at achieving gravimetric ones to the rapid non-destructive methods using
full and economical utilization of lignocellulosic biomass. various instruments (UV, FTIR, NMR; NIR is a secondary
These efforts include manipulating the lignification process by method that will not be discussed here).16–18 Overall, methods
mis-regulating genes within the monolignol biosynthesis for the measurement of lignin content can generally be
pathway in order to produce plants with desirable properties, grouped into two categories: direct and indirect quantitation.
including having low lignin contents and/or modified compo- The direct methods include the Klason lignin method and
sition and structure, developing efficient biomass pretreat- the recently proposed ALBTH method.19 These methods use
ment processes, and proposing new strategies focused on con- acids to hydrolyze and solubilize carbohydrates in samples
verting and utilizing the lignin stream up-front.7–10 In all these leaving the majority of the lignin as a solid residue to be deter-
activities, as well as in conventional pulping, irrespective of mined by gravimetric measurement. The Klason procedure
whether they are academic or industrial, lignin content uses 72% H2SO4 followed by more dilute acid hydrolysis to dis-
solve away carbohydrates, leaving lignin as an insoluble
residue.20 The small amount of lignin dissolved in the acidic
a
State Key Laboratory of Pulp and Paper Engineering, South China University of solution, called acid-soluble lignin (ASL), is determined by UV
Technology, Guangzhou, 510640, China spectrophotometry.21,22 The ALBTH method uses 60 wt% LiBr
b
Department of Biochemistry and The DOE Great Lakes Bioenergy Research Center,
solution containing 40 mM HCl to dissolve polysaccharides
The Wisconsin Energy Institute, University of Wisconsin, Madison, WI 53726, USA.
E-mail: fachuanglu@[Link]
leaving lignin as a solid residue, principally similar to the
† Electronic supplementary information (ESI) available. See DOI: 10.1039/ Klason method.19 The Klason procedure suffers from labor-
d1gc01507a intensive and tedious operation. Both the Klason method and

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the ALBTH method have some shortcomings including poten- it has not been used routinely for quantification of lignin in
tially insufficient hydrolysis of cellulose and the formation of whole plant samples.
humins from sugars, which often result in an overestimation Although many methods are available for directly and
of lignin.19,23,24 Additionally, a relatively large amount of indirectly measuring lignin contents in lignocellulosic
sample is required to produce reliable results by gravimetric biomass, new alternative methods with advantages over the
measurement and the acid-soluble lignin, which is significant ones currently used are rather desperately required to stream-
in hardwood and grass lignins, needs to be measured by UV line analyses. In this work, a new spectroscopic method, called
methods but from which the interference from furfurals is a the CASA lignin method, was established for measuring the
concern.11 Indirect methods for lignin quantitation include lignin content of lignocellulosic biomass by UV spectropho-
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invasive and non-invasive methods. Two invasive procedures tometry, based on the full dissolution of whole biomass in
(using thioglycolic acid or acetyl bromide) are based on the 72% sulfuric acid (SA) containing the amino acid cysteine.
Open Access Article. Published on 09 June 2021. Downloaded on 2/22/2022 [Link] AM.

complete solubilization of whole cell wall material or lignin

via sufficient derivatization, and the dissolved lignin in solu-
tion is measured by UV spectrophotometry.11 The thioglycolic Experimental
acid lignin method has not been widely used perhaps because
Materials
of the lengthy process, inconsistency with other methods, and
the lack of suitable lignin standards required for calibration. All chemicals and reagents are commercial products and used
The acetyl bromide method is the most popular indirect as supplied. L-Cysteine, microcrystalline cellulose and xylan
method for lignin quantitation, largely because of its relative were purchased from Sigma-Aldrich. Sulfuric acid (72%, 12 M,
simplicity and speed and the ability to use small sample SA) was prepared by careful dilution of concentrated (95–98%)
weights.25 The method has been modified several times to sulfuric acid purchased from Sigma-Aldrich or purchased from
determine the lignin content in non-woody plant Fisher Scientific. The lignocellulosic biomass materials were
samples.23,24,26 Briefly, a few milligrams of pre-extracted wood ground with a Wiley mill and sieved to collect the fraction
(CWR = cell wall residue after solvent extraction) are placed in between 40 and 80 mesh for analysis.
a glass vial containing 5 mL of 25% v/v AcBr in acetic acid,
sealed with Teflon-lined caps, and heated at 70 °C for 30 min. Methods
Lignin undergoes bromination of its α-hydroxy groups and Solvent extraction. To remove non-cell-wall extractives, the
acetylation of its γ-hydroxy groups and any free-phenolic groups ground biomass was Soxhlet-extracted with benzene/alcohol
and is consequently dissolved in acetic acid. The dissolved (v/v 2 : 1) for 4 h, followed by 95% ethanol extraction for 12 h;
lignin is subsequently quantified by UV spectrophotometry at toluene could replace benzene for safety reasons, and simply
280 nm. Dence27 cited the advantages of the procedure as being using 80% ethanol extraction alone is quite effective especially
rapid and simple, appropriate for small sample sizes (5–25 mg), for woody biomass.34 For monocot or grass biomass or non-
with no need to correct for acid-soluble lignin, providing precise woody dicots, extraction with water under sonication is rec-
absorbance values for determining total lignin content, and ommended first to remove proteins and other water-soluble
having less interference from non-lignin products. However, the components. Each solvent-extracted biomass sample was kept
furfural products derived from xylan interfere with the UV in a sealed glass bottle after drying in an oven at 50 °C for 48 h
absorbance of lignin at 280 nm and AcBr lignin values often and placed in a vacuum desiccator over P2O5.
differ from those from Klason lignin analysis that is still con- Synthesis of model compounds. Monolignols (coniferyl
sidered to provide the best lignin measure.11 alcohol, sinapyl alcohol, and p-coumaryl alcohol) were pre-
Noninvasive methods for lignin quantitation exploit the pared according to the published methods.35,36 Milled wood
properties of lignin to absorb radiation in specific regions of lignins (MWLs) were prepared from the corresponding
the electromagnetic spectrum. Because lignin has stronger biomass according to the procedure described by Björkman.37
absorbance to UV light at a wavelength of 280 nm than carbo- Preparation of model compounds and synthetic lignins
hydrates, UV-microspectrophotometry was applied to measure (dehydrogenation polymers, DHPs) is described in the ESI.†
lignin concentrations in cell walls of specific plant tissues.28,29 Klason method. The Klason method was performed to
Infrared spectroscopy has been considered as a method for measure the acid-insoluble lignin as well as acid-soluble lignin
quantifying lignin in samples, particularly with the application of biomass samples according to the NREL protocol (NREL/
of techniques such as diffuse reflectance Fourier transform TP-510-42618).13,38
spectrometry.30 Nuclear magnetic resonance spectroscopy Determination of the lignin content using the CASA lignin
(NMR) is a powerful tool frequently used to characterize struc- method. A cysteine stock solution (0.1 g mL−1) in 72% SA was
tural features of lignin, particularly when the lignin-containing prepared by dissolving 10 g L-cysteine in 100 mL SA (72% sul-
samples can be dissolved in, or swollen by, a suitable solvent furic acid). The solvent-extracted biomass sample (5–10 mg,
for solution-state NMR.31,32 The improved spectral resolution weighed to the nearest 0.01 mg) was placed in a 4 mL glass
of 13C-solid NMR, including cross-polarization/magic-angle- vial, and 1.0 mL of the prepared stock solution was added. The
spinning (CP/MAS) NMR, allows this technique to be used to mixture was sealed with a Teflon-lined screw-cap and stirred at
analyze lignin in lignocellulosic biomass samples;33 however, 24 °C (room temperature) using a magnetic stir bar (400 rpm)

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for 60 min, at which point the biomass had completely dis- wood meal was not complete in 30 min when the cysteine con-
solved. The solution was diluted with deionized water to a centration in SA was reduced to 0.05 g mL−1.
volume of 50 mL or 100 mL in a volumetric flask, depending The dissolving temperature was decreased to room tempera-
on the lignin content and biomass weight used, to allow the ture (24 °C in this study) in order to identify a milder con-
diluted solution to have an appropriate UV-absorbance (<1.0) dition, allowing convenient operation and minimizing interfer-
at 283 nm. The absorbance of the diluted solution was ences from carbohydrates. It was found that lignocellulosic
measured at 283 nm (A283) in a 1 cm quartz cell using a UV biomass samples were completely dissolved in the cysteine–SA
spectrophotometer against a blank solution (1 mL stock solu- solution containing 0.1 g mL−1 cysteine at 24 °C after stirring
tion diluted to the corresponding volume). If necessary, a UV- for 60 min. When the solution was diluted with DI water to a
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absorption spectrum was obtained by scanning the absor- volume of 100 mL to allow for appropriate UV absorbance
bance from 230 to 400 nm on the spectrophotometer at 1 nm values (0.1–0.9) at λ = 283, a clear colorless solution was
Open Access Article. Published on 09 June 2021. Downloaded on 2/22/2022 [Link] AM.

intervals. The lignin content (CASA_L%) of the lignocellulosic formed (Fig. 1). Although the exact mechanism behind the dis-
biomass was calculated based on the Beer–Lambert law using solution of lignocellulosic biomass in 72% H2SO4 containing
formula (1). All tests were performed at least in triplicate, and cysteine is not yet clear, discovering the extraordinary ability of
the mean and standard deviation are reported. the cysteine–SA combination to dissolve lignocellulosic
materials under otherwise mild conditions (24 °C in 60 min) is
Abs  V
CASA Lð%Þ ¼  100 ð1Þ striking, potentially allowing lignin contents to be measured
ε  ms  L
in a particularly convenient way that requires no heating, only
where Abs is the UV absorbance of the diluted solution at λ = a few mg of sample, and minimal amounts of reagents/
283 nm; V is the total volume (in L) of the diluted solution; ms solvents.
(g) is the mass weight of the extractive-free lignocellulosic The UV absorption spectrum of the dissolved loblolly pine
sample; L is the light path length, 1 cm here; ε (g−1 L cm−1) is wood had a local maximum absorption at λ = 283 nm, showing
the UV-absorption coefficient (absorptivity) of lignin at λ = the aromatic characteristic of lignin. The milled wood lignin
283 nm. (MWL) isolated from the loblolly pine wood was also readily
(ES & H considerations: sulfuric acid is corrosive and dissolved in the cysteine–SA solution under the same con-
should be handled with care.) ditions, forming a grey-colored solution. The UV absorption
spectrum of the dissolved MWL was similar to the one
obtained from the dissolved loblolly pine wood or a synthetic
Results and discussion guaiacyl lignin (G-DHP, dehydrogenation polymer) made from
coniferyl alcohol (Fig. 2A), suggesting that the UV absorption
Dissolution of lignocellulosic biomass of the dissolved wood was mainly contributed by lignin. When
Lignocellulosic biomass is normally not soluble in any solvent. microcrystalline cellulose, glucose, or xylose were dissolved in
Although isolated or purified cellulose can be dissolved in
many solvents and lignin can be partially extracted from ligno-
cellulosic biomass under various conditions, complete dis-
solution of woody materials requires severe conditions such as
intensive ball-milling and/or high temperature in ionic
liquids. Acetyl bromide (25%) in acetic acid has been used for
dissolving woody materials to measure lignin contents by UV
spectrophotometry. However, acetyl bromide is a corrosive,
volatile, and irritating compound such that the experiments
are required to be performed within a fume hood. Fig. 1 Illustration of the dissolution of wood meal in cysteine–SA for
In an initial trial to dissolve whole wood meal, 10 mg of lignin quantification.
loblolly pine wood powder of less than 0.5 mm size was stirred
in 1 mL of SA containing 0.1 g mL−1 of cysteine at 60 °C. A
homogeneous, purple-colored solution was formed in 30 min,
demonstrating that the woody biomass can be quickly and
completely dissolved in SA in which cysteine had been added.
When the solution was diluted to a volume of 10 mL, a clear
colorless solution was obtained. In contrast, the SA alone pro-
duced a fine-solid suspension of a black residue, exactly as
normally seen when performing the Klason lignin procedure
(Fig. S1, ESI†). When the cysteine concentration in SA solution
was reduced to 0.07 g mL−1, the wood meal could also be dis- Fig. 2 (A) UV spectra of dissolved G-DHP, loblolly pine MWL and
solved in 30 min at 60 °C although its diluted solution was loblolly pine wood. (B) UV spectra of loblolly pine MWL, xylan, glucose,
pale-yellow colored. The dissolution of the same amount of xylose, and cellulose dissolved in cysteine–SA.

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1 mL of the stock cysteine solution under the same conditions, MWLs,41 native lignin,42 and lignins isolated by acetyl bromide
their UV absorption spectra showed very low absorptivity at or acidic dioxane.43,44 The lignin absorptivity can also be esti-
283 nm (Fig. 2B). The UV-absorbance at 283 nm could there- mated by using wood samples of known lignin content, for
fore be used for the determination of lignin in lignocellulosic instance by Klason lignin contents or total lignin contents.27
biomass by spectrophotometry following the full dissolution of In this study, synthetic lignins, usually termed DHPs (dehydro-
the sample in cysteine–SA (followed by suitable dilution in genation polymers) made from monolignols, were used as
water). It was therefore decided to use 0.1 g mL−1 of cysteine in lignin standards to develop the standard curves. Using DHPs
SA as the stock solution to dissolve lignocellulosic biomass at as standards has some advantages: (1) the synthesized DHPs
24 °C and 60 min as treatment time to produce a stable solu- are pure and can be readily prepared in adequate quantities;
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tion for measuring lignin content with a spectrophotometer. (2) DHPs with varying molar ratios of units can be assessed to
study how G : S ratios affect the absorptivity of lignin. Thus,
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Correlation of UV absorbance with the lignin content five DHP preparations including one G-DHP, three GS-DHPs
Varying amounts (4–9 mg) of loblolly pine wood meals were with different G : S ratios, and one GSH-DHP (Table 1), were
stirred in the cysteine–SA (1 mL) solution at room temperature synthesized in a biomimetic system using peroxidase–H2O2
(24 °C) for 60 min and the resulting homogeneous solutions combination to produce phenolic radicals according to the
were diluted 100-fold to 100 mL. The absorbance of the conventional “Zutropf” protocol in which monomers are
diluted solution was measured at 283 nm (A283) in a 1 cm slowly dropped into the solution.45,46 The UV spectrum of the
quartz cell using a UV spectrophotometer. A very good linear G-DHP treated or dissolved by the cysteine–SA solution was
relationship was obtained (R2 = 0.9965) when a plot was made similar to that resulting from loblolly pine wood or its MWL
with the values of A283 against lignin concentrations calculated (Fig. 2A). The standard curves produced from the G-DHP had a
from the Klason lignins. When other lignocellulosic samples slope of 17.25, similar to that (17.61) obtained from loblolly
such as poplar and bamboo were applied to the cysteine–SA pine based on its Klason lignin content (Fig. 3). Therefore, the
dissolving system, similar results were obtained (Fig. 3A–C). absorption coefficient of 17.25 g−1 L cm−1 is recommended for
the CASA method to determine lignin contents in softwood
The absorptivity of lignin dissolved in cysteine–SA biomass.
As a spectrophotometric method, the CASA method requires a The UV spectra of the SG-DHPs (DHP-A, DHP-B and DHP-C)
reliable standard for developing standard (calibration) curves. dissolved in cysteine–SA had similar absorptivities at λ = 283
However, finding an appropriate lignin preparation is challen- although their absorptivities varied over wavelengths ranging
ging because lignin itself is not a pure compound, varying in from 300 nm to 350 nm (Fig. 4). As G : S increases, the absorp-
composition and inter-unit linkage distribution. In the past,
several materials have been tested as potential standards for
the acetyl bromide method, including processed lignins,39,40 Table 1 Molar ratios and absorptivities (ε, at 283 nm) of DHPs dissolved
in cysteine–SA

ε, at 283 nm
Molar ratios (g−1 L cm−1) Recommended ε

G-DHP G 17.25 ± 0.14 17.25 (G-lignin)

DHP-A G:S = 3:7 11.21 ± 0.14 11.23
DHP-B G:S = 5:5 11.26 ± 0.23 G : S ratios ≤1
DHP-C G:S = 7:3 12.20 ± 0.14 12.35
DHP-D G : S : H = 7.5 : 2 : 0.5 12.50 ± 0.08 G : S ratios ≥2

Fig. 3 (A–C) Linear correlations between UV absorbance (at 283 nm) of

the cysteine–SA-dissolved lignocellulosic biomass samples and their
lignin concentrations calculated based on their Klason lignin contents.
(D) Standard curve obtained from a G-DHP, showing its slope (absorp- Fig. 4 UV spectra of DHP-A (7 : 3 S : G), DHP-B (5 : 5 S : G), DHP-C (3 : 7
tivity) of 17.25 g−1 L cm−1. S : G) and DHP-D (2 : 7.5 : 0.5 S : G : H).

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tivity of the DHP in this range decreases. Although the absorp- mers tested. The spectra of H type monomeric compounds 5
tivity values for DHP-A and DHP-B are almost the same, DHPs and 8 treated with cysteine–SA showed local maximal absorp-
with higher G : S ratios generally had higher absorption coeffi- tion at around 276 nm in their UV spectra, although their
cients. Considering that the G : S ratios for the majority of absorption coefficients at 283 nm were relatively low compared
hardwood lignins (SG-lignin) or grass lignins (SGH-lignin) are to those of DHPs or lignins.
equal to or less than 1, the absorption coefficient of 11.23 g−1 Our model study indicated that guaiacyl type compounds
L cm−1 is recommended to be used for the CASA method have higher UV absorptivities (at 283 nm) than those of syrin-
quantification of lignin in hardwood or monocot ligno- gyl or p-hydroxyphenyl compounds, which is consistent with
cellulosic materials. the observation that softwood lignins have higher absorptivity
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than hardwood or monocot lignins.

Absorptivity of lignin-related model compounds
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Effects of cysteine–SA treatment on lignin spectral

To understand how much the various guaiacyl (G), syringyl (S),
characteristics
and p-hydroxyphenyl (H) units in lignin contribute to a
lignin’s UV absorptivity, eight lignin-related model compounds To investigate the effect of cysteine–SA treatment on the spec-
(Fig. 5) were treated with cysteine–SA following the same pro- tral characteristics of lignins, three isolated lignin preparations
cedure as used for lignin quantification. The three G-type com- (loblolly pine MWL, aspen MWL, and bamboo MWL) were dis-
pounds (1, 3, and 6) had similar spectra, each with a local solved in the cysteine–SA solution under the general con-
maximum absorption at around 280 nm and had the highest ditions and their UV spectra were compared with those of the
absorption coefficient at 283 nm among the three types of corresponding lignin samples dissolved in neutral aqueous
units (Table S2, ESI†); when dissolved in the cysteine–SA solu- dioxane solutions (Fig. 6). Generally, the cysteine–SA treatment
tion, syringaresinol 2 had a UV spectrum similar to those from changed lignin structures resulting in lower absorptivities,
hardwood lignins or GS-DHPs. The absorptivity at 283 nm for especially for bamboo lignin at λ from 275 nm to 350 nm. Two
syringaresinol dissolved in cysteine–SA is 11.02 g−1 L cm−1, local maximal absorptions were observed for bamboo MWL in
close to that obtained from DHP-A, although the S type mono- dioxane solution, one is at λ = 310 due to the conjugated
mers, S-glycerol 4 and ethyl sinapate 7, had the lowest absorp- double-bond of the hydroxycinnamates, mainly p-coumarate,
tion coefficients of 3.95 and 3.45 g−1 L cm−1 respectively. The present in the lignin; the other one at λ = 286 nm is caused by
local maxima at 280 nm were less pronounced for the S mono- aromatic rings of lignin units and hydroxycinnamates. After
being dissolved in the cysteine–SA solution, the local
maximum at 310 nm disappeared and the local maximal
absorption at 286 nm shifted to 282 nm, indicating that the
conjugated double-bond was saturated in the strong acid
environment with the strong nucleophile, cysteine. This result
is consistent with those from the model compounds noted
above. The UV spectrum of aspen MWL in dioxane had a local
maximum at 277 nm, which became a shoulder with lower
absorptivity after the CASA treatment. The UV spectrum of the

Fig. 5 Chemical structures of lignin-related compounds and UV

spectra of these compounds treated with cysteine–SA, showing the Fig. 6 UV spectra of three lignin preparations, showing the changes in
differences in their absorptivities at 283 nm. their spectral characteristics caused by treatment with cysteine–SA.

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needed; (4) the method provided results that are well corre-
lated to the total lignin contents obtained using the Klason
method; and (5) it is obvious that the method can be operated
in a high-throughput mode.

Conflicts of interest
There are no conflicts to declare.
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Fig. 7 Correlations between the lignin contents measured using the

CASA lignin method and the traditional Klason method.
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Acknowledgements
loblolly pine CEL, a G-type lignin, was slightly changed by the
cysteine–SA treatment, shifting the local maxima absorption The authors are grateful for the financial support for this work
from 281 to 283 nm but otherwise retaining its characteristics. by the National Natural Science Foundation of China
(31770621 and 31870560) and the DOE Great Lakes Bioenergy
Verification of the proposed CASA lignin method Research Center (DOE BER Office of Science DE-SC0018409).
The accuracy or reliability of an indirect analytical method
always needs to be verified by comparing or correlating to the
established method. To verify the reliability of the CASA lignin Notes and references
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