Journal of Chromatography A, 1147 (2007) 206–212

Gas chromatography–mass spectrometry characterization of the

varnish and glue of an ancient 18th century double bass
Francesco Caruso a,∗ , Santino Orecchio b , Maria Grazia Cicero c , Cosimo Di Stefano c
aIstituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche (CNR),
Via Ugo La Malfa 153, I-90146 Palermo, Italy
b Dipartimento di Chimica Inorganica ed Analitica “Stanislao Cannizzaro”, Università di Palermo,

Viale delle Scienze – Parco d’Orleans II, I-90128 Palermo, Italy

c Centro Regionale per la Progettazione ed il Restauro (CRPR), Via Cristoforo Colombo 52, I-90142 Palermo, Italy

Received 30 August 2006; received in revised form 8 February 2007; accepted 14 February 2007
Available online 21 February 2007

Abstract
A GC–MS investigation is conducted on the double bass “Panormus”, property of Conservatorio di Musica “Vincenzo Bellini” in Palermo. The
most important components of the varnish (fatty acids) and of the glue (proteinaceous amino acids), with which the musical instrument was treated
in the past, are determined. The analyses are carried out by prior derivatization of fatty acids by acidic methanol and of amino acids by acidic
methanol and trifluoroacetic anhydride (TFAA). Analytes identification is achieved by direct comparison with several reference materials and the
use of a digitized library.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Double bass; Art analysis; Derivatization; GC; Binding media; Varnish; Fatty acids; Amino acids; Resins

1. Introduction
A functional or conservative restoration work, well-designed
and -executed, should slacken the deterioration process of an
art object. Wooden artworks, although well preserved, suffer
from several kinds of deterioration attacks which are mainly
from biological origin. Physical and chemical agents which con-
tribute are moisture, temperature fluctuations, light, atmospheric
pollutants, etc.
Wood restoration techniques vary from work to work and
particular attention is needed for the handling of painted or
varnished material.
After a prior eventual disinfestation from insects or parasites,
preferably in a non-toxic gas chamber [1,2], and a surface clean-
ing, the pure restoration consists in the substitution of parts of
the ancient object trying to use the same kind of wood (ancient
or artificially aged, if possible) and the same kind of varnishes
and glues [3].
∗ Corresponding author. Tel.: +39 091 6809366; fax: +39 091 6809399.
E-mail address: caruso@pa.ismn.cnr.it (F. Caruso).
Usually, the restorer has to deal with two aspects of the var-
nish and glue problem [4]: what kind of varnish and glue were
originally used and which ones among the present products are
suited for the restoration work.
It is almost certain that a direct relationship exists between
varnish composition and quality of sound. Therefore, the makers
of stringed instruments, while trying to replicate lightness and
transparency effects and brightness of sounds, wanted to imitate
ancient masters and discover their secrets.
A comparison between ancient stringed instruments from
Cremona (Italy) and modern ones confirmed the existence of
differences in the aforementioned quality of sound and in the
timbre. The same difference was established between some
Stradivari’s undamaged instruments and other ones from which
the varnish was entirely removed (by scalpel or washing) and
re-varnished with non-original different products [5]. The var-
nish contribution to the quality of sound plays such an important
role that is able, in some cases, to compensate possible struc-
tural defects (i.e. the instruments made from Guarneri del Gesù
compared to Stradivari’s) [5].
Coloured varnishes used by the makers of stringed instru-
ments from the 17th to the 19th century can be roughly classified

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.02.048
 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212
Fig. 1. The ancient 18th century Vincenzo Trusiano’s double bass (a) and the
little label on the bottom of the instrument’s case (b).
as follows: (i) drying oils (linseed oil, walnut oil, poppy-seed
oil, etc.) based; (ii) essential oils (oil of turpentine, lavender oil,
etc.) based; (iii) alcohol based.
These varnishes, as demonstrated by elemental studies
involving X-ray fluorescence [6,7] and combined analyti-
cal techniques (optical and electronic microscopy, energy-
dispersive X-ray analysis, Raman) [8], often contained inorganic
dyes as aluminium coordinated with alizarin (extracted from
madder), iron or lead oxides [9], arsenic or mercury sulphides,
etc. [5].
It is, however, more opportune to distinguish the different
varnishes on the basis of the binding medium1 which can be
natural (exclusively used up to 30’s and 40’s) or artificial. Natu-
ral binding media are classified as follows [4]: (i) resinous; (ii)
proteinaceous; (iii) Siccative oil based; (iv) polysaccharides.
However, lute makers usually employed resinous and/or
siccative oil based varnishes [5] even if they often used to com-
bine different media for some applications.
The subject of this work is an investigation on an ancient
exquisite 18th century double bass made by Vincenzo Trusiano
(Fig. 1a).
A little label on the bottom of the case on which “Vin-
centius Trusiano fecit Panormi 1752” is written (Fig. 1b), is
a proof that the instrument is a wonderful hand made artefact
of the famous 18th century artisan from Monreale (Palermo,
Italy).
The aim of this paper is to describe a fast procedure to deter-
mine the main fatty acids contained in small varnish samples
and the proteinaceous amino acids in small glue samples by
gas chromatography–mass spectrometry, in order to understand
some relevant aspects of the ancient Trusiano’s varnish recipe
and to give precious information for the double bass restoration
treatment. As a matter of fact, a GC system, coupled with the use
of a capillary column and a MS detection, permits a high reliable
1 A binding medium is a compound present in every kind of varnish. Ideally,
its features should be the following:
• Functional (fixing dyes to the substrate);
• Aesthetic (chromatic power, lightness, etc.);
• Protective.
These binding medium’s characteristics are fundamental for the production of
a good paint [4].
207
separation and identification of these analytes if appropriately
derivatized.
Analytical derivatization of fatty acids has been extensively
treated in literature [10]. Esterification with diazomethane (DM)
reagent is still regarded as a useful and clean technique although
it has some negative aspects: gas explosive reagent to be prepared
in designed apparatus, toxic synthesis precursors and possible
reaction of the diazo group with double bonds. Trimethylsilyl-
diazomethane (TMSDM), although not recommended because
of slower reaction rates and lower derivatives yields than DM,
is a possible substitute of DM [10].
Alkyl chloroformates as general purpose derivatization
reagents in gas chromatography were studied by Hušek [11]. The
reaction of methyl (MCF) and ethyl (ECF) chloroformates with
fatty acids is quantitative (yield >98%), instantaneous and runs
at room temperature although the choice of solvent is critical
to avoid the presence of secondary reaction products. A simi-
lar approach is intended for amino acids though arginine and
citrulline are not fully derivatized. Ethyl chloroformate were
employed to identify proteinaceous and oil binding media in
paintings by Mateo Castro et al. [12,13]. Paik et al. [14] proposed
the alkylation of carboxylic group under alkaline (K2 CO3 ) and
hot (60 ◦ C) conditions by the use of bromoacetonitrile (BrACN).
The esters are sufficiently volatile so that even long chain fatty
acids can be determined by GC.
Hydrolysed fatty acids can be effectively converted
to N(O)-ter-butyldimethylsilyl esters with N-methyl-N-(ter-
butyldimethylsilyl) trifluoroacetamide (MTBSTFA) after an
alkaline hydrolysis with methanolic tetramethylammonium
hydroxide (TMAH) [15]. Similarly, hydrolysed amino acids
from proteinaceous binders of paintings can be effec-
tively derivatized by the use of MTBSTFA in pyri-
dine [16,17]. Recently, tubes of paint from the Arnold
Schönberg Center (Vienna) have been investigated using
GC–MS [18] and the corresponding fatty acids identified
using (m-trifluoromethylphenyl) trimethylammonium hydrox-
ide (TFTMAH), a methylation agent which is claimed to be
superior to TMAH although it does not make the esterification
of the hydroxy-group in hydroxy fatty acids occur. A good sepa-
ration of saturated fatty acids with a chain length between C12:0
and C26:0 was obtained by the use of flophemesyl chloride as
silylating derivatizing agent [19]. A method for the simultane-
ous analysis of fatty acids and glycerol in drying oil in artworks
using trimethylsulfonium hydroxide (TMSH) was described by
Rosenberg and coworkers [20].
Other typical alkylation methods for fatty acids derivatization
involve the use of short chain alcohols (e.g. methanol, ethanol,
isopropanol) with acidic or basic catalysts (as BF3 [21,22] or
sodium methoxide [23]).
Concerning protein analysis, a new proteomics methodol-
ogy has been able to identify proteins in microsamples from
two ancient Renaissance paintings (14th and 15th century).
This methods consists of an extraction with 1% aqueous tri-
fluoroacetic acid (measured efficiency by MALDI-TOF mass
spectrometry), an enzymatic hydrolysis with trypsin and the
analysis by nano-LC/nano-ESI/Q-q-TOF MS/MS using opti-
mized parameters [24].
208 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212
A novel GC–MS technique, combining different kinds of
derivatization, for the identification of lipids, proteins and other
analytes from painted artworks has been proposed by Colombini
and coworkers [25].
In the present work, we report on a procedure for the anal-
ysis of fatty and amino acids’ GC–MS profile identification,
based: (i) on the extraction of fatty acids with methanol and
derivatization with acidic methanol; (ii) on proteins hydrolysis
with aqueous HCl and a double step amino acids’ derivatization
with acidic methanol and TFAA. Our experimental procedures
are based on the choice of common and low-cost derivatiz-
ing reagents and solvents. Furthermore, our procedures can be
adopted by restoration and conservation laboratories to charac-
terize lipid binders and proteinaceous glues in artworks.
The whole procedures have been validated by trials on several
reference materials and by the direct comparison with proto-
cols available in literature regarding other kinds of artefacts
[12,16–18,20,26–32].
2. Experimental
2.1. Sampling
Seven samples of varnish and two samples of glue were taken
from the ancient artefact. Samples were taken by gently scraping
the surface of the instrument with a scalpel at nine points, thought
to be the most representative ones.
Samples collection is described in Table 1.
2.2. Experimental procedure for the analysis of fatty acids
Trials of fatty acids analyses and subsequent rigorous anal-
yses were carried out on several reference materials: three
siccative oils (linseed, boiled linseed and walnut) and seven dif-
ferent kinds of resins (Manila copal, Kios mastic tears, sandarac,
orange shellac, lemon shellac, white shellac and Kusmi shellac).
Afterwards, the seven varnish samples from the double bass were
analysed.
2.2.1. Extraction
For every solid reference material, a solubility test with
three different solvents (acetone (polar aprotic solvent), chlo-
roform (apolar solvent) and methanol (polar protic solvent))
was conducted for one week. Initial approximate visual anal-
Table 1
Sampling description and weight of the analyzed samples
Sample Sampling description
Sample 1 Varnish sample from the intern left f-hole.
Sample 2 Taken from intern right f-hole.
Sample 3 Taken from intern upper-right f-hole.
Sample 4 Taken from intern left f-hole
Sample 5 Taken from the bottom of the instrument.
Sample 6 Taken from the right edge of the f-hole.
Sample 7 Taken from the right edge of the harmonic case and from the bottom.
Sample 8 Glue taken from the intern left side of the harmonic case.
Sample 9 Glue taken from the intern bottom of the harmonic case.
ysis showed that in methanol solutions more quantity of every
material was dissolved (excluding Kios mastic tears and Manila
copal). Results of GC–MS analyses confirmed what stated
above.
The double bass varnish samples were treated with 0.50 mL
of methanol and left in a screwed cap tube in a dark and dry
place for about two weeks at room temperature.
2.2.2. Treatment
Every suspension of reference material was filtered through a
small column of anhydrous sodium sulphate to eliminate water
and solid residues. The column was previously packed with the
relative solvent. The filtered solutions were dried in a water
bath at 50–60 ◦ C under a gentle nitrogen stream. The resulting
residue was treated with 2.00 mL of hexane containing hexade-
cane (internal standard) at 20 ␮L/L. Subsequently, 2.00 mL of
about 1:5 (v/v) HCl in methanol were added to each tube to
produce a biphasic system which was heated in a 50–60 ◦ C
water bath for approximately 2 h. After cooling to room tem-
perature, the hydrocarbon phase was separated and filtered on
a small column of anhydrous sodium sulphate – again to elimi-
nate water and possible solid residues – previously packed with
hexane with hexadecane. Solutions were then injected into the
GC–MS.
Regarding the three oils, 50 ␮L of each were diluted with
2.00 mL of hexane with hexadecane, treated with 2.00 mL of
the aforementioned methylating reagent, esterified (or transes-
terified) and cooled. The hydrocarbon phase was then separated
and filtered.
The double bass samples were treated like the solid reference
materials.
2.3. Experimental procedure for the analysis of amino acids
Two GC–MS analyses were carried on each glue sample
taken as references: rabbit glue and bone glue (20 mg and
28 mg, respectively). Eight pure amino acids were used as
standards: aspartic acid, glutamic acid, phenylalanine, glycine,
4-hydroxyproline, proline, serine, valine, to allow identification
of these in test samples.
2.3.1. Hydrolysis
Following indications from the literature [33,34], each sam-
ple of modern and double bass glue was transferred into a 50 mL
Sample ID Mass of analyzed sample (mg)
VCB 1 0.7
VCB 2 0.4
VCB 3 0.7
VCB 4 5.1
VCB 5 2.8
VCB 6 1.8
VCB 7 2.3
CCB 8 12.1
CCB 9 49.7
 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212
round bottom flask, treated with about 5 mL of HCl 1:1 (v/v) in
water and refluxed for 24 h into a hot oil bath (100–110 ◦ C). Pure
amino acids were only treated with HCl 1:1.
2.3.2. Treatment
After quantitative recovery into a screwed tube, each sample
(pure amino acids included) was slowly dried in a water bath
at about 80 ◦ C under a gentle nitrogen stream. The dry residue
was treated with 2.00 mL of the methylating reagent used for
fatty acids derivatization. The suspension was heated in a water
bath at about 50–60 ◦ C for about 2 h. The suspension was sub-
sequently dried in a water bath at about 80 ◦ C and under a gentle
nitrogen stream. 2.00 mL of hexane with hexadecane (internal
standard) at 20 ␮L /L were added and the new suspension treated
with 200–250 ␮L of TFAA. The tube was screwed and heated
in a water bath at 50–60 ◦ C for about 1 h. The suspension was
again dried on a water bath at about 80 ◦ C under a gentle nitro-
gen stream, treated with 2.00 mL of hexane with hexadecane,
cooled to room temperature and filtered through a little col-
umn of anhydrous sodium sulphate to eliminate water and solid
residues, previously packed with hexane with hexadecane. Dou-
ble bass glue suspensions were filtered twice (with two different
columns) because the solutions filtered just once still showed
some turbidity. The solutions were then ready for the GC–MS
injection.
2.4. GC–MS instrumental conditions
Fatty and amino acids analyses were performed by a Shi-
madzu GC 5000 coupled with a quadrupole mass spectrometer
Shimadzu MS 5000 (Shimadzu Corporation, Kyoto, Japan). The
injector and interface temperatures were both 280 ◦ C. GC sep-
aration was achieved on a Supelco Equity-5 (30 m × 0.25 mm
i.d., 0.5 ␮m film thickness) fused-silica capillary column. The
following temperature program was set up: isothermal condi-
tions at 40 ◦ C for 2 min up to the final temperature of 250 ◦ C with
a rate increase of 11.5 ◦ C/min. Helium (85,7 kPa, inlet pressure)
was the carrier gas and its flow rate was 1.5 mL/min. Mass spec-
trometer conditions were as follows: 45.00–350.00, m/z range
of analysis; 1000 u/s, scan speed; 5.00 min, solvent cut; 1,80 kV,
detector potential. The injection volume was 1.00 ␮L and the
injection mode was splitless (sampling time = 0.61 min). Anal-
ysis was conducted in scan mode.
2.5. Materials
The fatty acids methyl esters solution (Supelco 37 Com-
ponent FAME Mix), used as main reference, was purchased
from Supelco (Supelco, Sigma–Aldrich Corporation (St. Louis,
MO, USA)). The internal standard was hexadecane (puriss.
p.a., standard for GC, ≥99.8%), purchased from Fluka (Fluka,
Sigma-Aldrich Corporation (St. Louis, MO, USA)).
As reference materials for fatty acids analysis, the seven
resins and the three oils were provided by CRPR. As refer-
ence materials for amino acids analysis, eight pure amino acids
(99%) were purchased from Carlo Erba (Milano, Italy) and
from Sigma–Aldrich; the two different kinds of glue were also
209
provided by CRPR. Reference materials were chosen on the
basis of historical sources of old violins varnish composition
[5].
3. Results and discussion
3.1. Fatty acids determination in varnish samples
Fatty acids derivatization involves the mineral acid-catalysed
esterification of the carboxylic groups by methanol.
Total ion chromatogram (TIC) of the VCB 6 sample is shown
in Fig. 2a.
A direct comparison of the peaks retention times of the TIC of
the Supelco 37 Component FAME Mix, the use of the digitized
library of the instrument (NIST 62 Library) and the interpretation
of the corresponding mass spectra of each eluted component
allow the identification of fatty acids derivates peaks. Identified
fatty acids in samples VCB 1, VCB 2, VCB 3, VCB 4, VCB 6
and VCB 7 are caproic, heptanoic, caprylic, pelargonic, capric,
lauric, myristic, pentadecanoic, palmitic, heptadecanoic, stearic
and azelaic.
Azelaic (nonanedieoic) acid is an important indicator of the
use of a siccative oil. In fact, the siccative power of a vegetable
oil is strictly connected to the presence of unsaturated fatty acids.
In a good siccative oil, their weight concentration should be at
least 65%. The hardening of linseed oil is due to a series of
slow oxidation reactions of fragmentation and of cross-linking
of unsaturated fatty acids and that the length of the chain of low
molecular weight oxidative products depend on the position of
the unsaturation [27,35–37].
As a matter of fact, it is not correct to compare chromatograms
of the original varnish with ones of modern varnishes because
of the aforementioned modification processes. Instead, it is rea-
sonable to consider the ratio between the areas of peaks relative
to palmitic acid methyl ester and to stearic acid methyl ester
(P/S). This ratio permits to recognize the kind of siccative oil
[38,39,27]. P/S ratio ranges for the most common siccative oils
are: 1.2–2.4 for linseed oil (our measured value is 1.4); 2.2–3.3
for walnut oil (our measured value is 2.8); 7.2–8.0 for poppyseed
oil.
In Table 2, P/S ratios for double bass varnish samples are
reported.
The mean value – and the comparison with the experimental
value of the reference sample (P/S ratio equal to 2.8) – evidences
the use of walnut oil as a component of the varnish binding
medium. Walnut oil is employed in oil paint, especially in wood
manufacture, as effective binding medium. This oil – although
less common than linseed oil – is regarded as a good paint thin-
ner for its quickness of drying, lack of yellow tint and non-
toxicity.
Sample VCB 5 chromatogram is shown in Fig. 2b. A high
concentration of unsaturated fatty acids (presence of palmi-
toleic, oleic and linolenic acids) is found and a P/S ratio equal
to 1.52, in the ratio range of linseed oil. This evidence con-
firms our first visual hypothesis which stated that this sample
had been taken from a reconstruction on the bottom of the inst-
rument.
210 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212

Fig. 2. TIC of methanol extracted and derivatized solution of VCB 6 sample (a); TIC of methanol extracted and derivatized solution of VCB 5 sample (the one from
the restored bottom of the instrument) (b). The internal standard (hexadecane) is labelled as “Rif. C16H34”.

In order to further confirm this evidence, the ratio between unsaturated fatty acids in the same siccative oil and, as a conse-
the areas of peaks relative to azelaic acid dimethyl ester and quence, the contemporaneity of different samples [16].
to palmitic acid methyl ester (Azel/P) is considered. This Azel/P ratios for double bass varnish samples are also
ratio allows to establish the oxidative degradation degree of reported in Table 2.

Table 2
Experimental P/S and Azel/P ratios of the double bass varnish microsamples 3.2. Proteinaceous amino acids determination in glue
and arithmetic means of the values samples
Sample Experimental P/S ratio Experimental Azel/P ratio
Amino acids derivatization involves, apart from the esterifica-
VCB 1 2.76 ± 0.01 0.31 ± 0.01
VCB 2 3.34 ± 0.01 0.31 ± 0.01
tion of the carboxylic groups by acid methanol, the quantitative
VCB 3 3.43 ± 0.01 0.35 ± 0.01 acetylation of the amino groups with TFAA.
VCB 4 2.88 ± 0.01 0.34 ± 0.01 The two TICs of the glue double bass samples are shown in
VCB 6 3.40 ± 0.01 0.32 ± 0.01 Fig. 3a and b.
VCB 7 2.81 ± 0.01 0.36 ± 0.01 The identification of amino acids derivates peaks is achieved
Mean 3.1 ± 0.3 0.33 ± 0.02
VCB 5 1.52 ± 0.01 0.08 ± 0.01
by the direct comparison of retention times of the chro-
matograms peaks and by mass spectra of the eight amino acids
 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212 211

Fig. 3. TIC of hydrolyzed and derivatized solution of CCB 8 sample (a); TIC of hydrolyzed and derivatized solution of CCB 9 sample (b); TIC of hydrolyzed and
derivatized solution of bone glue sample (c); TIC of hydrolyzed and derivatized solution of rabbit glue sample (d). The internal standard (hexadecane) is labelled as
“Rif. C16H34”.

used as references. The amino acids found in the analyzed sam-
ples are listed in Table 3.
A mass fragment at 69 m/z is present in each mass spec-
trum of the considered derivatized amino acids. This fragment is
assigned to the formation of the CF3 + cation in agreement with
literature [40].
The relative amino acids percentage composition of the two
double bass glue samples is obtained by integration of the peaks
areas (Table 3).
Table 3
Relative amino acids weight percentage for double bass glue samples CCB 8
and CCB 9 and for modern bone glue (BG) and rabbit glue (RG) samples
Amino acid Relative weight percentage
Both double bass glue samples (CCB 8 and CCB 9) show
high relative concentration in glycine (Gly), proline (Pro) and
4-hydroxyproline (Pro-OH). It is well known that an abundance
of these three amino acids reveals the presence of collagen-like
proteins amounting an animal glue [41]. In fact, the presence
of hydroxyproline, an amino acid very rarely found in other
proteins, prevents collagens from an early folding during biosyn-
thetic process [42].
The results of the analysis of the two available samples of
modern animal glue used in wooden artworks restoration (bone
glue and rabbit glue) confirms the presence of collagen deriving
amino acids (Fig. 3c and d and Table 3).
4. Conclusions

CCB 8 CCB 9 BG RG The use of the little destructive (in terms of sample mass)
Gly 21 32 15 17 procedure permits the identification of fatty and amino acids
Val – 5 2 1 in samples of varnish and glue of the 18th century double bass
Ser 7 – 3 3 Panormus by Vincenzo Trusiano and gives useful information
Asp 3 6 6 4
for restorer, historians of art and luthiers, in good agreement with
Pro 35 23 19 20
Pro-OH 19 15 21 22 historical references about the construction of ancient musical
Glu 4 10 18 18 instruments. Furthermore, data analyses shed some relevant light
Phe 11 9 17 14 on the ancient formulations (the workshop secrets) of varnishes
In sample CCB 8, valine derivate peak area was not detected while, in CCB 9, and glues of the makers of stringed instruments. In particular, the
serine derivate peak area was not detected. Amino acids are ordered according use of walnut oil as a component of the binding medium for this
to their elution times. ancient double bass varnish is detected; a previous restoration
212 F. Caruso et al. / J. Chromatogr. A 1147 (2007) 206–212
treatment on the bottom of the ancient instrument is confirmed
by the experimental data; the use of animal glue as adhesive in
the construction of the instrument is detected.
Acknowledgements
The authors wish to thank the Conservatorio di Musica “Vin-
cenzo Bellini” (Palermo, Italy) for providing the ancient musical
instrument and the whole staff of CRPR (in particular, Mr. Gio-
vanni Garofalo) for the technical support in the sampling and
working operations on the musical instrument.
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