A fast track for the accurate determination of methoxyl and ethoxyl groups in lignin

Article abstract of DOI:10.1039/c7ra00690j

A method based on headspace-isotope dilution (HS-ID) GC-MS for the quantitative analysis of methoxyl and ethoxyl groups in any kind of lignin has been developed. The method involves the application of an isotopically labeled internal standard (4-(methoxy-

Full text of DOI:10.1039/c7ra00690j

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A fast track for the accurate determination of
methoxyl and ethoxyl groups in lignin
Cite this: RSC Adv., 2017, 7, 22974
*
Ivan Sumerskii, Thomas Zweckmair, Hubert Hettegger, Grigory Zinovyev,
*
Markus Bacher, Thomas Rosenau and Antje Potthast
A method based on headspace-isotope dilution (HS-ID) GC-MS for the quantitative analysis of methoxyl
and ethoxyl groups in any kind of lignin has been developed. The method involves the application of an
isotopically labeled internal standard (4-(methoxy-d3)-benzoic acid and 4-(ethoxy-d5)-benzoic acid),
which, together with lignin, undergoes standard hydroiodic acid cleavage of methoxyl and ethoxyl
groups, followed by headspace GC-MS analysis of the respective deuterated and non-deuterated
iodomethanes and iodoethanes. As also the internal standard is generated in situ during ether cleavage,
any variations in the procedure are levelled out. The application of the isotopically labeled internal
standard essentially increased the robustness of the overall approach. The accuracy and precision of
methoxyl and ethoxyl group quantification in lignins have been assessed, and the data were compared to
Received 16th January 2017
Accepted 8th April 2017
the classical Zeisel–Viebock–Schwappach method for methoxyl groups in lignins. The novel approach
¨
DOI: 10.1039/c7ra00690j
for methoxyl and ethoxyl group determination showed a more satisfactory precision, accuracy, and also
a much higher sample throughput, with approximately 40 samples per day.
rsc.li/rsc-advances
distinctly accelerated reaction rates when methoxyl groups were
present.7
Introduction
Currently, many endeavors are devoted to lignin demethy-
lation in order to increase the number of hydroxyl groups and
thus to improve its overall reactivity in specic applications.
Various chemical and biological approaches for demethylation
have been proposed.8–10 With regard to analysis in most cases
the authors avoided application of conventional methods for
direct methoxyl group analysis which admittedly are rather
tedious and time-consuming – and opted for rough estimation
of the hydroxyl increase or methanol release according to
spectroscopic or chromatographic methods.
Overall, reliable quantication of the methoxyl group
content provides not only lignin structural information, but
also indicates structural changes during processing or lignin
modication, and can thus serve as an internal reference in the
analysis of other lignin functional groups.11
Within ongoing developments of ethanol-based organosolv
or related biorenery approaches, the analysis of the ethoxyl
group content in technical lignin plays an important role as
well.12 Knowledge of their content offers opportunities to
characterize and design the conditions of the overall process,
or, for example, to serve as a marker in lignin value-added
products. In contrast to methoxyl groups that are genuinely
contained in native lignin, ethoxyl groups are mainly intro-
duced by processing.
Lignin is one of the most abundant renewable aromatic biopoly-
mers, which is inevitably generated in large quantities in different
industrial conversion processes of plant matter. The presence of
vast amounts of lignin has recently triggered research in academia
and industry in order to create commodities such as biofuels and
bio-based chemicals, and also materials of higher value.
Taking a considered view on possible lignin utilization,
signicant progress in fast lignin analytics is necessary before it
becomes a versatile and widely used feedstock for future
industries. In this regard, one of the key parameters of lignin is
the content of methoxyl groups.
Methoxyl groups contained in lignin have been reported to
act as a stabilizer for phenoxyl radicals formed during scav-
enging of free radicals.1–4 This phenomenon is closely con-
nected to the antioxidant capacity of lignin, which has been the
focus of many studies.1–4
Recently, plant methoxyl groups were discovered to have
both distinct stable hydrogen and carbon isotope values upon
which studies of environmental and paleoclimate processes can
be based as well as investigations of the geographical origin and
authenticity of biomaterials.5,6
Recent studies on b-O-4 bond cleavage of lignin model
compounds during alkaline pulping processes have demonstrated
In investigations dealing with biomass lignin content is
a fundamental parameter. Most common methods for lignin
content analysis are acetyl bromide or Klason procedures.13,14
Division of Chemistry of Renewable Resources, Department of Chemistry, University of
Natural Resources and Life Sciences, Konrad-Lorenz-Str. 24, A-3430 Tulln, Austria.
E-mail: ivan.sumerskii@boku.ac.at; antje.potthast@boku.ac.at
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However, there are cases when the biomass undergoes multiple reaction and extraction of analytes with pentane or tetra-
transformations, rendering those common methods inappli- chloromethane, followed by subsequent GC-FID anal-
cable to correctly report the actual lignin content. This is the ysis.16,18,32,33 This approach allowed researchers to achieve values
case for all processes that generate covalent bonding between similar to the titrimetric method at a comparably low relative
lignin and secondary components, which can be carbohydrates, standard deviation (RSD, 2.5%). It was adapted for the analysis
carbohydrate condensation products, or graed polymers and of ethoxyl groups in lignin by application of propyl iodide as the
copolymers.
internal standard.16 In order to minimize the number of steps in
Methoxyl groups' cleavage by the classical hydroiodic treat- sample preparation – and hence to decrease possible errors
ment15 and quantication by a subsequent GC measurement16 caused by sample handling – a headspace (HS) GC-FID
were recommended as an alternative approach for evaluating approach was suggested.15 The peculiarity of the method con-
the lignin content in mulch17 and compost18 because of the high sisted in carrying out the demethylation reaction driven by
stability of methoxyls upon biodegradation. The lignin content hydroiodic acid and the analysis of released methyl iodide in
was calculated based on the assumption that one methoxyl the same headspace vial sealed with a septum. The authors
group corresponds to one 200 Da monolignol C6–C3 unit. The suggested external standard calibration of the HS-GC-FID
results were signicantly more accurate compared to conven- measurement and with that demonstrated decently low RSD
tional approaches.13,14
(<0.69%) conrming similar values for selected lignins by the
During decades of lignin research, many approaches were conventional titrimetric method.15 However, the suggested
proposed for quantitative methoxyl group determination. methodology, involving a neutralization step prior to GC anal-
However, only few of the most trustworthy and reliable ones ysis and thus side reactions of the analyte, raises doubts on the
remained and are in use at present. They belong to so-called stability of the analyte generated and therefore on the accuracy
destructive wet chemistry methods, where methoxyl functional and precision of the entire method.
groups are cleaved off by an acid treatment and the analyte
Currently, the development of lignin characterization
released is further quantied. Methods differ by the type of acid methods involving chemometry and hence statistical models is
applied and by the technique of subsequent analyte determina- also of great importance. Aiming at predicting the methoxyl
tion. One of the most widely used methods proposed by Zeisel, content in lignin, a simple empirical correlation based on lignin
¨
Viebock, and Schwappach involves the quantitative cleavage of ultimate analysis has been developed according to multiple
the methoxyl groups with 57% aqueous hydroiodic acid, followed regression methods.23 Though the average absolute error of the
by the formation of volatile methyl iodide, which is distilled off proposed correlation was rather high (14%), it required only
and “trapped” in a bromine solution by its conversion to the hydrogen and oxygen contents in lignin as inputs.
respective iodic acid.19–22 By reaction with potassium iodide, the
In light of recent developments, also spectroscopic, nonde-
iodic acid is further converted to iodine, which is then analyzed structive methods for the analysis of methoxyl content are worth
by standard iodometric titration.22,23 The formation of six atoms mentioning. Various FTIR34 and NMR4,35 approaches were
of iodine for each methoxyl group allowed the method to possess proposed, but due to the complexity of lignins and certain
a relatively high degree of accuracy. Although the method instrument limitations, they could only give either a relative value
involves many steps in addition to the application of special or just a rough estimation of methoxyl group content.36 Over the
custom-made gas-tight glassware and toxic chemicals, it became last few decades, the development of 13C-NMR techniques,
rmly entrenched in lignin research.1,24–27
providing well-resolved spectra in which methoxyl group signals
Analysis of lignin content in acid and alkaline wood hydro- are not overlapped, and the application of appropriate non-
lysates by means of lignin chlorination followed by GC-FID overlapping internal standards have permitted facile integra-
quantication of the methanol formed from methoxyl groups tion and methoxyl group quantication.37 Further developments
has been demonstrated to give more credible results compared have led to signicant decreases of experimental time38 and
to conventional methods.14 However, the overall error of this demonstrated a strong correlation with values determined by the
method was rather large at approximately 10%.
conventional wet chemistry GC-FID approach.32 Nevertheless, the
Aiming at expanding the frontiers of methoxyl group anal- availability of NMR hardware, the necessity of high purity, and
ysis, numerous attempts to utilize gas chromatography (GC) sufficient solubility of lignin samples in an appropriate solvent,
have been made. The most promising one suggested hydrolytic as well as still rather long experimental time, do not allow
cleavage of methoxyl groups by reuxing lignin in concentrated considering these approaches as routine methods for simple
sulfuric acid, followed by distillation of formed methanol and methoxyl group content determination.
its GC-FID detection.28 This approach was further modied by
In order to generate a high-throughput and robust method,
the implementation of butanol as an internal standard.29 we have developed a new headspace-isotope dilution (HS-ID)
Despite the uncertainties with respect to method precision, GC-MS approach for methoxyl and ethoxyl group analysis
accuracy, and reproducibility, which were not reported in involving the application of in situ-generated, isotopically
literature, this method is still applied.30,31
labeled internal standards upon hydroiodic acid treatment. A
A real pragmatic step toward method simplication and time fast-track protocol was developed to analyze various technical
saving has been done just recently: it was suggested to perform lignins, such as kra, organosolv or lignosulfonates, in much
standard lignin demethylation with hydroiodic acid in a closed shorter time than hitherto possible, while showing superior
vial, injection of an internal standard (ethyl iodide) aer the analytical gures of merit.
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a white solid (12.3 mmol, 90%). TLC (DCM/EtOH 8 : 1 v/v): 4-
Experimental
Materials and chemicals
methoxybenzoic acid-d3 Rf ¼ 0.79; FTIR: 3500–2000 (O–H), 1675
(C]O), 1601, 1575, 1513, 1429, 1299, 1270 cmꢁ1 1H NMR
;
(400.13 MHz, DMSO-d6) d ¼ 12.54 (br. s, 1H, COOH), 7.88 (d, J ¼
8.6 Hz, 2H, CarH), 6.99 ppm (d, J ¼ 8.9 Hz, 2H, CarH); 2H NMR
(61.42 MHz, DMSO-d6) d ¼ 3.77 ppm (s, 3D, CD3); 13C NMR
(100.61 MHz, DMSO-d6) d ¼ 167.2 (C]O), 163.0 (Cq–O), 131.5
(CarH), 123.2 (Cq), 114.0 ppm (CarH), n.d. (OCD3); ESI-MS
(8 ppm in MeOH, positive ionization mode): 156.0 m/z ([M +
H]+, C8H6D3O3); melting point: 184–186 ꢀC (water); purity
Ethyl 4-hydroxybenzoate, iodomethane-d3 (99.5 atom% D)
iodoethane-d3 (99.5 atom% D), HI (57%), HCl, acetone, ethyl
acetate, methanol, diethyl ether, sodium hydroxide, potassium
carbonate, magnesium sulfate, and sodium bicarbonate were
obtained from Sigma-Aldrich GmbH (Schnelldorf, Germany).
Indulin AT lignin was from MeadWestvaco (Charlston, SC,
USA). Other technical lignin samples were provided by collab-
oration partners. Lignin samples were puried in accordance
with procedures described in the literature.27,36 Prior to all
analyses, lignin samples were freeze-dried followed by vacuum
1
according to H-NMR: 99%.
Ethyl 4-(ethoxy-d5)-benzoate (3)
ꢀ
oven drying at 40 C until complete dryness.
Ethyl 4-(ethoxy-d5)benzoate was synthesized according to
the procedure described for compound (1). TLC (hexane/
EtOAc 7 : 3): ethyl 4-hydroxybenzoate Rf ¼ 0.43, ethyl 4-
(ethoxy-d5)-benzoate Rf ¼ 0.80; FTIR: 1707 (C]O), 1605, 1508,
Ethyl 4-(methoxy-d3)-benzoate (1)
Ethyl 4-hydroxybenzoate (2.50 g, 15.0 mmol) was dissolved in
dry acetone (50 mL), and K2CO3 (3.22 g, 23.3 mmol) was added
upon constant stirring. CD3I (1.40 mL, 22.5 mmol) was added to
the suspension, and the reaction mixture was allowed to stir for
48 h at RT. The progress of the reaction was controlled by TLC
(hexane/EtOAc 7 : 3 v/v). The white suspension was ltered,
acetone was evaporated, and aer dissolution in CH2Cl2, the
product was washed with saturated aqueous NaHCO3 solution
followed by water until the washings were neutral. Aer
washing with saturated brine, the organic phase was dried over
anhydrous MgSO4, ltered on a sintered glass lter with appli-
cation of a weak vacuum, and evaporated to dryness. The crude
product (transparent oil) was puried by ash column chro-
matography with hexane/EtOAc (7 : 3 v/v) as the isocratic eluent.
Fractions containing the pure product were combined, ltered,
and evaporated to dryness under reduced pressure. The evap-
oration procedure was repeated twice aer the addition of
CHCl3 (app. 10 mL) yield: 2.65 g of transparent oil (14.5 mmol,
97%). TLC (hexane/EtOAc 7 : 3 v/v): ethyl 4-hydroxybenzoate Rf
¼ 0.43, ethyl 4-(methoxy-d3)benzoate Rf ¼ 0.77; FTIR: 2982 (C–
1255, 1167, 1097, 846, 769, 696 cmꢁ1 1H NMR (400.13 MHz,
;
CDCl3) d ¼ 7.99 (dt, J ¼ 8.9, 2.5 Hz, 2H, CarH), 6.90 (dt, J ¼ 8.9,
2.5 Hz, 2H, CarH), 4.35 (q, J ¼ 7.1 Hz, 2H, CH2), 1.38 ppm (t, J ¼
2
7.1 Hz, 3H, CH3); H NMR (61.42 MHz, CDCl3) d ¼ 4.06 (s, 2D,
CD2), 1.39 ppm (s, 3D, CD3); 13C NMR (100.61 MHz, CDCl3) d ¼
166.4 (C]O), 162.7 (Cq–O), 131.5 (CarH), 122.7 (Cq), 114.0
(CarH), 60.6 (CH2), n.d. (CD2), 14.4 ppm (CH3), n.d. (CD3); purity
1
according to H NMR: 98%.
4-(Ethoxy-d5)benzoic acid (4)
4-(Ethoxy-d5)benzoic acid was synthesized according to the
procedure described for compound (2). TLC (DCM/MeOH 5 : 1):
4-(ethoxy-d5)benzoic acid Rf ¼ 0.72; FTIR: 3200–2300 (O–H),
1668 (C]O), 1605, 1432, 1300, 1268, 1175, 1100, 988, 932, 848,
772, 620 cmꢁ1; 1H-NMR (400.13 MHz, DMSO-d6) d ¼ 7.87 (d, J ¼
9.0 Hz, 2H, CarH), 6.97 ppm (d, J ¼ 9.0 Hz, 2H, CarH); 2H-NMR
(61.42 MHz, DMSO-d6) d ¼ 4.04 (s, 2D, CD2), 1.29 ppm (s, 3D,
CD3);13C-NMR (100.61 MHz, DMSO-d6) d ¼ 166.9 (C]O), 162.1
(Cq–O), 131.2 (CarH), 122.7 (Cq), 114.0 ppm (CarH), n.d. (CD2),
H), 1706 (C]O), 1604, 1509, 1257, 1168, 1096, 769 cmꢁ1 1H
;
ꢀ
n.d. (CD3); melting point: 197–199 C (water); purity according
1
NMR (400.13 MHz, CDCl3) d ¼ 8.01 (d, J ¼ 9.0 Hz, 2H, CarH),
6.92 (d, J ¼ 9.0 Hz, 2H, CarH), 4.35 (q, J ¼ 7.2 Hz, 2H, CH2),
1.39 ppm (t, J ¼ 7.2 Hz, 3H, CH3); 2H NMR (61.42 MHz, CDCl3)
d ¼ 3.84 ppm (s, 3D, CD3); 13C NMR (100.61 MHz, CDCl3) d ¼
166.4 (C]O), 163.2 (Cq–O), 131.5 (CarH), 122.9 (Cq), 113.5
(CarH), 60.6 (CH2), 54.6 (sept, J ¼ 21.3 Hz, 3D, OCD3), 14.4 ppm
to H NMR: 99%.
HI acid treatment and sample preparation
Approximately 1 mL of HI acid (57%) was added to a 10 mL
headspace screw cap vial (ND18 magnetic, Bruckner Analy-
sentechnik, Linz, Austria) containing the lignin sample (5–10
mg), internal standards 4-(methoxy-d3)-benzoic acid (4–5 mg)
and 4-(ethoxy-d5)-benzoic acid (2–3 mg), and a small magnetic
stirring bar. The vials were tightly closed with screw caps
1
(CH3); purity according to H NMR: 95%.
4-(Methoxy-d3)-benzoic acid (2)
Ethyl 4-(methoxy-d3)benzoate (2.50 g, 13.7 mmol) was dissolved equipped with PTFE-covered silicon septa (1.3 mm; Bruckner
in MeOH (20 mL), and a solution of NaOH (0.87 g, 21.8 mmol) in Analysentechnik, Linz, Austria) and placed in a heating/stirring
water (20 mL) was added. MeOH was added until complete module (Pierce Reacti-Therm III) for 3 h at 110 ꢀC. Subse-
dissolution of all reagents. The clear solution was stirred for 3 h quently, the vials were cooled to room temperature, and 4 mL of
at reux conditions. The reaction mixture was diluted with cold water were injected through the septa. In experiments on the
ꢀ
distilled water (20 mL), cooled to 0 C, and then acidied with pH-dependent stability of analytes, in which excess HI was
concentrated HCl to approximately a pH of 2. The product partially or completely neutralized, 4 mL of 1.1 or 2.2 M NaOH
immediately precipitated as a white solid. The solid was ltered were injected. Thus, the volume of the liquid phase in all
and washed with cold distilled water until the washings were experiments was adjusted to approximately half of the total vial
neutral. The product was then lyophilized. Yield: 1.91 g of volume. This decreased the phase ratio and, together with
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heating of the vials in the headspace sampler, enriched the hydroiodic acid treatment of their corresponding precursors, 4-
headspace with volatile analytes.
(methoxy-d3)-benzoic acid (2) and 4-(ethoxy-d5)-benzoic acid (4).
4-(Methoxy-d3)-benzoic acid (2), the actual component used
for in situ generation of iodomethane-d3, was synthesized from
ethyl 4-(methoxy-d3)-benzoate (1) by simple alkaline ester
cleavage and re-acidication (Fig. 1). 1, in turn, was obtained by
alkylation of the phenolic hydroxyl of ethylparaben with
deuterated iodomethane in potassium carbonate/acetone
(Claisen conditions). 4-(Ethoxy-d5)-benzoic acid (4) was synthe-
sized analogously. The structures of the intermediates and the
nal products were conrmed by FTIR and NMR.
As reported earlier, quantitative cleavage of methoxyl groups
in lignins can be achieved by heating 10–20 mg of samples in
0.5 mL of HI (57%) at 130–140 ꢀC for 30 min.25 In this study, we
have applied those established conditions with minor changes.
The volume of HI was increased to 1 mL in order to enhance
method robustness, so that methoxyls and ethoxyls of
completely unknown lignin samples – which might contain, for
example, high amounts of impurities or acid quenchers – would
still be quantitatively cleaved. Besides this, excess amounts of
HI improve homogenization of the reactants and thus lower the
probability of non-reacted matter being le over.
The sample amount applied in the method was set to about 5–
10 mg, which is sufficient for the analysis and at the same time
large enough for accurate weighing. The parameters of the HS-
GC-MS analysis, such as HS loop size, GC split ratio, tempera-
ture, and time program, were optimized by experiments with
vanillin as the source of methoxy groups to be split off (cf. Fig. 2).
As the standards undergo the same cleavage reaction as the
actual analyte samples, any variations in the cleavage procedure
are corrected for. In the subsequent HS-GC-MS analysis, the
methyl and ethyl iodides and the respective deuterated
compounds are separated and detected in SIM mode. Two time
windows with pairs of m/z-values (monoisotopic and isotopi-
cally labeled) were dened: 0–1.9 and 1.9–3.5 min as well as m/z
142/145 and 156/161, respectively, to detect iodomethanes and
iodoethanes (Fig. 2).
Headspace GC-MS
GC-MS analysis was carried out on an Agilent 6890N gas chro-
matograph coupled to an Agilent 5975B inert XL mass selective
detector (MSD). The GC was equipped with a split/splitless inlet
and DB5-ms column (30 m ꢂ 0.25 mm i.d. ꢂ 0.25 mm lm
thickness; J&W Scientic, Folsom, CA, USA). The split/splitless
inlet operated under the following conditions: constant
column ow: 1.0 mL minꢁ1 using helium carrier gas, split-ratio:
1 : 50; injector: 250 ꢀC. Oven temperature gradient prole: 40 ꢀC
(2 min), 10 ꢀC minꢁ1 to 150 ꢀC (3 min) and back to initial values.
The MSD was operated in EI mode at 70 eV ionization energy
and 1.13 ꢂ 10ꢁ7 Pa. Ion source temperature: 230 C, quadru-
ꢀ
ꢀ
ꢀ
pole: 150 C, transfer line: 280 C. The data were acquired in
SIM mode at 50 ms dwell time for each ion group. A closed loop
headspace sampler (Agilent Technologies 7697A equipped with
a 20 mL loop) was used for injection. The sampler operated
under the following conditions: vial temperature: 50 ꢀC, loop
temperature: 60 ꢀC, transfer line temperature: 70 ꢀC; vial
equilibration time: 3 min, vial pressurization time: 0.2 min,
loop ll time: 0.18 min, loop equilibration time: 0.05 min,
injection time: 1 min.
Calibration of the headspace GC-MS method
Calibration of the HS-GC-MS method was performed by adding
various amounts of vanillin (1–7 mg; 10 data points) and 4-
ethoxybenzoic acid (0.05–3 mg; 10 data points) into a set of
headspace vials containing known amounts (ꢃ4 and 2 mg) of the
internal standards (2) and (4), respectively, and then subjecting
them to HI acid treatment (see above). The GC-MS response
ratios of methyl and ethyl iodides and deuterated methyl and
ethyl iodides were plotted against concentration ratios.
¨
Zeisel–Viebock–Schwappach method
Even though the application of the in situ-generated, isotopi-
cally labeled internal standard levels out all possible measure-
ment errors, such as analyte leakage through the septa with
resulting loss of alkyl iodides, it is still strongly recommended—
due to the high toxicity of alkyl halides—to ensure that headspace
Determination of methoxyl groups was done by the Zeisel–
¨
Viebock–Schwappach procedure described in the literature for
comparison.22 A known amount of lignin (15–20 mg) was
weighed directly into a round-bottom 25 mL reaction ask; 300–
350 mg of phenol and 4 mL of 57% HI were added, and the ask
was attached to the custom-made apparatus. All the ground
glass joints were properly sealed with PTFE O-rings (Glinde-
mann, Sigma-Aldrich, Austria). During the reaction of lignin
with HI (1.5 h), the released methyl iodide was transferred by
a ow of N2 carrier gas through the scavenger containing 1% of
¨
red phosphorous into the Viebock–Schwappach apparatus lled
with bromine solution. Released iodine was determined by
titration with sodium thiosulfate solution (0.05 M).
Results and discussion
The developed HS-ID GC-MS method uses the standard HI
cleavage procedure22 as a starting point. Appropriate isotopi-
Fig. 1 Synthesis of the internal standard 4-(methoxy-d3)-benzoic acid
(2). Conditions: (a) CD3I, K2CO3, acetone, RT, 48 h; (b) NaOH, MeOH,
cally labeled internal standards are generated in situ upon H2O, reflux, 3 h; (c) HCl, H2O.
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Fig. 3 Effect of HI acid treatment time on kraft lignin and internal
standard (4-(methoxy-d3)-benzoic acid) on methoxyl groups
conversion and formation of MeI and MeI-d3. Stabilization of the ratio
between generated MeI and MeI-d3 occurred after about 30 min.
Fig. 2 Chromatogram: separation of both analytes, methyl iodide and
ethyl iodide, and detection of the natural and isotopically labeled
counterparts.
vials are tightly closed. Additionally, it is important that the
applied septa do not degrade and produce artifact signals in the
chromatogram. Therefore, several types of septa (different thick-
nesses, silicone, and cover types) were tested. Silicon PTFE-
covered septa were found to be the most stable and reliable.
The 3 mm septa of the crimp cap could easily withstand the vapor
pressure generated at the cleavage temperature of 130 ꢀC.21,22,30 As
a next step, screw caps with headspace vials were implemented in
order to allow an automated methoxyl group determination by
robot derivatization. However, 130 ꢀC was too high for the 1.3 mm
septa applied in screw caps. Therefore, lower temperatures of HI
ꢀ
acid treatment were tested. At 110 C, the 1.3 mm septa remain
stable and do not give additional peaks in the chromatogram.
It was conrmed that a time of 60 min was sufficient for
complete cleavage of methoxyl groups present in both isolated,
puried kra lignins and in lignosulfonates as well as in orga-
nosolv lignin (Fig. 3). The ratio between deuterated and non-
deuterated methyl iodide generated in all cases remains
stable aer 30 min reaction time (Fig. 3). However, in the case of
organosolv lignin, complete cleavage of ethoxyl groups and
stabilization of the ratio between the corresponding ethyl
iodide and deuterated ethyl iodide required 3 h (Fig. 4). Since
longer reaction times did not affect the methyl iodide yield and
its ratio of analyte to internal standard, it was decided to
establish the universal method for any kind of lignin with an HI
treatment for 3 h.
Fig. 4 Ratio between analytes (MeI/MeI-d3 and EtI/EtI-d5) generated
upon HI acid treatment from organosolv lignin and internal standards
(4-(methoxy-d3)-benzoic acid and 4-(ethoxy-d5)-benzoic acid).
Stabilization of the ratio between generated EtI and EtI-d5 occurred
after about 180 min.
whole calibration curve (Table 1). The limit of detection (LOD)
and limit of quantication (LOQ) of compounds were calculated
according to the 3s and 10s criteria, i.e., the threefold or tenfold
standard deviation of the noise quantied by single point
height calibration.39
The accuracy of the methoxyl and ethoxyl groups content in
technical lignins was evaluated by spiking known amounts of
vanillin and 4-ethoxybenzoic acid into known amounts of
a sample matrix. The kra lignin, lignosulfonate (both before
and aer purication), and organosolv lignin yielded a methyl
and ethyl iodide recovery in the range of 99.6–103.7% (Table 2).
The optimized HS-ID GC-MS method was tested on various
technical lignins, in particular kra, organosolv, lignosulfonates,
hydrolysis lignins, and lignins aer chemical modications. The
present HS-ID GC-MS method, compared to the conventional
Precision and accuracy
The precision of the new method was investigated by quintu-
plicate independent analysis of methyl and ethyl iodide
released from vanillin and 4-ethoxybenzoic acid, respectively.
The RSD was evaluated at the lower, middle, and higher ends of
the calibration curves.
It was conrmed that the precision of the analysis of
methoxyl and ethoxyl groups was decently high, and that the
RSD for both functional groups did not exceed 3% over the
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Table 1 Analytical figures of merit of the headspace-isotope dilution GC-MS method
RSD (n ¼ 5), %
LOD, mmol LOQ, mmol Lower end Middle Upper end
R2, 2nd
Internal standard Analyte
4-Methoxy-benzoic
m/z order polynom Calibrated range, mmola
145
acid-d3
Methoxyl- 142 0.999
0.71 ꢂ 10ꢁ3 to 45.0 ꢂ 10ꢁ3 0.21 ꢂ 10ꢁ3 0.71 ꢂ 10ꢁ3 2.56
0.59
2.04
2.08
1.15
4-Ethoxy-benzoic
acid-d3
161
Ethoxyl-
156 1.000
0.42 ꢂ 10ꢁ3 to 17.4 ꢂ 10ꢁ3 0.13 ꢂ 10ꢁ3 0.42 ꢂ 10ꢁ3 1.83
a
Considering LOQ and the highest standard calibrated.
Table 2 Accuracy of headspace-isotope dilution GC-MS method for
the analysis of methoxyl and ethoxyl groups in lignins
was shown by the HS-ID GC-MS method, those samples also
contained additional ethoxyl groups.
When comparing the sum of methoxyl and ethoxyl groups
obtained by HS-ID GC-MS and values attained by the Zeisel–
Lignin sample
Accuracy, %
¨
Viebock–Schwappach method, good agreement was found
Kra
Puried (1)
Non puried (1)
Puried (2)
Non puried (2)
Indulin AT
99.64
103.76
101.77
103.63
101.89
(Fig. 5). However, for some lignins, such as sample 5 (Table 3
and Fig. 5), containing relatively higher numbers of ethoxyl
groups, the conventional method was not able to provide
correct numbers for either methoxyl or ethoxyl contents
(Fig. 5). This phenomenon can be explained by the fact that
the HI treatment step in the titrimetric method was opti-
mized only for the quantitative cleavage of methoxyl groups
present in lignin, and, therefore, ethoxyl groups were cleaved
only partially.
Lignosulfonate
Puried
Non puried
101.35
100.55
Organosolv
OMe
OEt
103.74
100.99
Robustness and stability
In order to evaluate the overall robustness of the method, the
inuence of variations of method parameters, such as the
application of different headspace vials closed with screw or
crimp caps, the pH of the reaction medium aer methoxyl and
ethoxyl group cleavage, time, and temperature of samples
storage, etc., were carefully investigated.
titrimetric method, provides methoxyl group contents with lower
RSD (Table 3).
Comparison of the methoxyl group content determined by
¨
HS-ID GC-MS and the Zeisel–Viebock–Schwappach method
showed the latter to overestimate in some cases (Table 3). As
Table 3 Comparison of average methoxyl and ethoxyl group content in lignin samples analyzed by HS-ID GC-MS and the conventional Zeisel–
Viebock–Schwappach method
¨
¨
Zeisel–Viebock–Schwappach
Headspace-isotope dilution GC-MS
Lignin sample
OMe, mmol gꢁ1
RSD (n ¼ 3), %
OEt, mmol gꢁ1
RSD (n ¼ 3), %
OMea, mmol gꢁ1
RSD (n ¼ 5), %
1
2
3
4
5
6
7
8
4.43
6.33
5.13
3.28
3.78
3.66
4.27
4.47
4.00
5.91
4.17
4.43
4.14
4.44
4.40
0.71
0.63
2.51
1.93
0.63
0.83
1.39
1.28
1.01
2.43
0.06
3.13
2.97
3.74
0.99
1.62
—
—
0.46
—
1.45
—
—
—
0.17
—
0.48
0.25
0.21
0.44
0.50
—
—
1.67
—
0.53
—
—
—
2.05
—
1.07
1.98
0.89
2.46
5.24
1.99
4.24
6.20
5.30
3.20
4.42
3.77
4.59
4.43
4.18
5.70
4.47
4.65
4.74
4.75
4.65
3.43
1.70
2.22
0.68
3.31
6.79
4.74
3.99
2.93
1.66
0.43
2.06
5.05
3.69
0.18
2.86
9
10
11
12
13
14
15
Average :
a
Due to low method selectivity, i.e., inability to distinguish methoxyl and ethoxyl groups, these numbers are overestimated for ethoxyl-containing
lignins.
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Fig. 6 Stability of the analytes (MeI/MeI-d3) generated in the reaction
medium upon standing for six d at RT (LOQ ¼ limit of quantification).
Fig. 5 Comparison of OMe group content analyzed by HS-ID GC-MS
vs. the Zeisel–Viebock–Schwappach method.
¨
The application of different headspace vials with screw or
crimp caps, together with the aforementioned different
temperatures in the HI cleavage step, had no noticeable effect
on the precision and accuracy of methoxyl group
determination.
Several Indulin AT lignin samples prepared for the HS-ID
GC-MS analysis were subject to additional heating at 50 ꢀC
and freezing at ꢁ80 ꢀC for 24 h aer hydroiodic acid treatment.
Further analysis did not reveal any noticeable difference in the
methoxyl group content and RSD compared to samples
measured directly aer the cleavage step.
Fig. 7 Change of HS-ID GC-MS methoxyl group quantification when
analytes (MeI/MeI-d3) remain in the test vials upon standing for six
d at RT.
A different outcome was observed in case of pH variation in
the reaction medium aer HI cleavage with respect to the
stability of the analytes generated. Three sets of experiments
with six different lignins (two parallel repetitions each) were
conducted. The residual hydroiodic acid aer the cleavage
process was either le as is or was partially or completely
neutralized with NaOH solution, as recommended in the liter-
ature.15 In all three cases, the analytes generated were degrading
with time apart from slow migration through the sample cap
(Fig. 6 and 7). It was obvious that the degradation of the alkyl
halides was caused by simple SN2 nucleophilic substitution by
a hydroxyl ions when partial or complete HI neutralization was
applied.40
Application of complete or even partial neutralization of
excess HI caused a steep decrease in analyte concentration.
Aer 4 d, their concentration reached the limit of quantication
of the HS-ID GC-MS method. In addition, also the deuterated/
non-deuterated peak area ratio was affected (Fig. 7). In case of
complete neutralization, the isotopically labeled methyl iodide
degraded faster than the non-deuterated one, showing a clear
reverse isotope effect. Hence, due to the different degradation
speeds, the peak ratio cannot be used to level out differences
induced by the side reaction caused by neutralization (Fig. 7).
Partial neutralization did not affect the methoxyl/ethoxyl ratios,
but, probably due to the gradual decrease of the analytes'
concentrations, accuracy and precision of the HS-ID GC-MS
quantication dropped (Fig. 7).
Only in case of non-neutralized HI was the degradation of
analytes comparatively slow, and the ratio between them (MeI/
MeI-d3 and EtI/EtI-d5) remained satisfyingly stable (Fig. 8 and
9), thus causing only minor inuence on accuracy and preci-
sion. Over 6 d, the average relative change of methoxyl and
ethoxyl group content determined in case of kra, lignosulfo-
nate, and organosolv lignins decreased by just 3–4%, and the
average RSDs did not exceed 3% (Fig. 8 and 9).
Based on these tests, a neutralization as recommended in
the literature15 will denitely lead to erroneous results. This is
especially important when larger numbers of samples are
analyzed subsequently and hence remain waiting in an auto-
sampler for different times. The solution must remain non-
neutralized at acidic pH prior to injection, otherwise not even
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lignin, including isolated, puried, and non-puried lignins,
has been developed, optimized, and tested on model
compounds and on a range of different technical lignins. The
method is distinguished from previous approaches by the
application of isotopically labelled internal standards, in
particular 4-(methoxy-d3)-benzoic acid and 4-(ethoxy-d5)-ben-
zoic acid, allowing for signicantly reduced measurement
errors and thus largely increasing the robustness of the overall
analysis.
By varying method parameters, such as application of
different caps for proper sealing, time, and temperature of
sample storage among others, we have validated the HS-ID GC-
MS method and conrmed its analytical advantages. Indepen-
dent of the sample matrix, the new method showed higher
precision, better accuracy and improved selectivity compared to
Fig. 8 Content of OMe group determination in kraft lignin (left axis)
and stability of the analytes (MeI/MeI-d3) remaining in the test vials
upon standing for 6 d at RT without neutralization (right axis).
¨
the conventional titrimetric Zeisel–Viebock–Schwappach
variant. Most notably, it allowed for a much higher sample
throughput. Due to the optimized chemical steps that can be
performed relatively fast in an automated way, the actual GC-MS
analysis might become the bottleneck of the whole analytical
method, because of the temperature program and because of
cooling and temperature stabilization of the GC oven between
individual runs. However, the latter step can be shortened, for
example, by performing oven cryo-cooling. Including all prep-
aration steps, the present approach allowed us to run at least 40
samples per day.
The application of a neutralization step to get rid of excess HI
– aer methoxyl and ethoxyl group cleavage and before the
actual HS-GC-MS analysis, as recommended in the literature,
was shown to be detrimental regarding the stability of the
analytes and internal standards generated in situ. Only when HI
was not neutralized, analyte and standard degradation was
acceptably low and the ratio between labelled standard and
sample remained stable, allowing a precise and accurate
methoxyl and ethoxyl quantication even aer 6 d of storage.
This conrmed that even a high number of HI-treated samples,
requiring several days of chromatographic measurement, can
be safely analyzed with the new approach.
Fig. 9 Content of OMe and OEt groups determined in organosolv
lignin (left axis) and the stability of the individual analytes (MeI/MeI-d3
and EtI/EtI-d5) remaining in test vials upon standing for six days at RT
without neutralization (right axis).
Overall, it was demonstrated that the developed HS-ID GC-MS
method is very reliable, i.e., having fewer factors inuencing
methoxyl and ethoxyl group quantication in comparison to the
the isotopically labeled standard is able to correct for the
degradation.
Corrosion of the headspace sampler transfer line due to HI
remaining in the samples—as stated in the literature15—or other
possible damages to the headspace sampler and GC-MS instru-
ment components were not conrmed or did not pose a problem.
The robustness of the HS-ID GC-MS method proposed involving
cleavage of methoxyl and ethoxyl groups without further
neutralization of remaining HI was conrmed. A precise and
accurate measurement of samples was performed over a suffi-
ciently long period. However, the best results are obtained within
2 d aer HI treatment, which is plenty of time to complete the
analysis, e.g., of a whole sample rack with 111 vials.
¨
conventional titrimetric Zeisel–Viebock–Schwappach method,
and also to published HS GC-MS approaches. On a routine basis,
the number of parallel measurements to achieve fully robust
values was two to three repetitions, while in case of the titrimetric
approach, the number required was four to six.
The analytical approach opens new possibilities to quickly
and accurately characterize lignin with regard to the important
parameter of methoxyl group content (and ethoxyl group
content for special lignins). It is applicable to all types of lignin
and is able to cope with large sample amounts without trade-off
in quality – a property that hitherto available approaches lack.
The simultaneous quantitative analysis of ethoxy groups –
besides the “usual” methoxy group analysis – is an additional
benet that proves benecial when ethanol-based biorenery
Conclusions
A new headspace-isotope dilution (HS-ID) GC-MS method for concepts are dealt with.
the analysis of methoxyl and ethoxyl groups in any type of
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¨
19 S. Zeisel, Monatshee fur Chemie und verwandte Teile anderer
Acknowledgements
Wissenschaen, 1885, 6, 989–997.
We gratefully acknowledge the FLIPPRꢀ project, supported by
the Austrian Research Promotion Agency, FFG, and the associ-
ated companies SAPPI, MONDI, Heinzel Pulp, and Norske Skog,
for the nancial support of the work.
¨
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