Microchemical Journal 127 (2016) 102–112
Contents lists available at ScienceDirect
Microchemical Journal
j ourna l homepage: www.e lsev ie r .com/ locate /mic rocCIM® monolith chromatography-enhanced ELISA detection of proteins
in artists' paints: Ovalbumin as a case studyTanja Špec a,⁎, Sebastijan Peljhan b, Jana Vidič b, Nika Lendero Krajnc b, Marko Fonović c,d,
Črtomir Tavzes a, Polonca Ropret a,e
a Research Institute, Conservation Centre, Institute for the Protection of Cultural Heritage of Slovenia, Poljanska 40, 1000 Ljubljana, Slovenia
b BIA Separations, Mirce 21, 5270 Ajdovščina, Slovenia
c Jožef Stefan Institute, Department of Biochemistry and Molecular Biology, Jamova 39, 1000 Ljubljana, Slovenia
d Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, SI-1000 Ljubljana, Slovenia
e Museum Conservation Institute, Smithsonian Institution, 4210 Silver Hill Rd., Suitland, MD 20746, USA⁎ Corresponding author at: Research Institute, Conser
Protection of Cultural Heritage of Slovenia, Poljanska 40, S
E-mail address: tanja.spec@zvkds.si (T. Špec).
http://dx.doi.org/10.1016/j.microc.2016.02.004
0026-265X/© 2016 Elsevier B.V. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:
Received 17 December 2015
Accepted 4 February 2016
Available online 13 February 2016ELISA (Enzyme-linked ImmunoSorbent Assay), as one possible application of antibody-based methods, has
proved to be a good candidate for the determination of proteins in works of art due to the low limit of detection,
high sensitivity (in the nanogram range), specificity and micro invasiveness of required sampling. Despite the
advantages of this method, interference by metal ions present in pigments, lipids, saccharides and in other
components of paints, grounds, and varnishes may cause several problems for incontestable identification of
proteins. To overcome the drawbacks of ELISA, a novel way to purify the extracted mixtures to predominantly
protein fractions, by the DEAE-modified (weak anion exchange) CIM® (Convective Interaction Media)
chromatographic monoliths was introduced. Utilisation of monolithic chromatographic supports greatly
enhanced the ELISA detection of ovalbumin in the model paint samples (aged and non-aged), for at least
one tenfold dilution of a sample. In several cases the purification enabled detection of the target protein,
otherwise not possible with non-purified samples. For the first time in the field of Cultural Heritage, the
combined approach of ELISA andCIM®monolith-supported extractionwas successfully tested also for the detection
of protein in real-case paint layers.
© 2016 Elsevier B.V. All rights reserved.Keywords:
Paints
Ovalbumin
CIM® chromatographic monoliths
Chromatography protein purification
ELISA1. Introduction
Amongmaterials present in art objects, polymer organic compounds
(e.g. proteins) were used as adhesives, protective coatings, and especially
as bindingmedia for colouringmaterials [1,2]. An investigation of proteins
used for such purpose can be challenging due to their low concentration
present in solid matrix (paints), co-presence of different organic sub-
stances (complexmixtures, derived from different animal/plant sources),
pre-existing conservation treatments, etc. Furthermore, their propensity
to undergo alterations during ageing (fragmentation, alteration of
amino acids such as oxidation, deamination, hydrolysis, and biodeteriora-
tion) represents additional difficulty in their specific identification in dif-
ferent works of art. These processes can be accelerated by environmental
conditions (heat, excessive light exposure, variations in humidity, etc.) in
which particular cultural heritage objects can be stored [3–7].
When investigating proteins, such as casein (milk), ovalbumin (egg
white), or collagen (bone, skin), which are themost abundant in the artvation Centre, Institute for the
I-1000 Ljubljana, Slovenia.materials, classical instrumental methods, such as FTIR, py-GC, GC–MS,
HPLC [8–15] can therefore stand out not to be enough specific or can
give ambiguous results due to the sample size, the sample mixture,
protein degradation, as well as the influence of omnipresent micro-
organisms.
In scientific analysis ofworks of art, there is constant focus on getting
as much information as possible either non-invasively or by taking the
smallest possible representative sample, therefore the analytical
techniques advance in both sensitivity and specificity. For that reason,
the implementation of immunological analytical techniques, such as
ELISA [16–22] or Immunofluorescence Microscopy (IFM) [16,23–25]
shows the potential to become essential in the determination of
proteins in works of art. They offer high specificity of the interaction
between the antibody and antigen, which enables distinction between
different types of proteins, as well as lowering the limit of detection,
and increasing its sensitivity and accuracy. In addition, immunological
methods are faster, easier, and do not require large amounts of sample
and expensive instrumentation.
However, there are still several issues with the ELISA identification
of proteins in art and cultural heritage objects. The obstacles, challenges,
and someof the possible avenues of improvementhave been extensively
Fig. 1. Schematic presentation of themodel paint layers, prepared in left: egg yolk binder;
right: eggwhite binder. Both paint layerswere a.) not coated or coatedwith b.) eggwhite,
c.) egg white and mastic. This pattern was repeated for all five selected pigment-binder
systems.
103T. Špec et al. / Microchemical Journal 127 (2016) 102–112and comprehensively reviewed by the group from the Metropolitan
Museum of Art [17]. Protein modifications such as cross linking,
hydrolysis and oxidation can be catalysed by metal ions present in
some pigments [21]. These ions can also change the protein structure
and affect the recognition of a target protein by an antibody. Additionally,
interference by lipids and saccharides, present as components of paints,
grounds and varnishes may cause several problems for incontestable
identification of proteins. Several improvements in the technique
(extraction reagents, micro-titre plates, and reporting enzyme selection
optimization) were proposed and implemented with various degrees of
success; however the influence of above-mentioned impurities remains
a factor of concern [18–21]. To improve ELISA-based detection of proteins
in heterogeneous matrices, such as paint layers, purification by chroma-
tography was proposed as one of the possible alternatives.
In recent years, porous monoliths were used as sorbents in sample
preparation and provided impressive results [26]. Convective Interaction
Media CIM® chromatographic monoliths are porous methacrylate-
based polymers composed of highly interconnected channels, and further
modificationof themonolithmatrix surface enables toproducemonoliths
with different functional groups. The structure of CIM® chromatographic
monoliths enables fast and flow independent operation, high chromato-
graphic resolution, as well as high dynamic binding capacities for macro-
molecules [27–29]. Moreover, because of straightforward preparation of
themonolith in any size or shape (in-mould polymerisation) this support
is very convenient for preparation of microscale devices, such as pipette
tips or similar. That makes CIM® chromatographic monoliths a preferred
choice for solid phase extraction and purification of binder proteins from
milligramme-range amounts of samples taken from art objects.
For the first time in the field of Cultural Heritage, advantages of both,
ELISA and CIM® monolith-supported extraction were synergistically
employed for significantly improved detection of ovalbumin in artists'
paints in this investigation of model and real-case paint layers.
2. Materials and methods
2.1. Chemicals
The extraction of protein fraction from bulk samples was performed
by using Tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) (Fluka),
EDTA (Sigma-Aldrich), urea (Sigma-Aldrich), sodium dodecyl sulphate
(SDS) (Sigma-Aldrich) and NaHCO3 (Merck, p.a.). The samples purified
by CIM® chromatographic monoliths were extracted in NaHCO3
(Merck) and chromatographically purified using Tris–HCl (Sigma-
Aldrich) and (NH4)HCO3 (Alfa Aesar) buffers.
Anti-chicken egg albumin antibody produced in rabbit (Sigma-
Aldrich, C6534) as primary polyclonal antibody and anti-rabbit IgG
(whole molecule) alkaline phosphatase-conjugated antibody
produced in goat (Sigma-Aldrich, A0418) as secondary antibody
were used in the ELISA procedure. Alkaline phosphatase yellow
p-NPP (Sigma-Aldrich, P7998) was employed as the reporting dye
for colorimetrical detection (O.D. at 405 nm) of the immune-complex.
Antibodies were diluted in 5% new born calf serum (Sigma-Aldrich),
which served also as a blocking reagent. During the ELISA procedure,
several washing steps were performed, using 0.05% Tween-20 in
phosphate buffer saline (PBS), both purchased from Sigma-Aldrich.
Calibration curve of standard reference protein (ovalbumin) was
prepared with albumin, from chicken egg white (Sigma-Aldrich,
A5503-1G). All listed chemicals were used without further purification.
2.2. Model painting preparation
Canvas (30 × 30 cm of effective surface area) was stretched to a
wooden sub-frame and treated with a hot rabbit glue solution. Subse-
quently, white gesso ground was applied, dried, and the surface was
polished (this application procedure was repeated three times).For the preparation of paint layers on model paintings, egg tempera
was employed in separate squares (6 × 6 cm) on the gesso ground
in two different variations regarding the binders used. For the first
mock-up series, the selected pigments were bound in egg yolk exclu-
sively, while for the second the same pigments and egg white binder
were used. Egg yolk tempera was derived from whole hen's egg
(purchased at the local market) which was mixed with the same
volume of water, and a drop of apple cider vinegar. Egg white binder
was prepared the same way as glair (see below). Each square (paint
layer) was divided into three sections, depending on the used
finishing protective layer/varnish. First division was left uncoated,
the second was coated by glair only, and the third by a combination
of glair and mastic. A schematic diagram of paint and varnish layers
is provided in Fig. 1. To prepare glair varnish, the egg white was
separated from the yolk and beaten, until foam was formed. After
overnight incubation (2–8 °C) in the fridge, the foam was separated
from the liquid part, to which 2 g of sugar per one egg was added [2].
Gummi Mastix was purchased from Kremer Chemicals Nr. 60050
and mixed with Fir turpentine, purchased from Kremer Chemicals Nr.
70010 (1:3 ratio).
2.3. Ageing protocol
One set of the preparedmodel paintingswas exposed to accelerated,
artificial ageing in climatic chambers with well-defined and controlled
temperature, relative humidity, and light conditions. Lightfastness was
carried out according to ASTM D 4303-03 standard: “Test Methods for
Lightfastness of Colorants Used in Artists Materials”, with appropriate
modifications. For the simulation of UV–Vis radiation, metal halide
lamp (irradiance 100 W/m2) was used, equipped with window glass
filters, which is a good simulation of the sunlight. 30 days of light expo-
sure was followed by another 30 days exposure in climatic chamber,
with oscillations of temperature and relative humidity (from 0 °C to
50 °C and from 20% to 90%, respectively). One set of model painting
was left non-aged and served as a control.
2.4. Real samples
In order to test the reliability of the novel purification system and
its further application to the ELISA method, two micro-samples
(approximately 1 mg, extracted by scalpel) from real artworks were
investigated: a paint layer from 16th Century painting of Vittore
Carpaccio, Presentation at the Temple (Fig. 10 — left), and a paint
layer of the 17th century painting from Pietro Liberi, Saint Nicholas
between Saint Hermagoras and Saint Fortunatus (Fig. 10 — right).
Fig. 2.Monolith support inside plastic pipette tip.
104 T. Špec et al. / Microchemical Journal 127 (2016) 102–1122.5. Instrumentation
2.5.1. ELISA
ELISA test was performed using micro-titre plates (NUNC-Immuno™
96 MicroWell™ Plates, MaxiSorp™; Thermo Scientific). Sample absor-
bance at λ = 405 nm, expressed as optical density (OD405 nm), was
measured by Synergy H1, BioTek Instruments, USA.
2.5.2. FTIR
FTIR analyses of samples were carried out with a Perkin Elmer
Spectrum 100 FTIR spectrophotometer coupled to a Spotlight FTIR
microscope equipped with nitrogen-cooled mercury-cadmium telluride
(MCT) detector. The spectrawere collected in a transmissionmode as 64
scans in the range between 4000 and 600 cm−1, at 4 cm−1 spectral
resolution using a diamond anvil cell.
2.5.3. Monolith columns
A single use CIM® diethylaminoethyl (DEAE) weak anion exchange
custom prepared 4 μL monolithic columns packed into plastic micropi-
pette tips (monolith tip)were obtained fromBIA Separations (Ajdovščina,
Slovenia). Compared to C18 hydrophobic columns that are being used for
peptide (shorter fragments of proteins) extraction, and a research done
by Lee et al. [22] where C4 hydrophobic column was used for depletion
of metal ions from extracted materials, DEAE column not only allows
usage of completely inorganic solvent/buffer components, but also
enables to purify very large peptides and intact proteins, which are
necessary for successful determination of target proteins by ELISA.
Additionally, monoliths yield great recovery of large biomolecules,
which makes them exceptionally suitable for analysis of sub-
milligramme amounts of samples [30,31], and can bind multifold
higher amounts of extracted protein (80 μg of protein per monolith
tip used in this study). Furthermore, to enable greater experimental
flexibility, solvent buffer system in this study was chosen to match
the requirements of both, ELISA and LC–MS/MS techniques simulta-
neously, without any sample post-processing. Lastly, monoliths are
highly porousmaterialswith rigid structure andwell definedpore radius
(monoliths with 1.5 μm pore diameter were used in this study) so they
can serve as filters, which minimises sample preparation and handling.
2.6. Protocols
2.6.1. Sample protein extraction
Each of the binders was mixed with 5 different pigments and inves-
tigated in different combinations of varnish, ageing (see Section 2.2 and
Fig. 1) and purification pre-analysis, for a total of 24 combinations for
each paint layer and 120 in total.
Approximately 1mg samples of each of the 24model paints/varnish/
ageing combinations were collected from the canvas and, before the
application onto ELISA microtitre plate, extracted by the following
steps:
∙For the direct determination of proteins by ELISA (12 of the 24 sam-
ples), the extraction was done according to slightly modified procedure
by Heginbotham et al. [16] 1 mg of the sample taken from canvas
painting was diluted in a 3.33 ml buffer (10 mM Tris–HCl, 1 mM
EDTA, 6 M urea, 0,1% SDS), mixed well on a vortex and incubated at
37 °C for 5 h. Afterwards, 6.67 ml of 100 mM NaHCO3, pH 9.6, was
added to the extraction buffer, maintaining 2:1 ratio.
∙For the series of samples (the other 12 of 24), which were chro-
matographically purified and concentrated before ELISA procedure,
1 mg of model paint each was suspended in 100 μL of 0.1 M NaHCO3.
The suspensionwas alternately treated by ultrasonic bath to aid dissolu-
tion and vigorously shaken on a vortex mixer for 1 h. The suspension
was centrifuged 1 min at 11,000 g to remove any possible undissolved
material, and approximately 90 μL of supernatant was loaded onto
previously equilibrated monolith-equipped tip (Fig. 2).2.6.2. Purification by CIM® chromatographic monoliths
CIM® chromatographic monolith tips were equilibrated by washing
with 100 μL deionised water followed by washing with 50 μL of 20 mM
Tris buffer, pH 7.4, 50 μL of 20 mM Tris +2 M ammonium carbonate
buffer, pH 7.4, and finally again with 50 μL of 20 mM Tris buffer,
pH 7.4. The 90 μL of sample (Section 2.6.1) was loaded onto the tip, ∙
washed with 50 μL of 20 mM Tris buffer, pH 7.4, and the proteins
were finally eluted with 50 μL of 20 mM TRIS +2 M ammonium
carbonate buffer, pH 7.4. Each washing/loading/eluting step required
10 min of centrifugation at 1700 g.
2.6.3. ELISA protocol
The ELISA protocol was also adopted from Heginbotham et al. [16],
and used with minor modification.
The purified samples (Section 2.6.1, followed by Section 2.6.2) were
re-suspended with bicarbonate buffer (100 mM NaHCO3, pH 9.6) to
achieve the same theoretical concentration of the total protein in
purified and non-purified samples (Section 2.6.1, first bullet point).
The Bradford Ultra protein assay [32] was performed to establish a
“perceived” actual total protein concentration of each sample (purified
and non-purified). As this and other protein quantification tests are
influenced by impurities that are regularly found in paint layers
(model or real), this test was not used as an absolute measure in this
study (therefore not reported and data not shown), but more as a
guide or check-up tool to verify that the total protein concentrations
were within the same order of magnitude for all purified and non-
purified samples' pairs. Each of the samples was further diluted in four
steps (10-fold dilutions), all the dilutions were applied onto microtitre
plates (each dilution into three wells (triplicates); application in
triplicates was used also for the calibration curve, and all the control
samples), and incubated overnight (2–8 °C). Afterwards, 300 μl of 5%
calf serum albumin (CSA) blocking solution was added to prevent
unspecific binding of antibodies in the further steps of the procedure.
The indirect ELISA method was performed by using rabbit anti-
ovalbumin primary polyclonal antibodies (100 μl, 1 h incubation at
room temperature), followed by the addition of alkaline phosphatase-
conjugated (AP) goat-anti-rabbit secondary antibody (100 μl, 1 h incuba-
tion at room temperature). To obtain enzyme-substrate reaction, 100 μl of
colourless p-NPP was added to the wells and incubated for 30 min at the
room temperature. The indicator was, in the case of a positive reaction,
converted into a coloured product by the reporting enzyme. Finally, the
results were read by a spectrophotometer (optical density at 405 nm).
Between each step, the wells were washed twice using 300 μl of 0.02%
Tween-20 in PBS, and twicewith PBS only. After 30min, the chromogenic
enzymatic reaction was stopped by adding 50 μL of 2 M NaOH.
3. Results and discussion
3.1. Model paintings
The main aim of the study was to investigate the effects of pre-
purification of the samples by CIM® chromatographic monoliths on
the enhancement of the ELISA detection of ovalbumin used as a binder
or finishing protective layer in model and real paintings. To that end, a
wide variety of samples were prepared, a half of them artificially aged.
The selection of binders was intended to provide different quantities
Table 1
Pigments used for the preparation of the model paintings.
Pigment Chemical formula Product no.
Verdigris (Cu(CH3COO)2. [Cu(OH)2]3. 2H2O Kremer 44450
Dragon's blood C18H18O4 Kremer 37000
Malachite Cu2CO3(OH)2 Kremer 10300
Lead white Pb(CO3)2·Pb(OH)2 Kremer 46000
Green earth Al-, K-, Mg-, Ca-, Fe-silicates Schminke 18519
105T. Špec et al. / Microchemical Journal 127 (2016) 102–112of ovalbumin in the samples. Whereas there is very little ovalbumin to
be expected in the egg yolk tempera (in theory, ovalbumin is present
only in egg white) [33], an abundance of the protein is expected in
egg white tempera. Moreover, to study the influence of the different
concentration of the protein and the presence of other organic macro-
molecules, two finishing protective layers (glair and mastic) were
applied. Pigments used (Table 1) were selected with the expectation
of different influence on the integrity of ovalbumin (conformation
change due to binding of metal ions to the protein, its degradation
catalysed by these metals before or during ageing, etc.) [34] and/or
their direct effect on the ELISA detection system [35]. Indeed, it is
known from the conservation practice (real paintings) that paint layers
containing verdigris are often severely degraded and that lead white-
containing layers can exhibit instability. Conversely, good stability is
reported with green earth and malachite (although, as verdigris, it is
a copper-based pigment) paint layers. As a contrast to these metal-
based, dragon's bloodwas used as an organic pigment, andwas expected
to show the least interference either with the binder or the ELISA detec-
tion system. Therefore, the effect of binder-pigment interaction, ageing,
and combination of the two on the ELISA detection was also studied in
this work.
Indeed, a visual inspection of the agedmodel paintings, compared to
the non-aged revealed striking differences between the two. The most
affected paint layers were those containing verdigris (striking colour
change from almost turquoise blue to black) and dragon's blood (very
strong fading of the red colour and/or pronounced browning thereof).
Lead white and malachite coloured surfaces showed moderate fading
and varnish darkening. The least prominent visual changewas observed
in the green earth paint layers.
In preliminary tests, a series of step-wise tenfold dilutions (starting
with the 0.1 mg/ml of initial dried ovalbumin standard) was used as a
template to ascertain the optimal primary and secondary antibody
dilutions. Recommendations of the antibody manufacturers of 1:500
and 1:600, respectively, were confirmed as optimal, and the mentioned
step-wise tenfold dilutions range was used as the calibration curve forFig. 3. ELISA optical densities obtained for (from left to right): Calibration curve concentration o
non-aged paint layers of dragons' blood in egg tempera with glair — non purified and purified
deviation.ovalbumin concentration, as well as a positive control in all subsequent
ELISA assays. Furthermore, primary antibody specificity/possible cross-
reactivity (false positive results)was controlled by tests against collagen
(several terrestrial and fish species), casein, and several other binders
commonly used in cultural heritage, all in 0.1mg/ml standard solutions.
Additionally, to evaluate possible false positive results, different types of
negative controls were made (combinations of buffers and antibodies
used in the assay, in the absence of ovalbumin) on every assay plate.
The highest result of their respective means plus three times their
standard deviation (SD) was assumed as limit of detection (LOD).
To illustrate how the results of all the 120 combinations of pigment,
binders, protective coatings, and ageing and purification procedures
are presented, always relative to the standard calibration curve of
ovalbumin and taking into consideration OD values of the negative
controls, only one investigated paint layer (dragon's blood prepared in
egg yolk and coated with glair; reference (non-aged), non-purified/
purified) (Fig. 3) is presented in detail. The horizontal line
(OD405 nm = 0.1) indicates the limit of positive detection in that
assay. The columns that extend above this threshold present positive
detection of ovalbumin in that sample dilution. Numbers 1, 10, 100,
and 1000 on the horizontal axis (above paint samples) present the
step-wise tenfold dilutions steps of the initial (1 mg/ml) sample solu-
tions. To enable comparison, results of the same, but aged, paint layer
(non-purified/purified) are presented in Fig. 4 in the same manner.
Positive detection of ovalbumin was possible in both, non-purified
and purified reference (non-aged) samples, but for the latter, the posi-
tive response was obtained in the 100 times more diluted sample
(100 times lower limit of detection) (Fig. 3). For the non-purified aged
samples, the positive identification of ovalbumin was possible only in
themost concentrated solution. In comparison, in the accordingpurified
sample, the signal (OD reading) was much higher. Even more impor-
tantly, the identificationwas positive also in the ten-fold diluted sample
(Fig. 4).
Due to the large number of investigated model paint samples, the
rest of the results are not presented in such a detailedmanner. However,
the same rigorous procedure of results evaluation (calibration curve/
positive control, negative controls, LOD determination) as described in
detail above (also Figs. 3 and 4) was used for all of the tested samples'
combinations. The results are summarised in charts, separately for
each of the studied pigments (Figs. 5-9). In these charts, the pairs of
bars representing the ELISA response of the non-aged samples are
situated on the left side of each section of a diagram, and of the aged
on the right. It should be noted that darker columns in the pairs
represent response of the ELISA test of the purified samples. Each bar
in Figs. 5–9 therefore represents a series of tenfold dilutions of onef standard ovalbumin solution (0.1mg/ml of initial dried ovalbumin standard), followed by
. The horizontal line indicates limit of detection (LOD). The error bars represent standard
Fig. 4. ELISA optical densities obtained for (from left to right): Calibration curve concentration of standard ovalbumin solution (0.1 mg/ml of initial dried ovalbumin standard) followed by
agedpaint layers of dragons' blood in egg temperawith glair—non purified and purified. The horizontal line indicates limit of detection (LOD). The error bars represent standarddeviation.
106 T. Špec et al. / Microchemical Journal 127 (2016) 102–112binder/pigment/ageing/purification sample combinations (one four-bar
series presented in detail in Figs. 3 and 4). The results are given as the
number of step-wise tenfold dilutions for which a positive reaction was
obtained. Therefore the higher the bar, the more diluted sample was
positively identified containing ovalbumin. In other words, the higher
the bar, the lower concentration of ovalbumin was detected, the lower
amount of initial paint layer sample would have been needed for positive
identification of the said binder.
In all investigated non-aged paint layers of dragons' blood, it was
evident that the purification enhanced the ELISA response for at least
one tenfold dilution (Fig. 5). Interestingly, the detection of ovalbumin
was possible even on the samples prepared with egg yolk, although it
is known that ovalbumin is present there in a very low concentration.
[33] The ageing processes clearly affected the protein in the paint
layer, which was noticed as the decrease in ELISA response. However,
this unfavourable effect was at least partially offset by the purification
of samples, as it was possible to detect the ovalbumin in these parallels
for up to one tenfold dilution higher than the respective non-purified
(Fig. 5).
For the paint layers prepared with green earth (Fig. 6), the detection
of ovalbumin was not possible where only egg yolk was used as a
binder, probably due to the low concentration of ovalbumin in eggFig. 5. The summarised ELISA response results (expressed in the number of step-wise tenfold d
containing samples. Combinations of two binders and three protective layers (along the x-axis
purified, aged— non purified, aged— purified) were assayed.yolk. [33] However, when the samples contained also glair (mostly
egg white, abundant in ovalbumin — see Section 2.2) surface coating,
it is evident, that even thin layer of varnish, contained enough ovalbu-
min for “classical” ELISA detection, although having an almost negligible
mass share in the entire paint layer sample. Purification enhanced the
ELISA response in all cases, but especially so on aged samples of coatings
(glair or mastic) on pre-prepared egg tempera paint layer. In these
samples, the detection became possible only after the purification, no
response was obtained with the “traditional” ELISA. The enhancement
of ELISA response was obtained also for the purified paint layers
prepared in egg white, egg white with glair or egg white with two
coatings, but it was less pronounced (one tenfold dilution further) (Fig. 6).
In the case of malachite paint layers (Fig. 7) prepared only in egg
yolk tempera, the ovalbumin was detected on both, purified and non-
purified samples (the same ELISA response), but after ageing the detec-
tion of the ovalbumin was impossible even in the purified samples. Due
to the higher amount of the ovalbumin present in the egg white, [33]
the ELISA response is generally higher on the samples which contains
glair, especially on the purified ones. As in the case of the two previously
described binder/pigment systems, artificial ageing negatively influ-
enced the ELISA detection across the board. However, in both aged
and non-aged samples, the purification enhanced the ELISA response,ilutions for which a positive reaction was obtained) of variously prepared dragons' blood-
), subjected to ageing and purification procedures (reference — non purified, reference —
Fig. 6. The summarised ELISA response results (expressed in the number of step-wise tenfold dilutions for which a positive reaction was obtained) of variously prepared green earth-
containing samples. Combinations of two binders and three protective layers (along the x-axis), subjected to ageing and purification procedures (reference — non purified, reference —
purified, aged— non purified, aged— purified) were assayed.
107T. Špec et al. / Microchemical Journal 127 (2016) 102–112except for aged paint layer prepared with egg white only and the non-
aged sample of egg white binder and glair varnish, where it was equal
for purified and non purified samples (Fig. 7).
On the lead white paint layers (Fig. 8), the influence of the pigment
on the detection of protein (ovalbumin) was more prominent, regardless
whether ageing procedure was applied. According to the results of the
ELISA test, the general response to ovalbumin was lower in comparison
to above-described paint layers (dragon's blood, malachite, and green
earth). None of themore than 100-fold diluted samples yielded a positive
ELISA response. Moreover, a positive response for 100-fold dilutions, and
a general improvement of detection by the CIM® chromatographicmono-
lith purification was obtained only for the samples prepared in egg white
binder (higher amount of the initial target protein) (Fig. 8) [33].
The most varied results were obtained when verdigris was used as
the pigment for the preparation of paint layers (Fig. 9). There were
two sets of samples, reference egg tempera and aged egg white
tempera, where ovalbumin could not be detectedwith ELISA. Converse-
ly to the other four tested pigment/binder systems, there was also no
generally consistent trend (either positive or negative) observed on
either the influence of ageing, or monolith purification on the abilityFig. 7. The summarised ELISA response results (expressed in the number of step-wise tenfold
containing samples. Combinations of two binders and three protective layers (along the x-axis
purified, aged— non purified, aged— purified) were assayed.to detect ovalbumin by ELISA. Such a result was not completely unfore-
seen, as it is well-documented that verdigris paint layers (blue-green
copper acetate) can undergo alteration, mostly browning, which causes
irreversible, non-esthetical degradation of the paintings. This phenome-
non has been subject of much research in the last decade, but themech-
anism details are not yet completely understood (Fig. 9) [36–39].
It is also known that some metal ions (especially copper) can influ-
ence the catalytic activity of enzymes used in ELISA and thusly interfere
with the detection of binding media. Copper ions are also a well-known
catalyst in Fenton-like chemical reactions, which yield oxygen-based
radicals (and other oxygen reactive species). [40–42] These can, in
turn, damage the protein structure in several ways, including causing
unspecific oxidation, and depolymerisation. Moreover, these ions can
also change the structure (folding) of proteins by linking to the cysteine
amino acid residues [43] that can therefore possibly affect its ability to
bind to the microtitre plate walls and/or hinder the recognition of a
target protein by an antibody, thusly interfering with ELISA on another
level. As discussed above, the effect of metal ions in extracted solution
was successfully eliminatedwith other paint samples by using the puri-
ficationwith CIM® chromatographicmonoliths. However, in the case ofdilutions for which a positive reaction was obtained) of variously prepared malachite-
), subjected to ageing and purification procedures (reference — non purified, reference —
Fig. 8. The summarised ELISA response results (expressed in the number of step-wise tenfold dilutions for which a positive reaction was obtained) of variously prepared lead white-
containing samples. Combinations of two binders and three protective layers (along the x-axis), subjected to ageing and purification procedures (reference — non purified, reference —
purified, aged— non purified, aged— purified) were assayed.
108 T. Špec et al. / Microchemical Journal 127 (2016) 102–112verdigris paint layers, the problemof protein–pigment interaction could
not be completely eliminated, most likely because of considerably
higher solubility of verdigris in water-based buffers, and consequently
higher concentration of Cu2+ in samples being analysed. One of the
indicators that this is the case is an observation that the monolith
columns were coloured blue (the general colour of diluted verdigris)
after the extraction mixture was loaded onto them. They remained
blue even after elution of proteins from the CIM® monolith chromato-
graphic tip using high salt concentration buffer. Such an effect was not
so pronounced when purifying paints which contained other pigments.
Moreover, also the eluent from the chromatographic tip, which was
supposed to contain the purified ovalbumin, was coloured lightly blue.
Due to the combined effect of relatively high solubility of verdigris and
good chelating capability of proteins, probably a good portion of copper
remains attached onto the protein even after purification (with the un-
desired consequences towards the ELISA effectiveness and reliability
described in this paragraph above). In order to overcome this problem,
further research will be focused on the implementation of various
extraction and elution buffers, with the goal to eliminate copper ions
from the protein solution (extract, during chromatography, or eluent).Fig. 9. The summarised ELISA response results (expressed in the number of step-wise tenfol
containing samples. Combinations of two binders and three protective layers (along the x-axis
purified, aged— non purified, aged— purified) were assayed.Overall, the amount of ovalbumin, theoretically present in the
studied samples, was a significant factor in obtaining a positive
ELISA response. Generally, a better response was obtained for the
samples that were intended to contain a higher amount of this
protein (egg white tempera, and especially all samples containing
glair). Nevertheless, that ovalbumin was detected in samples
containing egg yolk tempera testifies to the strength and robustness
of the ELISA method, to its ability to detect even extremely low
concentrations of targeted molecules. Namely, any ovalbumin
present in such samples is considered to be an impurity, and a conse-
quence of “contamination” from egg white during themanual separation
of the two during binder preparation.
According to the above-presented results purification enhances the
detection of the ovalbumin by ELISA in majority of the investigated
paint layers, most likely due to the removal of the impurities. Moreover,
since the purification improves detection, it is possible to successfully
detect also aged proteins that cannot be detected by conventional
methods. This finding is significant while the samples that are taken
from the real works of art are mostly deteriorated and the detection of
the proteins can be very difficult.d dilutions for which a positive reaction was obtained) of variously prepared verdigris-
), subjected to ageing and purification procedures (reference — non purified, reference —
Fig. 11. FTIR spectra of the Sample 1 (Vittore Carpaccio, The Presentation at the Temple).
Identified compounds are marked in the spectra.
Fig. 10. (Left) Vittore Carpaccio, The Presentation at the Temple, 16th Century; 1 indicates area of sampling. (right) Pietro Liberi, Saint Nicholas between Saint Hermagoras and Saint
Fortunatus, 17th Century; 2 indicates area of sampling.
109T. Špec et al. / Microchemical Journal 127 (2016) 102–112To our best knowledge, the only similar research on the pre-
purification of the protein containing paint samples that were
further tested by ELISAwas done by Lee et al. [22]. The authors observed
that purification step using C4 columns did not have an effect on ELISA
detection. Since proteins express great tendency towards formation of
stable metalloprotein complexes [44], extraction of proteins from C4
support using 0.1% TFA in 50% acetonitrile probably does not disturb
metalloprotein complexes sufficiently, which leads to incomplete remov-
al of metal ions and consequently does not improve ELISA sensitivity. On
the other hand, Tris,which is present in both,washing and elutionbuffers,
has strong complexing affinity towards metal ions [45]. During the sam-
ple preparation procedure, monolith tips were washed with 25 column
volumes of buffers containing Tris (0.25 mg of Tris, compared to approx-
imately 1 mg of paint sample, from which the majority of the material
remained undissolved). Therefore at least a part of metal ions were
extracted from proteins to Tris buffers, which may have lead to an
increase of detection sensitivity of ELISA. Additionally, preliminary
SDS-PAGE investigation (data not shown) could not show consistently
the amount of protein loss in the purification step, probably due to inter-
ference with metal ions which were present with non-purified samples.
Even with purified samples, it was apparent that the binders mixed
with lead white, and particularly so with verdigris, were either still
complexed with the metals (not fully solubilised) or a fraction of them
was strongly degraded, or combination thereof, as some of their bandlines were also smeared. Therefore any eventual loss of investigated
protein was not due to the lack of binding capacity of the monolith
(Section 2.5.3) or recovery yield, which is reported to be high in the
Fig. 12. FTIR spectra of the Sample 2 (Pietro Liberi, Saint Nicholas between Saint
Hermagoras and Saint Fortunatus). Identified compounds are marked in the spectra.
110 T. Špec et al. / Microchemical Journal 127 (2016) 102–112monoliths for large biomolecules [30,31]. Inconsistencies in the enhance-
ment of the ELISA response in some cases of lead white, andmore gener-
ally in the case of verdigris-containing paints are therefore likely due to
enhanced degradation of the binder influenced by the metals contained
in the said pigments and not due to possible sample loss in the purifica-
tion step.
3.2. Paints in art works
After successfully testing the coupling of the twopowerful techniques,
ELISA and CIM® monolith-supported extraction, and proving that their
combination significantly improves the ability to detect ovalbumin in
the model paint samples, further tests were made to validate this proce-
dure on realworks of art. In order to transfer thenovel purification system
and its further application to ELISA method into conservation science
analytical practice, the micro-samples (approximately 1 mg) collect-
ed from the real artworks were investigated. Two art samples were
analysed; from a paint layer from 16th Century painting of Vittore
Carpaccio, Presentation at the Temple (Sample 1, Fig. 10 — left),
followed by the paint layer of the 17th Century painting from Pietro
Liberi, Saint Nicholas between Saint Hermagoras and Saint
Fortunatus (Sample 2, Fig. 10 — right).
To obtain the initial, general information on the materials' composi-
tion present in the paint layers, a FTIR analysis was done on the both
collected samples. According to the analysis in the Sample 1, lipids, [46]
waxes, [47] cellulose (present in the textile support/canvas), [48] calcite,Fig. 13. ELISA optical densities obtained for (from left to right): Calibration curve concentration o
paint samples of V. Carpaccio and P. Liberi. The horizontal line indicates limit of detection (LOD[49] and proteins [46]were present (Fig. 11), while in the Sample 2 lipids,
proteins, and leadwhite [50]were determined (Fig. 12). In the both cases
of the investigated bulk samples, proteinswere detected (Figs. 11 and 12;
marked as P), but due to the unspecific character of this method, it was
impossible to determine the protein type. To overcome this challenge
and to determine the source of protein found in these paintings, the
ELISA test was performed, synergistically with purification by CIM®
chromatographic monoliths. Since the use of egg tempera (as binder) or
egg white (as finishing protective layer) had been frequently used as
the masters` technique in the 16th and 17th century, the presence of
protein ovalbumin (present in egg) [33] was suspected.
In the investigated paint samples, the suspected source of ovalbumin
can be either egg tempera or glair. [2,33] Therefore it was expected that
the relative share of ovalbumin in the mass of the removed sample
would be very small, almost negligible. Moreover, the paints were
naturally aged for centuries and a mixture of compounds was found in
the investigated samples (see FTIR analysis), which can influence the
ELISA test. However, the presented results confirm the sensitivity of
the ELISAmethod, especiallywhen using CIM® chromatographicmono-
liths purification. Positive results for the ovalbumin can be obtained
even in the 10 fold dilution of the initial sample solutions (1 mg/ml),
relative to the standard ovalbumin calibration curve (Fig. 13).
Finally, with the analytical results obtained by ELISA onmodel paints
as well as on the authentic samples of V. Carpaccio (The Presentation at
The Temple) and Pietro Liberi (Saint Nicholas between Saint Hermagoras
and Saint Fortunatus) it was demonstrated that the synergistical
employment of CIM® chromatographic monolith-supported extraction
and ELISA is very effective for the detection of ovalbumin in paints.
4. Conclusions
Outstanding characteristic of the ELISAmethod (low limit of detection,
differentiation between types of proteins, high specificity, sensitivity,
accuracy and cost-efficiency) is paving the way for it to become a very
important tool for the detection of proteins in different works of art.
Nevertheless, the specific recognition of a target protein by an antibody
(a prerequisite for a successful and most of all correct ELISA result) can
be severely compromised, mostly due to the complexity of the materials
present in the samples taken from the objects of Cultural Heritage and
effects of ageing thereof.
To overcome these issues, chromatographic purification using CIM®
chromatographicmonolithswas tested as a complement to ELISA in this
work, and broad synergistic action of the two powerful techniques was
clearly demonstrated on a comprehensive set of 120 investigated
samples. Moreover, the joint CIM® monolith chromatography/ELISA
approach was successfully validated by the investigation of real works
of art.f standard ovalbumin solution (0.1mg/ml of initial dried ovalbumin standard) followedby
). The error bar represents standard deviation.
111T. Špec et al. / Microchemical Journal 127 (2016) 102–112The results also confirmed that ageing of proteins in cultural heritage
binders bears a negative impact on their ability to bind antibodies raised
against their recently isolated counterparts. However, since the
purification improves detection, it is possible to successfully detect
also aged proteins that cannot be detected by conventional methods.
In practical terms thatmeans that purification by CIM® chromatographic
monoliths improves/in some cases enables the detection of aged
samples. Therefore, it is strongly believed that simple prepurification
and concentration of protein samples using CIM®monolith chromatog-
raphy can enhance the detection also for other immunology-based
(protein) identification and characterisation techniques (other types
of ELISA).
The presented work is a significant breakthrough in antibody-based
identification of cultural heritage material components (robustness,
reliability, limit of detection). With further optimisation of the CIM®
monolith chromatography purification and its integration with various
types of immunology-based techniques, an expansion is foreseen for the
identification of not only proteins, but also other organic components
extracted from works of art.Acknowledgements
The financial support of the Slovenian Research Agency (grant L6-
4217 and P4-0369) is gratefully acknowledged. This paper is also a re-
sult of doctoral research, in part financed by the European Union,
European Social Fund and the Republic of Slovenia,Ministry for Education,
Science andSportwithin the frameworkof theOperational programme for
human resources development for the period 2007 – 2013 (operation no.
3330-13-500221).References
[1] J.S. Mills, R. White, The Organic Chemistry of Museum Objects, second ed. Butterworth
Heinemann Ltd, Oxford, 1994.
[2] K.Wehlte, TheMaterials and Techniques of Painting, Kremer Pigments Incorporated
1975.
[3] M.R. Schilling, H.P. Khanjian, L.A.C. Souza, Gas chromatographic analysis of amino
acids as ethyl chloroformate derivatives. Part 1, composition of proteins associated
with art objects and monuments, J. Am. Inst. Conserv. 35 (1996) 45–59.
[4] M.P. Colombini, F. Modugno, S. Giannarelli, R. Fuoco, M. Matteini, GC–MS characteriza-
tion of paint varnishes, Microchem. J. 67 (2000) 385–396.
[5] J.J. Boon, S.L. Peulvé, O.F. van den Brink, M.C. Duursma, D. Rainford, in: T. Bakkenist,
R. Hoppenbrouwers, H. Dubois (Eds.), In Early Italian Painting Technique, Limburg
Conservation Institute, Maastricht 1997, pp. 32–47.
[6] O.F. van den Brink, J.J. Boon, P.B. O'Connor, M.C. Duursma, R.M.A. Heeren, Matrix-
assisted laser desorption/ionization Fourier transform mass spectrometric analysis of
oxygenated triglycerides and phosphatidylcholines in egg tempera paint dosimeters
used for environmental monitoring of museum display conditions, J. Mass Spectrom.
36 (2001) 479–492.
[7] M.P. Colombini, F. Modugno, E. Menicagli, R. Fuoco, A. Giacomelli, GC–MS characteriza-
tion of proteinaceous and lipid binders in UV aged polychrome artifacts, Microchem. J.
67 (2000) 291–300.
[8] F. Casadio, L. Toniolo, The analysis of polychrome works of art: 40 years of infrared
spectroscopic investigations, J. Cult. Herit. 2 (2001) 71–78.
[9] I. Bonaduce, M.P. Colombini, Gas chromatography/mass spectrometry for the
characterization of organic materials in frescoes of the Momumental Cemetery of
Pisa (Italy), Rapid Commun. Mass Spectrom. 17 (2003) 2523–2527.
[10] G. Chiavari, G. Lanterna, C. Luca, M. Matteini, S. Prati, I.C.A. Sandu, Analysis of
proteinaceous binders by in-situ pyrolysis and silylation, Chromatographia 57
(2003) 645–648.
[11] M.P. Colombini, F. Modugno, Characterisation of proteinaceous binders in artistic
paintings by chromatographic techniques, J. Sep. Sci. 27 (2004) 147–160.
[12] A. Andreotti, I. Bonaduce, M.P. Colombini, G. Gautier, F. Modugno, E. Ribechini,
Combined GC/MS analytical procedure for the characterization of glycerolipid, waxy,
resinous, and proteinaceous materials in a unique paint microsample, Anal. Chem. 78
(2006) 4490–4500.
[13] M.T. Doménech-Carbó, Novel analytical methods for characterising binding media
and protective coatings in artworks, Anal. Chim. Acta 621 (2008) 109–139.
[14] E. Mazzeo, S. Joseph, A. Prati, Millemaggi, attenuated total reflection–Fourier transform
infrared microspectroscopic mapping for the characterisation of paint cross-sections,
Anal. Chim. Acta 599 (2007) 107–117.
[15] I. Karapanagiotis, D. Mantzouris, C. Cooksey, M.S. Mubarak, P. Tsiamyrtzis, An
improved HPLC method coupled to PCA for the identification of Tyrian purple in
archaeological and historical samples, Microchem. J. 110 (2013) 70–80.[16] A. Heginbotham, V. Millay, M. Quick, The use of immunofluorescence microscopy
and enzyme-linked immunosorbent assay as complementary techniques for protein
identification in artists' materials, J. Am. Inst. Conserv. 45 (2006) 89–105.
[17] J. Schultz, J. Arslanoglu, C. Tavzes, K. Petersen, Immunological techniques: a different
approach to the analyses of proteins in cultural heritage. Part I: basics explained, Z.
Kunsttechnologie Konservierung 23 (2009) (2009) 129–139.
[18] L. Cartechini, M. Vagnini, M. Palmieri, L. Pitzurra, T. Mello, J. Mazurek, G. Chiari,
Immunodetection of proteins in ancient paint media, Acc. Chem. Res. 43 (2010)
867–876.
[19] J. Arslanoglu, J. Schultz, J. Loike, K. Peterson, Immunology and art: using antibody-based
techniques to identify proteins and gums in artworks, J. Biosci. 35 (2010) 3–10.
[20] M. Palmieri, M. Vagnini, L. Pitzurra, P. Rocchi, B.G. Brunetti, A. Sgamellotti, L.
Cartechini, Development of an analytical protocol for a fast, sensitive and specific
protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA),
Anal. Bioanal. Chem. 399 (2010) 3011–3023.
[21] M. Palmieri, M. Vagnini, L. Pitzurra, B.G. Brunetti, L. Cartechini, Identification of
animal glue and hen-egg yolk in paintings by use of enzyme-linked immunosorbent
assay (ELISA), Anal. Bioanal. Chem. 405 (2013) 6365–6371.
[22] H.Y. Lee, N. Atlasevich, C. Granzotto, J. Schultz, J. Loike, J. Arslanoglu, Development
and application of an ELISA method for the analysis of protein-based binding
media of artworks, Anal. Methods 7 (2014) 187–196.
[23] M. Vagnini, L. Pitzurra, L. Cartechini, C. Miliani, B.G. Brunetti, A. Sgamellotti, Identifica-
tion of proteins in painting cross-sections by immunofluorescence microscopy, Anal.
Bioanal. Chem. 392 (2008) 57–64.
[24] G. Sciutto, L.S. Dolci, A. Buragina, S. Prati,M. Guardigli, R.Mazzeo, A. Roda, Development
of amultiplexed chemiluminescent immunochemical imaging technique for the simul-
taneous localization of different proteins in paintingmicro cross-sections, Anal. Bioanal.
Chem. 399 (2010) 2889–2897.
[25] G. Sciutto, L.S. Dolci, M. Guardigli, M. Zangheri, S. Prati, R. Mazzeo, A. Roda, Single and
multiplexed immunoassays for the chemiluminescent imaging detection of animal
glues in historical paint cross-sections, Anal. Bioanal. Chem. 405 (2012) 933–940.
[26] X. Huang, D. Yuan, Recent developments of extraction and micro-extraction
technologies with porous monoliths, Crit. Rev. Anal. Chem. 42 (2012) 38–49.
[27] M. Barut, A. Podgornik, L. Urbas, B. Gabor, P. Brne, J. Vidič, S. Plevčak, A. Štrancar,
Methacrylate-based short monolithic columns: enabling tools for rapid and efficient
analyses of biomolecules and nanoparticles, J. Sep. Sci. 31 (2008) 1867–1880.
[28] A. Podgornik, S. Yamamoto, M. Peterka, N.L. Krajnc, Fast separation of large biomole-
cules using short monolithic columns, J. Chromatogr. B 927 (2013) 80–89.
[29] R.N. D‘Souza, A.M. Azevedo,M.R. Aires-Barros, N.L. Krajnc, P. Kramberger, M.L. Carbajal,
M. Grasselli, R. Meyer, M. Fernández-Lahore, Emerging technologies for the integration
and intensification of downstream bioprocesses, Pharma. Bioprocess. 1 (2013)
423–440.
[30] B. Gabor, U. Černigoj, M. Barut, A. Štrancar, Reversible entrapment of plasmid
deoxyribonucleic acid on different chromatographic supports, J. Chromatogr. A. 1311
(2013) 106–114.
[31] M. Segura, Chromatography purification of canine adenoviral vectors, Hum. Gene
Ther. B Methods 23 (2012) 182–197.
[32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem.
72 (1976) 248–254.
[33] T. Yamamoto, Hen Eggs: Basic and Applied Science, CRC Press, Boca Raton, 1997.
[34] E.R. Stadtman, R.L. Levine, Free radical-mediated oxidation of free amino acids and
amino acid residues in proteins, Amino Acids 25 (2003) 207–218.
[35] Q.-X. Chen, W.-Z. Zheng, J.-Y. Lin, Y. Shi, W.-Z. Xie, H.-M. Zhou, Effect of metal ions
on the activity of green crab (Scylla serrata) alkaline phosphatase, Int. J. Biochem.
Cell Biol. 32 (2000) 879–885.
[36] L. Cartechini, C. Miliani, B.G. Brunetti, A. Sgamellotti, C. Altavilla, E. Ciliberto, F.
D'Acapito, X-ray absorption investigations of copper resinate blackening in a XV
century Italian painting, Appl. Phys. A Mater. Sci. Process. 92 (2008) 243–250.
[37] C. Santoro, K. Zarkout, A.-S.L. Hô, F. Mirambet, D. Gourier, L. Binet, S. Pagès-Camagna,
S. Reguer, S. Mirabaud, Y. Le Du, P. Griesmar, N. Lubim-Germain, M. Menu, New
highlights on degradation process of verdigris from easel paintings, Appl. Phys. A
Mater. Sci. Process. 114 (2014) 637–645.
[38] M. Gunn, G. Chottard, E. Rivière, J.-J. Girerd, J.-C. Chottard, Chemical reactions between
copper pigments and oleoresinous media, Stud. Conserv. 47 (2002) 12–23.
[39] E. Ioakimoglou, S. Boyatzis, P. Argitis, A. Fostiridou, K. Papapanagiotou, N. Yannovits,
Thin-film study on the oxidation of linseed oil in the presence of selected copper
pigments, Chem. Mater. 11 (1999) 2013–2022.
[40] K. Ahn, A. Hartl, C. Hofmann, U. Henniges, A. Potthast, Investigation of the stabilization
of verdigris-containing rag paper by wet chemical treatments, Herit. Sci. 2
(2014) 12.
[41] M. Strlic, J. Kolar, B. Pihlar, The effect of metal ion, pH and temperature on the yield
of oxidising species in a Fenton-like system determined by aromatic hydroxylation,
Acta Chim. Slov. 46 (1999) 555–556.
[42] M. Strlič, J. Kolar, V.S. Šelih, D. Kočar, B. Pihlar, A comparative study of several transition
metals in Fenton-like reaction systemsat circum-neutral pH, Acta Chim. Slov. 50 (2003)
619–632.
[43] N. Russo, D.R. Salahub, M. Witko, Metal–Ligand Interactions, Kluwer Academic,
Dordrecht, 2002.
[44] W.J. Goux, P.N. Venkatasubramanian, Metal ion binding properties of hen ovalbumin
and S-ovalbumin: characterization of the metal ion binding site by phosphorus-31
NMR and water proton relaxation rate enhancements, Biochemistry 25 (1986)
84–94.
[45] J. Nagaj, K. Stokowa-Sołtys, E. Kurowska, T. Frączyk, M. Jeżowska-Bojczuk, W. Bal,
Revised coordination model and stability constants of Cu(II) complexes of tris buffer,
Inorg. Chem. 52 (2013) 13927–13933.
112 T. Špec et al. / Microchemical Journal 127 (2016) 102–112[46] R. Mazzeo, S. Prati, M. Quaranta, E. Joseph, E. Kendix, M. Galeotti, Attenuated total
reflection micro-FTIR characterisation of pigment-binder interaction in reconstructed
paint films, Anal. Bioanal. Chem. 392 (2008) 65–67.
[47] M.T. Doménech-Carbó, A. Doménech-Carbó, J.V. Gimeno-Adelantado, F. Bosch-Reig,
Identification of synthetic resins used in works of art by Fourier transform infrared
spectroscopy, Appl. Spectrosc. 55 (2001) 1590–1602.
[48] D. Ciolacu, F. Ciolacu, V.I. Popa, Amorphous cellulose— structure and characterization,
Cellul. Chem. Technol. 45 (1–2) (2011) 13–21.[49] S. Gunasekaran, G. Anbalagan, S. Pandi, Raman and infrared spectra of carbonates of
calcite structure, J. Raman Spectrosc. 37 (2006) 892–899.
[50] K. Catalli, J. Santillán, Q. Williams, A high pressure infrared spectroscopic study of
PbCO3-cerussite: constraints on the structure of the post-aragonite phase, Phys.
Chem. Miner. 32 (2005) 412–417.