Journal of Analytical and Applied Pyrolysis

43 (1997) 41 – 57

Analysis of oxidised diterpenoid acids using thermally

assisted methylation with TMAH
I. Pastorova a,*, K.J. van der Berg a, J.J. Boon a, J.W. Verhoeven b
a
FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, Netherlands
b
Department of Organic Chemistry, Uni6ersity of Amsterdam, Nieuwe Achtergracht 129,
1018 WS Amsterdam, Netherlands
Received 7 March 1997; accepted 2 June 1997

Abstract

An aged intermediate varnish sample from ‘The Girl with the Pearl Earring’ by J. Vermeer
was analysed by thermally assisted methylation with tetramethylammoniumhydroxide
(TMAH) followed by gas chromatography in combination with mass spectrometry. Using
library search of the available mass spectra, only three diterpenoid acids were identified. In
addition to methyldehydroabietate, 7-oxo-dehydroabietic acid methyl ester and 7-oxo-15-hy-
droxy-dehydroabietic acid methyl ester were identified. There was reason to believe that the
remaining unidentified compounds might be related to the oxidised derivatives of dehydroa-
bietic acid and are formed upon methylation with TMAH. In order to investigate the
behaviour of pure 7-oxo-dehydroabietic acid and 7-oxo-15-hydroxydehydroabietic acid
under these specific derivatisation conditions, these compounds were synthesised and iso-
lated. On derivatisation with TMAH, several methylation products, with varying degrees of
methylation were formed from each of the pure compounds. Their structures were elucidated
using specific fragment ions present in their mass spectra. Unusual products formed by
methylation on a carbon atom under these conditions were observed. Based on these
comparative studies, 7-oxo-dehydroabietic acid and 7-oxo-15-hydroxy-dehydroabietic acid
were identified as the main components of the varnish. © 1997 Elsevier Science B.V.

Keywords: Thermally assisted methylation; Tetramethylammoniumhydroxide; Diterpenoid

acids; Dehydroabietic acid

* Corresponding author. Present address: Kiwa Research and Consultancy, Groningenhaven 7, 3430
BB Nieuwegein, Netherlands. Tel.: + 31 30 6069511.

0165-2370/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 5 - 2 3 7 0 ( 9 7 ) 0 0 0 5 8 - 2
42 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

1. Introduction

Diterpenoid acids are the most abundant components of diterpenoid natural

resins. The most important sources are the Coniferae and Leguminosae families.
Most widely used are the resins derived from the Pinaceae subfamily, which
includes pine (Pinus), larch (Larix), spruce (Picea), fir (Abies). These trees, espe-
cially pine, have been valuable source of rosin (colophony), oil of turpentine, tar
and pitch, materials that have found many applications in the past [1]. Tars and
pitches have been used for centuries for impregnation and sealing purposes [2,3]. At
present, colophony is utilised on a commercial scale, often chemically modified, as
an additive to manufacture products used as adhesives, printing inks etc. [4].
Diterpenoid acid containing resins and balsams were also applied by artists in
paintings for copper resinate pigments, additive to paint and on paintings as a
varnish [5]. Remedial conservators often used rosin as a component of wax/resin
linings to strengthen the original support of the painting, particularly in the
Netherlands [6].
Acids with pimarane and abietane skeletons (Fig. 1) are the most abundant
components of the resins derived from the Pinaceae subfamily. Whereas pimaranes
are known to be relatively stable towards isomerisation and oxidation, acids with
an abietane structure are less stable. During heating, they are known to isomerise
readily, predominantly to abietic acid [7]. The abietic acids then oxidise in air to the
aromatic dehydroabietic acid (Fig. 2) [8,9]. This acid is relatively stable, but on
longer exposure to air and light oxidises further to produce several highly oxidised
products, of which 7-oxodehydroabietic acid, 15-hydroxy-dehydroabietic acid and
7-oxo-15-hydroxy-dehydroabietic acid are the most abundant [5,10,11] (Fig. 2).
7-Oxodehydroabietic acid has been found frequently in aged samples from works of

Fig. 1. Important components of Pinaceae diterpenoid resins which have been often used in and on
paintings.
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 43

Fig. 2. Oxidation scheme of abietanes.

art [5,12,13]. In addition, 15-hydroxydehydroabietic acid and 7-oxo-15-hydroxy-de-

hydroabietic acid have been found in high amounts in similar samples [5,13].
Gas chromatography in combination with mass spectrometry has proved to be a
powerful tool for the analysis of complex mixtures, e.g. samples from works of
painted art [12 – 15]. With this technique, derivatisation of polar groups of the
molecular components is often required. For diterpenoid acids, methylation of the
carboxylic groups with diazomethane is the most common method for this purpose
[12,16]. However, this method is limited by the solubility of the samples in solvents
used for diazomethane methylation. For non-soluble components, wet chemical
pretreatments such as hydrolysis are then also required.
44 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

An alternative workup method is in situ thermally assisted methylation with

TMAH, which has been developed for the analysis of carboxylic acids [17]. This
method has also been succesfully applied for the analysis of other classes of
componds such as phenols and amines. By mixing the analysed sample with the
solution of TMAH and evaporating the water, tetramethylammonium salts are
formed (Fig. 3). The salts decompose at higher temperatures to yield methylated
acids, phenols and side products formed by decomposition of TMAH [17]. These
side products are volatile, elute at the very begining of the chromatogram and do
not interfere with the analysis. Hydrolysis and methylation of hydrolysable poly-
meric material has been reported by Challinor [18]. Anderson and Winans [19]
and Challinor [20] have applied the method for the analysis of diterpenoid resins
and resinites. Winans and Anderson concluded that this approach is suitable for
the analysis of diterpenoid acids, but less so for their oxidised derivatives, con-
taining additional carbonyl and/or hydroxyl groups. For these compounds, side
reactions such as eliminations and incorporation of nitrogen were reported to
occur, complicating the interpretation of the results.
Thermally assisted methylation in combination with pyrolysis is very often
used for the analysis of intrinsically minute and complex painting samples
[14,15,21]. This derivatisation technique is not only less time-consuming, but is
also more reliable and inherently more sensitive than wet chemical workup tech-
niques. The combination with pyrolysis enables the analysis of low molecular
weight components as well as the polymeric material (in-situ hydrolysis of the
hydrolysable polymers and thermal decomposition of the non-hydrolysable poly-
mers) in one measurement.
In this paper, the suitability of this method for analysing samples containing
oxidised diterpenoids of aged works of art is demonstrated. An intermediate
varnish sample from ‘The Girl with the Pearl Earring’, known to contain oxi-
dised diterpenoids was analysed. Two of the major oxidation products of diter-
penoid acids, 7-oxo-dehydroabietic acid and 7-oxo-15-hydroxydehydroabietic acid
were synthetised, isolated and used as standards. Their behaviour under ther-
mally assisted methylation with TMAH was studied and the results were com-
pared with the results of the varnish analysis.

Fig. 3. Proposed mechanism of methylation of carboxylic acids with TMAH [17].

 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 45

2. Experimental

2.1. Synthetic part

Detailed description of the syntheses was out of the scope of this paper and
was published elsewhere [28]. The literarure used is mentioned below in the form
of references.

2.2. Isolation of DHA

DHA (dehydroabietic acid) was isolated from commercially degraded resin

(731D, containing 50 – 60% of DHA, a kind gift of Abieta Chemie, Germany) as
its aminoethanol salt, using the method of Hallbrook and Lawrence [22]. The
purity was 85% (determined by GC-MS, the remaining 15% were composed of
dihydroabietic acids). This purity was considered suitable for further synthetic
purposes.

2.3. Synthesis and isolation of 7 -oxodehydroabietic acid

For the synthesis and isolation of this compound, the method described by K.
Krohn et al. [10] was used with little or no modification. The yield of 7-oxo-de-
hydroabietic acid was 17.5%.

2.4. Synthesis and isolation of 7 -oxo-15 -hydroxydehydroabietic acid methyl ester

This acid was prepared by further oxidation of 7-oxo-dehydroabietic acid

methyl ester [28]. The yield was 8%.

2.5. Varnish sample

The varnish was a scraping (about 10 mg) from the very brown varnish near
the top edge of ‘The Girl with the Pearl Earring’ by Jan Vermeer (Mauritshuis,
The Hague, cat. no. 1687) [23]. It was covered by an extensive overpaint, which
was applied in 1960 in a restoration treatment. It is therefore at least 36 years
old.

2.6. Direct temperature-resol6ed mass spectrometry-DTMS

DTMS was performed on a JEOL SX-102 double focusing mass spectrometer

(B/E), using a direct insertion probe equipped with a Pt/Rh (9/1) filament. Ions
were generated by electron impact (16 eV) in an ionisation chamber kept at
180°C and accelerated to 8 kV. The scan range was m/z 20–1000 with a 1 s
46 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

Fig. 4. GC–MS measurement after thermally assisted methylation with TMAH of an intermediate
varnish sample from ‘The Girl with the Pearl Earring’. TIC of the diterpenoid region. Peaks relevant for
this study are marked with their MW.

cycle time and mass resolution of 3000. The probe filament was temperature
programmed at a rate of 0.5 A min − 1 to final temperature of 800°C. Data were
aquired using a JEOL MP-7000 data system.

2.7. Thermally assisted methylation-gas chromatography-mass spectrometry

The standards were dissolved and the varnish suspended in methanol and
applied to a pyrolysis wire. A drop of 2.5% TMAH (or deuterated TMAH in
D2O) solution in water was then added. The solvent was evaporated under
reduced pressure. The wire was placed into a glass liner, flushed with argon and
inserted into the FOM-4LX pyrolysis unit. Py-GC-MS was performed on the
HRGC 8565 Mega 2 series gas chromatograph (FISONS) equipped with a CP-
CF 818 pressure/flow control box and with the FOM-4LX pyrolysis unit. The
pyrolysis conditions were as follows: Curie-point temperature 358° or 610°C
temperature rise time 1 s (600°C s − 1), total pyrolysis time was 6.0 s, and the
temperature of the pyrolysis chamber 190°C. The unit was flushed with helium,
which was also used as carrier gas, with a constant linear flow velocity of 26 cm
s − 1. The evaporated sample was directly introduced into a 25 m SGE (Scientific
Glass Engineering, Australia)-BPX5 fused silica capillary column (d= 320 mm,
0.25 mm film). The temperature of the GC oven was kept at 50°C until 5 min
after the pyrolysis and subsequently heated at a rate of 6°C min − 1 to 350°C, at
which it was held for an additional 4 min. The GC column was directly intro-
duced into the ion source of a JEOL DX-303 double focusing mass spectrometer
via a home built heated interface kept at 300°C. The compounds were ionised
with electron impact at 70 eV, the accelerating voltage was 3 kV and the ions
were post accelerated to 10 kV before detection. The cycle time (in seconds) was
0.54 (scan) + 0.1 (rescan). The mass range was m/z 40–800. The mass spectra
were processed with a JEOL MP-7000 data system (JEOL).
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 47

3. Results and discussion

3.1. TMAH-GC-MS analysis of an intermediate 6arnish

Fig. 4 shows the total ion current (TIC) of a diterpene varnish from the painting
‘The Girl with the Pearl Earring’ by J. Vermeer. Only the diterpenoid region is
shown. The indicated molecular weights were determined from the m/z values of
the molecular ions present in the mass spectra of the eluting compounds.
Using library search of the available mass spectra, only three diterpenoid acids
could be identified. In addition to methyldehydroabietate with MW 314, the
structures of compounds with MW 328 and 344 (trace amounts) were elucidated.
Their mass spectra correspond to 7-oxo-dehydroabietic acid [24,25] and 7-oxo-15-
hydroxy-abietic acid methylesters [10]. There was reason to believe [5] that the
remaining unidentified compounds might be related to the oxidised derivatives of
dehydroabietic acid and are formed upon methylation with TMAH. In order to
investigate the behaviour of pure 7-oxo-dehydroabietic acid and 7-oxo-15-hydrox-
ydehydroabietic acid under these specific derivatisation conditions, these com-
pounds were synthesised and isolated.

3.2. TMAH-GC-MS of the standard compounds

3.2.1. Products with different degree of methylation

On reaction with TMAH, 7-oxo-dehydroabietic acid (I) with a molecular weight
(MW) 314, yields three abundant methylation products. Their molecular weights
were determined from the m/z values of the molecular ions present in their mass
spectra (see further in text). Based on their molecular weights, 328, 342 and 356, the
number of methyl groups incorporated in these molecules is proposed as 1, 2 and
3, respectively. Although the relative amounts of the compounds are not repro-
ducible, the compound with MW 342 is always more abundant than the methylation
products with MW 328 and 356.
Seven different methylation products of 7-oxo-15-hydroxydehydroabietic acid
(II) with MW 330, contribute with abundant peaks to the total ion current obtained
by GC-MS measurement. Their molecular weights are 326, 340, 344, 354, 358, 372
and 386. Compounds with MW 344, 358, 372 and 386 contain 1, 2, 3 and 4 methyl
groups respectively. From the MW of the remaining three compounds we concluded
that elimination of water or methanol had taken place. The MW values 326, 344
and 354 can be obtained by subtracting either 18 or 32 from the MW of the first
four compounds. The proposed number of methyl groups incorporated in these
methylation products is 1, 2 and 3, respectively. Although once again, the relative
abundances of these compounds are not reproducible and differ from measurement
to measurement, the compounds with MW 340 and 372 are always the most
abundant components.
The number of methyl groups proposed for all the methylation products men-
tioned above was verified by experiments with perdeuterated TMAH (TMAH-d13).
The predicted mass shifts in molecular weights were indeed observed for all the
48 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

compounds (not shown). The experiments with deuterated TMAH confirmed that
the mass increments of 14 U correspond to an extra methylene group and not to
incorporation of nitrogen. No evidence for nitrogen incorporation, described by
Anderson and Winans [19], was found.

3.2.2. Interpretation of mass spectra of the methylation products

The structures of the methylation products were assigned by interpretation of
their mass spectra. None of the spectra shown in Fig. 5 and Fig. 6 can be found in
the literature, with the exception of the compounds with MW 344 and 328. Their
spectra are shown in Fig. 5a and 6g. The first is the spectrum of 7-oxo-15-hydroxy-
dehydroabietic acid methyl ester IIg, published by Krohn et al. [10]. The second
spectrum belongs to 7-oxo-dehydroabietic acid methylester, Ia and was described in
the literature [10,23,24].
The assignment of the structures of the remaining compounds was based on the
comparison of the mass shifts in the spectra of the above mentioned compounds
and the remaining methylation products. The fragmentation reactions observed in
their spectra are quite similar. Cleavage of the (substituted) functional groups,
leading to ions [M-15] + ’ for the loss of the methyl group on C10, [M-75] + ’ for the
loss of formic acid methylester and the loss of methyl on C 10, and [M-59] + ’ from
the loss of the methyl ester group (the m/z values are listed in Table 1), can be

Fig. 5. The 70 eV mass spectra of 7-oxo-dehydroabietic acid methylation products obtained with
thermally assisted methylation by TMAH.
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 49

Fig. 6. The 70 eV mass spectra of 7-oxo-15-hydroxydehydroabietic acid methylation products obtained

with thermally assisted methylation by TMAH.
 50

Table 1
Compounds formed under thermally assisted methylation with TMAH conditions and their main fragment ionsa

Ion 328 (Ia) 342 (Ib) 356 (Ic) 358 (IIa) 372 (IIb) 386 (IIc) 326 (IId) 340 (IIe) 354 (IIf) 344 (IIg)

M 328 342 356 358 372 386 326 340 354 344
–CH3 313 327 341 340 357 371 311 325 339 329
–HCOOR; – 253 267 281 283 297 311 251 265 279 269
CH3
–COOR 269 283 297 299 313 327 267 281 295 285
a 199 213 227 243 243 257 197 211 225 213
b 187 201 201 231 231 231 185 199 199 201
c 216 230 232 246 260 214 228
–CH3OH 340 354
–CH3OH; –CH3 325 339
–H2O 340

a
Structures of these compounds are shown in Fig. 8 and Fig. 9
I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 51

Fig. 7. (a) Structure of fragment ion c, formed on fragmentation of methylated enol tautomers of
7-oxo-dehydroabietic acid and 7-oxo-15-hydroxy-dehydroabietic acid; (b) formation of fragment ions a
and b, important for determining the position of carbon methylation by TMAH.

observed in the mass spectra of all the methylation products, Fig. 5a–c and Fig.
6a–g. In addition to these ions, there are some ions present in the spectra that are
very structure-specific and can be used for structural elucidation.
The compound with MW 342, containing two methyl groups was identified as the
methylated enol form of Ia. The tautomerisation of the keto group occurs quite
easily if the newly formed double bond is conjugated with the aromatic ring. The
52 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

Fig. 8. The structures of identified methylation products formed from 7-oxo-dehydroabietic acid upon
methylation by TMAH.

enol structure, compound Ib is then quite favourable. Methylation of the enol

tautomer by TMAH has been observed before [17]. The mass spectrum of com-
pound Ib contains an abundant even-mass fragment ion at m/z 216. A proposed
structure of this ion c is shown in Fig. 7a and can only be formed from the
methylated enol tautomer Ib. It includes rings B and C of the original structure. An
analogous even-mass (14 mass U higher) ion is present in the spectrum of the
compound with MW 356 at m/z 230. This suggests that the extra methyl group is
located on either ring B or C. Its exact location can be determined by comparing
the m/z values of fragment ions formed by cleavage in rings A and B. The
formation of these ions upon ionisation of dehydroabietic acid and its decarboxyla-
tion products was described by Enzel [26,27] and used by Proefke et al. [25] to
identify 7-oxo-dehydroabietic acid methyl ester in a Egyptian Mummy sample. Fig.
7b shows a simplified picture of their formation together with the m/z values of ions
a and b for dehydroabietic acid and 7-oxo-dehydroabietic acid methylesters (Ia).
For the methylated enol form of 7-oxo-dehydroabietic acid Ib the m/z values of
ions a and b are 201 and 213, respectively. The fragment a still contains C5,
whereas fragment ion b does not (Scheme 3). Therefore any substitution on C5 will
be visible in the mass spectra by a mass shift corresponding to the mass of the
substituent only for the fragment ion a and not the fragment ion b. Fig. 5c shows
that the mass spectrum of compound Ic indeed contains fragment ions a: m/z 201
and b: m/z 227 (213+14), indicating the presence of a methyl group on C5. To the
best of our knowledge, this is the first time that methylation on a carbon under
thermally assisted methylation with TMAH is described. This particular methyla-
tion reaction occurs only in the enol tautomers. As a consequence of the newly
formed double bond, the hydrogen on C5 becomes allylic and therefore relatively
acidic and reactive. Under strongly basic conditions, it probably reacts in an
acid-base reaction with TMAH and substitution occurs. Whether the reaction
mechanism is the same as proposed for the methylation on a carboxylic or phenolic
oxygen (Fig. 3), has not been investigated. The structures of all the identified
methylation products formed from 7-oxo-dehydroabietic acid under TMAH condi-
tions are shown in Fig. 8.
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 53

The methylation products formed from 7-oxo-15-hydroxy-dehydroabietic acid

were identified in a similar way. Compounds analogous to those described above
compound IIb, methylated enol form with MW 372 and compound IIc with MW
386, corresponding with the enol form with an additional methyl group on C5.
Compound IIa with MW 358, is the methylated enol form, with a free hydroxy
group on C15. The characteristic m/z values of the fragment ions are listed in Table
1 and Fig. 7.
The tertiary alcohol group on C15 introduces a reactive center into the molecule.
The free hydroxyl group and/or methoxy group eliminates easily, leading to
compounds with a double bond in the isopropyl group with MW 326, 340 and 354,
IId, IIe and IIf, respectively. The structures of the identified compounds are shown
in Fig. 9.

3.3. Py-GC-MS of 7 -oxo-15 -hydroxy-dehydroabietic acid methyl ester without

TMAH

7-oxo-15-hydroxy-DHA methyl ester was pyrolysed (evaporated) under similar

conditions to those used above, but with no TMAH added. This experiment was
performed to investigate the influence of the experimental conditions on the
formation of the different methylation products. Two compounds can be identified,
IIg and its dehydrated form IId. The dehydration product is the more abundant
component, but the relative amounts of the two compounds is not exactly repro-
ducible and differs from measurement to measurement.

Fig. 9. The structures of identified methylation products formed from 7-oxo-15-hydroxy-dehydroabietic

acid upon methylation by TMAH.
54 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

Fig. 10. Sum spectrum obtained by DTMS measurements of (a) 7-oxo-15-hydroxy-dehydroabietic acid
methylated with TMAH; (b) 7-oxo-15-hydroxy-dehydroabietic acid methyl ester, no TMAH added.

3.4. DTMS of 7 -oxo-15 -hydroxy-dehydroabietic acid

The sum spectrum obtained from 7-oxo-15-hydroxy-dehydroabietic acid methy-

lated with TMAH by direct temperature resolved mass spectrometry (DTMS) is
shown in Fig. 10(a). The molecular ions of compounds IIa through IIg (m/z 358,
372, 386, 326, 340, 354 and 344, observed in the GC-MS measurements) are all
visible in the spectrum, with varying relative intensities. The most abundant ion is
m/z 372, corresponding to the molecular ion of compound IIb. The compounds
with unsaturation in the isopropyl group (IId–IIf) are of very low abundance. Fig.
10B shows the spectrum of 7-oxo-15-hydroxy-dehydroabietic acid methyl ester
obtained by a DTMS experiment with no TMAH added. This is a spectrum of one
compound and corresponds to the spectrum of compound IIg, Fig. 6g. There is no
evidence for dehydration of the isopropanol group in the sum spectrum.
Comparison of these results with those described above from the GC-MS
measurements, indicates that the methylation in different positions of the oxidised
diterpenoid acids occurs on mixing the sample with TMAH and is not dependent
on the instrument. However, the elimination of water or methanol from the
7-oxo-15-hydroxy-dehydroabietic acid derivatives occurs more readily under gas
chromatographic conditions and is not dependent on the presence or absence of
TMAH. While no dehydration of 7-oxo-15-hydroxy-dehydroabietic acid methyl
 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57 55

ester is observed under DTMS conditions, dehydration of up to 90% of this

compound is observed when measured with GC-MS. From the fact that two
separate peaks were observed in the TIC, and they are very sharp with no tailing,
it can be concluded that the elimination reactions take place before the separation
and not on the GC column or the source of the mass spectrometer.
One of the main differences between the different methods is the material of the
wire on which the sample is deposited. The material of the wire used for GC-MS
analysis may have a catalytic influence on the elimination reactions. In addition,
under GC conditions, before the separation, the compounds may come into contact
with an active surface, in the glass liner, or an activated part at the entrance of the
GC column, which may also have a catalytic effect on the elimination reactions.
This is not the case under DTMS conditions, where compounds are evaporated
from a Pt/Rd wire, directly ionised and analysed. Further investigations on the
influencing factors are currently in progress.

4. Conclusions

The originally unknown compounds observed in the TIC of the thermally

assisted methylation with TMAH combined with GC-MS of the varnish from ‘The
Girl with the Pearl Earring’ by Vermeer were identified (Table 2). The relative
retention times, molecular weights and mass spectra are identical with methylation
products of pure 7-oxo-dehydroabietic acid and 7-oxo-15-hydroxy-dehydroabietic
acid formed under similar derivatisation conditions.
Based on these results, it can be concluded that most of the peaks in the oxidised
diterpenoid region of the intermediate varnish (Fig. 4) represent only two oxidised
derivatives of dehydroabietic acid, namely 7-oxo-dehydroabietic acid and 7-oxo-15-
hydroxy-dehydroabietic (Table 2). The determination of the oxidation state of

Table 2
Oxidised diterpenoid acids identified by thermally assisted methylation — gas chromatography, mass
spectrometry of an intermediate varnish from the painting ‘The Girl with the Pearl Earring’

Retention time Molecular weight Structure Methylation product of:

39.29 314 Methyl DHA Dehydroabietic acid (DHA)

41.12 312 MethylD DHA Dehydro-DHA
41.34 342 Ib 7-Oxo-DHA
42.17 344 15-Hydroxy-dehydroabietic acid
43.14 340 IIe 7-Oxo-15-hydroxy-DHA
43.21 356 Ic 7-Oxo-DHA
43.83 328 Ia 7-Oxo-DHA
44.01 372 IIb 7-Oxo-15-hydroxy-DHA
44.72 358 IIa 7-Oxo-15-hydroxy-DHA
44.97 354 IIf 7-Oxo-15-hydroxy-DHA
45.32 326 IId 7-Oxo-15-hydroxy-DHA
45.91 386 IIc 7-Oxo-15-hydroxy-DHA
56 I. Pastoro6a et al. / J. Anal. Appl. Pyrolysis 43 (1997) 41–57

diterpenoid materials was described by K.J.v.d. Berg et al. [5]. It should be noted
that at that time, not all the methylation products of 7-oxo-dehydroabietic acid and
7-oxo-15-hydroxydehydroabietic acid were identified, the oxidation state described
in this publication [5] is somewhat underestimated. The relevant peaks are inte-
grated and in this case the area of the peaks which correspond to different
methylation products of the same oxidised diterpenoid acid were summed. The
oxidation state is then described by the contribution of abietic, dehydrobietic,
7-oxo-dehydroabietic and 7-oxo-15-hydroxydehydroabietic acids to the diterpenoid
acid fraction. By this method, the oxidation state of the intermediate varnish from
‘The Girl with the Pearl Earring’, which is characterised by the percentages
0:5:26:69, can be described as very high [5].
Studies with pure 7-oxo-dehydroabietic acid and 7-oxo-15-hydroxyabietic acid
show that several side reactions occur upon methylation of higher oxidised diter-
penoids with TMAH, especially under Py-GC-MS conditions. Products with a
varying degree of methylation are formed. Unusual methylation on a tertiary
carbon atom was observed. Under GC-MS conditions, elimination of water and/or
methanol occurs. The structures of all the methylation products were elucidated.
Despite the relative complexity of the results, thermally assisted methylation with
TMAH followed by GC-MS proved to be a powerful method for analysing
oxidised diterpenoids. More experiments, using this method and the spectra ob-
tained in this study for analysis of various diterpene containing samples are
presently in progress.

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