Journal of Cultural Heritage 14 (2013) 499–508

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Original article

Tannins characterization in historic leathers by complementary analytical

techniques ATR-FTIR, UV-Vis and chemical tests
Lina Falcão , Maria Eduarda M. Araújo ∗
Chemistry and Biochemistry Centre, Faculty of Sciences of the University of Lisbon, Campo Grande, Edifício C8, 1749-016 Lisboa, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: This paper presents a complementary analytical approach to characterize vegetable tanning materials
Received 16 December 2011 in historic leathers. It is described the application of two molecular spectroscopic techniques, ATR-FTIR
Accepted 7 November 2012 and UV-Vis, and three specific chemical tests to analyse tannins present in leathers. Acid butanol, nitrous
Available online 14 December 2012
acid and rhodanine colorimetric tests, evaluated both visually and spectrophotometrically, were used
to identify condensed tannins, ellagitannins and gallotannins, respectively. Ten samples of commercial,
Keywords: or laboratory prepared, vegetable tannins and seven new vegetable tanned leathers were also analysed
Leather
and obtained results were used for comparison. The complete analytical procedure was performed, in a
Tannins
ATR-FTIR
semi-micro-destructive scale, using fibres collected from leather. Analysis of ATR-FTIR and UV spectra of
UV-Vis commercial and laboratory prepared vegetable tannins allowed the establishment of the characteristic
Chemical analysis bands of condensed and hydrolysable tannins and, more specifically, gallotannins. These data were used
to confirm the type of vegetable tanning agents used in new leather extracts. The same approach was
used in cultural heritage leathers, supported by the colorimetric tests, since protein degradation products
were co-extracted in aged leathers and interfered in IR spectra.
© 2012 Elsevier Masson SAS. All rights reserved.

1. Research aims 2. Introduction

Vegetable tanned leathers, i.e. leathers produced with plant
materials rich in tannins, are the commonest heritage leathers
in western museum and archival collections. Identification of
leather making process and tanning agents is important to compre-
hend leather technology, degradation susceptibility and condition.
The aim of this study is to improve the knowledge about his-
toric vegetable tanned leathers by presenting a methodology that
overcomes limitations of spot tests previously described for the
characterization of vegetable tanning agents [1]. The use of leather
extracts avoided interferences of coloured degradation products
of finishing materials. ATR-FTIR spectra allowed inequivocal iden-
tification of not only condensed tannins but more important
gallotannins, which was a limitation of the previous work. Ellagi-
tannins were positively identified by a chemical reaction monitored
in the visible region of the electromagnetic spectrum. This com-
bined analytical approach using ATR-FTIR supported by chemical
tests, if necessary, provided a reliable identification of tannins
type.
∗ Corresponding author. Tel.: +351 217500968; fax: +351 217500188.
E-mail address: mearaujo@fc.ul.pt (M.E.M. Araújo).
Vegetable tannins are polyphenolic compounds widely dis-
tributed in plants which have the property to precipitate proteins
[2,3]. Since ancient times, this property has been empirically
explored to transform animal skins, a proteinaceous biomaterial,
into leather [4,5]. The process, termed vegetable tanning, is one
of the oldest known leather making processes and it can be suc-
cinctly described as a treatment of hides/skins with powdered
barks, leaves, wood, fruits, pods or galls, or their extracts, obtained
from different vegetable sources [6]. With this treatment, tradi-
tionally performed in pits, a chemical interaction between collagen
protein (the main constituent of dermis) and tannins present in
vegetable materials is slowly established, generating a very useful
and remarkably non-putrescible material under moist and warm
conditions, termed vegetable tanned leather [5,7].
Vegetable tanned leather was one of the most important pre-
industrial materials in Western and Mediterranean Europe, very
much appreciated and demanded due to its versatility. It was
the main material of a wide range of artefacts and adapted to
very diverse functional needs such as footwear, bookbindings, sad-
dles, harness, liquid vessels, cases (étuis) and caskets coverings or
seating furniture and carriages upholstery. Beyond its utilitarian
function, it was also used as support material for artistic and dec-
orative paintings, wall hangings and screen coverings. Different
ornamental techniques such as dyeing, painting, gilding, moulding,

1296-2074/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.culher.2012.11.003
500 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508
tooling, embroidering, cutting-out, scorching or sewing, have been
often incorporated transforming vegetable tanned leather into a
noble, luxurious and valuable material. One of the European finest
examples is gilt leather (Spanish leather, guadameci, cuoio d’oro), a
silvered and then “golden” varnished, painted and tooled/moulded
vegetable tanned leather, mostly produced between the 16th and
18th centuries [8,9]. Despite a great number of utilitarian, deco-
rative and artistic purposes may be depicted by vegetable tanned
leathers, reflecting changes of custom, taste, economic and even
social organization it is often disregarded and neglected its impor-
tance as heritage material.
The versatility of vegetable tanned leathers is a consequence
not only of the different and possible combinations of raw materi-
als used (skins from different animal species and tanning materials
from diverse vegetable sources) but also of the leather making pro-
cess itself (operations and time requirements). Vegetable tanned
leather can be thick, rigid and compact (heavy leather), such as sole
leather, or thin and pliable (light leather), such as morocco leather
(i.e. grained goat skin commonly tanned with sumac leaves). Heavy
leathers are made from hides (skins from large animals) and require
the absorption of about 30–40% (per skin dry weight) of tannins,
which, before the 19th century industrialization, might take about
12 to 24 months to accomplish. Light leathers are produced with
skins from young and small animals requiring the absorption of
15–20% of tannins (per skin dry weight), which was often achieved
in few days or weeks [10–12].
The vegetable sources with importance for leather making were
confined to a limited number of plant materials. Different indige-
nous plants materials have been traditionally used in Europe: barks
from birch (Betula spp.), willow (Salix spp.), larch (Larix spp.) and
spruce (Picea spp.) were used in northern Europe and Russia; barks
from various species of oaks (Quercus spp.) widely used throughout
Western Europe; leaves from sumac shrub (Rhus coriaria), valonia
(Quercus aegilops) acorns and oak galls from Quercus infectoria in
Mediterranean region [13,14]. Nevertheless, oak bark and sumac
were the most important/esteemed tanning materials in Europe,
the former in the production of firm leather with a wide range
of common uses (footwear, saddlery, bookbinding, etc.), the later
mainly used in the manufacture of fancy, pliable and light coloured
leathers, such as morocco leathers, appreciated in exclusive items
[10,12,15].
In European post-medieval period, the increasing demand for
leather led to a shortage of native tanning materials encouraging
the search and importation of alternatives plants. Among others,
the use of mimosa (Acacia mearnsii) bark and quebracho (Schinopsis
lorentzii) wood had became significant throughout the 19th–20th
century remaining even nowadays, together with chestnut (Cas-
tanea sativa) wood, the main used vegetable tanning materials
[5,13,16].
Vegetable tanning materials differ greatly in chemical consti-
tution and tanning properties. Chemically, tannins are complex
and heterogeneous group of polyphenolic secondary metabolites
biosynthesized by higher plants with molecular weights ranging
from 500 to over 20,000 Da [3]. According to the main polyphe-
nolic compounds present, they are classified in condensed tannins
(also named proanthocyanidins) and hydrolysable tannins, which
comprise two subclasses, gallotannins and ellagitannins [16–18].
In general, only one class of tannins is predominant in each veg-
etable tanning material (Table 1). Oak bark is an exception [1,18].
Condensed tannins are oligomeric or polymeric flavonoids and
hydrolysable tannins consist of a polymer containing a polyol core
(d-glucose is the commonest) multi-esterified with gallic acid (gal-
lotannins) or its oxidized derivative, ellagic acid (ellagitannins).
Classification and examples of different chemical structures of
tannins were extensively reviewed by Khanbabaee and van Ree
[3].
This study aims to improve the knowledge about historic veg-
etable tanned leathers and vegetable tanning materials used in
leather making. This information is essential to understand leather
composition, technology, degradation susceptibility or condition
in order to carry out suitable conservation and treatments, and
if necessary, to select appropriate conservation vegetable tanned
leathers.
On our ongoing work on the chemical class identification of
tannins in historic leathers we analysed aqueous-acetone leather
extracts by complementary analytical approach that combines
information provided by Fourier transform infrared spectroscopy
(FTIR) in attenuated total reflectance (ATR) mode and ultraviolet-
visible spectroscopy (UV-Vis), with the results of three chemical
tests. The analysis of leather extracts by an adaptation of rhoda-
nine, nitrous acid and acid butanol chemical tests, initially used to
analyse tannins in plant tissues [2,17] and formerly modified to be
used as spot tests in leather fibres [1], is also here described.
3. Experimental
3.1. Chemicals
The reagents used were sodium nitrite (Fluka), potassium
hydroxide (Fluka), rhodanine (2-thio-4-ketothiazolidine, Alfa
Aesar), ferric ammonium sulphate (Merck) and 37% hydrochloric
acid (Panreac). Gallic, ellagic and tannic acids were provided by
Sigma-Aldrich. All solvents used were of analytical grade.
3.2. Equipment
UV-Vis spectra were acquired on Shimadzu 1603 double beam
spectrophotometer with 1 cm quartz cells. FTIR analyses were
carried out using a Nicolet 6700 spectrometer (Thermo, Italy)
equipped with a Smart Multi-Bounce Horizontal Attenuated Total
Reflectance (HATR), a device with a zinc selenide (ZnSe) crystal.
3.3. Samples and extracts preparation
3.3.1. Vegetable samples
Different vegetable tanning materials used for leather produc-
tion considered to be representative of the main chemical classes
of tannins were analysed (T1-T10, Table 1).
Seven tannins (T1-T7), all in powder form, were kindly provided
by different institutions, as indicated in Table 1. Three vegetable
tanning materials (T8-T10) were prepared at the laboratory. T8 and
T9 were obtained from plants collected in the northeast of Portu-
gal, in Foz Côa and Penamacor, respectively. T10 was obtained from
oak tree belonging to the Botanical Garden of the University of Lis-
bon. The botanical species that provided T8-T10 were identified by
Dr. Otília Correia from Plant Biology Department, Faculty of Sci-
ences of the University of Lisbon (FCUL). Collected plant materials
were dried at room temperature, protected from light, for at least
4 weeks and then milled. Lyophilized aqueous extracts were pre-
pared (extraction for 24 hours, at room temperature, followed by
centrifugation and supernatant filtration).
Each tanning material was dissolved in distilled water
(10 mg/mL), then filtered through a 0.45 ␮m syringe PTFE filter and
filtrates collected for analysis.
3.3.2. New leather samples
Seven vegetable tanned leathers (L1-L7) recently produced
with diverse known vegetable sources were also kindly supplied
(Table 1). Five leather samples were produced with one single veg-
etable tanning material: mimosa (L1), valonia (L2), sumac (L3), tara
(L4) and oak bark from Quercus petraea (L7). L5 was tanned with
sumac and retanned with a mixture of chestnut and quebracho. L6,
 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508 501

Table 1
Samples description.

Sample Description Chemical class of tannins

[15,16]

Vegetable tanning T1 Mimosa, sulphited commercial extract, orange-brown powder, FILK, Germany Condensed tannins
materials T2 Mimosa, commercial extract, pale yellow powder, CTIC, Portugal Condensed tannins
T3 Quebracho, sulphited commercial extract, dark red powder, FILK, Germany Condensed tannins
T4 Chestnut, commercial extract, dark brown granulate powder, CTIC, Portugal Ellagitannins
T5 Valonia, commercial extract, pale grey powder, FILK, Germany Ellagitannins
T6 Tara, milled pods of Caesalpinia coriaria, pale yellow powder, FILK, Germany Gallotannins
T7 Sumac, milled leaves of Rhus sp., pale green granulate, FILK, Germany Gallotannins
T8 Sumac, lyophilized aqueous extract from milled Rhus coriaria leaves collected in Foz Côa Gallotannins
(Portugal), pale green powder
T9 Rebollo (syn pyrenean) oak bark, lyophilized aqueous extract from milled Quercus pyrenaica Ellagitannins, condensed
bark collected in Penamacor (Portugal), brown powder tanninsa
T10 English (syn pedunculate) oak bark, lyophilized aqueous extract from milled Quercus robur Ellagitannins, condensed
bark collected in Lisbon (Portugal), brown powder tanninsa

New vegetable tanned L1 Calf skin tanned with mimosa, UK

leathers L2 Calf skin tanned with valonia, FILK, Germany
L3 Goat skin tanned with sumac, FILK, Germany
L4 Calf skin tanned with tara, FILK, Germany
L5 Calf skin tanned with sumac, retanned with chestnut and quebracho, UK
L6 Calf skin tanned with sumac, retanned with aluminium salts, UK
L7 Cattle grain split tanned with oak bark from Quercus petraea, FILK, Germany

Historical leathers HL1 Bookbinding, 19th century, private owner, Portugal

HL2 Upholstery, dark brown plain leather, 19th century, Germany
HL3 Upholstery, brown morocco leather, 19th century, National Palace of Ajuda, Portugal
HL4 Upholstery, dark green morocco leather, 19th century, National Railway Museum, Portugal
HL5 Upholstery, gilt leather, 18th century, National Palace of Sintra, Portugal
HL6 Upholstery, dark brown tooled leather, 18th century, Palace of the Dukes of Bragança, Portugal
a
Experimental data.

the only available commercial sample, as archival quality leather,
was tanned with sumac and retanned with aluminium salts.
3.3.3. Historic samples
Six different heritage leathers dated from the 17th to 19th
century were analysed (Table 1, Figs. 1–3): one 19th century
bookbinding leather (HL1) and five different types of upholstery
leathers — a plain one (HL2), two morocco leathers (HL3, HL4), a
gilt leather (HL5) and a tooled decorated leather (HL6). As indi-
cated in Table 1, HL3-HL6 samples were collected from seating
furniture of different Portuguese museum institutions. In partic-
ular, both HL3 and HL4 leather objects belonged to King Luis I of
Portugal (1861–1889).
were recorded from 4000 to 600 cm−1 by averaging 128 or 512
scans, T1-T10 and L1-L7 or HL1-HL6 respectively, at a spectral res-
olution of 4 cm−1 .
3.5. UV-Vis spectra
UV-Vis spectra were recorded between 200–700 nm. Tannins
spectra were acquired using 10 ␮L aliquot of a aqueous tannin
solution (10 mg/mL) diluted in 5 mL of distilled water and, if nec-
essary, re-diluted. Spectra from leather extracts were acquired
using the aqueous solution obtained after evaporation of aliquots
of aqueous-acetone extracts, 20 or 100 ␮L for new and historic
leathers respectively, and re-dissolution in 5 mL of distilled water.

3.3.4. Tannins extraction from leather samples
Extracts were prepared by using finely cut leather fibres col-
lected in an inconspicuous area of the flesh side (fibres from the
reticular layer). Tannins were extracted from leather fibres with
aqueous-acetone solution (1/1, v/v) in capped vials (ratio: 10 mg
leather fibres per mL of solution), under continuous magnetic stir-
ring, at room temperature for 48 hours. The extracts were then
filtered through 0.45 ␮m PTFE syringe filters and filtrates kept
refrigerated.
3.3.5. Extract hydrolysis
For hydrolysis, 1 mL extract aliquot was collected and evapo-
rated to dryness. The residue was dissolved in 1 mL of 2 N H2 SO4
and heated for 10 hours at 100 ◦ C in a nitrogen atmosphere. The
obtained hydrolysed extract was analysed by nitrous acid test and
rhodanine test.
3.6. Qualitative chemical tests on extracts
3.6.1. Acid butanol test for condensed tannins detection
Acid butanol test was performed according to the procedure
described in literature [2] with a slight modification. Briefly two
solutions were prepared: acidic butanol by mixing 95 mL of n-
butanol with 5 mL of 37% HCl and 2% (w/v) ferric ammonium
sulphate [FeNH4 (SO4 )2 .12H2 O] in 2 N HCl.
The test was performed in a small capped glass vial. Firstly,
2.4 mL of acidic butanol was added to the residue obtained from
the evaporation of 400 ␮L extract. Then 40 ␮L of ferric ammonium
sulphate solution was added, the mixture vortexed and spectrum
was collected between 400–700 nm. Then, the mixture was heated
for 50 minutes at 100 ◦ C and, after cooling, the spectrum was col-
lected again. The appearance of a band with a maximum in the
range 530–550 nm indicates the presence of condensed tannins.

3.4. ATR-FTIR spectra
An aliquot of filtrates with 20 ␮L was required for each mea-
surement. Each liquid sample was deposited on the surface of ATR
crystal and evaporated to dryness under nitrogen flow. Spectra
3.6.2. Nitrous acid test for ellagic acid and ellagitannins detection
Nitrous acid test was adapted from the procedure developed
by Wilson and Hagerman [19]. Both aqueous-acetone extract and
hydrolysed extract of leathers were tested for ellagic acid by nitrous
acid test. Extracts were cooled in an ice bath for 10 minutes to
502 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508
Fig. 1. Morocco leathers upholstery: a: “chaise longue” from the National Palace of
Ajuda (HL3); b: wing chair from the National Railway Museum (HL4).
allow the precipitation of ellagic acid. 150 ␮L aliquot of the lower
layer (enriched with ellagic acid) was collected and evaporated for
analysis. In a small glass vial, 2 mL of pyridine was added to the
residue and vortexed. Then 150 ␮L of 37% HCl was added and the
Fig. 2. Gilt leather upholstery from the National Palace of Sintra (HL5).
Fig. 3. Portuguese tooled leather from the Palace of the Dukes of Bragança (HL6).
mixture heated for 5 minutes at 30 ◦ C. Five minutes later, 150 ␮L of
1% (w/v) sodium nitrite (NaNO2 ) was added, the reaction mixture
vortexed to complete homogenization and absorbance was imme-
diately read at 538 nm (Abs538 , t0 ). The reaction mixture was kept
at 30 ◦ C and 36 minutes later absorbance (Abs538 , t36 ) was recorded
again. The difference between Abs538 ,t0 and Abs538 ,t36 is dependent
on the ellagic acid content in the sample. An increase of ellagic acid
content in analysed extracts indicates the presence of ellagitannins.
Positive result:
[(Abs538 ,t36 – Abs538 ,t0 ) hydrolysed/(Abs538 ,t36 – Abs538 ,t0 )
non-hydrolysed] > 1.
3.6.3. Rhodanine test for gallic acid and gallotannins detection
Rhodanine test was adapted from the procedure developed
by Inoue and Hagerman [20]. Both aqueous-acetone extract and
hydrolysed extract of leathers were tested for gallic acid by rhoda-
nine test. In a small glass vial 150 ␮L of 0.667% (w/v) rhodanine in
methanol were added to the residue obtained from the evaporation
of 100 ␮L extract. The mixture was vortexed and 5 minutes later
150 ␮L of 0.5 N aqueous KOH were added. To the hydrolysed extract
0.5 N KOH was added until a basic pH was obtained. Then, the mix-
ture was diluted with distilled water to a final volume of 2.5 mL
and kept at room temperature. Five minutes later, the absorbance
was read at 520 nm. The unequivocal presence of gallotannins is
indicated by a significant difference (> 4) between the absorbance
of the hydrolysed extract regarding the non-hydrolysed extract.
3.7. Chemical spot tests
Powdered vegetable tannins and fibres of leathers were charac-
terized by spot tests as described elsewhere [1].
4. Results and discussion
4.1. Spectroscopic analysis
4.1.1. ATR-FTIR
FTIR in the mid-IR region has been described as useful analyti-
cal tool for the molecular characterisation of commercial available
tannins, both for tanning [21,22] and oenology [23]. In cultural her-
itage field, FTIR studies of tanning materials have been performed
in bookbinding leathers [24,25] and parchment [26] samples.
 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508
Fig. 4. Mid-IR spectra obtained in ATR of vegetable tanning materials: a: commercial
mimosa (T1); b: commercial valonia (T5); c: sumac (T8); d: rebollo oak bark (T10).
Some authors [21,23,24,27] showed that the fingerprint region
of hydrolysable tannins presents an absorption pattern distinct
from condensed tannins. In this study, the region 1750–700 cm−1
was considered the most informative and carefully examined.
Tables 2 and 3 list the principal bands of ATR-FTIR spectra.
Regarding vegetable tanning materials (Table 2) and new leathers
extracts (Table 3), all showed five main common absorption bands
in the 1750–700 cm−1 range: 1615-1606, 1518-1507, 1452-1446,
1211-1196, 1043-1030 cm−1 (Fig. 4).
Vegetable tanning materials classified as condensed tannins,
T1-T3, showed the following characteristic bands: 1288-1282,
1162-1155, 1116-1110, 976 and 844-842 cm−1 . All hydrolysable
tannins, T4-T8, presented bands at 1731-1704 and 1325-
1317 cm−1 . The gallotannins sub class, T6-T8, presented three
distinctive bands at 1088-1082, 872-870 and 763-758 cm−1 .
Results agreed well with literature data, showing that tannins
from different vegetable sources present characteristic common
bands: four strong bands, two of them 1615-1606 cm−1 and
1452-1446 cm−1 assigned to aromatic ring stretch vibrations and
the other two at 1211-1196 cm−1 and 1043-1030 cm−1 assigned
to stretch vibrations of C–O bond. In T4 and T5 samples, the
∼1200 cm−1 band of the C–O stretch vibration was overlapped
by an intense band at 1184 and 1177 cm−1 respectively. Tannins
also present another weak band at 1518-1507 cm−1 due to skeletal
vibration of the aromatic rings [21,22,28].
Condensed tannins are clearly identified by the presence of
three strong bands at 1288-1283 cm−1 , 1160-1155 cm−1 and 1116-
1110 cm−1 and two other weak bands at 976 and 844-842 cm−1 .
These bands are absent in the spectra of gallo- and ellagitan-
nins. The 1288-1283 cm−1 indicates a characteristic feature for
503
the flavonoid-based tannins. These bands can be assigned to the
ethereal C–O asymmetric stretching vibration arising from the
pyran-derived ring structure of this class of tannins [27,28]. Spectra
of L1 confirmed the presence of condensed tannins (Table 3).
Hydrolysable tannins, gallo- and ellagitannins, present two
characteristic strong bands at 1731-1704 cm−1 and 1325-
1317 cm−1 , the former assigned to the stretching vibration of the
carbonyl function and the later to the symmetric stretching of the
C–O bond of the ester function [27,28]. Further, in all samples where
gallotannins were present it was evident three more characteristic
bands of medium to weak intensity and which can be considered
marker bands for this subclass of tannins: 1088-1082, 872-870 and
763-758 cm−1 .
When the leather is tanned with tannins from the three classes,
the first vegetable tanning agent used tends to dominate the leather
character [5]. This is quite evident in L5 extract leather, which was
firstly tanned with sumac and than retanned with a mixture of
chestnut and quebracho.
Ellagitannins (T4, T5, T9 and T10) showed the mentioned
characteristic bands of vegetable tannins (1615-1606, 1518-1507,
1452-1446, 1211-1196, 1043-1030 cm−1 ) and the characteristic
bands of the hydrolysable chemical class (1731-1704 cm−1 and
1325-1317 cm−1 ). However, no particular band was found for this
class. Identification only can be made by checking the absence of
the characteristic bands of gallotannins.
ATR-FTIR spectra of historic leathers extracts were also
investigated (Table 3) and HL1-HL4 showed unequivocally the
characteristic bands of tannins at 1610-1612, 1447-1448, 1204-
1207 and 1043-1032 cm−1 . The weak band near 1518 cm−1 is
visible only in the spectra of HL2, HL3 and HL4 and is hidden by
other more intense bands in HL1, HL5 and HL6. In an attempt
to identify the type of tannin the two regions characteristic of
hydrolysable tannins, 1731-1704 and 1325-1317 cm−1 , were inves-
tigated. Both HL3 and HL4 samples have bands in these two regions
of the spectra, which allow classifying them as hydrolysable (Fig. 3).
The examination of HL1 and HL2 spectra reveals the existence
of a band characteristic of the hydrolysable tannins, 1707 and
1704 cm−1 respectively. A shoulder at 1325 cm−1 was visible in HL2
spectra. HL1-HL4 spectra present the three characteristic bands of
gallotannins (Table 3, Fig. 5).
Despite the lack of evidence of the band near 1325 cm−1 for HL1,
it is possible to classify these four samples as hydrolysable, possi-
bly of the gallotannin type, although the presence of ellagitannins
cannot be excluded. In three samples, HL1, HL3 and HL4 the char-
acteristic bands of condensed tannins are absent. In sample HL2,
two bands of medium intensity at 1287 cm−1 and 1114 cm−1 , char-
acteristic of condensed tannins, are visible, so it can be assumed
that in HL2 a mixture of tannins is present. Regarding samples HL5
and HL6 the careful examination of the spectra does not allow to
find all the characteristic bands of the tannins. A very strong band
at 1642 and 1646 cm−1 in HL5 and HL6 respectively, attributed to
the amide band of the peptides due to collagen degradation, also
co-extracted, hides the tannin band at 1612 cm−1 , leading only to
the visibility of the band at 1452 and 1441 cm−1 respectively. This
fact indicates that these two leathers are deteriorated and the FTIR
bands of those peptides overlap the bands of the tannins. Since
it was not possible to unequivocally identify the existence of veg-
etable tannins, the weak bands at 1707, 1712, 1319 and 1322 cm−1 ,
which could indicate the presence of hydrolysable tannins, they can
also derive from fats used in lubrication that were extracted in a
small extent.
4.1.2. UV-Vis
UV-Vis spectroscopy has been previously used to analyse
vegetable tannins and to distinguish hydrolysable tannins from
condensed tannins [21,22].
504 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508

Table 2
ATR-FTIR characteristic bands of vegetable tanning materials.

Main vibrational bands of vegetable tanning materials (wave numbers, cm−1 , intensity)

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Marker bands spectral

mimosa mimosa quebracho chestnut valonia tara sumac sumac oak bark oak bark intervalsa

Tannins’ compounds
1612 s 1614 s 1608 vs 1606 m 1606 ms 1609 ms 1609 m 1610 m 1612 s 1609 vs 1615-1606 (m-vs)
1507 m 1508 m 1518 ms 1508 m 1507 w 1516 w (sh) 1513 mw 1516 w (sh) 1517 w 1518 mw 1518-1507 (w-m)
1452 ms 1452 ms 1451 s 1451 ms 1448 m 1446 m 1448 m 1446 m 1448 m 1448 m 1452-1446 (m-s)
1200 ms 1196 ms 1209 s (sh) a) a) 1204 vs 1202 vs 1200 vs 1211 m 1207 m 1211-1196 (m-vs)
1030 vs 1030 vs 1044 s 1038 vs 1043 vs 1034 ms 1037 vs 1033 s 1042 s 1041 s 1043-1030 (m-vs)

Condensed tannins
1285 m 1289 m 1283 vs 1287 m (sh) 1288 m 1288-1282 (ms-vs)
(sh) (sh)
1157 s 1155 s 1160 s 1162 s 1162 s 1162-1155 (s)
1111 s 1115 s (sh) 1116 vs 1116-1110 (s-vs)
976 w 976 w 976 w 976 (w)
842 m 842 m 844 m 844-842 (w)

Hydrolysable tannins
1731 m 1731 s 1704 m 1713 m 1716 m 1716 w 1718 w (sh) 1731-1704 (m-s)
1320 m 1317 s 1321 s 1325 m 1321 s 1320 s (sh) a) 1325-1317 (m-s)

Gallotannins
1088 m 1083 m 1083 ms 1088-1082 (m)
872 w 871 w 870 w 872-870 (w)
761 m 759 w 758 mw 763-758 (w-m)

vs: very strong; s: strong; m: medium; w: weak; sh: shoulder; a): overlapped.
a
Collected data.

All analysed samples were transparent in the visible region. Data

Fig. 5. Mid-IR spectra obtained in ATR of: a: sumac (T8); b: HL3; c: HL4.
obtained in this study shows the existence of a characteristic UV
absorption pattern for the different types of tannins analysed. The
wavelength of the maximum absorbance (␭max ) and the respective
inflection point (␭min ) are presented in Table 4. Their characteristic
absorption bands are consistent with those described in literature
[21].
Spectra of mimosa and quebracho samples, T1-T3, classified as
condensed tannins, presented strong absorption around 200 nm,
an inflection point (␭min ) between 258–259 nm and ␭max between
279–281 nm. Spectra of ellagitannins samples, T4 and T5, are not
very informative only presenting a strong broad absorption around
200 nm with a shoulder around 235 nm. Gallotannins, T6-T8, pre-
sented two characteristic absorption maximums, ␭max1 around
212 nm and ␭max2 around 275 nm, with distinctive inflection point
around 242 nm. These values are almost identical to the tannic acid
(pure gallotannin), which presents a ␭max1 at 214 nm and ␭max2 at
275 nm. Extract obtained from rebollo oak bark (T9) and English oak
bark (T10) present strong absorption near 200 nm and a shoulder
around 277 nm.
L1 extract obtained from mimosa tanned leather, showed a sim-
ilar absorption pattern to condensed tannins, T1-T3. L2 extract
obtained from valonia tanned leather, unexpectedly presented a
␭max at 214 nm e two shoulders, around 263 and 361 nm. This
spectrum can be interpreted as the result of a degradation process
of the valonia tannins. Ellagic acid, the monomer of ellagitannins,
absorbs strongly around 200 nm with two ␭max at 255 and 365 nm.
As a consequence, the spectra recorded of L2 extract is the sum of
the absorption of all species present i.e., the tannin, the monomer
ellagic acid and free gallic acid (␭max1 = 214 nm and ␭max2 = 276 nm)
from the hydrolysis of tannins, leading to a spectra without dis-
tinct bands. L3 extract, obtained from a leather tanned with
sumac, present ␭max1 = 213 nm, ␭max2 = 275 nm and ␭min = 242 nm.
Similar results were obtained with L4 and L5 samples. Neverthe-
less, L5 spectrum presented some deviation in inflection point
(␭min = 250 nm) that could be interpreted to the presence of quebra-
cho condensed tannins, since this material exhibits a ␭min = 258 nm.
L6 extract, obtained from a leather sample tanned with sumac
and a mineral agent, showed a UV absorption spectrum similar
Table 3
ATR-FTIR characteristic bands of new and historical leather extracts.

L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508

Marker bands spectral Vibrational bands of leather extracts (wave numbers, cm−1 , intensity)
intervals
L1 L2 L3 L4 L5 L6 L7 HL1 HL2 HL3 HL4 HL5 HL6

Tannins’ compounds
1615-1606 (m-vs) 1615 vs 1610 s 1611 m 1610 ms 1611 ms 1610 m 1613 s 1612 vs 1611 vs 1611 ms 1612 ms a) a)
1518-1507 (w-m) 1507 m 1516 w 1516 w (sh) 1515 w (sh) 1514 w 1512 w 1520 w a) 1518 m 1518 w (sh) 1514 w (sh) a) a)
1452-1446 (m-s) 1453 ms 1448 m 1447 mw 1447 ms 1448 m 1448 m 1452 s 1448 s 1447 ms 1447 ms 1448 m 1447 m 1448 ms
1211-1196 (m-vs) 1196 m 1205 vs 1199 vs 1205 vs 1203 vs 1203 vs 1196 vs 1207 vs 1206 s 1205 vs 1204 vs 1205 mw 1203 m (sh)
1043-1030 (m-vs) 1031 s 1041 ms 1031s 1035 s 1034 s 1035 s 1045 s 1040 s 1041 m 1035 s 1032 s

Condensed tannins
1288-1282 (ms-vs) 1285 ms 1285 s 1287 ms 1283 m
1162-1155 (s) 1159 s 1157 w (sh) a)
1116-1110 (s-vs) 1114 s 1111 mw (sh) 1110 ms 1114 m 1114 w (sh) a)
976 (w) 976 w 976 w (sh) 979 w
844-842 (w) 843 842

Hydrolysable tannins
1731-1704 (m-s) 1705 w (sh) 1712 s 1708 m 1705 ms 1711 ms 1712 ms 1711 s 1707 s 1704 m 1708 ms 1704 ms 1707 sh
1325-1317 (m-s) 1326 s (sh) 1320 s 1321 s 1319 s 1326 m (sh) a) 1325 s (sh) 1324 s 1322 s 1319 1322 sh

Gallotannins
1088-1082 (m) 1088 mw 1094 m 1096 m 1094 m 1097 ms 1096 m 1092 m 1093 mw 1094 ms
872-870 (w) 870 w 875 w 873 w 874 w 873 w 874 w 871 w 870 w
763-758 (w-m) 763 w 758 mw 762 w 763 w 765 w 761 mw 765 w 762 m 760 w

vs: very strong; s: strong; m: medium; w: weak; sh: shoulder; a): overlapped.

505
506 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508

Table 4
UV and chemical tests data of vegetable samples and leathers.

Samples UV (nm) Chemical analysis

␭max1 , ␭min , ␭max2 , (sh)
Acid butanol Nitrous acid Rhodanine
spot test (extract) spot test (extract) spot test (extract)

T1 (mimosa) –, 259, 279 ++ (++) – –

T2 (mimosa) –, 259, 279 ++ (++) – –
T3 (quebracho) –, 258, 281 ++ (++) – –
T4 (chestnut) –, –, – (235) – (–) + +
T5 (valonia) –, –, – (235) + (tr) + +
T6 (tara) 215, 241, 274 + (tr) – ++
T7 (sumac) 207, 243, 277 – (–) – ++
T8 (sumac) 212, 243, 275 + (tr) – ++
T9 (rebollo oak bark) –, –, – (278) + (+) + +
T10 (English oak bark) –, –, – (277) + + –

L1 (mimosa) –, 260,279 ++ (++) – (–) – (–)

L2 (valonia) 212, –, – (263,361) – (–) + (++) – (+)
L3 (sumac) 213, 242, 275 – (–) – ++
L4 (tara) 213, 241, 273 + (tr) – +
L5 (sumac + chestnut + quebracho) 204, 250, 275 + (+) + (++) + (++)
L6 (sumac + Al salts) 212, 252, 278 – (–) – (–) + (++)
L7 (oak bark) –, –, – (279) + (+) + (+) – (–)

HL1 207, 240, 267 (363) + (+) – (++) + (++)

HL2 –, 253, 276 + (+) – (–) – (+)
HL3 214, 241, 275 (365) – (–) – (–a ) ++ (++)
HL4 213, 241, 274 – (–) – (–a ) ++ (++)
HL5 –, –, – (256,368) + (–) – (–a ) – (–)
HL6 –, –, – (284) – (–) – (–) – (–)

++: strongly positive; +: positive; tr: traces; –: negative.

a
Ellagic acid detected.

to the gallotannins with ␭min and ␭max2 auxochromically dis-
placed (higher wavelengths) possibly due to the presence of a small
amount of aluminium salts that were co-extracted. The extract of
L7, an oak bark tanned leather, only presented a shoulder around
279 nm, similar to T9 and T10.
Regarding historic samples spectra (Table 4) all samples pre-
sented a strong absorbance around 200–214 nm. In that range, only
HL3 and HL4 showed a ␭max1 . HL1 to HL4 spectra presented a ␭min
and a ␭max2 in the region ranged from 260 nm to 280 nm. In of
HL5 and HL6 spectra, only shoulders were registered. From these
results, it is possible to state that only HL3 and HL4 spectra show
the presence of gallotannins.
Results here presented demonstrate that UV spectra clearly
indicates the presence of gallotannins and condensed tannins in
aqueous-acetone extracts obtained from leathers tanned exclu-
sively with one of these types of tannins. UV spectra obtained from
samples containing ellagitannins, mixtures of different types of
tannins or combined tannages are more difficult to interpret.
4.2. Qualitative chemical analysis
The results of chemical tests — acid butanol, nitrous acid and
rhodanine — are presented in Table 4. These chemical and col-
orimetric tests have been previously described as spot tests to
characterize the chemical class of tannins present in leather fibres
[1]. In this study, all powdered vegetable tanning materials and
leather fibres collected from the reticular layer samples were tested
by spot tests.
However, since results are evaluated only visually, the identi-
fication of tannins is difficult in coloured or aged leathers [1]. In
fact, the existence of coloured substances in the leather, either
a colorant or a degradation product of the leather itself, may
give ambiguous results. To overcome these spot tests limitations,
chemical analysis of historic leathers, HL1-HL6, was also per-
formed on aqueous-acetone extracts and evaluation was achieved
by absorbance reading at established wavelengths (Experimen-
tal, 3.6.). When necessary, for comparative purposes or when spot
test results were unexpected, vegetable tannins samples were also
investigated using this methodology.
4.2.1. Acid butanol test for condensed tannins detection
Acid butanol test is specific for the detection of condensed
tannins [2,17]. In this test, the formation of red-coloured antho-
cyanidins with an absorbance maximum around 530–550 nm is
an indication of the presence of condensed tannins. This coloured
product is specifically formed due to the oxidative depolymerisa-
tion of proanthocyanidins. The red colour can vary significantly
depending on the anthocyanidin formed [2]. Nevertheless, all
species absorb at the mentioned wavelength.
Acid butanol spot test gave, as expected, unambiguous positive
and strong result with T1-T3 and positive with T9 and T10 (Table 4).
In samples T5, T6 and T8, which were described in literature as
a source of hydrolysable tannins [1,18], traces of condensed tan-
nins were detected. Positive results were observed in L1, L5 and L7.
When historic leathers were investigated by spot tests, condensed
tannins could only be clearly identified in HL1 and HL2. HL5 and HL6
results were difficult to interpret since they are very dark coloured
materials. Reinvestigation of historic samples using the aqueous-
acetone extracts showed unequivocally that condensed tannins are
only present in HL1 and HL2 (Table 4).
4.2.2. Nitrous acid test for ellagitannins detection
Ellagitannins are characterised by the presence of hexahydrox-
ydiphenoyl (HHDP) esters of a glucose polyol (the most common).
HHDP is formed through oxidative coupling between two gallic
acid units which spontaneously lactonize to form ellagic acid. Acid
hydrolysis of ellagitannins will release HHDP units with the subse-
quent formation of ellagic acid [2].
Ellagitannins react slowly with nitrous acid forming a purplish
or bluish product [1,19]. The same reagent can also be used to
specifically detect ellagic acid and indirectly identify ellagitannins
[2,17,19]. In this later case a red product is formed which can be
detected spectrophotometrically at 538 nm. Since ellagic acid can
be present in aged samples as a degradation product of tannins,
 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508
particularly as result of gallotannins and ellagitannins hydrolysis
and free gallic acid oxidation [29], it is important to assure that, if
detected, ellagic acid is due only to ellagitannins presence. Extracts
were therefore hydrolysed and a positive result is assumed if the
content of ellagic acid is greater in the hydrolysed sample.
As expected, ellagitannins were detected by spot test in T4, T5,
T9 and T10 samples. The use of this test to investigate new leathers
extracts showed an unequivocal positive result with L2, L5 and
L7 sample extracts, and a negative result with the other samples.
Regarding historic leathers, detection of ellagitannins by spot test
on leather fibres proved to be unfruitful.
Reinvestigation of the same leathers but using the water
acetone extract and monitoring the reaction at ␭ = 538 nm
allowed to detect the formation of the red ellagic acid deriva-
tive, and therefore the existence of ellagitannins in HL1.
With the same methodology ellagic acid was detected in
HL3-HL5 extracts. However, in this case, the ellagic acid
content was similar in the hydrolysed and non-hydrolysed
sample and its presence a result of tannins degrada-
tion.
4.2.3. Rhodanine test for gallotannins detection
Rhodanine reacts specifically with the vicinal hydroxyl groups
of gallic acid to produce a red complex that can be detected
spectrophotometrically at 520 nm [20]. Rhodanine spot test only
allows the detection of free gallic acid [1]. Positive results were
obtained for T4-T9 and L3-L6 (Table 4). Since free gallic acid
can be present due to the hydrolysis of all types of tannins,
a positive result by itself is not enough to assume the pres-
ence of gallotannins. It has to be examined together with the
other two chemical tests above described [1]. Regarding historic
leathers, free gallic acid was detected in HL1, HL3 and HL4 sam-
ples.
The test can be used to detect gallotannins if the amount of gallic
acid is compared before and after hydrolysis of the extract [2,20].
Reinvestigation of historic leathers extracts showed the presence
of gallotannins in HL1-HL4 samples.
4.3. Combined evaluation of historic leathers
Regardless of their aesthetical qualities, historic leathers here
studied, HL1-HL6, were produced with utilitarian purposes. They
have been not only handled and naturally aged, but also exposed
to physical, chemical and biological factors, such as moisture or
pollutants, which may have contributed differently to their degra-
dation.
These factors affect the condition of the main constituents of
vegetable tanned leathers, the collagen protein and the vegetable
tanning compounds used to stabilize and preserve the skin protein,
mainly due to hydrolysis and oxidation degradation reactions that
may have occurred with time [29]. Bearing this in mind, the iden-
tification of tanning materials can be a difficult task requiring the
joint use of different analytical techniques.
To the authors, the best approach was to firstly record the
ATR-FTIR spectra of leathers extracts. This spectroscopic technique
indicated clearly the presence of gallotannins in HL1-HL4. How-
ever, the existence of ellagitannins could not be excluded, since
this type of compounds does not have distinctive band(s) in the
mid-IR region. UV spectroscopy was also important since it sup-
ported FTIR identification. The identification of ellagitannins was
only achieved by a more laborious procedure, the nitrous acid
test; and with this test it was possible to detect them in HL1. On
what condensed tannins concerns, the presence of three of the five
bands attributed to these compounds in the ATR-FTIR spectra of
HL2, indicated that they might have been used to manufacture
this leather. The acid butanol test performed both on the fibres
507
as spot test and on the aqueous-acetone extract confirmed their
presence.
The same approach was used with the older samples HL5 and
HL6. The absence of the all five characteristic bands of tannins indi-
cated that the samples deterioration of these two samples was
greater than visually perceived. Chemical analysis of these two
samples only identified ellagic acid in HL5.
5. Conclusions
This study highlights the potential of an analytical approach
based on molecular spectroscopy and chemical analysis to char-
acterize tannins in new and historic leathers. Each analytical
technique gives different information of complementary nature,
which is useful for the knowledge and research of vegetable mate-
rials formerly used for leather production.
Notwithstanding it may be difficult to interpret ATR-FTIR spec-
tra of historic samples, the aim of this study is also to provide
the scientific community some reference data of vegetable tanning
materials for future studies.
ATR-FTIR indicates straightforwardly the presence of vegetable
tannins, particularly, condensed tannins and gallotannins. This
technique proved to be very useful to identify the chemical class of
the tanning materials in leather extracts, mostly in well-preserved
samples. In aged samples this technique must be complemented
with chemical analysis. Chemical analysis, if performed on leather
extracts, can provide unequivocal identification of the different
classes of tannins but has the disadvantage to be laborious, time
consuming and the use of unpleasant chemical reagents.
Acknowledgments
The authors acknowledge the Portuguese Foundation
for Science and Technology (FCT) for funding project PEst-
OE/QUI/UI0612/2011. Lina Falcão acknowledges FCT for PhD
grant SFRH/BD/62704/2009. The authors gratefully acknowledge
Dr. Bernhard Trommer, Forschungsinstitut für Leder und Kun-
stsoffbahnnen (FILK, Freiberg, Germany) for kindly supply T1,
T3, T5-7, L3, L4, L7 samples; Dr. Andreas Schulze, Landensamt
für Denkmalpfleg Sachsen of Dresden, for kindly provide L2 and
HL2 samples; Katia Bittencourt for supplying HL1 sample; Centro
Tecnológico da Indústria do Couro (CTIC, Portugal) for T2 and T4.
The authors thank to Dr Isabel Henriques and Dr Otília Correia for
kindly providing and identifying, respectively, botanical species.
The authors are also grateful to the Portuguese National Palace of
Ajuda, National Railway Museum, National Palace of Sintra and the
Palace of the Dukes of Bragança for permission to collect historic
samples.
References
[1] L. Falcão, M.E.M. Araújo, Tannins characterisation in new and historic vegetable
tanned leathers fibres by spot tests, J. Cult. Herit. 12 (2) (2011) 149–156.
[2] W. Vermerris, R. Nicholson, Phenolic compound biochemistry, Springer, Dor-
drecht, 2006.
[3] K. Khanbabaee, T. van Ree, Tannins: classification and definition, Nat. Prod. Rep.
18 (2001) 641–649.
[4] Z. Goffer, Archaeological chemistry, Wiley-Interscience, Hoboken, 2007.
[5] A.D. Covington, Tanning chemistry: the science of leather, The Royal Society of
Chemistry, Cambridge, 2009.
[6] R. Thomson, Leather, in: E. May, M. Jones (Eds.), Conservation science: heritage
materials, The Royal Society of Chemistry, Cambridge, 2006, pp. 92–119.
[7] E. Haslam, Vegetable tannage – where do the tannins go? J. Soc. Leather Tech.
Chem. 81 (1997) 45–51.
[8] G. Gall, Leder im europäischen Kunsthandwerk, Klinkhardt & Biermann, Braun-
schweig, 1965.
[9] J.H. Waterer, Leather craftsmanship, G. Bell and Sons, London, 1968.
[10] A. Watt, Leather manufacture, Crosby Lockwood and Son, London, 1906.
[11] H.R. Procter, The making of leather, University Press, Cambridge, 1914.
[12] H.G. Bennett, The manufacture of leather, Constable & Company, London, 1920.
508 L. Falcão, M.E.M. Araújo / Journal of Cultural Heritage 14 (2013) 499–508
[13] R. Thomson, The manufacture of leather, in: M. Kite, R. Thomson (Eds.), Con-
servation of leather and related materials, Butterworth Heinemann-Elsevier,
Oxford, 2006, pp. 121–129.
[14] H. Kühn, Conservation and restoration of works of art and antiquities, Butter-
worth, London, 1986.
[15] J.P.H. Azéma, Moulins du cuir et de la peau : moulins à tan et à chamoiser en
France xiie – xxe siècle, Éditions Créer, Nonette, 2004.
[16] F.L. Hilbert, Tanning materials (vegetable), in: R.E. Kirk, D. Othmer (Eds.), Ency-
clopedia of chemical technology, vol. 13, The Interscience Encyclopedia, New
York, 1954, pp. 578–586.
[17] A. Scalbert, Quantitative methods for the estimation of tannins in plant tissues,
in: R.W. Hemingway, P.E. Laks (Eds.), Plant polyphenols: synthesis, properties,
significance, Plenum Press, New York, 1992, pp. 259–278.
[18] J.C. Bickley, Vegetable tannins, in: C. Calnan, B. Haines (Eds.), Leather, its compo-
sition and changes with time, The Leather Conservation Centre, Northampton,
1991, pp. 16–23.
[19] T.C. Wilson, A.E. Hagerman, Quantitative determination of ellagic acid, J. Agric.
Food Chem. 38 (8) (1990) 1678–1683.
[20] K.H. Inoue, A.E. Hagerman, Determination of gallotannins with rhodanine, Anal.
Biochem. 169 (1988) 363–369.
[21] K. Nakagawa, M. Sugita, Spectroscopic characterization and molecular weight
of vegetable tannins, J. Soc. Leather Tech. Chem. 83 (5) (1999) 261–264.
[22] H. Ozgunay, O. Sari, M. Tozan, Molecular investigation of valonea tannin, J. Am.
Leather Chem. Assoc. 102 (2007) 154–157.
[23] L. Laghi, G.P. Parpinello, D. Del Rio, L. Calani, A.U. Mattioli, A. Versari, Fingerprint
of enological tannins by multiple techniques approach, Food Chem. 121 (3)
(2010) 783–788.
[24] M. Giurginca, N. Badea, L. Miu, A. Meghea, Spectral techniques for identify-
ing tanning agents in the heritage leather items, Rev. Chim. 58 (9) (2007)
923–928.
[25] N. Melniciuc Puica, A. Pui, M. Florescu, FTIR Spectroscopy for the analy-
sis of vegetable tanned ancient leather, Eur. J. Sci. Theology 2 (4) (2006)
49–53.
[26] M. Bicchieri, M. Monti, G. Piantanida, F. Pinzari, A. Sodo, Non-destructive spec-
troscopic characterization of parchment documents, Vib. Spectrosc. 55 (2011)
267–272.
[27] A. Edelmann, B. Lendl, Toward the optical tongue: flow-through sensing of tan-
nin protein interactions based on FTIR spectroscopy, J. Am. Chem. Soc. 124
(2002) 14741–14747.
[28] K. Fernández, E. Agosin, Quantitative analysis of red wine tannins using
Fourier transform mid-infrared spectrometry, J. Agric. Food Chem. 55 (2007)
7294–7300.
[29] R. Larsen, The chemical degradation of leather, Chimia 62 (11) (2008)
899.