Evaluation of errors associated with d 13 C analysis of lignin-derived TMAH thermochemolysis products by gas chromatography–combustion–isotope ratio mass spectrometry L.E. Beramendi-Orosco a, * , C.H. Vane b , M. Cooper a , C.G. Sun a , D.J. Large a , C.E. Snape a a Nottingham Fuel and Energy Centre, School of Chemical, Environmental and Mining Engineering, University of Nottingham, University Park, Nottingham NG7 2RD, UK b British Geological Survey, Keyworth, Nottingham NG12 5GG, UK Received 17 July 2005; accepted 19 July 2005 Available online 14 November 2005 Abstract This study reports the compound specific stable carbon isotope compositions (d 13 C) of lignin tetramethylammonium hydroxide (TMAH) chemolysates obtained with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS). The possible sources of the errors associated are considered. Off-line (TMAH) thermochemolysis was performed on wood samples and the d 13 C values of the chemolysates were compared with the bulk d 13 C of the native woods and their Klason lignins. For the four woods investigated, the d 13 C values, corrected for derivative carbons added, were spread over a wide range of values, ranging from 40 to 25% and were, on average, depleted in 13 C by ca. 9% relative to the native woods and by ca. 7% relative to the Klason lignins. This large variability can be partially attributed to overlapping chromatographic peaks and to the low intensity of some of the peaks. However, isotopic fractionation cannot be ruled out, especially in compounds resulting from C–C bond cleavage in the propyl side chain. The uncertainties associated with the correction for carbons added by derivatisation were found to be high, especially for compounds having high contribution of derivative carbons. # 2005 Elsevier B.V. All rights reserved. Keywords: Tetramethylammonium hydroxide; Thermochemolysis; Lignin; Lignin derivatives; Stable carbon isotope; Gas chromatography–combustion– isotope ratio mass spectrometry 1. Introduction Lignin-derived products have the potential to be used as geochemical indicators because lignin is a specific component of vascular plants and is relatively refractory [1,2]. In particular, stable carbon isotope composition (d 13 C) of lignin and its derivatives can be used to complement palaeoenvir- onmental studies based on structural data obtained from its pyrolysates and phenols generated by CuO oxidation [3–7]. The combination of detailed molecular information with stable carbon isotope analysis in palaeoenvironmental studies allows the assessment of whether variations in d 13 C values are due to the presence of polysaccharides, or due to alterations suffered by the lignin polymer [8,9]. A key issue in obtaining reliable d 13 C data of lignin derivatives by gas chromato- graphy–combustion–isotope ratio mass spectrometry (GC– C–IRMS) is to determine if these compounds reflect the isotopic composition of the parent macromolecule. Analytical pyrolysis and CuO oxidation, both widely used methods for lignin structural studies, have been combined with GC–C–IRMS to study past vegetation changes [5], sources and distribution of organic matter in marine sediments [6,10] and genesis of humic substances www.elsevier.com/locate/jaap J. Anal. Appl. Pyrolysis 76 (2006) 88–95 * Corresponding author. Present address: Instituto de Geofisica, Univer- sidad Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico DF 04510, Mexico. E-mail address: laura@geofisica.unam.mx (L.E. Beramendi-Orosco). 0165-2370/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jaap.2004.07.010
Transcript
Evaluation of errors associated with d13C analysis of lignin-derived
TMAH thermochemolysis products by gas
chromatography–combustion–isotope ratio mass spectrometry
L.E. Beramendi-Orosco a,*, C.H. Vane b, M. Cooper a, C.G. Sun a,D.J. Large a, C.E. Snape a
a Nottingham Fuel and Energy Centre, School of Chemical, Environmental and Mining Engineering, University of Nottingham,
University Park, Nottingham NG7 2RD, UKb British Geological Survey, Keyworth, Nottingham NG12 5GG, UK
Received 17 July 2005; accepted 19 July 2005
Available online 14 November 2005
Abstract
This study reports the compound specific stable carbon isotope compositions (d13C) of lignin tetramethylammonium hydroxide (TMAH)
chemolysates obtained with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS). The possible sources of the
errors associated are considered. Off-line (TMAH) thermochemolysis was performed on wood samples and the d13C values of the
chemolysates were compared with the bulk d13C of the native woods and their Klason lignins. For the four woods investigated, the d13C
values, corrected for derivative carbons added, were spread over a wide range of values, ranging from �40 to �25% and were, on average,
depleted in 13C by ca. 9% relative to the native woods and by ca. 7% relative to the Klason lignins. This large variability can be partially
attributed to overlapping chromatographic peaks and to the low intensity of some of the peaks. However, isotopic fractionation cannot be ruled
out, especially in compounds resulting from C–C bond cleavage in the propyl side chain. The uncertainties associated with the correction for
carbons added by derivatisation were found to be high, especially for compounds having high contribution of derivative carbons.
Samples were run in triplicate and the reported values are
the mean � S.D. relative to the Vienna PeeDee Belemnite
(vPDB) standard. Accuracy and precision of the bulk
measurements were evaluated by analysing a reference
material of known isotopic composition (IAEA-CH-7 polye–
thylene, d13C = �31.80%), precision was found to be better
than �0.06% (1s, n = 5) and accuracy better than 0.1%.
2.5. Gas chromatography–combustion–isotope ratio
mass spectrometry
Compound specific isotope analyses of TMAH thermo-
chemolysis products were performed on a Trace GC
(Thermo Finnigan, Milan, Italy) gas chromatograph coupled
L.E. Beramendi-Orosco et al. / J. Anal. Appl. Pyrolysis 76 (2006) 88–9590
to a Thermo Finnigan Delta plus XP isotope ratio mass
spectrometer (Thermo Finnigan) via a GC combustion III
interface. The GC was fitted with a DB-5 fused silica
capillary column (30 m � 0.25 mm i.d., film thickness
0.25 mm). Samples were injected in splitless mode with
helium as carrier gas and an oven temperature program from
50 8C (held for 3 min) to 320 8C at 5 8C min�1 (held for
25 min) applied. As components eluted from the column
they were passed to a combustion interface set at 940 8C, and
then a reduction reactor held at 600 8C; finally, the CO2
produced was transferred to the isotope mass spectrometer.
The standard deviation of d13C measurements, based on
pulses of reference CO2 of known isotopic composition
(d13C = �33.92%, Air Products, UK), was determined to be
Fig. 1. Structures of the TMAH thermochemolysis products from l
�0.05%; however, it has been reported that chromato-
graphic inefficiencies may increase the standard deviation to
ca. �0.3% for compound specific d13C analyses [24].
3. Results and discussions
3.1. Identification of TMAH thermochemolysis products
from wood
Gas chromatography–mass spectrometry analysis (GC/
MS) was performed to identify the TMAH thermochemo-
lysis products prior to GC–C–IRMS. The chemical
structures of the TMAH products are illustrated in Fig. 1
ignin. G denotes guaiacyl units and S denotes syringyl units.
L.E. Beramendi-Orosco et al. / J. Anal. Appl. Pyrolysis 76 (2006) 88–95 91
Fig. 2. Partial chromatograms of the TIC for the TMAH thermochemolysis products of beech wood (top) and oak wood (bottom). Peak assignments are listed in
Table 1.
and the partial chromatograms of the total ion current (TIC)
for beech and oak are presented in Fig. 2. Product
identifications (Table 1) were made by comparison of their
mass spectra and relative retention times with those reported
in the literature [20–23].
The chromatograms indicate that the TMAH thermo-
chemolysis products consist of very complex mixtures of
compounds, including dimethoxy- and trimethoxy-substi-
tuted products. These monomers are derived from guaiacyl
(G) and syringyl (S) units with propyl side chains linked
mainly via b-O-4 bonds [25]. These results are supported by
recent work by Nakawaga-izumi et al. [26] reporting that
TMAH products derived from b–b linked lignin structures
included mainly di-O-methylresinols and that monomeric
compounds, such as those found here, were present only in
very low yields.
The presence of components derived from both types of
lignin units (G and S) is consistent with the fact that the
analysed woods are angiosperm, with lignin composed of
guaiacyl and syringyl units. The product distribution
patterns for all the samples investigated are very similar.
3.2. GC–C–IRMS of TMAH thermochemolysis products
from wood
The m/z 44 chromatograms obtained by GC–C–IRMS
(Fig. 3) are analogous to the TIC chromatograms obtained
by GC/MS; however, as expected, the chromatographic
resolution in GC–C–IRMS is impaired. The reason for the
lower chromatographic separation is a consequence of
different factors. One is that after the GC column and prior
to the IRMS the sample stream is passed through furnaces
and dryers, generally reducing the chromatographic
efficiency. Another factor is that CO2 disperses more freely
within the carrier gas stream than the parent organic
compounds, resulting in broader CO2 peaks [27,28]. A third
factor is that a 30 m column, with a slightly different
temperature program, was used in the GC–C–IRMS instead
of the 50 m column used in the GC/MS analysis and this
may have contributed to the lower chromatographic
separation.
The d13C values of the identified TMAH products
(uncorrected for the derivative carbons) are spread over a
L.E. Beramendi-Orosco et al. / J. Anal. Appl. Pyrolysis 76 (2006) 88–9592
Table 1
Identified TMAH thermochemolysis products of angiosperm woods
The mass balance was performed using Eq. (1) where x is
the fractional carbon contribution of the underivatised
Fig. 3. Partial trace of the m/z 44 GC–C–IRMS chromatogram (bottom) and 45/44 r
d13C values are tabulated in Table 2.
compound to the methylated product (C in original
compound/C in original compound + OCH3 added,
Table 2). The d13CTMAH value was determined by bulk
isotope analysis (�34.21 � 0.25%, n = 6). Corrected values
for the identified TMAH products are listed in Table 2,
together with the bulk d13C values of the corresponding
wood and Klason lignin.
The corrected d13C values of the TMAH products
obtained from wood are still spread over a very wide range,
from �40 to �25% (Table 2). For all the cases the TMAH
products are, on average, depleted in 13C relative to wood
and to the corresponding Klason lignin. For example, for oak
atio (top) for oak wood TMAH products. Identified peaks and their corrected
L.E. Beramendi-Orosco et al. / J. Anal. Appl. Pyrolysis 76 (2006) 88–95 93
Table 2
d13C values of wood, Klason lignin and TMAH thermochemolysis products (corrected for derivative carbons added)
Compound OCH3 added C in original compound d13C (%)
Ash Beech Oak Silver birch
Wood – – �25.23 �23.40 �24.13 �25.86
Klason lignin – – �27.49 �25.71 �26.56 �28.37
S1 1 8 n.d. �28.52 �29.82 �30.46
G7 2 9 �37.73 �33.63 �32.91 �34.85
G8 2 9 �38.11 �34.97 �34.41 �35.86
G10 2 10 �29.85 �29.94 �29.37 �31.31
S5 1 10 �28.91 �26.56 �27.22 �27.66
S6 2 9 �31.16 �25.48 �26.00 �28.04
S7 2 10 �35.22 �33.48 �31.90 �36.19
S8 2 10 �36.54 �33.76 �33.46 �36.21
G14 4 10 �36.88 �37.37 �35.88 �38.33
S10 2 11 �36.20 �38.74 �34.41 �38.60
S14 4 11 �41.98 �38.62 �37.99 �40.27
S15 4 11 �35.35 �37.67 �34.03 �34.50
Samples were analysed in triplicate. S.D. were less than �3.6% for TMAH products and �0.2% for bulk d13C of wood and Klason lignins. n.d., not detected.
wood, the TMAH products average �32.35 � 5.79%, and
range from �26.82 to �37.99%, whereas its Klason lignin
has a bulk d13C value of �26.56 � 0.20%.
As already mentioned, this large variability in the d13C
values of TMAH products may be a consequence of different
factors. The first one is related to the poor chromatographic
resolution, which results in peaks having contributions from
more than one compound. In addition, overlapping peaks
have been observed to be systematically distorted in isotope
ratio [27,29]. Goodman and Brenna [27] reported errors in
accuracy of up to �10% even for compounds with d13C
values differing by only 0.30%. This may explain the
difference in d13C values for S14 and S15, which are R and S
isomers but have values differing by up to ca. 6.63%(Table 2; Fig. 3).
Another source of error can be related to the low intensity
of some of the peaks. Errors in accuracy approaching �3%(D13C = d13Cmeasured � d13Cknown) have been reported for
peaks of intensities less than 0.5 of the intensity of the
reference peaks [24].
There is also the possibility of isotopic fractionation
caused during TMAH thermochemolysis. It has been
reported that TMAH thermochemolysis induces C–C bond
cleavage in the propyl side chain, generating a number of
products, some of which are oxidised on the remaining side
chain carbons [25,30]. In accordance with these reports,
some of the compounds detected among the TMAH products
from the studied woods have less than three carbons in the
side chain, and some of them also exhibit oxidation; e.g. G4,
S4, G5, S5, G6 and S6 (Fig. 1). On the other hand, solid-state13C NMR spectra of various milled lignins extracted from
fresh woods show small resonances from lignin-derived
carbonyl/carboxyl groups that could be present in small
amounts in the parent polymer [31], suggesting that these
type of compounds could be derived from lignin units.
However, if these compounds are, in fact, products of C–C
bond cleavage and oxidation reactions in the side chain
induced by the TMAH thermochemolysis, then the
possibility of some isotopic fractionation occurring would
yield TMAH products isotopically lighter, and not
representative of the parent polymer. Despite it having
been reported that TMAH thermochemolysis preserves the
isotopic composition of standard phenols [17], this has not
been hitherto explored in lignin preparations. Clearly, more
work is needed to assess the possible isotopic fractionation
during thermochemolysis of native lignin and lignin
preparations.
In addition to the potential problem of isotopic
fractionation, another source of error associated with the
TMAH thermochemolysis relates to derivatisation. The
mass balance used for calculating the d13C values of the
underivatised TMAH products (Eq. (1)) did not consider the
errors (or uncertainties) associated with the measurements
of d13CTMAH and d13CTMAHproduct. The uncertainty asso-
ciated with the calculation of d13Cunderivatised can be
estimated from the equation of error propagation (Eq. (2))
[32,33]:
e2underivatised ¼ e2
TMAH
�nTMAH
nunderivatised
�2
þ e2TMAH product
�nunderivatised þ nTMAH
nunderivatised
�2
(2)
where e is the uncertainty and nunderivatised and nTMAH are the
number of moles of carbon in the underivatised compound
and TMAH, respectively. The smaller the relative contribu-
tion of derivative carbons to the derivatised compound, the
smaller the eunderivatised [25]. The uncertainties calculated
with Eq. (2) for d13C values of the underivatised TMAH
products are tabulated in Table 3, the errors used for the
TMAH products were the standard deviations for each peak
found by triplicate injection.
As expected, the compounds with more than one deri-
vative carbon added are those with higher uncertainties. For
L.E. Beramendi-Orosco et al. / J. Anal. Appl. Pyrolysis 76 (2006) 88–9594
Table 3
Uncertainty in the estimation of d13Cunderivatised of TMAH thermochemo-
lysis products, calculated using Eq. (2) with eTMAH = �0.26% and eTMAH -
product corresponding to the S.D. for each peak after triplicate injection,
which was less than 3.6% for all cases
Compound eunderivatised
Ash Beech Oak Silver birch
S1 n.d. 0.65 0.95 0.56
G7 0.79 0.81 2.01 1.39
G8 1.17 0.97 1.51 0.76
G10 2.48 3.47 0.97 0.69
S5 1.08 0.89 0.53 0.76
S6 0.56 0.98 1.11 0.75
G7 1.61 1.30 0.63 1.08
G8 0.46 2.92 0.94 1.13
G14 1.18 5.03 2.13 3.96
S10 1.69 2.52 1.87 1.85
S14 2.57 3.05 1.79 1.73
S15 1.87 3.73 1.72 1.30
n.d., not detected.
example, G14 has 10 carbons in the original compound and
4 added carbons, the uncertainties found for this compound
are 1.18, 5.03, 2.13 and 3.96 for ash, beech, oak and silver
birch, respectively.
For the reasons discussed above, in order to obtain
meaningful information from isotope data of TMAH
products, only the chromatographic peaks that are relatively
intense and well resolved should be considered. In addition,
it would be preferable to consider only those compounds
with a complete propyl side chain, like G10 and S10, in
order to minimise the effect of possible isotopic fractiona-
tion caused during thermochemolysis, and finally, com-
pounds with high relative contribution of derivative carbons,
such as G14, S14 and S15, should be avoided to minimise
imprecision in d13C calculations.
In the m/z 44 chromatograms of the samples investigated,
S5 and S6 are peaks with high intensity, and S14 is a peak
with relatively good chromatographic resolution (Fig. 3);
however, these peaks have some problems associated. For
S5, the poor shape seems to indicate front co-elution
although there is no peak between G10 and S5 in the GC/MS
trace (Fig. 2); whereas for S6, it can be confirmed in Fig. 2
that there is back co-elution. Despite these compounds
having d13C values close to the bulk value of the
corresponding Klason lignin, they do not possess a three-
carbon side chain (Fig. 1), and therefore isotopic fractiona-
tion induced by C–C bond cleavage in the side chain during
thermochemolysis cannot be ruled out, yielding compounds
that are not representative of the original lignin signal. In
terms of reducing the uncertainty derived from the
introduction of derivative carbons, S5 and S6 are among
the compounds with lower relative contribution of derivative
carbons per carbon in the original compound (Tables 2 and
3).
Regarding S14, the chromatographic resolution is better
and isotopic fractionation induced by C–C bond cleavage
can be ruled out, as this compound has a complete side
chain. However, the uncertainty associated with the
introduction of derivative carbons is quite high because
this compound has 4 TMAH-derived carbon atoms per 11
carbons in the original compound (Tables 2 and 3). This may
be reflected in the fact that the d13C values for S14 are
depleted in 13C, relative to the bulk values of the
corresponding Klason lignin, by more than 11.43%.
4. Conclusions
In spite of TMAH thermochemolysis being a powerful
method for identifying variations in lignin composition, it
seems unreliable as a preparation method for lignin isotopic
analysis, owing to the limitations encountered through GC–
C–IRMS analysis of TMAH products, and the increased
uncertainties for compounds with more than one derivative
carbon atom added.
In addition to these issues, potential isotopic fractionation
caused by C–C bond cleavage during the thermochemolysis
may occur. In order to establish if lignin-derived TMAH
chemolysates are representative of the parent polymer and
can be used in isotope studies, more research is needed in
assessing the degree of isotopic fractionation produced by
this thermal technique, because only preliminary results on
standard phenols have been reported [17]. One possible
route to explore is to perform TMAH thermochemolysis on
dimeric lignin model compounds. This would help to
elucidate if the wide range of d13C values found for lignin-
derived TMAH chemolysates results from isotopic fractio-
nation associated with the thermal technique, or is a result of
isotopic heterogeneity of the lignin macromolecule.
Acknowledgements
Financial support and the Ph.D. scholarship from the
Mexican Council for Science and Technology (CONACyT)
for L.E. Beramendi-Orosco are gratefully acknowledged.
