Volume 8 Number 24 28 June 2016 Pages 4773–4966

Analytical
Methods
www.rsc.org/methods

ISSN 1759-9660

PAPER
V. Antunes, A. Candeias, M. L. Carvalho et al.
A multidisciplinary approach to the study of the brightening effects
of white chalk ground layers in 15th and 16thcentury paintings
Analytical
Methods
PAPER

A multidisciplinary approach to the study of the

brightening effects of white chalk ground layers in
15th and 16thcentury paintings
Cite this: Anal. Methods, 2016, 8, 4785

Vanessa Antunes,*ab António Candeias,cd João Coroado,e Vitor Serrão,a

Mário Cachãof and Maria L. Carvalhob

This paper employs various techniques to analyze the mixture of chalk and binder materials used, by
Portuguese and Flemish painters in the 15th and 16th centuries, to enhance the reflection of light in
paintings. The cases studied show evidence of the search by painters for light effects created when
combining specific fillers and binders to obtain absorbent or non-absorbent ground layers in order to
reflect superficial or deep light in paintings. These brightening effects are largely provided by
microscopic coccoliths and calcispheres – the main constituents of chalk. The composition, size and
slightly concave-convex shield-like shape of calcareous nannofossils (micrometrical dimensions) also
facilitate application, thereby increasing the speed of handling. These calcareous nannofossils are
crucial proof that chalk was used in the ground layers of Portuguese paintings. They have proved to be
important in defining the various stages of Portuguese painting workshops, such as the Viseu Workshop
(1501–1569), which used powdered chalk in the first phase and powdered limestone in the second
phase in the production of a ground layer. A two-layer structure has been verified in some Flemish
paintings of the period, but the use of different binders to provide different levels of light absorption
and reflection in these artworks had not been previously identified. The results showing this two-layer
Received 12th February 2016
Accepted 3rd May 2016
ground structure are significant in making the connection between Flemish and Portuguese art in the
context of a specific painting technique. The use of calcium carbonate ground layers was verified by
DOI: 10.1039/c6ay00435k
SEM-EDS and confirmed by m-XRD, and m-Raman, while binders were analyzed by m-FTIR and optical
www.rsc.org/methods microscopy, using staining tests.

similar ground layers in various paintings from the 15th and

1. Introduction
Investigation of painting techniques demands a signicant
understanding of the types of material used as well as the
possible combinations of these in order to understand an
artist's reason for choosing such materials. This research, using
analytical practices, alongside several protocols found in the
literature concerning the study of painting materials, compares
a
ARTIS-Instituto História da Arte da Faculdade de Letras da Universidade de Lisboa
(ARTIS-FLUL), Alameda da Universidade, 1600-214 Lisboa, Portugal. E-mail:
vanessahantunes@gmail.com
b
LIBPhys-UNL, Laboratório de Instrumentação, Engenharia Biomédica e Fı́sica da
Radiação, Departamento de Fı́sica, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516, Caparica, Portugal
c
Laboratório José de Figueiredo / Direcção-Geral do Património Cultural (LJF-DGPC),
Rua das Janelas Verdes 37, 1249-018 Lisboa, Portugal
d
Laboratório HERCULES, Escola de Ciências e Tecnologia, Universidade de Évora,
Largo Marquês de Universidade de Évora, Largo Marquês de Marialva 8, 7000-676
Évora, Portugal
e
Instituto Politécnico de Tomar (IPT)/GeoBioTec ID&T unit, 2300-313 Tomar, Portugal
f
Instituto Dom Luiz, Faculdade de Ciências, Universidade de Lisboa, Campo Grande,
1749-016 Lisboa, Portugal
16th centuries. These paintings have been deeply studied in
order to identify different techniques used by painters during
this period.1–6 When dealing with materials which are unde-
tectable to the naked eye, as it is the case of the painting ground
layers, analytical procedures require a detailed and exhaustive
research plan to understand this “invisible matter”.7 In Portu-
gal, the study of ground layers in paintings from the 15th and
the 16th centuries has proved that two different types of material
exist in these layers: calcium sulphate and calcium
carbonate.1–4,7–9 The existence of calcium carbonate in the
ground layers of Flemish paintings in Portugal and in Portu-
guese paintings has been proved by earlier studies8,9 but the
possible reasons that led to Portuguese painters selecting this
particular material is a little studied issue7 and this paper aims
to bring some new data on this subject. In some cases, the
limestone was powdered and used as a ground layer while in
other cases a special kind of limestone, i.e. pure chalk, was
used. Chalk is a so limestone composed mainly of small
biogenic calcium carbonate particles (biomicrite). Typically it
dates from the Cretaceous period and was usually taken from
the sea cliffs of England and France, but can also be found in

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 4785–4797 | 4785
Analytical Methods
Paleogene (Danian) deposits (North Sea, Denmark, Maastricht).
Its genesis is associated with deep marine deposits and is
characterized by the presence of calcareous nannofossils of
unicellular marine phytoplankton organisms, the coccolitho-
phores. Coccoliths are microscopic calcium carbonate shells of
various shapes, the most common morphotype being the
discoidal placoliths. These are produced by the coccolitho-
phores, unicellular organisms where the cell is enclosed in an
exoskeleton of calcite, the coccosphere.10
Today, chalk is extracted from several regions and its
primary characteristics include neness, soness and
whiteness.
A two-layer glue/oil structure has been veried in some
Flemish paintings of the period,11 indicating that this technique
might have been taught in artist workshops at the time.
However, the use of different binders to provide different levels
of light absorption and reection in these art works had not
previously been discussed. This two-layered ground technique
was proved, during a study of Rembrandt's paintings, to have
been used in the 17th century. Rembrandt had worked on panels
since c. 1624 and had used ground layers that fundamentally
followed the Flemish technique. Treatises such as the
de Mayerne Manuscript, of 1620–1646, refer to the use of glue
and chalk ground layers,12 but chalk in oil as a ground layer
is reported very uncommonly, being found in a German
19th century treatise.13,14 Some panel preparations used by
Rembrandt, and other Flemish, French and Italian painters,
had a two-layered ground.12 In Rembrandt's panel paintings the
rst ground layer might consist of glue and chalk (with or
without pigments) in order to seal the wood's porosity; a second
oil-containing layer could be added, obtaining a smooth,
yellowish (or other color surface) on which to paint. This second
layer might contain chalk mixed with other pigments.12
The present study reveals that the two-layer structure used in
the ground, and the phenomenon of using chalk material,
provided largely by calcareous nannofossils (mainly coccoliths
and calcispheres), to reect light, could have been a practice
that had been taught in painting workshops since the
15th century. Calcareous nannofossils are calcite skeletons of
very small dimensions (typically between 2 and 20 mm),
including coccoliths, nannoliths and calcispheres. Its excellent
workability and lubricating qualities come from physical and
mechanical properties such as neness and soness, as well as
the rock being naturally pulverulent.7,15,16 The composition and
size of the slightly concave-convex shield-shaped micro-
fossiliferous limestone, facilitate the speed of application of the
substances. The quick application of the chalk as a painting
ground layer allows its homogenization before drying, and
therefore facilitates the aggregation of particles and their
subsequent attening.
The unique properties of chalk allow for the identication of
different phases of work in a painting workshop. This was
observed in the rst decades of the so-called Viseu Workshop,
led by the painter Vasco Fernandes (Grão-Vasco) (c. 1475–1542)
in the north of Portugal. This painter was the precursor to the
Portuguese Renaissance, being distinguished among other
painters due to his technique of polishing the painting ground
4786 | Anal. Methods, 2016, 8, 4785–4797
Paper
layer with oil, to facilitate a smoother nish with the
paintbrush, as is referenced in the 17th century Portuguese
treatise Breve tratado de iluminaçam.17
In addition to its neness and smoothness, chalk has
a special feature which was sought aer and exploited by the
artists who used it – a particular ability to reect light. From this
perspective, it is possible to identify a distinctive ground layer
stratigraphy in these paintings. This ground layer is thought to
contain a proteinaceous binder in the lower layer and in the
upper layer an oily binder, as can be seen in the historical case
studies of Flemish and Portuguese paintings.7,18,19 The mixture
of chalk and animal glue has characteristics of “supercial
light”, since chalk elements are kept in suspension by adhesive
bonds, allowing the light to penetrate through the layers and
cause a reection mainly at the surface of the layer.
When mixed with oil, chalk provides the phenomenon of
“deep light”, with the decrease in the coating power of the
mixture and the slow-drying oil oxidation and polymerization
that encapsulate the particles. This factor allows light to pene-
trate deeply at the same time as being partially absorbed by the
ground layer.20,21 However, the absorption of the oil by the
pigment or ller (material added to a substance to modify its
physical properties) depends on the quality of both materials,
requiring differing amounts according to this inherent charac-
teristic. An excess of oil can cause yellowing of the ground.
According to the oil absorption index required for each pigment
or ller, a certain amount of oil is required to obtain a homo-
geneous and efficient mixture. These values have been studied
and tabulated. It takes approximately 18.9 g of oil to 100 g of
chalk, stirring white chalk into the oil until it turns yellow-gray
in color and eventually becomes a hardened paste.21
Thus, the ideal percentage of oil for each pigment or ller is
closely related to the proper protection of its particles and with the
accurate degree of plasticity required for the mixture. When the
amount of oil is too high this causes the yellowing of the paintings,
their hardening, and consequently, the detachment of the layers.
Complex reactions between the pigments and the binder affect the
drying of the mixture, its consistency, drying speed, extent of
oxidation and the exibility, hardness, durability and color
stability of the resulting paint lm.21 To adjust the degree of drying
in the mixture artists might mix the linseed oil with other
materials such as poppy oil which has a slower drying rate.21
As evidenced by the panels studied in this work, the reec-
tion of light was caused by the presence of the minute calcite
crystals of the coccoliths in the chalk/binder mix.
This combination of materials was inuenced by Northern
European tradition and was selected for the earlier works of this
renaissance workshop due to the long-lasting nature of the
mixture. The results showing this two-layer ground structure
(chalk/binder mix) are signicant in making the connection
between Flemish and Portuguese art in the context of a specic
painting technique.
2. Experimental
The studied ground layers were analysed by complementary
techniques, in order to conrm and full the different results:
This journal is © The Royal Society of Chemistry 2016
Paper
Optical microscopy (OM) was carried out using a Leitz
Wetzlar optical microscope coupled with digital camera Leica
DC 50 equipped with visible light, dark and light eld. Samples
were observed by OM with visible and ultraviolet light. In UV,
samples were observed with an emission lter of 490 nm aer
a staining test had been performed. This test was achieved with
a highly sensitive uorescent staining protein (Orange Sypro©)
observable between 300 and 472 nm (excitation) and
490–570 nm (emission) following established protocols.22–24
SEM imaging (secondary electrons (SE) and back-scattered
electrons (BSE) modes) were executed using a Hitachi S-3700N
scanning electron microscope coupled with a Bruker XFlash
5010 SDD detector. This technique conrmed the exact location
of the ground layers between the support and the painting
layers.
SEM-EDS identied elemental composition of inorganic
calcium carbonate compounds in the ground layers. In some
cases samples were covered with a conductive lm of carbon to
improve measurement of calcium carbonate.
Micro X-ray diffraction (m-XRD), performed on a Bruker AXS
D8 Discovery diffractometer with Cu Ka radiation and Gadds
detector, permitted the detection of crystalline phases through
the EVA soware. This procedure established the presence of
calcium carbonate mineral calcite rich ground layers.
Micro-confocal Raman spectroscopy (m-Raman) was per-
formed using an Xplora (Horiba) spectrometer equipped with
a 785 nm laser diode, a 100
objective and a 1200 l mm1
optical grating with a spectral resolution of about 4 cm1. The
incident laser power on the samples was 5 mW and the
compounds were detected comparing to literature and data-
bases (RRUFF, Crystal Sleuth, and Spectral ID).25
Micro-Fourier transform infrared spectroscopy (m-FTIR) was
analysed in transmission mode aer the sample was
compressed in a Spectra-Tech (Sample Plan micro-compression
diamond cell). A Thermo Nicolet Nexus spectrometer coupled to
a Nicolet Continuum microscope, equipped with a Nicolet
mercury–cadmium–telluride (MCT-A) detector (working range:
4000–650 cm1) was used. For each spectrum 256 scans were
recorded with a spectral resolution of 4 cm1. Samples were
mounted in an epoxy polymeric resin and cross-sectioned with
a Leica microtome in a thickness of 20 mm in order to remove
micro-samples of the upper and lower areas of each
ground layer.
Ground layer reconstructions were set according to the
recommendations of treatises, oral tradition and other docu-
mentation referring to chalk ground layers.20,26–28 Reconstruc-
tions were made taking into account the maximum thickness
found in the studied historical samples (c. 300 mm). On the
wooden oak support a sizing layer of animal glue was rst
applied (from the brand Manuel Riesgo, Spain). It was followed
by a layer consisting of glue/chalk or oil/chalk or both, over-
laying this last to rst. This superposition was made with chalk
that had been more nely powdered, mixed with oil and applied
aer the rst layer was dry and polished with silicon carbide,
the thickness of each layer being c. 150 mm in this case. In some
of these last reconstructions a sizing between both layers was
applied. The chalk (Belgian chalk powdered and exported by
This journal is © The Royal Society of Chemistry 2016
Analytical Methods
Omya company, average size of the particles 2.6 mm) was mixed
with a warmed solution of animal glue (with previous 10%
dilution in H2O, animal glue) in 1 : 1 and 3 : 1 v/v proportions,
respectively. The same proportions were used in the mixture of
oil/chalk (Windsor&Newton drying linseed oil). These recon-
structions had a curing time of 3 months at room temperature.
Specimens were characterized by their brightness and color
index using the CIE systems, from the initials of the Commis-
sion Internationale de l'Eclairage, the following parameters:
CIE L* a* b* (L* represents the lightness, a* the redness-
greenness, b* the yellowness-blueness) and CIELCH (L*C*h – L*
species lightness, C* denotes chroma and h denes hue
angle, with angular measurement).29
The spectrophotometer used was a Datacolor CHECK®
II-Plus with the apertures Large Area View (LAV), measured
values at 11 mm aperture and Ultra-Small Area View (USAV),
measured values at 2.5 mm aperture, scanning from 60 to
700 nm at 10 nm intervals for LAV and USAV, Lab at
D65/10 .White tile calibration was used with the label in the
upright position, centered over the port opening before start
measuring. The illuminant/observer conditions were kept in
order to collect comparable data from the reconstructions.
3. Results and discussion
3.1. Chalk-raw material characterization
White chalk is composed mostly of calcite (from 90 to 98%
CaCO3), less than 1% of minerals such as micas, quartz and
heavy minerals which have been derived mainly from the
erosion of metamorphic and igneous rocks, and a small
percentage of clay minerals (circa 1%).30 Its content is a volume
of between 20% and 50%, composed of the inorganic skeletal
remains of marine phytoplankton cells with micrometrical
dimensions (less than 62 mm) and overall denominated calcar-
eous nannofossils.30 These microfossils occurred in past marine
environments such as ocean sediment and are important lith-
ogenetic contributors in that they are abundant and
geographically widely distributed. They are biostratigraphic
indicators dating from the Mesozoic and Cenozoic rocks since
they rst appeared in the Triassic period 220 million years ago.31
The fact that individualized calcareous nannofossils are of
small dimensions, typically between 5 mm and 20 mm, offers the
advantage of their shells being well preserved and in large
numbers even within a small sample size.31 The ner the chalk,
the greater number of calcareous nannofossils which can be
found.30 Despite the workability and processing capacity of this
material, these microfossil rocks have good mechanical
strength against being powdered.31
The natural function of these coccolith shells is to protect
the coccolithophore, as well as playing a role in the regulation of
the light by acting as reectors.31 This fact is of utmost impor-
tance when dealing with the theme of chalk ground layers in
painting. A major reection of light was one of the maximum
objectives in the execution of a ground layer for the studied
epochs.
Calcareous nannofossils found in the studied paintings have
different types, corresponding to different levels of reection
Anal. Methods, 2016, 8, 4785–4797 | 4787
Analytical Methods
existing in the ground layers. Its presence in the ground layers
suggests that the material was used in its natural form, without
the articial synthesizing of calcium carbonate by calcination or
recarbonation. The species of these calcareous nannofossils,
usually identiable by OM and SEM techniques, are difficult to
recognize in ground layers, since some are partly macerated and
immersed in protein or oily binders which have been used as
a mixture for the application of the ground layer. The OM
analysis usually requires the disintegration of the chalk sample
in order to individualize and thus identify the coccoliths.
Therefore, it was decided to identify them using the SEM
analytical technique in order to preserve the historical samples,
assuming the impossibility of classifying many of the cocco-
liths. Calcareous nannofossils mostly found in these ground
layers are divided into two distinct groups: coccoliths and cal-
cispheres. In coarser granulometry ground layers also contain
larger microfossils with morphology compatible with forami-
nifera, along with other indeterminate bioclast fragments with
possible connections to bivalve remains. Foraminifera are
unicellular marine protists that typically produce chambered
shells. These shells also exist in the chalk, and although they are
larger than calcareous nannofossils they are still microfossil
remains of marine organisms. Bivalves (marine shelled
molluscs), serpulids (tube-building annelid shelled worms),
bryozoans (aquatic invertebrate animals that may produce
mineralized exoskeletons), and equinoderms can also be found.
Coccoliths found in the painting of São Pedro of Tarouca
Monastery church, c. 1530–1535, Viseu, Portugal and attributed
to Vasco Fernandes and Gaspar Vaz (1515–1568) who were
both painters of the Viseu Workshop, resemble a typical
morphotype of Watznaueria britannica, the dominant coccolith
of Jurassic and Cretaceous periods, although rarer in the lower
Cretaceous32 (Fig. 1).
This species is found in various ancient deposits of
calcareous nannofossils, such as the formation of the Upper
Jurassic Kimmeridge Clay of Wessex in southern England, and it is
considered a good indicator of paleoproductivity, promoting low
diversity of the species.33 Some of the specimens of W. britannica32
studied in the upper Jurassic deposits of southern Germany,
show that this coccolith is abundant in the stratigraphic Balingen-
Tieringen section and varies in size, morphology and degree of
mineralization of the species, according to the progressive changes
of paleoenvironmental factors.34 Abundance and diversity of these
Fig. 1 (a) Painting of “São Pedro”, Tarouca Monastery church, c. 1530–1535, Viseu, Portugal, attributed to Vasco Fernandes and Gaspar Vaz
(image of painting © Vanessa Antunes 2016, taken with permission of Governo de Portugal); (b) SEM image (SE mode) of coccolith found in the
cross-section of sample 1a, painting “São Pedro”, Tarouca Monastery church, illustrates a morphology compatible with the type “Watznaueria
Britannica”; (c) SEM image (SE mode) of coccosphere with identical type of coccoliths found in the same sample.
4788 | Anal. Methods, 2016, 8, 4785–4797
Paper
calcareous nannofossil species have been used to characterizes oil
stratigraphic sections, based on the geological sequence.33 It
occurs abundantly in deposits in southern England and southern
Germany, despite its presence in other outcrops during these
periods. The presence of calcareous nannofossils of the placolith
type in the ground layer of the painting of São Pedro of Tarouca
Monastery church shows that the concentration of these coccoliths
is high in the chalk that was used. This might suggest that
the chalk for this painting was sourced from southern England
or southern Germany. Fig. 1 shows SEM images (SE mode) of
coccoliths found in the cross-section of sample 1a of the
painting of São Pedro of the Tarouca Monastery church, illustrating
coccoliths and a coccosphere comprising a morphology compat-
ible with W. britannica.
The clear rings of the coccoliths were also disclosed in the
altarpiece of Viseu Cathedral (1501–1506), and attributed to
Vasco Fernandes and the Flemish painter Francisco Henriques
(Flanders, ?-Portugal, 1518). Identiable in the SEM image
(BSE mode) of Fig. 2, which shows the cross-section of sample
2a of the Apresentação no Templo painting from the altarpiece of
Viseu Cathedral, are the rounded forms of calcispheres in the
ground layer; cross-sections from the samples 3a and 3b of the
Adoração dos Magos painting from the same altarpiece show
unrated coccoliths. These nannofossils were also found in the
altarpiece of the main chapel of Lamego Cathedral, contracted
by Vasco Fernandes and constructed between 1506 and 1511,
(nowadays the remaining paintings are in Lamego Museum
(ML)) and in the Assunção da Virgem painting (National Museum
of Ancient Art (MNAA), Lisbon),attributed to Vasco Fernandes
between 1511 and 1515. These were also found in Flemish
paintings such as the old Flemish polyptych in Évora Cathedral
and the Esporão Chapel altarpiece of the same cathedral, both
in Museu de Évora (ME) and attributed to the circle of Gerard
David (aer 1495).
Calcispheres occur most frequently in the lower levels of the
ground layers studied, as proved by the SEM image (BSE mode)
of the cross-section from sample 1b of the painting of São Pedro
of Tarouca Monastery church (Fig. 3a). They were produced
using calcareous dinoagellates and constitute the exoskeleton
of these unicellular microorganisms of marine phytoplankton.
They may occur in limestone from the Devonian to the Creta-
ceous. In this case, most are probably from the Cretaceous,
considering that they were found in the chalk and are the
This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

Fig. 2 (a) SEM image (BSE mode) of cross-section of sample 2a, painting “Apresentação no Templo”, altarpiece of Viseu Cathedral, with rounded
forms of calcispheres in ground layer; (b) SEM image (BSE mode) of cross-section of sample 3a with unrated coccolith of the painting “Adoração
dos Magos” of the same altarpiece; (c) SEM image (BSE mode) of sample 3b with unrated coccolith of the same painting.

second most abundant group of microfossils in rocks of the painting, belonging to the altarpiece of Viseu Cathedral also
Upper Cretaceous.35 With the shape of a hollow sphere, their shows macrofossil remnants in the ground layer; the Raman
walls consist of calcite crystals, bodies with a diameter of spectrum obtained for the last sample compound exhibits the
400 mm or less. These microorganisms oen form two circular increasing intensity of calcite peaks (1088 cm1, 715 cm1, and
calcareous shells, one that protects the body and another that 281 cm1), constituent of the chalk calcium carbonate ground
grows in parallel, bonded thereto. These specimens were found layer (Fig. 4).
in the same pictorial groups that disclosed coccoliths.
In the ground layers studied, fragments of foraminifera are 3.2. Chalk-processing technology
also present with macrofossils of possible bivalves or other
A feature of major importance in the context of our study is that
marine organisms (serpulids, bryozoans). These bioclasts
the higher the content of these calcareous nannofossils, the
present various shapes and dimensions although typically less
coarser the chalk.30 This aspect suggests the need for grinding,
than 1 mm in size.30
and thereby partly destroying the shells of macrofossils in order
Such bioclasts were also found in the ground layers of
to apply a smoother and thinner material to the upper layer of
paintings from the Viseu workshop, such as the painting of
the ground. The studied cases suggest that chalk needs to be
São Pedro of Tarouca Monastery church, the Viseu Cathedral
processed by grinding before being mixed with a binder thereby
altarpiece, the old Flemish polyptych of the Évora Cathedral and
enabling a thinner ground layer technology, a different method
the Esporão Chapel altarpiece of the same cathedral. Fig. 3b
from it being used directly aer mining and washing, as some
SEM image (BSE mode) of the cross-section from sample 1a of
authors state.36 Fig. 5 illustrates the SEM image (BSE mode) of
the painting of São Pedro of Tarouca Monastery church, shows
the cross-section from sample 2a of the Apresentação no Templo
the remnants of macrofossil, and a detailed image (SE mode) at
painting from the altarpiece of Viseu Cathedral showing a thin
a higher magnication. Fig. 3c also presents a diffractogram of
ground layer with coarser granulometry in the lower part, and
sample 1c from the same painting, highlighting the increased
thinner in the upper area; the SEM image (BSE mode) of the
intensity of calcite (CaCO3) peaks, the main component of the
cross-section from sample 1b of the painting of São Pedro of
chalk calcium carbonate ground layer.
Tarouca Monastery church shows an increased amount of
The SEM image (BSE mode) of the cross-section from sample
calcareous nannofossils having the same granulometry
4a of the Cristo perante Pilatos painting from the Esporão
throughout the ground layer. The analysis made of the chalk
Chapel altarpiece of Évora Cathedral (ME) demonstrates the
from the altarpiece of Viseu Cathedral, when compared to the
presence of macrofossil remnants; the SEM image (BSE mode)
one from the painting of São Pedro of Tarouca Monastery
of the cross-section from sample 3b of the Adoração dos Magos
church, deserves further interpretation. It presents a nely

Fig. 3 (a) SEM image (BSE mode) of cross-section of sample 1b, painting “São Pedro”, Tarouca Monastery church, showing calcisphere; (b) SEM
detail image (SE mode) showing a remnant of macrofossil in sample 1a of the same painting, at high magnification; (c) diffractogram of sample 1c
emphasizing the increased intensity of calcite (CaCO3) peaks, principal component of the chalk calcium carbonate ground layer.

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 4785–4797 | 4789
Analytical Methods
Fig. 4 (a) SEM image (BSE mode) of cross-section of sample 4a of painting “Cristo perante Pilatos”, Esporão chapel altarpiece of Évora Cathedral
(ME), with macrofossils remnants; (b) SEM image (BSE mode) of cross-section of sample 3b, painting “Adoração dos Magos”, altarpiece of Viseu
Cathedral, with macrofossils remnants; (c) Raman spectrum of the compounds analyzed in sample 3b, increasing the intensity of calcite peaks
(1088 cm1, 715 cm1 and 281 cm1) constituent of the chalk calcium carbonate ground layer.
Fig. 5 (a) SEM image (BSE mode) of cross-section of sample 2a,
painting “Apresentação no Templo”, altarpiece of Viseu Cathedral, with
a thin ground layer and different granulometry, coarser in the lower
part and thinner in the top area; (b) SEM image (BSE mode) of cross-
section of sample 1b of painting “São Pedro”, Tarouca Monastery
church, with increased amount of calcareous nannofossils and same
granulometry across the ground layer.
Fig. 6 (a) Cross-section of sample 1b, painting “São Pedro”, Tarouca
Monastery church, with 624.2 mm thick, having an heterogeneity in the
absorption of the binder coming from painting in yellowish color (b)
cross-section of sample 5a of painting “Circuncisão”, Viseu Cathedral
altarpiece (MGV), with a ground layer measuring about 45 mm thick.
ground aspect (sample 5a from the Circuncisão painting from
the Viseu Cathedral altarpiece (MGV), measuring about 45 mm
thick) and apparently with a lower quantity of fossil in the upper
layer while the one of São Pedro of Tarouca Monastery church,
presents a greater thickness (sample 1b has 624.2 mm thick) and
a coarser chalk in the ground layer (Fig. 6).
Chalk material is also an identier of the technique used in
a specic epoch of certain Portuguese painting workshops, as in
the case of the Viseu Workshop. Aer a primary phase of
working with chalk material and using a ground layer tech-
nology with coarser grains in the lower part and ner powder in
4790 | Anal. Methods, 2016, 8, 4785–4797
Paper
the upper area of the ground layer, the second phase of this
workshop technique used calcium carbonate ground layers. The
absence of coccoliths or other microfossils in some of the cases
studied, however, led to the conclusion that powdered
limestone was used instead of chalk, as in the case of the
altarpiece of the church of Freixo-de-Espada-à-Cinta, Bragança
(about 1530, a possible partnership between Vasco Fernandes
and António Vaz (act. 1537–1569)).37 This altarpiece is made of
chestnut oak and prepared with powdered limestone, coarser in
the rst layer and ner in the second layer. Fig. 7 shows an OM
image of the cross-section of sample 6a of the Ecce Homo
painting, presenting a saturated darker tone in the upper area
of the ground layer; the SEM image (BSE mode) of the cross-
section from the same sample presents coarser calcium
carbonate (bigger grains of CaCO3) in the rst layer and ner
powder in the upper area. The altarpiece was recently restored
due to its extreme state of degradation.7,38–40
Despite limestone being an important natural resource in
Portugal, this powdered material was only found in two of the
groups studied. This move, made by the Viseu Workshop, into
using chalk could be a consequence of the difficulty in getting
chalk overseas, since it does not exist as a natural resource in
Portugal, but also in an attempt to explore natural resources
within the geographical proximity, such as the Ançã stone,
Coimbra. This oolitic limestone, much used by the 15th and
16th century sculptors, contains silica (SiO2), since the oolites
are formed around quartz grains.7 The maps of elemental
analysis by SEM-EDS from powdered calcite in the ground layer
of the sample 6a of the Ecce Homo painting from the altarpiece
of the church of Freixo-de-Espada-à-Cinta shows the detected
Ca and Si elements (Fig. 8).
The later work of the workshop evidenced a move towards
the use of gesso sottile in the ground layers of their subsequent
paintings, as in the case of the São Pedro palla made for Viseu
Cathedral (MGV) or the two paintings of saints São Vicente and
Santo António, c. 1550, from Cavernães Church, Viseu, and
attributed to António Vaz, follower of Vasco Fernandes. In Fig. 9
both the OM images of cross-section from sample 7a and
the SEM image (BSE mode) of the Santo António painting in
Cavernães Church show an homogeneous mixture of ller and
binder ground layer; the detailed SEM image (BSE mode) of the
This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

Fig. 7 (a) OM image of cross-section of sample 6a of the painting “Ecce Homo”, altarpiece of the church of Freixo-de-Espada-à-Cinta showing
a saturated darker tone in the top area of the ground layer; (b) SEM image (BSE mode) of cross-section of the same sample; (c) detailed SEM
image (BSE mode) of cross-section of the same sample with calcium carbonate coarser (bigger grains of CaCO3) in the first layer and thinner in
the top area.

3405 cm1 correspond to an elongation nOH of water molecules

Fig. 8 (a) Map of elemental analysis by SEM-EDS from grinded calcite
in the ground layer of the sample 6a of the painting “Ecce Homo”,
altarpiece of the church of Freixo-de-Espada-à-Cinta, detecting Ca
element; (b) map of elemental analysis by SEM-EDS of the same
sample detecting Si element.
same sample shows acicular particles characteristic of
gesso sottile. The semi-quantitative measurements of m-XRD
conducted in the areas of gypsum and anhydrite peaks revealed
the presence of 67% to 91% of gypsum and 9% to 33% of
anhydrite. This analysis also detected the presence of hydro-
cerussite, certainly corresponding to Pb found by the SEM-EDS
analysis. The presence of Pb may be linked with the addition of
lead carbonate to the ground layer. This Pb is associated with
aluminosilicates Mg, K and Fe. Iron, besides being associated
with aluminosilicates, also coexists in grains whose major
constituent is Fe but which also contain a large amount of Pb,
with traces of Mg, Al, S, P, S, K, Ca and Cu; these grains corre-
spond to red ocher, as already veried by OM,41 being the main
factor responsible for the color of the ground layer. In Fig. 10
the spectrum obtained by m-FTIR of the coarser lower part of the
ground layer of sample 7a is largely dominated by bands
related to the presence of dehydrated gypsum 3539 cm1 and
present in the gypsum structural network; 1621 cm1 is due to
deformation of the dOH group of water molecules of the
structural network; the bands at 1139 cm1 match an anti-
symmetric group nSO4 elongations of the band to 1008 cm1
corresponds to a symmetrical elongation nSO4 group, and the
band at 670 cm1 is attributable to deformation of the sulfate
group, dSO4. The proteins with a weak band at 1448 cm1 and
two minor bands at 1546 cm1 and 1683 cm1, are covered by
the coexistence of a typical gypsum band in the spectra; these
two bands relating to N–H deformation and elongation C–N
(amide II) and elongation amides of C]O (amide I), respec-
tively, bands associated with the presence of protein. The
presence of alkyl group bands, are evident in a wide range of
organic compounds, suggesting in this case the occurrence of
oil, material stated in Portuguese treatises as a polisher of
gypsum ground layers;17 the oil is identiable in the bands at
2926 cm1 and 2857 cm1 corresponding to an antisymmetric
and symmetric elongation, respectively nCH alkyl groups
linked, the band related to the elongation nC]O z 1740 cm1
is less detectable, and is only visible with a small shoulder.
These paintings show a homogeneous mixture of gypsum and
binder in the ground layer.
3.3. Binders-absorbent and non-absorbent systems
The difference in coarseness of the powder from the lower part
to the upper part of the ground layer, while having the
mechanical aim of sustaining the panel oscillations, also has
aesthetic intentions with specic absorbency qualities.
The upper area of the chalk ground layer in painting has
been noted in recent studies.19,42 The increased transparency in

Fig. 9(a) OM image of cross-section of sample 7a of painting “Santo António”, Cavernães Church; (b) SEM image (BSE mode) of the same sample
showing an homogeneous mixture of and binder ground layer; (c) detailed SEM image (BSE mode) of the same sample showing acicular particles
characteristic of “gesso sottile”.

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 4785–4797 | 4791
Analytical Methods Paper

Fig. 10 Spectrum obtained by m-FTIR of the coarser lower part of the ground layer of sample 7a largely dominated by bands related to the
presence of dihydrated gypsum: 3539 cm1 and 3405 cm1, 1621 cm1 1139 cm1 match antisymmetric group nSO4 elongations of the band,
1008 cm1 and the band at 670 cm1. The proteins have a weak band at 1448 cm1 and two minor bands at 1546 cm1 and 1683 cm1 while oil
can be identifiable by the bands at 2926 cm1 and 2857 cm1, having a small shoulder at 1740 cm1.

Table 1 Refractive index (n) of ground layer materials; adapted from is activated when it is mixed in an aqueous solution of glue,
ref. 20 which in a concentration of about 10% has a refractive index of
around n ¼ 1.35 (Table 1).20
Refractive
Type Material index (n) When chalk is placed in a mixed solution of linseed oil with
a refractive index n ¼ 1.48, its coating power decreases due to
Pigment/ller Chalk 1.50–1.64 the close refractive index of both materials (of about 0.07).43 On
Aqueous binder Animal glue (diluted to 10%) 1.348 the other hand, if chalk is mixed with animal glue, with
Oil binder Linseed oil 1.484
a greater difference in the refractive index, the mixture improves
its coating power, even while wet. Fig. 11 exemplies the
reconstruction of mixture of chalk with animal glue showing
this upper level when compared to the lower part of the ground the drying of the rst ground layer, even while wet, with an
layer is an optical quality which can be attributed to the improved coating power. The SEM images (BSE mode) of the
penetration of the oil used as medium in the painting layers. reconstruction of Portuguese powdered calcareous rock and of
This oil can be absorbed through the ground layer, due to less the cross-section of the sample 6a of the Ecce Homo painting
porosity in the upper area when compared to the lower part of from the altarpiece of the church of Freixo-de-Espada-à-Cinta
the ground.42 The upper area is also responsible for slow drying, puts has evidence of powdered calcite on its angular faces. The
in order to obtain the singular transparency seen in Flemish coating power of the mixture continues to improve upon drying
paintings.7,19 of the surface since the chalk remains primarily surrounded by
Previous studies show that chalk has a lower refractive index air.20
(n ¼ 1.50–1.64) than the remaining pigments. Its coating power The color parameters were registered in both apertures
LAV and USAV for ground layer reconstructions of glue/chalk,

Fig. 11 (a) Reconstruction of mixture of chalk with animal glue showing the drying of the first ground layer, even while wet, with an improved
coating power (b) SEM image (BSE mode) of reconstruction of Portuguese grind calcareous rock; (c) SEM image (BSE mode) of cross-section of
sample 6a, painting “Ecce Homo”, altarpiece of the church of Freixo-de-Espada-à-Cinta, with calcite grinded showing angular faces.

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Paper Analytical Methods

Table 2 Spectrophotometric analysis parameters registered in apertures LAV for ground layer reconstructions of animal glue (g)/chalk (c), oil (o)/
chalk or both with their amounts (volume (v/v) or grams (gr)). Brightness and color index where analyzed using the CIE systems: CIE L* a* b* (L*
represents the lightness, a* the redness-greenness, b* the yellowness-blueness) and CIELCH (L*C*h – L* specifies lightness, C* denotes
chroma and h defines hue angle, with angular measurement). Lightness has higher values in the chalk/animal glue reconstructions but color
parameters (a* b* and C*) are higher in chalk/oil reconstructions, giving the ground layers a grayish half-tone for painting.

CIE Lab Ch D65/10Deg

Ingredients Amounts Sample CIE L CIE a CIE b CIE C CIE h

Chalk/animal glue 1 : 1 (v/v) 1 83.31 1.54 11.88 11.98 82.62

Chalk/animal glue 3 : 1 (v/v) 2 90.55 0.96 6.91 6.98 82.07
Chalk/oil over chalk/animal glue with 1 : 1 (v/v) 3 72.13 3.79 21.31 21.64 79.91
sizing layer between them
Chalk/oil over chalk/animal glue 1 : 1 (v/v) 4 71.11 3.8 21.41 21.75 79.93
Chalk/oil over chalk/animal glue with 3 : 1 (v/v) 5 71.65 4.59 19.72 20.25 76.91
sizing layer between them
Chalk/oil over chalk/animal glue 3 : 1 (v/v) 6 71.96 4.63 19.64 20.18 76.72
Chalk/oil over chalk/animal glue with 25 gr c + 4.72 gr o; 1c:1/2g (v/v) 7 73.2 4.75 19.04 19.63 75.98
sizing layer between them
Chalk/oil over chalk/animal glue 25 gr c + 4.72 gr o; 1c:1/2g (v/v) 8 75.09 4.36 19.09 19.58 77.14
Chalk/animal glue 1 : 1/2 (v/v) 9 89.7 1.09 7.34 7.43 81.52
Chalk/oil 25 gr c + 4.72 gr o 10 69.98 4.48 19.1 19.62 76.79
Chalk/oil 1 : 1 (v/v) 11 69.22 4.19 20.9 21.32 78.66
Chalk/oil 3 : 1 (v/v) 12 71.86 4.05 19.94 20.35 78.52
Chalk/strong animal glue mixture — 13 clear surface 77.27 2.18 23.66 23.76 84.75
Chalk/rabbit glue — 14 clear surface 78.71 1.28 24.68 24.71 87.02
Chalk/strong animal glue mixture — 13 dark surface 58.93 9.38 24.53 26.26 69.07
Chalk/rabbit glue — 14 dark surface 58.22 9.41 25.16 26.86 69.5

oil/chalk or both, overlaying this last to rst, in order to evaluate
color differences between the samples and to investigate the
reectance percentage. In Table 2 we verify the calculated values
so that: L* > 0, the sample became more luminous; L* < 0, the
sample darkened; a* > 0, the sample became redder; a* < 0, the
sample became greener; b* > 0, the sample became more yellow;
b* < 0, the sample became more blue,29 C* > 0, the sample
became more chromatic and h denotes hue angle. The results
presented in Table 2 show that samples that contain oil are less
luminous, having lower reectance percentage, than animal
glue samples. The colorimetric results showed that the total
change in color is signicant when mixing chalk with oil or with
glue. They conrmed the decreasing refractive index of the
grayish mixture oil/chalk (lower values of a* when comparing to
b*) in contrast with the whitish mixture of glue/chalk (lower
values of a*and b* when comparing to oil/chalk mixture).
Lightness has higher values in the chalk/animal glue recon-
structions but color/chroma parameters (a* b* and C*) are
higher in chalk/oil reconstructions, giving the ground layers
a grayish half-tone for painting.
Animal chalk/glue historical reconstructions made in 1973
by Reis Santos in Laboratório José de Figueiredo (samples 13
and 14) when measured and compared to the above mentioned
reconstructions made by us show that the lightness of the
mixture decreases, probably due to the yellowing of the binder,
also visible by naked eye. These samples are heterogeneous in
color when comparing to the reconstructions made by us.
Results show that darker surfaces of the mixture are less
luminous than clearer areas.
In the reconstructions with overlapping layers, glue/chalk on
the base and oil/chalk on the top, it is possible to achieve an
average slight improvement of lightness when compared to the
oil/chalk single layer reconstruction, proong the utility of this
2-layer structure. Results conrm also data brought by histor-
ical texts referring to the prominence of the white ground layers
with the aging of the paintings.11,44 With aging, shadows and
half-shadows disappear (probably due to the aging of the
binders in the ground layer, as shown by the results of
decreasing lightness of the historical reconstructions analyzed),
and as such, it was probably required by the painters the
search of a grayish half-tone ground, preventing these aging
problems.11,44 These results corroborate the need for a two-
layered glue/chalk and oil/chalk ground structure.
The rst ground layer applied over the panel, aer being
sealed with a sizing layer, is a mixture of chalk and animal glue,
promoting the adhesion of the ground layer to the support.3,4,7,19
Although the distinction between these two layers is sometimes
difficult to recognize in OM, there are cases where this differ-
ence is evident, as in sample 6a of the Ecce Homo painting from
the altarpiece of the church of Freixo-de-Espada-à-Cinta
(powdered limestone) (Fig. 7) or sample 4a of the Cristo perante
Pilatos painting from the Flemish altarpiece of the Esporão
Chapel (ME) (chalk). Fig. 12 contains a diagram which has been
adapted from a pictorial layer structure.21 The diagram shows
the reection of “supercial light” on a white ground layer, thus
bringing a greater level of brightness and luminosity to the
pictorial layer, which is equivalent to a lower layer of chalk and
glue (Fig. 12a); the reection on the “deep light” translucent
background reduces the brightness of the color layer (Fig. 12b);

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Analytical Methods
Fig. 12 (a) Diagram of the reflection of “superficial light” on white
background, giving brightness and luminosity to the pictorial layer,
equivalent to the bottom of chalk and glue, adapted diagram from;21
(b) diagram of the reflection on the “deep light” translucent back-
ground, reducing the amount of reflection and the brightness of the
color layer, adapted diagram from;21 (c) cross-section of sample 4a of
painting “Cristo perante Pilatos”, Altarpiece of the Esporão Chapel (ME)
showing the reflection scheme of “superficial light” of the bottom
proteic ground layer and “deep light” in the oil binder top area of the
chalk ground.
Fig. 13 (a) Cross-section of sample 4a of painting “Cristo perante
Pilatos”, Altarpiece of the Esporão Chapel (ME), showing the thicker
(105.9 mm thick) proteic bottom versus the thinner (30 mm thick) oil
binder top area of the ground layer; (b) OM image cross-section of
sample 4a under ultraviolet light stained (Orange Sypro©) in fluores-
cent orange focusing the proteic area of ground layer.
the cross-section of sample 4a of the Cristo perante Pilatos
painting from the altarpiece of the Esporão Chapel (ME) shows
an historical case of the reection scheme of “supercial light”
from the bottom proteinaceous ground layer and “deep light”
from the oil binder upper layer of the chalk ground. The
example shows the distinction between binders in the ground
layer, oil for the upper layer and animal glue for the level below,
reproducing the search for a different refractive index of the
materials to cover the surface of the support and to obtain
4794 | Anal. Methods, 2016, 8, 4785–4797
Paper
Fig. 14 (a) Cross-section of sample 8a of painting “Morte da Virgem”,
Évora Cathedral old Flemish polyptych, showing the thicker proteic
bottom versus the thinner oil binder in top area of the ground layer; (b)
OM image cross-section of sample 8a under ultraviolet light and
stained (Orange Sypro©) in fluorescent orange, focusing the proteic
area of ground layer.
distinct reecting light effects. In this sample, which is
considered to be complete (due to the wood ber remains of the
support) the thickness of the ground layer mixed with animal
glue is about 105.9 mm, with the thickness of the upper part
mixed with oil being about 30 mm (Fig. 13a). This technical
aspect is understandable, given the fact that particle size, in
conjunction with the concentration, inuences the refractive
index of the covering capacity of the material. In general, the
ner the particles the greater the coating power.20 By applying
a rst layer of coarser chalk bonded with animal glue, the artist
achieved a ground with hydrophilic characteristics i.e. having
a strong affinity for water. At the second stage, to give the
necessary transparency and facilitate the drawing and painting
of the future composition, a powder layer was applied with an
oil binder, providing a compatible surface to optimize the
integration between the painting and the ground layer. This
non-absorbent ground allows for the painting of the following
layers maintaining the hydrophobic effect and promoting the
union between lms, contributing to the cohesion of the
painting.
When observed by OM with ultraviolet light and an emission
lter of 490 nm, sample 4a highlights a medium-rich layer of
higher density in the upper area of the chalk ground, marking
the area of the corresponding ground layer with animal glue
proteins in a uorescent orange color. This test was performed
using a highly sensitive uorescent protein dye observable
between 300/472 nm (excitation) and 490/570 nm (emission)
(Orange Sypro©).22,23 Fig. 13b and 14b show, via OM image,
historical cross-sections from sample 4a of the Cristo perante
Pilatos painting from the altarpiece of the Esporão Chapel (ME)
and sample 8a of the Morte da Virgem painting from the old
Flemish polyptych in Évora Cathedral, under ultraviolet light
and stained in uorescent orange (Orange Sypro©). These
gures focus on the proteinaceous area of the ground layer, thus
distinguishing the thicker proteinaceous bottom from the
thinner oil binder of theupperareas of the ground layers.
This non-absorbent surface area of the chalk ground layer,
when saturated, has the ability to control the penetration of the
descendant oil coming from the painting layers, maintaining
a homogeneous and regular distribution of the deep light effect.
In the examples studied we observe that paintings with different
This journal is © The Royal Society of Chemistry 2016
Paper
granulometry in the ground layer, using oil in the upper area,
retain the binder homogeneously. Painting with a thicker
proteinaceous ground layer using the same granulometry has
a heterogeneity in the absorption of the binder coming from
painting, priming or polishing layers, as is the case with the
altarpiece of Viseu Cathedral, when compared with the painting
São Pedro of Tarouca Monastery church (Fig. 6a).
Mixing the pigments with the binder is essential since it
disintegrates the agglomerates, surrounding each particle of the
binder and thus obtaining a homogeneous mixture, creating
greater plasticity and stability.21
A highly accurate technique of ground layer execution was
required, based on the expertise and traditional knowledge of
Flemish painters and passed to Portuguese masters, as in the
case of the Viseu Workshop.
4. Conclusions
From this work we can conclude that one of the main objectives
of the ground layer is to create the best possible reection of
light, using thin transparent layers to enhance the vibrancy of
the colors painted upon them. This technique was used by
Flemish painters and seems to have been adapted by Portu-
guese painters during the early stages of the Viseu Workshop to
achieve translucent and thin layers with an aesthetic
appearance.
Furthermore, it was observed that coarser calcium carbonate
(chalk or powdered limestone) and glue emulsion are the rst
phase of a ground layer to be applied, aer sizing the panel. A
second, thinly powdered ground layer of the same ller material
was then applied with oil to achieve specic light effects. This
granular difference between the upper area and the lower area is
essential to increase resistance to mechanical movements of the
panel and to control light effects of the ground layer. In addi-
tion, using a thinner ground layer technology facilitates the ease
of application, thereby increasing the speed of handling.
In addition, it was veried that the aim of this ground layer
technique, using oil and proteinaceous binders separately, was
to create a saturated system of a non-absorbent ground layer
with a reective capacity of supercial and deep light. Provided
by the intentional use of oil as a binder, the upper ground layer
was received by an aqueous system of the absorbent ground
layer (using glue as a binder) and permitting a supercial light
reection. Both systems (absorbent/non-absorbent) led to the
reection of supercial and deep light and might have been
used intentionally by the artists to obtain the thinness and
translucency of the grounds. Therefore, it is consistent that the
oily upper layer was nely powdered, and upon being mixed
with oil, its coating power was reduced and this made possible
the reection of the underlying coarser protein ground layer
which was thick and white, taking advantage of “deep light” and
“supercial light” brought by both binders. The results of
the reconstructions corroborate the need for a two-layered
glue/chalk and oil/chalk ground structure, as found for
historical samples of the studied paintings.
The reconstructions prove the less luminous (deep light)
grayish tone achieved when combining oil and chalk when
This journal is © The Royal Society of Chemistry 2016
Analytical Methods
compared to the glue/chalk mixture (supercial light). By
overlapping both layers, with oil/chalk on the top, it is possible
to achieve a slight improvement of lightness when compared to
the oil/chalk single layer, proong the utility of this 2-layer
structure (deep and supercial light). A grayish half-tone of
ground layers combining supercial and deep light for painting
could have been an achievement of the painters of the 15th and
16th centuries.
Finally, to accomplish the durability of the layers, and to
conserve its light, careful grinding combined with specic
quality and compatibility between materials was required.
This constructive technology has enabled these panel
paintings to preserve their quality until today: a Baltic oak
panel, isolated by an aggregating sizing layer and chalk ground
layers, coarser in the base and nely powdered in the upper area
to achieve mechanical resistance and translucent light effects.
We can also conclude that different granulometry of the
powdered limestone medium forming a ground layer did not
bring about the desired results of supercial and deep light
required by the artists since it was only used at one point in
time. Also the use of gesso sottile suggests characteristics of
smoothness and capture of the light closer to chalk provided by
its shield-shaped particles, being the next most used ground
material at the Viseu Workshop, aer chalk.
The characterization of inorganic and organic materials
existing in ground layers, conrmed by the new analytical
results obtained, have led to novel conclusions about the tech-
niques applied to Flemish and Portuguese paintings of the 15th
and 16th centuries. This ground layer technique could have
been the origin of the canvas priming technique, used in the
16th and 17th centuries and thereaer, where the use of oil
overlies the use of proteinaceous ground layers.
Acknowledgements
The authors acknowledge Fundação para a Ciência e Tecnologia
for nancial support (Post-doc grant SFRH/BPD/103315/2014)
through program QREN-POPH-typology 4.1., co-participated by
the Social European Fund (FSE) and MCTES National Fund.
Also wish to acknowledge Dr Irina Sandu for staining protocols,
Dr José Mirão for the text revision, Dr Maria José Oliveira for the
assistance at the m-XRD, Dr Luı́s Dias for the assistance at the
SEM-EDS, Dr Stéphane Longelin for the assistance at the
m-Raman, Dr Catarina Miguel and Dr Ana Cardoso for the
assistance at the m-FTIR.
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