Multifaceted Approach for 

Determining the Absolute 

Values for Lignin Subunits in 

Lignocellulosic Materials

Daniel J. Yelle

United States Department of Agriculture

Forest

Service

Forest Products

Laboratory

Research Note

FPL–RN–0384

August

2020

MeO

O

OMe

HO

O

HO

OMe

MeO

Lignin

β α 4

γ

NMR

+

GC-MS
August 2020

Yelle, Daniel J. 2020. Multifaceted approach for determining the absolute 

values for lignin dubunits in lignocellulosic materials. Research Note 

FPL-RN-0384. Madison, WI: U.S. Department of Agriculture, Forest 

Service, Forest Products Laboratory. 7 p.

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Abstract

Quantifying the cell wall polymers in wood and plants has 

been a tremendous challenge because no single method can 

achieve absolute quantification of any cell wall polymer. 

Two-dimensional NMR spectroscopic techniques have 

advanced this area with the introduction of wood dissolution 

and cryogenically cooled probes, allowing for semiquantification. However, a more robust technique had to 

be developed to allow for a more absolute quantification 

of cell wall polymers. This study explored a multifaceted 

approach to accurately quantify lignin methoxyls using gas 

chromatography and mass spectroscopy so as to normalize 

the methoxyl peak in the two-dimensional NMR spectra. 

This made it possible for any well-dispersed peak in the 

spectra to be accurately quantified. Several lignin model 

compounds were analyzed to test the accuracy of the 

methoxyl analysis, and several lignocellulosic materials 

were tested using this approach. The results showed that 

the methoxyl analyses of the model compounds were 

useful in confirming the accuracy of the method, which 

allows for faster and more robust methoxyl analysis of 

the lignocellulosic materials. The importance of this study 

relates to a fundamental understanding of what specific 

cell wall polymers are being modified during chemical, 

biological, or other modification of lignocellulose.

Keywords: Lignin, methoxyls, gas chromatography, 

mass spectroscopy, NMR spectroscopy, absolute value, 

quantification

Contents

Introduction..........................................................................1

Experimental........................................................................2

Results and Discussion ........................................................3

Conclusions..........................................................................6

Literature Cited ....................................................................6

Acknowledgment

The author expresses sincere gratitude to Kolby Hirth, 

Forest Products Laboratory, Madison, Wisconsin, for the gas 

chromatography and mass spectroscopy analyses described 

in this paper.
Multifaceted Approach for Determining 

the Absolute Values for Lignin Subunits 

in Lignocellulosic Materials

Daniel J. Yelle, Research Forest Products Technologist

USDA Forest Service, Forest Products Laboratory, Madison, Wisconsin, USA

Introduction

Visualizing changes in the chemistry of wood and plant cell 

walls after some kind of modification (such as chemical, 

hydrothermal, biological) using one-dimensional (1D) NMR 

spectroscopy is not greatly informative. In many cases, the 

1D NMR peaks in 13C or 1H spectra of cell wall polymer 

structures are not well dispersed and overlap heavily 

(Ralph et al. 1999). Significant improvements over the 1D 

NMR experiments were made with the utilization of twodimensional (2D) heteronuclear single-quantum coherence 

(HSQC) applied to ball-milled wood or plants (Lu and Ralph 

2003; Yelle et al. 2008a; Kim et al. 2008; Kim and Ralph 

2010). These advanced techniques, in conjunction with 

cryogenically cooled triple-resonance probes, have allowed 

for significant advances in our understanding of whole cell 

wall chemistry in wood and plants and their modifications 

(Yelle et al. 2008b; Yelle et al. 2011a; Yelle and Ralph 

2016). Yet, with these existing techniques there remains the 

challenge of absolute quantification of cell wall structures. 

With the introduction of adiabatic pulse sequences (Kupče 

and Freeman 2007), the HSQC experiment has become 

more quantitative. Thus, semi-quantitative information can 

now be obtained from these adiabatic HSQC experiments, as 

long as the cell wall material is prepared properly and some 

rules are followed in the NMR acquisition and processing 

(Zhang and Gellerstedt 2007; Yelle et al. 2011a; Yelle et al. 

2013). 

The semi-quantitative data that is achieved with these 2D 

NMR techniques are not without their own limitations. 

For example, polymeric structures in the cell wall can be 

quantified only relative to some large peak (or peaks) in 

the spectra. The aromatic guaiacyl unit (C2) and syringyl 

unit (C2 and C6) peaks are known to be commonly used 

as internal references in 2D NMR spectra because they 

are indicative of native lignin (Lu and Ralph 2003; Yelle 

et al. 2008a). Thus, selecting the guaiacyl C2 or syringyl 

C2/C6 aromatic peaks as internal references to help semiquantify other peaks in the 2D NMR spectra sounded 

promising. However, researchers have revealed that when 

lignin condensation occurs at the C5-position of a guaiacyl 

unit during a plant cell wall modification, the newly 

condensed bond at C5 created a broadened group of peaks 

surrounding the C2 peak that could not be completely 

attributable to the native guaiacyl C2 peak (Yelle et al. 

2008b). Furthermore, peaks arising from condensation 

at the C5-position in wood or plants with both guaiacyl 

and syringyl units may potentially be counted as part of 

syringyl aromatic peaks at C2 and C6, thus overestimating 

the amount of syringyl units and interjecting unwanted 

error in any subsequent quantification (Zhang et al. 2016). 

The lignin methoxyl group is a common peak that was 

chosen as an internal reference because of its good chemical 

stability (Lundquist and Lundgren 1972). However, in 

certain cell wall modifications, the lignin methoxyls are not 

always stable and may cleave or become demethoxylated 

during certain biological treatments. One such treatment 

is brown-rot decay of wood, where the ethers in lignin can 

become cleaved because of the presence of nonselective 

hydroxy-radicals formed during Fenton chemistry (Yelle 

et al. 2008b; Goodell et al. 1997). Nevertheless, even 

when the lignin methoxyl group is cleaved to a substantial 

degree during a biological treatment, methoxyl analysis 

using a chromatographic method has been shown to be 

accurately determined (Li et al. 2017). Collectively, the 

lignin methoxyl is currently the most promising functional 

group in wood and plants to be the representative internal 

reference for quantifying cell wall structures. Quantifying 

the chemical structures in wood requires an approach that 

involves 2D NMR experiments (such as adiabatic HSQC) 

and methoxyl analysis (Yelle et al. 2011b). With methoxyl 

analysis, the chemical structures in the NMR spectra can 

be quantified to give absolute values because the methoxyl 

peak in the NMR spectra can be normalized to this value, 

but this of course requires an accurate and robust methoxyl 

analysis method. 

The methoxyl analysis of lignin not only is a fundamental 

characterization method of lignin but can provide indicative 

information on modification of the cell wall polymer 

chemistry. The original method was introduced by Zeisel, 

in which methoxyl or ethoxyl groups in compounds were 

treated and cleaved with refluxing hydriodic acid (HI), 

giving off ethyl or methyl iodide (Zeisel 1885). The 

most widely used modification of the Zeisel method was 

developed by Viebӧck and Schwappach (1930) because of 

its incorporation of titration, rather than precipitation, which 

increased precision and accuracy. However, this method 
Research Note FPL–RN–0384

2

is time consuming, involving an intricate apparatus, with 

distillation of the volatile alkyl iodide that must be trapped 

and then titrated. 

The use of gas chromatography in methoxyl content analysis 

of lignin model compounds, lignin, and lignocellulosics 

has been developed by several researchers to help simplify 

the method. Shtreis and Bobkova (1973) studied methoxyl 

analysis of lignin model compounds, lignin, and wood 

using gas-liquid chromatography. Hodges et al. (1979) used 

hydroxypropyl methyl cellulose to study the precision of a 

Zeisel gas chromatographic method for methoxyl analysis 

and found the method gave low variability. Girardin and 

Metche (1983) studied the methoxyl content of poplar 

milled-wood lignin using gas chromatography and found 

alkyl iodide recovery to be >97%. Baker (1996) performed 

methoxyl analysis of industrially prepared lignins using 

gas chromatography and found that values were within 

2% of those obtained by titrimetric methods. All these 

gas chromatographic methods have demonstrated the 

advantages of saving time and increasing sample throughput 

over those by Zeisel (1885) and Viebӧck and Schwappach 

(1930).

Research by Goto et al. (2005, 2006) demonstrated 

that traditional hydriodic acid (HI) methoxyl analyses 

of lignocellulosic materials may be flawed in that the 

polysaccharides in the material may also release methyl 

iodide in a similar fashion to that of lignin, thus obscuring 

the true number of lignin methoxyls. However, they found 

that selected monosaccharides produced methyl iodide to 

the level of 5–10 μmol/g, whereas selected wood species 

produced 1,400–1,800 μmol/g. Therefore, the contribution 

of saccharides to methoxyl content was found to be less than 

0.7% of the total methoxyl content. For example, if wood 

has 4% methoxyl content, then the highest contribution from 

saccharides would be 0.03%.

This paper focuses on the iodometric method and 

quantifying the released methyl iodide from lignin model 

compounds, wood, and plants. The goal is to determine 

the absolute values for polymeric cell wall structures in 

lignocellulosic materials using a gas chromatographic–mass 

spectroscopic (GC–MS), combined with a solution-state 

NMR spectroscopic, approach. Obtaining absolute values 

of chemical structures in wood and plants is vital, so that 

when modifications are performed, the specific structures 

involved in a cell wall modification can be quantified to a 

high level of confidence. Specific steps in the approach to 

achieve this goal are as follows:

1. Use accessible lignin model compounds to test the 

viability of the gas chromatographic methods described 

in the literature on how to determine methoxyl groups 

and modify methods as necessary (Hodges et al. 1979; 

Girardin and Metche 1983; Baker 1996; Goto et al. 2005, 

2006).

2. With viable and accurate methoxyl contents of the lignin 

model compounds using gas chromatography and mass 

spectroscopy, measure methoxyl contents of several 

lignocellulosic materials.

3. Run two-dimensional 1H–13C correlation NMR 

experiments on selected lignocellulosic materials and 

integrate the major lignin subunit peaks and the methoxyl 

region.

4. Use the determined methoxyl content values to normalize 

the methoxyl peak in the spectra and calculate the 

absolute values for major lignin subunit polymers in 

wood and plant materials.

Experimental

Materials

All wood and plant samples were provided by the 

Forest Products Laboratory. The wood species, common 

North American commercial species, included Populus 

tremuloides (trembling aspen), Liriodendron tulipifera 

(yellow poplar), Acer saccharum (sugar maple), Pinus taeda 

(loblolly pine), and Picea glauca (white spruce). All wood 

materials were from kiln-dried lumber. The plant species, 

herbaceous grasses commonly used as biomass for biofuels, 

were Triticum aestivum (wheat straw) and Heteropogon 

contortus (tanglehead). The wood and plant materials were 

dried in a vacuum oven at 40 °C until their masses were 

constant. Ball-milling of the dry wood and plant materials 

followed a similar method as described in Yelle et al. 

(2008a). Briefly, the dry sound wood or plant stem material 

was weighed out to approximately 500 mg and then cut 

into approximately 5- by 5- by 5-mm (125-mm3) cubes 

and ball-milled using a Retsch PM-400 planetary ball-mill 

(Newtown, Pennsylvania, USA) with a 50-mL ZrO2 jar with 

three 20-mm ZrO2 balls at 200 rpm for 10 h (20 min milling, 

10 min pause). The pre-milled samples were then further 

ball-milled using approximately 200–300 mg of material 

into a 50-mL ZrO2 jar with ten 10-mm ZrO2 balls and milled 

at 300 rpm for a total of 10 h (20 min milling, 10 min pause) 

to give 400 min actual milling time. Model compounds and 

other chemicals used were obtained from Aldrich Chemical 

Company (Milwaukee, Wisconsin, USA), unless otherwise 

noted. 

Methoxyl Analysis

The procedure for determining methoxyl content of the 

lignin model compounds and lignocellulosic samples 

was similar to that described by Hodges et al. (1979), 

Girardin and Metche (1983), Baker (1996), and Goto et al. 

(2005, 2006), but with modifications. Briefly, to a 40-mL 

head-space vial, approximately 5–50 mg of each model 

compound or ball-milled lignocellulosic sample was added, 

followed by 5 mL of 57% hydriodic acid via a volumetric 

pipette. A few carborundum crystals were added to each 
Multifaceted Approach for Determining the Absolute Values for Lignin Subunits in Lignocellulosic Materials

3

vial (to suppress bumping), and the vials were purged 

with nitrogen. The vials were sealed with Teflon/silicone 

septa (with crimped caps), and reaction vials were heated 

in a welled hot-plate to 140 °C for 60 min. Immediately 

after this reaction period, the reaction vials were cooled 

in an ice-water bath, and 10.0 mL of carbon tetrachloride 

(organic solvent) was added through the septa via a 10.0-mL 

syringe (no. 20 size needle) to extract the organic phase. 

As an internal standard, 100 μL of an ethyl iodide solution 

(89.7 mg/mL in carbon tetrachloride) was added through 

the septa via a 100-μL glass syringe so that each sample 

vial contained 10.0 mL + 0.1 mL carbon tetrachloride with 

0.888 μg ethyl iodide. The vials were shaken vigorously 

for 1 min using a mistral mixer and stored in a freezer 

(approximately –20 °C) to allow complete phase separation. 

The organic phase (uppermost layer containing methyl 

iodide) was then subjected to GC–MS analysis. 

Gas Chromatography–Mass Spectroscopy

GC–MS quantitative analyses were performed using a 

Varian (Walnut Creek, California, USA) model 3800 gas 

chromatograph interfaced to a Varian model 4000 ion trap 

mass detector, operated in full scan EI mode at 70 eV in the 

range m/z = 135–175. The transfer line was maintained at 

250 °C. A Restek (Bellfonte, Pennsylvania, USA) Rxi-1MS 

column (30 m × 0.25 mm × 0.25 μm) was temperature 

programmed as follows: 35 °C to 45 °C at 2 °C/min, to 

250 °C at 50 °C/min. A CombiPal autosampler (CTC 

Analytics, Zwingen, Switzerland) equipped with a 10-μL 

liquid syringe was used for 1-μL injections. The injector was 

operated in 50:1 split mode at 200 °C. Quantification was 

performed on peak areas of quant ion m/z = 142 for CH3I 

using Varian MS Workstation software v6.9. Processing was 

performed using triplicate injections of external standards 

with response factor RSD 0.03% and correlation coefficient 

0.999%. Each lignin model compound sample analysis was 

replicated twice with three replicate injections per sample 

to give six total injections. Each lignocellulosic sample 

analysis was replicated three times with three replicate 

injections per sample to give nine total injections.

Solution-State NMR Spectroscopy

The polymeric structures in the lignocellulosic materials 

were further analyzed using solution-state NMR 

spectroscopy. Approximately 30 mg of each ball-milled 

sample was dissolved directly into a 5-mm diameter NMR 

tube (7 in. (178 mm) length) using 500 μl of DMSO-d6

and sonicated at 35 °C for 1 h. In some cases, the ballmilled samples required 1-methylimidazole-d6 (NMI-d6) 

to facilitate dissolution; a solution of 400 μl of DMSO-d6

with 100 μl of NMI-d6 was added directly to the sample 

in a 5-mm NMR tube. The dissolved samples were 

homogeneous and clear solutions. NMR spectra were 

acquired on a Bruker-Biospin (Rheinstetten, Germany) 

AVANCE III HDTM 500 MHz spectrometer fitted with a 

nitrogen-cooled 5-mm ProdigyTM TCI gradient cryoprobe 

with inverse geometry. One-bond 1H–13C correlation 

(HSQC) spectra were acquired using adiabatic Bruker 

pulse program hsqcetgpsisp2.2 and processed as previously 

described (Yelle et al. 2008a) using Bruker TopSpin 3.6.2 

software. 

Quantification of Methoxyls

Quantifying the methoxyls (OMe) in each sample was 

performed using the data from gas chromatography. 

Methoxyl content was calculated by the following 

equations:

(1) OMe (mg) = [Ave μg/μL CH3I found with GC] × 

[10.1 mL total volume] × [1 mmol CH3I/141.95 mg CH3I] 

× [1 mmol OMe/1 mmol CH3I] × [31.03 mg OMe/1 mmol 

OMe]

(2) % OMe = [OMe (mg)] / [weight of wood sample or 

compound (mg)] × 100

(3) μmol OMe/g wood = [OMe (mg)] × [mmol OMe/

31.03 mg] × [1000 μmol/1 mmol] × [integral of subunit 

peak relative to OMe peak] / [weight of wood sample (g)]

Results and Discussion

GC-MS Analysis

Lignin Model Compounds

The purpose of measuring the methoxyl content of lignin 

model compounds was to test the accuracy of the GC–MS 

technique with low-molecular-weight structures that are 

similar to structures found in lignin. Table 1 shows the 

model compounds selected for these initial experiments 

along with the methoxyl analysis results. These model 

compounds represent a variety of functionality at the Cα, 

Cβ, and Cγ positions as well as the potential hydroxyls or 

methoxyls at the C3, C4, and C5 positions. From the 

GC–MS results of these model compounds, the theoretical 

values for methoxyl concentration were compared to the 

measured values. The comparison showed that the technique 

was accurately measuring the methoxyl content for all 

the model compounds tested here with relative standard 

deviation (RSD) values of less than or equal to 3.6.

Lignocellulosics

With the accuracy of lignin model compounds established, 

the focus shifted to the analysis of lignocellulosic materials, 

such as isolated lignins, holocellulose, and ball-milled 

wood and plants. Table 2 summarizes the methoxyl analysis 

results for several lignocellulosic materials. The results of 

the analysis gave low variability overall, with the highest 

variability seen in aspen. Aspen is known to have high 

amounts of tension wood, a reaction wood tissue that is 

high in cellulose content and low in lignin. This may lead 

to higher variability because it is difficult to control where 

reaction wood tissues reside in lumber, and once milled, all 

anatomical features are destroyed.
Research Note FPL–RN–0384

4

Table 1—Lignin model compounds analyzed for methoxyl content using GC–MSa

Model 

compound

Formula 

weight 

(g/mol)

Weight 

(mg)

CH3I 

(theory) 

(μg/μL)b

CH3I 

(average, 

measured) 

(μg/μL)

OMe 

(theory) 

(%)

OMe

(average, 

measured) 

(%)

RSD 

(%)

Vanillin methyl 

ether

166.18 9.62 1.627 1.536 

(0.0281)

37.35 35.25 

(0.64)

1.8

Syringaldehyde 

methyl ether

196.20 10.44 2.244 2.374 

(0.0833)

47.45 49.71 

(1.70)

3.5

4-hydroxy3-methoxy 

cinnamaldehyde

192.21 10.19 0.745 0.792 

(0.0121)

16.14 16.99 

(0.26)

1.5

Ferulic acid 208.21 5.31 0.358 0.361 

(0.0130)

14.90 15.01 

(0.54)

3.6

Syringaldehyde 182.17 10.78 1.663 1.641 

(0.0110)

34.07 33.61 

(0.23)

0.7

Vanillin 152.15 11.16 1.031 1.073 

(0.0318)

20.39 21.23 

(0.63)

3.0

Syringic acid 

methyl ether

212.20 10.06 1.999 2.018 

(0.0307)

43.87 44.29 

(0.67)

1.5

Veratric acid 182.17 10.87 1.677 1.646 

(0.0508)

34.07 33.43 

(0.10)

3.1

Benzyl alcohol 

methyl ether

198.22 6.24 1.327 1.225 

(0.0490)

46.96 43.34 

(1.73)

1.4

Vanillyl alcohol 154.16 11.78 1.074 1.064 

(0.0376)

20.13 19.94 

(0.70)

3.5

a

Each model compound sample was replicated twice, with three separate injections per sample, to give six total injections. 

Standard deviations are given in parentheses.

b

Theory = (mg weighed/10.1 mL) × (1/formula weight of compound) × (no. OMe) × (formula weight of methyl iodide).

Table 2—Ball-milled lignocellulosics analyzed for 

methoxyl content using GC–MSa

Sample

Weight 

(mg)

CH3I 

(average, 

measured) 

(μg/μL)

OMe 

(average, 

measured) 

(%)

RSD 

(%)

Trembling aspen 

(Populus 

tremuloides)

49.65 1.072 

(0.1606)

4.8 

(0.33)

6.9

Yellow poplar 

(Liriodendron 

tulipifera)

30.09 0.723 

(0.0248)

3.5 

(0.10)

3.0

Sugar maple 

(Acer saccharum)

29.40 0.698 

(0.0153)

5.2 

(0.11)

2.2

Loblolly pine 

(Pinus taeda) 

30.30 0.507 

(0.0062)

3.7 

(0.05)

1.5

White spruce 

(Picea glauca)

49.06 1.049 

(0.0518)

4.7 

(0.27)

5.8

Wheat straw 

(Triticum aestivum)

10.83 0.151 

(0.0161)

3.1 

(0.17)

5.6

Tanglehead 

(Heteropogon 

contortus)

30.60 0.347 

(0.0032)

2.5 

(0.09)

3.7

a

Each lignocellulosic sample was replicated three times, with three 

separate injections per sample, to give nine total injections. Standard 

deviations are given in parentheses.
Multifaceted Approach for Determining the Absolute Values for Lignin Subunits in Lignocellulosic Materials

5

The syringyl/guaiacyl (S/G) ratio of hardwoods can 

substantially influence the amount of lignin methoxyls. 

Hardwoods have both syringyl units (two methoxyls) and 

guaiacyl units (one methoxyl), but the lignin content of 

hardwoods (18% to 25% of dry wood) are typically lower 

than softwoods (25% to 35% of dry wood). Therefore, 

methoxyl contents of softwoods can be equivalent to those 

found in hardwoods, unless the syringyl content is high (that 

is, S/G ratios are >2).

The monocotyledon grasses wheat straw and tanglehead 

showed the lowest methoxyl contents. Grasses also have the 

least amount of lignin (averaging 15% to 22% of dry grass). 

Grasses, like hardwoods, have syringyl units. However, 

they also have p-coumaryl units, or H-units, that have no 

methoxyls. They also have hydroxycinnamates, such as 

ferulate (one methoxyl) and p-coumarates (no methoxyls), 

and tricin (two methoxyls), a flavonoid that was found to be 

covalently incorporated into the lignin polymer in monocots 

(Lan et al. 2015). 

Solution-State NMR Spectroscopy

Two-dimensional (2D) NMR analysis was performed 

on nonderivatized samples to visualize the structural 

chemistry of the lignin. For this study, trembling aspen, 

loblolly pine, and wheat straw were selected for spectral 

analyses. Figures 1 to 3 show the 1H–13C HSQC spectra 

for these lignocellulosic samples. For simplicity, only the 

aliphatic regions are displayed in the figures because the 

aliphatic region is where that portion of the lignin subunits 

are visualized. In the aliphatic region, the major lignin 

subunit linkages (that is, β-aryl ethers, phenylcoumarans, 

and resinols) and the lignin methoxyls were analyzed by 

integrating the contour peaks representing the Hα-Cα 

positions of these subunits. Figure 4 displays the chemical 

structures of these three lignin subunits that are referenced 

in the spectra in Figures 1 to 3. The methoxyl contour peak, 

typically one of the largest peaks in wood and plant cell wall 

NMR spectra, was normalized using the GC–MS data of 

methoxyl content for each lignocellulosic sample. With the 

methoxyl peak normalized, the lignin subunit contour peaks 

in the 2D NMR spectra were integrated to calculate the 

amounts of the major lignin subunits. Table 3 summarizes 

the NMR integral data and absolute quantification results 

for the three major lignin subunits found in trembling aspen 

wood, loblolly pine wood, and wheat straw. 

From the results in Table 3, the quantities of cell wall 

polymer structures in wood and plants can be easily 

compared to each other. The aspen was found to have more 

than double the amount of β-aryl ethers and approximately 

20 times as many resinols of both the pine and the straw. On 

the other hand, the pine was found to have approximately 

18 times and 8 times as many phenylcoumarans as aspen 

and straw, respectively. This analysis gives a clearer 

understanding of how these major lignin subunits are 

distributed in different wood and plant species. 

5.5 5.0 4.5 4.0 3.5 3.0 ppm

55

60

65

70

75

80

85

Trembling Aspen

OMe

Aα

Cα

Bα

Figure 1—Partial 2D NMR spectrum of nonderivatized 

trembling aspen wood cell walls. The horizontal axis 

represents the 1H dimension, and the vertical axis 

represents the 13C dimension. The letters A, B, and C 

(along with their respective darkened contour peak) 

represent the lignin subunit structures as shown in 

Figure 4. The methoxyl (OMe) Aα, Bα, and Cα peaks were 

integrated, and these values were placed into Table 3.

Figure 2—Partial 2D NMR spectrum of nonderivatized 

loblolly pine wood cell walls. The horizontal axis 

represents the 1H dimension, and the vertical axis 

represents the 13C dimension. The letters A, B, and C 

(along with their respective darkened contour peak) 

represent the lignin subunit structures as shown in 

Figure 4. The methoxyl (OMe) Aα, Bα, and Cα peaks were 

integrated, and these values were placed into Table 3.

5.5 5.0 4.5 4.0 3.5 3.0 ppm

55

60

65

70

75

80

85

Loblolly Pine

OMe

Aα

Cα Bα
Research Note FPL–RN–0384

6

Further research is needed to expand this analysis to explore 

more wood and plant species. Moreover, other tissues (such 

as earlywood, latewood, reaction wood), cell types (such 

as vessel, tracheid, parenchyma), and structures in the cell 

wall will need to be explored and quantified so that a more 

comprehensive understanding of cell wall chemistry can be 

established.

Conclusions

Quantifying the detailed chemistry of wood and plant cell 

walls has been one of the most challenging tasks faced by 

wood and plant chemists. The technique described here 

allows researchers to use the accuracy and precision of 

a well-established wet chemical method, the titrimetric 

methoxyl analysis, but use modern GC–MS instrumentation 

to increase sample capacity, speed, and robustness of the 

analysis. Several wood and plant cell wall species were able 

to be analyzed for methoxyl content with good accuracy 

and precision. When the GC–MS methoxyl analysis was 

combined with 2D NMR spectroscopy, the analysis of cell 

wall structures became much more quantitative. GC–MS–

NMR analysis of trembling aspen, loblolly pine, and wheat 

straw cell walls allows for a more quantitative approach to 

understanding the structural chemistry of the cell wall. With 

this technique, any major or minor changes that might occur 

to the cell wall during modifications (such as biological 

decay, chemical modification, biomass pre-treatments) can 

be determined.

Literature Cited

Baker, S.M. 1996. Rapid methoxyl analysis of lignins using 

gas chromatography. Holzforschung. 50(6): 573–574.

Girardin, M; Metche, M. 1983. Fast micro-determination 

of alkoxyl groups by gas chromatography: application to 

lignin. Journal of Chromatography. 264: 155–158.

Goodell, B.; Jellison, J.; Liu, J.; Daniel, G.; Paszczynski, 

A.; Fekete, F.; Krishnamurthy, S.; Jun, L.; Xu, G. 1997. 

Low molecular weight chelators and phenolic compounds 

isolated from wood decay fungi and their role in the fungal 

Figure 3—Partial 2D NMR spectrum of nonderivatized 

wheat straw plant cell walls. The horizontal axis 

represents the 1H dimension and the vertical axis 

represents the 13C dimension. The letters A, B, and C 

(along with their respective darkened contour peak) 

represent the lignin subunit structures as shown in 

Figure 4. The methoxyl (OMe) Aα, Bα, and Cα peaks were 

integrated, and these values were placed into Table 3.

5.5 5.0 4.5 4.0 3.5 3.0 ppm

55

60

65

70

75

80

85

Wheat Straw

OMe

Aα

Cα

Bα

Figure 4—The three major subunits found in lignin. These 

chemical structures are represented in Figures 1 to 3 

and in Table 3. Structure A corresponds to b-aryl ethers, 

structure B corresponds to phenylcoumarans, 

and structure C corresponds to resinols.

O

O

α

α

β β

γ

γ

HO

O

HO

OMe

α β

γ

4 O

HO

α

β γ

5

A B C

Table 3—Absolute quantification of lignin subunits from selected wood and 

plant cell wall lignocellulosicsa

Trembling aspen Loblolly pine Wheat straw

Lignin structure

Integral 

Hα/Cα 

peak

Absolute 

value 

(μmol/g 

wood)

Integral 

Hα/Cα 

peak

Absolute 

value 

(μmol/g 

wood)

Integral 

Hα/Cα 

peak

Absolute 

value 

(μmol/g 

wood)

β-aryl ether (A) 0.5157 797 0.2874 343 0.2919 291

Phenylcoumaran (B) 0.0018 2.8 0.0447 53 0.0063 6.3

Resinol (C) 0.0522 81 0.0036 4.3 0.0045 4.5

a

Absolute values were calculated by the following equation:

Lignin subunits (μmol/g wood) = [integral of Hα/Cα peak relative to OMe peak] × [ave. OMe content 

(μmol OMe/g wood)]
Multifaceted Approach for Determining the Absolute Values for Lignin Subunits in Lignocellulosic Materials

7

biodegradation of wood. Journal of Biotechnology. 53(2–3): 

133–162.

Goto, H.; Koda, K.; Tong, G.; Matsumoto, Y.; Meshitsuka, 

G. 2005. Formation of methyl iodide from methoxyl-free 

compounds by hydriodic acid treatment. Journal of Wood 

Science. 51: 312–314.

Goto, H.; Koda, K.; Tong, G.; Matsumoto, Y.; Meshitsuka, 

G. 2006. Interference of carbohydrates in the determination 

of the methoxyl content of lignin in woody samples. Journal 

of Wood Chemistry and Technology. 26: 81–93.

Hodges, K.L.; Kester, W.E.; Weiderrich, D.L.; Grover, J.A. 

1979. Determination of alkoxyl substitution in cellulose 

ethers by Zeisel-gas chromatography. Analytical Chemistry. 

51: 2172–2176.

Kim, H.; Ralph, J. 2010. Solution-state 2D NMR of ballmilled plant cell wall gels in DMSO-d6/pyridine-d5. 

Organic and Biomolecular Chemistry. 8(3): 576–591

Kim, H.; Ralph, J.; Akiyama, T. 2008. Solution-stage 2D 

NMR of ball-milled plant cell wall gels in DMSO-d6. 

Bioenergy Research. 1: 56–66.

Kupče, E.; Freeman, R. 2007. Compensated adiabatic 

inversion pulses: broadband INEPT and HSQC. Journal of 

Magnetic Resonance. 187: 258–265.

Lan, W.; Lu, F.; Regner, M.; Zhu, Y.; Rencoret, J.; Ralph, 

S.A.; Zakai, U.I.; Morreel, K.; Boerjan, W.; Ralph, J. 2015. 

Tricin, a flavonoid monomer in monocot lignification. Plant 

Physiology. 167(4): 1284–1295.

Li, H.; Yelle, D.J.; Li, C.; Yang, M.; Ke, J.; Zhang, R.; 

Liu, Y.; Zhu, N.; Liang, S.; Mo, X.; Ralph, J. 2017. 

Lignocellulose pretreatment in a fungus-cultivating termite. 

Proceedings of the National Academy of Sciences. 114(18): 

4709–4714.

Lu, F.; Ralph, J. 2003. Non-degradative dissolution and 

acetylation of ball-milled plant cell walls: high-resolution 

solution-state NMR. Plant Journal. 35: 535–544.

Lundquist, K.; Lundgren, R. 1972. Acid degradation of 

lignin. Part VII. The cleavage of ether bonds. Acta Chemica 

Scandinavica. 26(5): 2005–2023.

Ralph, J.; Marita, J.M.; Ralph, S.A.; Hatfield, R.; Lu, F.; 

Ede, R.M.; Peng, J.P.; Quideau, S.; Helm, R.F.; Grabber, 

J.; Kim, H.; Jimenez-Monteon, G.; Zhang, Y.S.; Jung, 

H.J.; Landucci, L.L.; MacKay, J.; Sederoff, R.R.; Chapple, 

C.; Boudet, A.M. 1999. Solution-state NMR of lignins. 

In: Argyropoulos, D. (ed.). Advances in lignocellulosics 

characterization. Atlanta, GA: TAPPI Press: 55–108.

Shtreis, G.B.; Bobkova, V.V. 1973. Determination of 

methoxyl groups in wood and lignin and its model 

compounds by gas-liquid chromatography. Khimiya 

Drevesiny 14: 72–75.

Viebӧck, F.; Schwappach, A. 1930. Eine neue Methode 

zur massanalytischen Bestimmung der Methoxyl- und 

Athoxylgruppe I. Makro-Analyse. Berichte der Deutschen 

Chemischen Gesellschaft. 63: 2818–2823.

Yelle, D.J.; Ralph, J. 2016. Characterizing phenolformaldehyde adhesive cure chemistry within the wood cell 

wall. International Journal of Adhesion and Adhesives. 70: 

26–36.

Yelle, D.J.; Ralph, J.; Frihart, C.R. 2008a. Characterization 

of nonderivatized plant cell walls using high-resolution 

solution-state NMR spectroscopy. Magnetic Resonance in 

Chemistry. 46: 508–517.

Yelle, D.J.; Ralph, J.; Lu, F.C.; Hammel, K.E. 2008b. 

Evidence for cleavage of lignin by a brown rot 

basidiomycete. Environmental Microbiology. 10: 

1844–1849.

Yelle, D.J.; Ralph, J.; Frihart, C. 2011a. Delineating pMDI 

model reactions with loblolly pine via solution-state NMR 

spectroscopy. Part 2. Non-catalyzed reactions with the wood 

cell wall. Holzforschung. 65(2): 145–154.

Yelle, D.J.; Wei, D.; Ralph, J.; Hammel, K.E. 2011b. 

Multidimensional NMR analysis reveals truncated lignin 

structures in wood decayed by the brown rot basidiomycete 

Postia placenta. Environmental Microbiology. 13(4): 

1091–1100.

Yelle, D.J.; Kaparaju, P.; Hunt, C.G.; Hirth, K.; Kim, H.; 

Ralph, J.; Felby, C. 2013. Two-dimensional NMR evidence 

for cleavage of lignin and xylan substituents in wheat 

straw through hydrothermal pretreatment and enzymatic 

hydrolysis. BioEnergy Research. 6: 211–221.

Zeisel, S. 1885. Über ein Verfahren zum quantitativen 

Nachweis von Methoxyl. Monatshefte für Chemie. 6: 

989–996.

Zhang, L.M.; Gellerstedt, G. 2007. Quantitative 2D HSQC 

NMR determination of polymer structures by selecting 

suitable internal standard references. Magnetic Resonance in 

Chemistry. 45: 37–45.

Zhang, W.; Sathitsuksanoh, N.; Simmons, B.A.; Frazier, 

C.E.; Barone, J.R.; Renneckar, S. 2016. Revealing the 

thermal sensitivity of lignin during glycerol thermal 

processing through structural analysis. RSC Advances. 

6(36): 30234–30246.