Journal of Chromatography A, 1307 (2013) 144–157

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Analysis of lignocellulose derived phenolic monomers by headspace

solid-phase microextraction and gas chromatography夽
Michaela Kolb a , Doris Schieder a,∗ , Martin Faulstich b,1 , Volker Sieber a
a
Chemistry of Biogenic Resources, Technische Universität München, Schulgasse 16, 94315 Straubing, Germany
b
Resource and Energie Technology, Technische Universität München, Petersgasse 18, 94315 Straubing, Germany

a r t i c l e i n f o a b s t r a c t

Article history: A headspace solid-phase microextraction method with subsequent GC–MS (HS-SPME/GC–MS) was estab-
Received 6 December 2012 lished for the quantitative analysis of volatile lignin derived phenolic monomers in complex aqueous
Received in revised form 24 July 2013 solutions. Extraction was done using a polyacrylate fiber. The optimization of HS-SPME – parame-
Accepted 25 July 2013
ters was performed using a multi component model solution of six representative phenolic monomers
Available online 1 August 2013
identified in liquid hot water (LHW) supernatants of hydrothermally treated lignocellulosic biomass:
p-coumaric acid, guaiacol, vanillin, acetosyringone, 4-hydroxy-3-methoxyphenylacetone, and acetophe-
Keywords:
none. Plackett–Burman design was applied for pre-evaluation and 23 central composite designs with
Gas chromatography
Headspace
star points for parameter optimization. LOQ (S/N > 10) and LOD (S/N > 3) were determined for 12 phenols
Solid-phase microextraction yielding LOQ of <0.005–618 nM and LOD of <0.005–412 nM. Within-day and between-day tests (n = 6)
Phenols showed different results for the tested phenols. RSD ranged from 2% to 30% and recovery rates from 99%
Lignocellulose to 160% in LHW matrix. Tests on storage of LHW supernatants for several weeks indicated a considerable
Lignin influence of temperature on the stability of the solutions which may even have to be taken into account
for auto sampler handling. All in all the method allows a fast and solvent free analysis requiring low sam-
ple volumes making it a powerful tool for screening or high-throughput analysis of aqueous solutions of
lignin derived aromatics.
© 2013 Published by Elsevier B.V.

1. Introduction may influence the subsequent utilization of the supernatants

Lignocellulosic biomasses like wood, corn stover, or wheat
straw are of interest for many technical applications including
the recovery of saccharides for subsequent chemical processing or
fermentation. The saccharide recovery usually requires the disinte-
gration of the lignocellulosic compounds, which can be performed
by mechanical, chemical, physico-chemical or biological methods.
Amongst them different hydrolysis methods using acids, alkaline,
steam, or hot water have been tested in the recent decades with
highly promising results [1]. Yet, besides the desired solubiliza-
tion of saccharides also various lignin derived compounds are
produced and transferred into the aqueous supernatants, what
夽 The article in part is based on a PhD thesis: M. Kolb, Einsatz einer Laccase
aus Trametes versicolor bei der enzymatischen Hydrolyse sowie der Detoxifizierung
von Lignocellulose-Aufschlüssen, Technische Universität München, published in:
Schriftenreihe Nachwachsende Rohstoffe in Forschung und Praxis, Band 6, 2013,
Verlag Attenkofer, Straubing, Germany.
∗ Corresponding author. Tel.: +49 9421 108; fax: +49 9421 310.
E-mail addresses: doris.schieder@wzw.tum.de, d.schieder@wz-straubing.de
(D. Schieder).
1
Current address: CUTEC Institute, Clausthal University of Technology, Leibnizs-
trasse 21+23, 38678 Clausthal-Zellerfeld, Germany.
and require additional purification procedures. Lignin derived
aromatic compounds may especially act as inhibitors toward
microorganisms or saccharolytic enzymes [2–9] thus affecting the
fermentation of the saccharide containing hydrolyzates. The range
of lignin derived compounds in lignocellulosic hydrolyzates cov-
ers a variety of monomers, oligomers, and polymers, depending
on feedstock as well as on pretreatment parameters. Pheno-
lic monomers have been reported to belong to the most toxic
lignin derived compounds of lignocelluloses [2,3]. Monomers like
vanillin, syringaldehyde, syringacid, acetosyringone, syringol, 4-
hydroxybenzaldehyde, 4-hydroxyacetophenone, or cinnamic acid
derivatives amongst others have been identified in pretreatment
products of wheat straw depending not least on the pretreatment
method [4,10].
Suitable analytics are required to identify and quantify the
solubilized lignin derived compounds. Size exclusion chromatog-
raphy may be used to determine the molecular size distribution
of lignin derivatives in solutions and to estimate the degree of
polymerization [11–13], while liquid chromatography methods
like HPLC-UV, LC–MS [14–17], or gas chromatography (GC–FID
or GC–MS) [5,14,18–20] are applied to quantify monomeric com-
pounds. GC–MS is probably the most frequently used method to
identify lignin derived aromatic monomers. Compared to liquid

0021-9673/$ – see front matter © 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.chroma.2013.07.094
 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157
chromatography, gas chromatography enables better separation of
the compounds and is less affected by matrix effects [14,15]. The
mass detection allows the identification even of “unknown” sub-
stances by their mass spectra and retention times thus providing
more detailed information on complex samples. Liquid–liquid-
extraction (LLE) or solid phase extraction (SPE) with subsequent
derivatization are the most common sample preparation methods
for analysis of lignin derived phenolics by GC–MS. The separation of
phenolics from aqueous samples by LLE is performed using organic
solvents, e.g. chloroform or ethyl acetate. By SPE sample prepara-
tion the target molecules can be separated by adsorption on the
solid phase matrix and elution at different pH. Klinke et al. sepa-
rated phenolic acids at pH 2 from the liquid products of alkaline
wet oxidized wheat straw while they applied pH 5 for the others
compounds [5]. After LLE or SPE the target molecules are usually
subjected to derivatization mainly by silylation. Sample prepara-
tions by LLE as well as by SPE are multi step procedures, which
makes them time consuming [21] and may lead to enhanced errors
and reduced recovery rates.
Nowadays, the principles of green chemistry become more and
more important not only in chemical synthesis and engineering
but also in analytical chemistry. Miniaturized analytical systems
as well as solvent free extraction methods may be used amongst
others to reduce the consumption of organic solvents in analytical
chemistry [22]. Headspace solid-phase microextraction (HS-SPME)
is a solvent free method of GC–MS sample preparation that can
both simplify and accelerate the overall analytical procedure. SPME
has been developed by Pawliszyn et al. [23,24] for the extraction
of low-molecular molecules for qualitative and quantitative GC
and LC analysis. Similarly to SPE the SPME target molecules are
adsorbed on a solid phase. SPME solid phases are thin fibers made
of polydimethylsiloxane (PDMS), polyacrylate (PA), or polyethy-
lene glycol (PEG). PDMS – fibers may also be doped with carboxene
(Car/PDMS) or divinylbenzene (DVB/PDMS) to provide pores in the
range of 20 up to >500 Å. SPME extraction of liquid samples can be
performed directly in the liquid phase (direct immersion) or indi-
rectly from the gaseous phase applying headspace (HS) technology.
HS is of special interest for complex samples, like lignocellulosic
hydrolyzates, as it prevents interfering matrix effects as well as
damages of the SPME – fibers by non-volatile sample compounds.
After extraction the fiber is desorbed in the GC injector or the LC
interface, respectively. The method can be performed with low
sample volumes and the handling is little time consuming enabling
an enhanced sample throughput and making it an ideal screening
method. If required, derivatization of the target compounds may be
performed, e.g. by adding the derivatization agent directly on the
fiber (in-fiber derivatization) prior to or after extraction [25–28].
HS-SPME/GC–MS thus can be a powerful tool for quick and high
throughput analysis of volatile compounds in complex liquid sam-
ples. It also meets the goals of green chemistry as only low sample
volumes and no solvents are required.
SPME has been applied to different analytical tasks, e.g. in
food technologies to the detection of flavoring agents in cheese,
honey, beverages, and fruits, or to the analysis of herbicides and
pesticides in oil, fish, or milk, respectively [29–31], and is also a
well known method for drug analysis [27,28,32]. Several phenolic
and additional aromatic monomers were still analyzed via SPME,
such as vanillin, ethyl vanillin, vanillic acid, 4-hydroxbenzaldehyde,
and coumarin in ethanolic extracts of vanilla beans [33], as well
as vanillin, syringol, acetophenone, acetovanillone, benzoic acid,
benzaldehyde, guaiacol, eugenol, and isoeugenol in smoked goat
cheese flavor [31], and phenol and catechol in urine [26].
Yet, to the knowledge of the authors, SPME methods had
not been used on a wider range of aromatic monomers derived
from lignocellulose degradation. Therefore we developed a HS-
SPME/GC–MS method for quick and high-throughput analysis of
145
lignin derived phenolic monomers in aqueous solutions. A first
application of a preliminary version of this method was published
recently studying the effect of laccase on monomeric delignifica-
tion products from liquid hot water pretreated wheat straw [34].
The present study reports the optimization of HS-SPME param-
eters using DOE and its application on storage experiments of
supernatants of liquid hot water pretreated (LHW) wheat straw.
It also includes the identification of the limits of detection (LOD)
and the limits of quantification (LOQ) of 12 selected lignin derived
aromatic monomers as well as their recoveries in the lignocellulosic
samples.
2. Materials and methods
2.1. Chemicals
All chemicals were purchased from Sigma–Aldrich (St. Louis,
USA), Neolab (Heidelberg, Germany), Carl Roth (Karlsruhe,
Germany), and VWR International (USA/Germany), at laboratory
or analytical grade quality.
2.2. Experimental
2.2.1. HS-SPME/GC–MS equipment and procedure
HS-SPME/GC–MS was performed using a Thermo Finnigan Trace
GC 2000 equipped with a Trace DSQ and a TriPlus auto sampler for
SPME fibers.
A SLB-5ms fused silica column (30 m × 0.25 mm i.d.) was used
coated with a 0.25 ␮m film of 5% phenyl cross-bond (Supelco, Belle-
fonte, USA). The oven temperature was held at 80 ◦ C for 2 min,
than increased at 10 ◦ C min−1 to 110 ◦ C, 3 ◦ C min−1 to 160 ◦ C, and
20 ◦ C min−1 to 280 ◦ C. This temperature was kept constant for
20 min. Constant temperature (CT) splitless injection mode (280 ◦ C)
was applied and the MS transfer line was heated to 250 ◦ C. Helium
was used as carrier gas with a flow rate of 1.0 mL min−1 . MS spectra
were recorded at 70 eV with a m/z range of 50–650. Identifica-
tion of lignocellulose derived phenols was done using reference
substances as well as the National Institute of Standards and Tech-
nology (NIST) library. For quantification the extracted ion current
(EIC) was used at one or two substance specific masses.
Headspace solid-phase microextraction (HS-SPME) was per-
formed using a polyacrylate (PA) fiber for auto samplers supplied by
Supelco (Bellefonte, USA). 20–100 ␮L of sample or standard were
transferred into a 10 mL headspace vial and the appropriate amount
of buffer (50 mM citric buffer at pH 5.0 if not indicated otherwise),
NaCl and – if required for quantification and identification tests –
the internal standard (10 ␮L of ethyl vanillin, 1 mM) were added to
give the desired volumes (0.1–3.0 mL). The vials were sealed with a
PTFE coated cap prior to analysis. The injection depth of the PA fiber
into the vials in the headspace oven was adjusted to 15 mm. After
each extraction the fiber has been cleaned in the second injection
port of the GC at 280 ◦ C for 10 min.
2.2.2. Design of experiments for HS-SPME/GC–MS parameter
optimization
Plackett–Burman design was used for screening experiments
and 23 -central composite designs with star points were applied for
HS-SPME parameter optimization. Experimental designs as well as
analysis were performed using Statgraphics Centurion, version XV,
Statpoint Incorporation (Virgina, USA).
2.2.3. Preparation of lignocellulose (wheat straw) samples
Wheat straw (<2 mm) was provided by Fraunhofer ICT (Pfinztal,
Germany). The liquid hot water (LHW) pretreatment was per-
formed in a 2-liter high pressure autoclave from Parr Instruments
at 180 ◦ C, 30 min, on samples of 10% wheat straw dry matter in
146 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

distilled water. The pretreated samples were centrifuged (6288

rcf, 20 min) and the liquid hot water (LHW) supernatant was used
for the experiments [34]. Storage experiments were performed in
100 mL serum flasks with sealed caps and 10 mL of LHW super-
natant samples. For experiments under argon atmosphere the
samples were flushed with argon for about 10 min prior to the
experiments. Stirring was done using magnetic stirring devices.

3. Theory

According to Risticevic et al. [24] 14 different parameters have

to be considered to develop or adopt a SPME method: fiber
type, extraction method, separation and detection devices, shak-
ing mode, sample volume, derivatization, pH, ion strength, water
content, content of organic solvents, sample temperature, extrac-
tion time, conditions of desorption, and calibration mode. SPME
extraction of liquid samples can be performed directly in the liq-
uid phase (direct immersion) or indirectly in the gaseous phase
applying headspace (HS) technology. HS is of special interest for
complex samples, as it prevents interfering matrix effects as well
as damages of the SPME-fibers by non-volatile sample compounds.
The distribution coefficient between sample and gaseous phase
restricts the mass transfer of a given substance from the gaseous
phase to the SPME-fiber, i.e. the concentration of low-volatile sub-
stances in the gaseous phase is lower than of high-volatile ones and
consequently they are extracted more slowly. Sample volume, ion
strength, extraction temperature, pH, and shaking mode thus are of
special importance for HS extraction. The fiber selection depends
on the type of substance to be analyzed, i.e. on its polarity and
volatility, but also on its molecular mass [23].
4. Results and discussion
4.1. Pre-selection of parameter settings
In the present work some parameter pre-settings have been
done prior to perform experiments to adopt SPME/GC–MS for anal-
ysis of lignocellulose derived phenolic monomers. Headspace (HS)
was chosen for sample extraction method with respect to the
complex matrix of supernatants from lignocellulose pre-treatment
containing saccharides, particles, lignin derived oligomers and
polymers, as well as other kinds of non-volatile compounds that
may damage the SPME-fiber or affect GC-analysis. For experiments
with LHW-supernatants of wheat straw internal standard calibra-
tion was applied. Ethyl vanillin was chosen for internal standard,
as it was not detected in LHW-supernatants, but its chemical struc-
ture is very close to the lignin derived target molecules in the
wheat straw LHW-supernatants. Polyacrylate (PA), which covers a
wide range of polarity and of volatility, was chosen for SPME-fiber
type following the results of several authors analyzing phenolic
substances from different matrices [26,31,33]. The GC–MS param-
eters applied for the present work for subsequent analysis of the
extracted compounds are given in Section 2.2.1.
In addition to the pre-selected settings, several parameters had
to be adjusted and optimized for application of HS-SPME/GC–MS on
lignocellulosic samples, i.e. pH, sample volume, ion strength (NaCl
content), extraction temperature, incubation, extraction and des-
orption time. The adaption and optimization of these parameters
were done using model solutions of selected phenolic monomers.
4.2. Selection of pH for HS-SPME-extraction
Fig. 1. Effect of pH on SPME extraction: relative peak areas (related to the maxi-
mum areas detected) of selected model substances in multi component solution.
Parameters: 10 min incubation, 40 min extraction at 60 ◦ C.
effect of pH was most prominent for the cinnamic acid derivates as
well as for acetosyringone and syringaldehyde. With the excep-
tion of eugenol, acetophenone and acetovanillone, all tested
monomers showed best extraction at pH 2.0. Although the overall
extraction efficiency was best at pH 2.0, pH 5.0 was selected for
further experiments, as the GC–MS peaks at pH 2 showed remark-
able tailing affecting peak integration. Additionally, pH 5 was in the
pH range of the lignocellulosic samples (pH 4–6) and can as well
be regarded to be suitable for application on (buffered) samples
of enzymatic hydrolysis or fermentation samples of lignocellulosic
substrates.
4.3. Optimization of HS-SPME extraction and desorption
parameters
A two step procedure was performed to optimize HS-SPME
extraction and desorption parameters using design of experiment
(DoE). First, a screening was done using a Plackett–Burman design
to evaluate the main factors of the method on the responses,
i.e. sample volume, concentration of NaCl (ion strength), incu-
bation time, extraction temperature and time, and desorption
time. With the results of the screening a response surface design
was applied for further parameter optimization. The number of
model substances was reduced to six monomers for these exper-
iments representing different types of lignin derived monomeric
delignification products detected in wheat straw LHW super-
natant: p-coumaric acid (cinnamic acid derivatives), vanillin
(methoxyphenols and phenolaldehydes), guaiacol (non-alkylated
phenols), acetosyringone (di-methoxy-phenols and phenolke-
tones), 4-hydroxy-3-methoxyphenylacetone (HMPA) representing
carbonyl-propyl-phenols, and acetophenone. Multi component
solutions were prepared for these experiments containing the
selected monomers at concentrations of 2 ␮M (acetosyringone,
HMPA), 3 ␮M (p-coumaric acid, vanillin, acetophenone), and 4 ␮M
(guaiacol).

The extraction of selected phenolic monomers was performed 4.3.1. Plackett–Burman design
at pH 2.0, 5.0, and 7.0. The results are given in Fig. 1, indicat- The Plackett–Burman design was performed as a folded plan
ing positive effects of acidic pH on the extraction efficiency. The choosing quite large test ranges for the selected parameters. The
 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157 147

Table 1 The screening design was applied to select the most important
Levels and units of the factors for Plackett–Burman design.
parameters, i.e. parameters exhibiting significant main effects on
Factor (−) (+) Unit the GC–MS peak areas. The evaluation of the relative areas served
A – desorption time 0.5 10.0 min especially to get a better insight into the impact of sample vol-
B – incubation time − 10.0 min ume on extraction quality. Figs. 2 and 3 summarize the results
C – extraction time 5.0 60.0 min of the standardized Pareto charts for absolute and relative peak
◦
D – extraction temperature 40.0 80.0 C areas, respectively. Enlarged sample volumes in the 10 mL GC-Vials
E – NaCl content − 50.0 % (w/v)
exhibited positive effects on the absolute peak areas and nega-
F – sample volume 0.1 3.0 mL
tives on the relative ones for all tested compounds. This had to be
expected, as the extracted amounts increase with increasing vol-
upper and lower limits of the parameters are summarized in ume resulting in larger absolute peak areas while the relative level
Table 1. The statistical analysis was done referring to absolute and of extraction decreased. The standardized Pareto charts indicated
relative GC–MS peak areas (A) of the tested compounds. For a given significant effects of the sample volume only on the relative areas
substance the relative peak area was calculated according to Eq. (1): of vanillin, p-coumaric acid, acetophenone, and guaiacol. The incu-
bation time as well as the desorption time showed no significant
Aabsolute effects on the peak areas, while the extraction time exhibited posi-
Arelative = (1) tive effects for all substances, which were significant on most of the
nsample
relative and several of the absolute areas. The effects of the extrac-
where Aabsolute = absolute peak area, nsample = quantity (mol) of tar- tion temperature proved to be positive for vanillin, p-coumaric acid,
get molecules in sample. acetosyringone, and HMPA, being significant on the relative as well

Fig. 2. Standardized Pareto charts of the main effects on absolute peak areas of the Plackett–Burman design. (A) Desorption time, (B) incubation time, (C) extraction time,
(D) extraction temperature, (E) NaCl content, and (F) sample volume.
148 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

Fig. 3. Standardized Pareto charts of the main effects on relative peak areas of the Plackett–Burman design. (A) Desorption time, (B) incubation time, (C) extraction time, (D)
extraction temperature, (E) NaCl content, and (F) sample volume.

as on most of the absolute areas of these substances. The concentra-
tion of NaCl was found to exhibit positive effects for all compounds
which were even significant in some cases (guaiacol, vanillin,
acetophenone). These findings correspond to the results of [35]
who reported significant positive effects of extraction time and
NaCl concentration on vanillin, guaiacol and several other phenols
at HS-SPME of smoke from biomass samples. Positive effects of the
extraction temperature on vanillin have already been reported by
several authors for various sample types [33,36,37]. Díaz-Maroto
et al. even established a slight improvement of the vanillin extrac-
tion by the enhancement of temperature and time, but also a
decrease at extraction times above 40 min at several temperatures,
indicating interacting effects between extraction temperature and
time [37].
NaCl was kept at its highest content tested (500 g/L) as this enables
comparable conditions of ionic strength for the measurement of
samples containing different amounts of ionic compounds.
Two 23 -central composite designs (CCD) with star points
(˛ = 1.6786) were performed (Table 2) to elucidate the impacts of
extraction time, extraction temperature, and sample volume on the
responses at GC analysis and to elaborate the optimum parame-
ter setting. For the subsequent statistical analysis the experimental
results of both designs were combined, as only the extraction tem-
perature ranges of the designs were slightly different.
Table 2
Parameters and limits of the 23 -central composite designs (CCD) with star points
(˛ = 1.6786).

Factor Levels Unit

4.3.2. Response surface design
With respect to the results of the screening, extraction time, (−) (+)
extraction temperature, and sample volume were selected for fur- Extraction temperature (plan A) 40 70 ◦
C
A ◦
ther optimization using response surface design. Incubation time Extraction temperature (plan B) 50 75 C
and desorption time were assigned to 0.5 min and 2 min, respec- B Extraction time 15 55 min
C Sample volume 1.0 2.5 mL
tively, as they had not shown any significant effects at the screening.
 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157 149

Table 3
Predicted significant main effects (ME) and interactions (IE) on peak areas, as well as particular coefficients of determination (R2 ).

Substances Acetophenone Guaiacol Vanillin HMPA p-Coumaric acid Acetosyringone

Relative areas
A −4.19 × 107 3.88 × 106 4.93 × 106 1.46 × 105 1.38 × 105
B 2.63 × 107 −6.09 × 105 2.55 × 106 2.08 × 106 – –
C −8.74 × 107 −1.14 × 107 −2.03 × 106 −2.42 × 106 −1.26 × 105 −1.11 × 105
AA – – – – – 5.64 × 104
BB −2.95 × 107 −3.87 × 106 – – – –
CC 4.55 × 107 4.14 × 106 – – – –
AB −3.24 × 107 – – – – –
AC 3.34 × 107 – – – −7.51 × 104 −8.12 × 104
BC – – – – – –
R2 90% 76% 68% 62% 71% 59%

Absolute areas
A −2.06 × 108 1.05 × 106 1.63 × 107 1.74 × 107 6.89 × 105 4.22 × 105
B 1.51 × 108 −4.65 × 106 1.06 × 107 7.11 × 106 – –
C 1.36 × 108 2.03 × 106 – – – –
AA – −1.32 × 107 – – – 1.70 × 105
BB −1.47 × 108 −1.84 × 107 – – – –
CC – −1.16 × 107 – – – –
AB −1.79 × 108 −1.56 × 107 – – – –
AC – – – – – –
BC – – – – – –
R2 92% 65% 72% 63% 67% 59%

A: ME of extraction temperature; B: ME of extraction time; C: ME of sample volume; AA: quadratic IE of extraction time; BB: quadratic IE of extraction time; CC: quadratic
IE of sample volume; AB: IE of extraction temperature and time; AC: IE of extraction temperature and sample volume; BC: IE of extraction time and sample volume.

The major results of the statistical analysis are summarized in 4.4. Application to lignocellulosic samples
Table 3. The sample volume (C) was confirmed to have significant
negative effects on the relative peak areas of all tested compounds, The developed method was applied to analyze lignin derived
while it had positive effects on the absolute areas of guaiacol and phenolic monomers in the supernatants of liquid hot water (LHW)
acetophenone. The extraction temperature (A) showed positive pretreated wheat straw. First application results of a prelimi-
main effects, except for acetophenone, which was negatively influ- nary version of this method were recently published studying
enced. Additionally, slight interactions between temperature and the removal of phenolic monomers from LHW supernatants of
sample volume (AC) on the relative areas of several compounds wheat straw by treatment with laccase [34]. In the present arti-
(actophenone, p-coumaric acid, and acetosyringone) could be iden- cle selected method validation procedures were performed for
tified being more prominent at low sample volumes (Fig. 4). No application on lignin derived phenols detection in LHW solutions.
interactions were detected between sample volume and extraction Moreover, the method was used to evaluate the impacts of different
time (BC) whereas the factors extraction temperature and time (AB) storage conditions on the concentration of phenolic monomers in
were detected to interact for acetophenone and guaiacol, which the supernatants of liquid hot water (LHW) pretreated wheat straw.
had to be expected. For guaiacol it showed an improvement with For these experiments parameter settings were applied according
increasing temperature at short extraction times (15 min) while to Table 4.
the influence of temperature was negative at extended extraction
(55 min). As already discussed, the temperature exhibited neg- 4.4.1. Method validation data
ative effects on the GC–MS response of acetophenone, but the LOD and LOQ were determined using S/N – ratios of 3 and 10,
impact was much more prominent at the longer extraction period respectively, according to [38]. Table 5 summarizes the calibration
(Figs. 4 and 5). data, LOD, and LOQ of 12 phenolic monomers. Besides, the table
The statistical calculation of the optimal conditions of extraction presents retention times, quantifiers, and concentration ranges of
temperature, extraction time, and sample volume for the entity several phenolic monomers identified in the tested LHW samples
of the tested model compounds was performed with respect to as well as data of some additional phenolic compounds which
the relative and the absolute peak areas. Yet, more importance could not be calibrated due to lack of appropriate standards. The
had been attached to the absolute areas as the direct effects of
the sample volume on the peak areas should not be underes-
timated. Fig. 6 presents the response surface plots at a sample
volume of 1.0 mL and an extraction time of 30 min, respectively. The Table 4
response surfaces indicate that the best conditions were achieved Optimum values of the HS-SPME/GC–MS parameters as well as parameter settings
at high temperatures and moderate extraction periods. The statisti- for application to lignocellulosic samples.
cally calculated overall optimum yielding a desirability of 0.48 was Parameter Unit Calculated Setting for
attributed to 80 ◦ C, 31 min, and to a sample volume of 0.88 mL. optimum application
With these results the parameters for application of the method Extraction temperature ◦
C 80 70
were appointed to 30 min (extraction time), 1.0 mL (sample vol- Extraction time min 31 30
ume), and 70 ◦ C (extraction temperature). The lower extraction Sample volume mL 0.88 1.0
temperature was applied as high temperatures forwarded the NaCl-content g/L – 500
pH – – 5.0 (citric
disadvantageous input of trace amounts of water into the gas
buffer 50 mM)
chromatograph affecting GC-column and MS-system. The assigned Incubation time min – 0.5
parameters for application as well as the estimated optimum Desorption time min – 2
parameters are summarized in Table 4. Internal standard (ethyl vanillin) ␮M – 10
150 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

Table 5
Retention time, quantifier, calibration range, coefficient of determination (R2 ) of the calibration as well as limit of detection (LOD) and limit of quantification (LOQ) of selected
substances identified in LHW wheat straw supernatants.

Substances Retention time (average) [min] Masses/quantifier (intensity %) [m/z] Concentration range in LHW samples (n = 4–6 samples)

Acetophenone 5.6 51 (15), 77 (74), 105 (100), 120 (29) 0.03–1.5 ␮M

Guaiacol 5.9 53 (11), 81 (43), 109 (100), 124 (80) 3–79 ␮M
Benzochinone 6.8 79 (17), 123 (68), 137 (40), 138 (100) n.d.
p-Coumaric acid 8.4 65 (13), 91 (41), 119 (30), 120 (100) 9–72 mM
Ethylguaiacol 9.9 122 (12), 137 (100), 138 (9), 152 (35) n.d.
Ferulic acid 11.0 77 (28), 107 (27), 135 (83), 150 (100) 1.5–18 mM
Eugenol 12.2 77 (34), 131 (32), 149 (42), 164 (100) n.d.
HBA 12.3 65 (30), 93 (36), 121 (100), 122 (93) 26–235 ␮M
Vanillin 13.4 81 (17), 123 (14), 151 (100), 152 (90) 2.4–811 ␮M
Ethylvanillin 15.3 109 (12), 137 (100), 138 (29), 166 (38) ISTD
Acetovanillone 16.2 123 (24), 151 (100), 166 (47) 0.08–12.7 ␮M
HMPA 17.6 121 (27), 122 (18), 137 (100), 180 (15) 4.0–27 ␮M
Syringaldehyde 21.9 111 (12), 167 (12), 181 (68), 182 (100) 1.1–249 ␮M
Acetosyringone 23.4 153 (14), 181 (100), 182 (10), 196 (45) 0.5–80 ␮M
Coniferylaldehyde 23.5 107 (33), 135 (59), 147 (42), 178 (100) 2.9–136 ␮M
UIP-1* 15.1 121 (26), 131 (36), 149 (45), 164 (100) n.d.
UIP-2* 17.0 57 (33), 191 (100), 192 (15), 206 (17) n.d.
UIP-3* 20.0 105 (18), 119 (31), 131 (28), 194 (100) n.d.

Substances Calibration

Range R2 LOQ [nM] LOD [nM]

(S/N > 10) (S/N > 3)

Acetophenone 15.0–0.2 ␮M 99.8% 3 0.2

Guaiacol 37.4–0.5 ␮M 99.5% 25 3
p-Coumaric acid 2.5–0.05 mM 99.6% 16 2
Ferulic acid 2.5–0.05 mM 99.8% 618 412
Eugenol 49.5–0.5 ␮M 99.6% 5 0.3
HBA 22.5–0.3 ␮M 99.5% 125 50
Vanillin 22.5–0.3 ␮M 99.8% 50 38
HMPA 9.9–0.1 ␮M 99.2% 125 25
Acetovanillone 22.5–0.3 ␮M 99.9% 100 25
Syringaldehyde 22.5–0.3 ␮M 97.0% <0.005
Acetosyringone 15.0–0.2 ␮M 92.0% 500 100
Coniferylaldehyde 37.4–0.5 ␮M 96.0% <0.005
*
UIP (unidentified phenol): unidentified peaks that were attributed to be phenols due to their mass spectra properties.

calibration curves of the 12 examined substances proved to be
linear within the applied ranges of measurement. LOD and LOQ
were in the nM – range or even lower, where syringaldehyde and
coniferylaldehyde exhibited the best and ferulic acid the lowest
sensitivity.
Fig. A1 in the appendix shows an exemplary gas chromatogram
of a LHW sample. Figs. A2–A4 show the mass spectra of six selected
phenols including the internal standard ethylvanillin.
According to Kromidas [38] the recovery rate may be taken
as a means to evaluate the accuracy of a method for a given
sample type (matrix) and given experimental conditions. In the
present work, the recovery rates of phenolic compounds were
determined to examine the accuracy of the method for appli-
cation on LHW pretreated wheat straw solutions as no reliable
reference methods or reference samples were available for this
type of samples. The recovery rate (R) was estimated according
to [38]:
(S1 − S2)
R= × 100% (2)
S3
where S1 equals the peak area ratio of the LHW sample including
calibration sample, S2 means the area ratio of the non-spiked LHW
sample and S3 the area ratio of the calibration sample. The recov-
eries as well as the precision data (relative standard deviation RSD
in spiked LHW samples) were determined within-day (n = 6) and
between-day (n = 6) for several phenols. The results summarized
in Table 6 yielded good precision and recoveries for acetophe-
none, guaiacol, ferulic acid, vanillin, HMPA, and acetovanillone
with almost no differences of within-day and between-day tests,
although the recoveries tended to be somewhat above 100%,

Table 6
Within-day and between-day precision and accuracy (recovery) data of phenols in spiked LHW samples. Samples contain 50 ␮L LHW solution per mL.

Compound Amount added (␮M) Within-day (n = 6) Between-day (n = 6)

Amount detected (␮M) RSD (%) Recovery (%) Amount detected (␮M) RSD (%) Recovery (%)

Acetophenone 10 11.3 3 107 10.8 3 106

Guaiacol 25.5 34.5 3 109 30.5 8 108
Ferulic acid 1476 2513 6 106 2520 5 111
HBA 15.0 34.8 25 136 35.1 18 157
Vanillin 15.0 27.9 5 108 24.7 7 107
HMPA 6.0 7.5 10 112 7.6 6 116
Acetovanillone 15.0 15.6 5 99 15.0 2 102
Syringaldehyde 15.0 31.2 26 136 36.1 22 160
Acetosyringone 10.0 14.9 28 120 24.0 30 145
Coniferylaldehyde 25.5 38.2 21 123 41.6 18 141

RSD = relative standard deviation.

 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157 151

but indicated high RSD and excessive recovery rates for HBA,
syringaldehyde, acetosyringone, and coniferylaldehyde. Neverthe-
less, these data can be considered to be fairly good results with
respect to the complex matrix of the pretreated wheat straw solu-
tion, the problems of the analysis of phenols in biogenic samples
described by other authors [39], and with respect to the objective
to provide a method for fast multi phenol analysis in lignocellullose
derived aqueous samples. For more precise analysis of individual
phenols the method has to be further adopted or complemented,
e.g. by derivatization.

4.4.2. Impacts of storage on the concentration of phenolic

monomers in LHW supernatant samples
Phenolic compounds are known to be instable in aqueous
samples and their concentrations may alter during storage. We
reported the reduction within seven days at elevated tempera-
ture (45 ◦ C) for several lignin derived monomers in wheat straw
LHW supernatants, while there were no decreases in single sub-
stance model solutions [34]. The instability of phenols in complex
aqueous matrices was published for p-coumaric acid in wine
fermentation samples as well [39]. The content of phenols in lig-
nocelluloses hydrolyzates is of importance e.g. for fermentation on
the hydrolyzates, as lignin derived phenols are known to act as
inhibitors toward microorganisms [3,4].

Fig. 5. Selected interaction plots of the absolute areas of guaiacol and acetophenone
determined within the CCD-23 -design.

Fig. 4. Selected interaction plots of the relative areas of p-coumaric acid, acetosy- Fig. 6. Response surface plots at a sample volume of 1.0 mL (1) and an extraction
ringone, and acetophenone determined within the CCD-23 -design. A: extraction time of 30 min (2).
temperature (◦ C), B: extraction time (min), C: sample volume (mL).
152 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

Fig. 7. Phenolic monomers in LHW pretreated wheat straw supernatant after storage for several weeks at different storage conditions. Experiments were performed in
duplicates. Error bars refer to 4 times HS-SPME/GC–MS analysis (2 times per sample). UIP (unidentified phenol): unidentified peaks that were attributed to be phenols due
to their mass spectra properties.

To get more insight in the storage properties of the wheat impact of mixing, samples were stirred during storage while ref-
straw LHW supernatants with respect to lignin derived phenolic erence samples were stored without mixing. An additional stirred
monomers, storage experiments were performed over a period of setup was kept under argon to check the effect of oxygen, as phe-
11 weeks at room temperature (RT) as well as at 4 ◦ C. To test the nols may be subjected to oxidation. Fig. 7 presents the results after
 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157
6 and 11 weeks of storage. No effect of the storing conditions
concerning mixing or presence of air could be established, except
for the 4 ◦ C samples after 6 weeks where the sum of phenols in the
stirred samples was reduced to a remarkable higher extend (around
−30%) than in not stirred ones (−18%). Generally, the storage at 4 ◦ C
seems to be more favorable to preserve the phenols than the storage
at room temperature where more than 80% of the total identified
phenols had disappeared. Concerning the individual compounds,
tremendous differences could be established. The concentration
of acetophenone, vanillin, and acetovanillone were hardly reduced
at 4 ◦ C. At room temperature, vanillin, acetovanillone, syringalde-
hyde, acetosyringone, and coniferylaldehyde, raised up to 50% at 6
weeks, but decreased again after 11 weeks. The increase preferably
happened in the stirred (and argonzied) samples, indicating that
these amounts might be produced by depolymerization of lignin
derived oligomers or polymers during storage. HBA also showed
a slight increase after 6 weeks at 4 ◦ C as well as at room tempera-
ture, but was reduced by more than 80% after 11 weeks. Ferulic and
p-coumaric acid, which were the major phenolic monomers iden-
tified in the LHW samples, decreased tremendously, especially at
room temperature. Ferulic acid also was the compound contribut-
ing most to the decrease at 4 ◦ C after 6 weeks.
All in all, temperature seems to be most important for the stor-
age of LHW pretreated lignocellulosic samples while oxidation
reactions caused by oxygen in the air seem to be of minor or no
importance. Freezing might be the best storage mode, although the
hydroxycinnamic acid derivates, like p-coumaric acid and ferulic
acid, which made the major part in the wheat straw LHW samples,
were found to be slightly lowered even after −18 ◦ C storage (data
not shown).
Anyhow, the inconsistency of the phenols has to be taken into
account when handling these types of samples, e.g. when apply-
ing the HS-SPME/GC–MS method. The easy and quick sample
preparation is clearly a benefit of this method, but the sample han-
dling time in the auto sampler – leading to some kind of interim
Fig. A1. Gas chromatogram of a LHW pretreated wheat straw supernatant sample. Assignment of retention times, see Table 5.
153
storage – also has to be limited to prevent errors caused by losses
of phenols. In our experiments, we limited the sample handling
in the auto sampler to 24 h, although aberrations up to 20% were
detected within this time frame. Additionally, we randomized the
measurement sequences of time dependent tests.
5. Conclusion
A HS-SPME/GC–MS method has been examined and success-
fully optimized for the quantitative analysis of several volatile
lignin derived phenolic compounds in complex aqueous solutions.
The method allows the detection of the target compounds in the
complex matrix of lignocellulose hydrolyzates containing various
interfering components, e.g. saccharides, organic acids, various
lignin derived phenolic monomers, oligomers and polymers, pro-
teins, and inorganics, without extensive sample preparation. This
means short sample handling times and a high throughput. Addi-
tionally, only small sample volumes (less than 1 mL) are required.
Thus, the method is especially suited for fast analysis, which makes
it an elegant screening method. In this work, the method was tested
for supernatants (hydrolyzates) of liquid hot water (LHW) pre-
treated wheat straw, but it may also be adopted to the hydrolyzates
of alternative lignocellulosic feedstock and alternative lignocel-
luloses pretreatment methods, e.g. steam explosion, dilute acid
hydrolysis, alkaline or organosolv pre-treatments.
Acknowledgements
The authors gratefully acknowledge the funding received from
Wacker Chemie AG and the Federal Ministry of Education and
Research, Germany (FKZ: 0315193). The authors also wish to thank
Petra Lommes for her excellent technical assistance.
Appendix 1.
See Figs. A1–A4.
154 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

Fig. A2. Mass spectra of (A) p-coumaric acid and (B) vanillin. Quantifier, see Table 5.
 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157 155

Fig. A3. Mass spectra of (A) guaiacol and (B) 4 hydroxy-3-methoxyphenylacetone (HMPA). Quantifier, see Table 5.
156 M. Kolb et al. / J. Chromatogr. A 1307 (2013) 144–157

Fig. A4. Mass spectra of (A) acetosyringone and (B) ethylvanillin (internal standard). Quantifier, see Table 5.

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