Review

Vegetable Tannins Used in the Manufacture of

Historic Leathers
Lina Falcão 1,2 and Maria Eduarda M. Araújo 2,*
1 Artistic Studies Research Centre, Faculty of Fine Arts, University of Lisbon,
Largo da Academia Nacional de Belas-Artes, 1249-058 Lisboa, Portugal; [email protected]
2 Centre of Chemistry and Biochemistry, Department of Chemistry and Biochemistry, Faculty of Sciences,

University of Lisbon, Campo Grande, Edifício C-8, 1749-016 Lisboa, Portugal

* Correspondence: [email protected]; Tel.: +351-217-500-968

Academic Editor: Hideyuki Ito, Tsutomu Hatano and Takashi Yoshida

Received: 28 March 2018; Accepted: 1 May 2018; Published: 3 May 2018

Abstract: In this review, a brief description of how animal skins were transformed in leathers in
Europe using different vegetable tannins will be presented. Special attention will be dedicated to
the description of the type of tannins and the characteristics of the most important type of historic
leathers thus obtained. The text will also focus on the description of the techniques used in the
identification of these tannins in historic objects: colorimetric tests and spectroscopic analysis.

Keywords: tannins; vegetable tanning; European historic leathers; colorimetric tests; spectroscopy;
UV-Vis; FTIR

1. Introduction
Natural tannins, stricto sensu, are polyphenolic compounds of vegetal origin with the property
to precipitate proteins. It is assumed that the oldest application of this tannins chemical property in
technology is the stabilization of animal skins protein against putrefaction [1].
According to some authors [2–5], it may have been by the end of Neolithic period and in the
eastern Mediterranean region that man began to use, incipiently, plant materials, such as leaves,
twigs, fruits, barks or roots, to prevent animal skins degradation, transforming them into a more
durable and useful material. Empirical development of this process during Classical Antiquity
resulted in one of the most important animal skin transformation technique: vegetable tanning. This
millenary technology, which is still in use, is one of the oldest processes used to produce leather. It
was introduced in the north western regions of Europe after the Roman conquest [6], reaching
thereafter a great importance in leather production which lasted until the end of the 19th century,
when mineral tanning based on chromium (III) salts was developed and implemented in tanneries.
Mineral tanning is nowadays the most used tanning process. In the early of the 20th century
synthetic tannins, based on different organic molecules, were introduced in leather industry with the
purpose of aiding vegetable tanning [1,7].
Succinctly, the vegetable tanning method can be described as the treatment of animal
skin—previously washed, limed, dehaired, fleshed, delimed-, with crushed vegetable materials, or
liquors/macerations (oozes), infusions, or decoctions, or extracts prepared with those materials. The
result is the transformation of animal skin into what is termed by leather: a stable and
non-putrescible material, resistant to deterioration promoted both by microorganisms and heat,
when wetted. This is due to the chemical bonds established between collagen, the main constitutive
protein of skin, and the tannins present in the vegetable materials. Collagen stabilization occurs

Molecules 2018, 23, 1081; doi:10.3390/molecules23051081 www.mdpi.com/journal/molecules

Molecules 2018, 23, 1081 2 of 20

when 15% to 40% of tannins, per dry weight of skins, are absorbed and incorporated into the
collagen fibres matrix, depending of the type of leather produced [1,8–10].
The origin of the term ‘tannin’ it is also close related to the vegetable tanning process and came
into use in France at the end of the 18th century by the French chemist Armand Séguin (1767–1835),
who had previously worked with Antoine Lavoisier. Séguin firstly used the word tannin to define
the organic substance present in the aqueous extracts of plants responsible for the transformation of
skin into leather. The word derives from the French word tan, which corresponds to the ground oak
bark, an important widespread and extensively used European vegetable source for leather making.
The term tanning (and tannage in French) itself has also a root in the word tan, and it was
originally reserved for leather production with tannins but, since the 19th century with the
development of other processes, the word has been applied for all different processes of leather
making [11,12].
Vegetable tanning could be performed in different ways mostly depending on the size of
animal skins, but since the medieval period three main techniques could be found in Europe: pit, vat
and bag tanning [13–16].
Tanning in pits—large tanks set on the ground—was a slow and static process mostly reserved
to the treatment of large animal skins (hides) and to produce heavy and strong leathers for shoe
soles, belting or harness. Succinctly, hides were set in horizontal layers inside the pits (or layaways)
with the ground vegetable material, one layer of hide another layer of crushed barks or leaves. Water
or oozes was then added to the pits until all the skins were immersed and left for about 6 to 12
months.
The tanning of small to medium skins was mostly performed in wooden or clay vats where they
were put together with the vegetable material, or an aqueous liquor of these materials, and stirred
together for a few weeks until the required amount tanning material had been absorbed.
The last process, bag tanning, mostly used in south of Europe, was a faster process since it took
only two to three weeks to complete. This method was used to produce light and fancy leathers that
were then applied in lavish items such as jewellery and cases coverings, etc. To obtain these
exquisite leathers, the skins were folded, sewed like a small bag and filled up with water and with
the ground vegetable material inside. Then “bags” were immersed in an identical infusion or liquors
used to fill it and shake to speed up the process of tanning. Basils (badanas, in Iberian languages),
cordovans and morocco are some examples of vegetable tanned leathers traditionally produced by
bag tanning technique [17–20].
With the Industrial Revolution, and the need of faster tanning processes, all vegetable tanned
leathers started to be produced in drums which consist of a wooden cylinder rotating around its
own axis filled with skins, tanning agents and water.
Leather produced with tannins, or vegetable tanned leather, was one of the most important
pre-industrial materials in Western world, very much appreciated and demanded due to its
versatility. Its characteristics, ranging from rigid to flexible, depend on the raw materials used, both
skins and vegetable tannins, and the tanning techniques. It was a fundamental material for the
production a wide range of artefacts such as footwear, garments, book bindings, saddlery,
wall-hangings, furniture upholstery, cases coverings, carriages or liquids containers [7,21]. Beyond
its utilitarian function, it was also used as support material for artistic and decorative paintings, wall
hangings or screen coverings. Different ornamental techniques such as dyeing, painting, gilding,
moulding, tooling, embroidering, cutting-out, scorching or sewing, have been often incorporated
transforming vegetable tanned leather into a valuable and luxurious material. Gilt leather (Spanish
leather, guadameci) is one of the finest examples created in Europe: it is a silvered and then “golden”
varnished, painted and tooled or moulded vegetable tanned leather, mostly produced between the
16th and 18th centuries [13,22,23].
Therefore, vegetable tanned leather is the most common type of heritage leather found in
museums and collections [24,25].
Molecules 2018, 23, 1081 3 of 20

2. Vegetable Tanning Materials: The Sources of Tannins

Vegetable tannins are polyphenolic secondary metabolites produced by higher plants. They can
precipitate not only proteins, as it was stated before, but also polysaccharides and alkaloids. They
are large molecules with a molecular weight comprised from 500 to 30,000 Da. However, not all
tannins are useful for tanning, only those with a molecular weight lower than 3000 Da are efficient in
leather making since large molecules are unable to penetrate into the skin’s fibre structure and tend
to be water insoluble [1,26].
Tannins can be divided in four classes: hydrolysable, condensed, complex and phlorotannins
[27–29]. Complex tannins have a molecular structure that can be considered a mixture of
hydrolysable and condensed, including gallic, ellagic and catechin sub-units. Phlorotannins are a
small group of tannins isolated mainly from brown seaweeds. None of the two groups is important
for leather tanning.
Hydrolysable tannins consist in a monosaccharide core, usually glucose, esterified with gallic
acid, forming the gallotannins, or with hexahydrodiphenoic acid, the precursor of ellagic acid, and
gallic acid, forming the ellagitannins. Upon heating in acidic aqueous medium, they hydrolyse to
yield gallic and ellagic acid. Thermal decomposition originates pyrogallol which gave the traditional
and former name to this class of compounds.
Condensed tannins have a flavonoid origin. They are oligo- or polymeric proanthocyanidins
were the phenolic hydroxyls are totally or partially esterified with gallic acid.
Many plants rich in tannins had been used over time. Table 1 lists the most important sources of
tannins used in European tanneries until development of mineral tanning since before the 18th
century the autochthonous plants were the main raw vegetable tanning materials. However, with
the growing need for leather and the increase in trading between Europe and other continents such
as Africa, Australia and South America, other exotic sources arrived at Europeans tanneries in large
amounts and low cost [15,30,31].
The selection of the appropriate vegetable tanning material was crucial to obtain leathers with
specific characteristics [32]. Leathers produced with hydrolysable tannins are light brown, yellow or
greenish, and are lightfast. Leathers produced with condensed tannins have a brown to reddish
colour, becoming darker if exposed to light. This latter type of leather tends to absorb more easily
atmospheric pollutants like sulphur dioxide, promoting the acidic hydrolysis of collagen, leading to
its degradation. The so-called red rot is an advanced stage of degradation where cohesion is lost due
to the disintegration of collagen fibres. Leather produced with condensed tannins is more prone to
this occurrence than the one produced with hydrolysable tannins [9,25].

2.1. Barks
Barks from different tree species were, and some still are, very important raw materials in the
production of vegetable tanned leathers.
In Europe, oak (Quercus spp.) barks were the most used. In the British Isles and other western
European countries, the prevailing species were English (pedunculated) oak (Quercus robur syn. Q.
pedunculata) and sessile oak (Quercus petraea syn. Q. sessiliflora), while in the south predominated
Pyrenean oak (Quercus pyrenaica) and Portuguese oak (Quercus faginea). In the Mediterranean and
Atlantic regions of south Europe, the bark of smaller and endemic species belonging to the Quercus
genus such as cork oak (Quercus suber), holm oak (Quercus rotundifolia syn. Q. ilex subsp. rotundifolia)
and kermes oak (Quercus coccifera) were also used since these species grow spontaneously [12,18,33].
Barks were removed from trees with 12 to 15 years old, between spring and early summer, since
it is the time when barks are richer in tannins and, at the same time, easier to separate from the
trunk. Barks were dried and then ground into a powder containing 6–17% weight of a mixture of
condensed tannins and ellagitannins [1,7].
Early analysis of Quercus robur and Q. petraea barks found that they contained
proanthocyanidins of the procyanidin and prodelphinidin type. Nevertheless only 23% of the
water-soluble oak bark tannins consisted of oligomeric proanthocyanidins [34,35].
Molecules 2018, 23, 1081 4 of 20

Quercus spp., namely Q. robur, such the other species known by the common name of oak, are
also a rich source of ellagitannins. The main ellagitannins that account for 40–60% of components of
the bark are vescalagin and castalagin. These are complex molecules comprising a linear glucose unit
with OH at the 4 and 6 positions esterified with a hexahydrodiphenoic acid (HHDP acid). At the
same time, it also exists a C-C coupling between C-1 of glucose and C-2 of a nonahydroxytriphenoyl
moyaty. This unit esterifies the remaining OH of glucose at the 3, 4 and 5 positions (Figure 1).
Vescalagin and castalagin are epimers at the C-1 position of glucose unit [26,36].

Figure 1. Structure of vescalagin and castalagin.

Steriochemistry of the C-C bond between the two galloyl unit, linked to C-3 and C-5 OH of
glucose has recently been reinvestigated [37].
Quercus suber, cork oak, was also used in tanning. The inner part of the bark was removed and
used in Portugal, Spain and Corsega. It was also exported to Ireland [20,33,38]. Investigation of cork
from Spanish specimen indicates that the main tannins were common to the other Quercus species,
the ellagitannins grandinin (Figure 2), vescalagin, and castalagin , being grandinin and castalagin the
main components [39].
Quercus coccifera, kermes oak, was very used in the French Provence where it was known as
garouille. Rusque, the name of the husk of the root, was used to produce shoe sole of high quality
[19,20].

Figure 2. Structure of grandinin.

Molecules 2018, 23, 1081 5 of 20

Table 1. Post-medieval and traditional vegetable tanning materials used in Europe [18–20,33].
Part of the
Botanical Name Common Name Origin and Distribution Main Tannins Geographical Uses Observations
Plant Used
Australia, cultivated in imported since second
Acacia mearnsii mimosa, wattle South Africa since 1864 barks condensed decade of 19th century,
and South America commercial extracts
northern Europe, used to produce Russia
Betula spp. birch northern Europe, Russia barks condensed
Russia leather
Central and South hydrolysable: imported since late 18th
Caesalpinia coriaria divi-divi pods
America gallotannins century
used mixed with other
hydrolysable: since 19th century,
Castanea sativa chestnut, sweet chestnut Mediterranean region wood vegetable materials to
ellagitannins commercial extracts
produce firm leather
Mediterrenean coriaria southern France and southern France and
leaves
Coriaria myrtifolia (emborrachacabras, redoul, Mediterranean coastal hydrolysable Mediterranean coastal
(redoul)
roldor, rodor) Spain Spain
leaves
Cotinus coggygria southern Europe, (Venetian or hydrolysable:
smoke tree southern Europe
(syn Rhus cotinus) Mediterranean region Turkish gallotannins
sumac)
Larix larch northern Europe bark condensed northern Europe
hydrolysable:
Mirtus communis myrtle southern Europe leaves Italic Peninsula
ellagitannins
Alps, Pyrenees, northern and central
Picea abies Norway spruce barks condensed
Germany, Scandinavia Europe
coastal areas of the
Pinus halepensis Aleppo pine western Mediterranean barks condensed northern Europe yields a reddish leather
region
eastern Mediterranean hydrolysable: Middle Ages in Turkey,
Quercus aegilops valonea oak, Turkish oak acorn cups
region ellagitannins Greece, Italy
husk of root
Quercus coccifera garouille Mediterranean region hydrolysable south of France
(rusque)
eastern Mediterranean galls (Allepo hydrolysable:
Quercus infectoria Aleppo oak Europe
region galls) gallotannins
central-western part of condensed and
Quercus ilex holm oak barks Iberian Peninsula
the Mediterranean hydrolysable
Quercus spp. (Q. oak Europe barks condensed and Europe
Molecules 2018, 23, 1081 6 of 20

ilex, Q. robur, Q. hydrolysable:

petraea, Q. ellagitannins
pyrenaica)
hydrolysable:
wood
ellagitannins
condensed and
Quercus suber cork oak inner bark hydrolysable: Iberian Peninsula
ellagitannins
yields light coloured, soft and
leaves
hydrolysable: supple leathers. Used to
Rhus coriaria sumac Mediterranean region (Sicilian southern Europe
gallotannins produce basil and cordovan
sumac)
leather.
yields a light coloured,
northern Europe,
Salix spp. willow northern Europe, Russia barks condensed yellowish-brown leather that
Russia
is soft and flexible
Schinopsis imported and used in
balansae, S. quebracho south America wood condensed Europe since last
lorentzii decades of 19th century
used in mixed tannages for
Terminalia chebula myrabolans India fruits hydrolysable British Islands
sole leather
Molecules 2018, 23, 1081 7 of 20

In northern Europe and Russia, the main vegetable tanning materials were the barks of local
trees like Betula spp., birch, Salix spp., willow, Larix spp., larch, or Picea spp., spruce. The use of these
barks was restricted to these regions [20,33].
One of the finest examples of vegetable tanned leathers produced in Eastern Europe was Russia
leather, which was obtained by tanning the hides or skins with birch or willow barks. It was a highly
valued leather, being exported from Moscow to Western Europe from the 17th to the 19th centuries
where it was very appreciated for furniture upholstery and lavish coverings [7,18].
The above species biosynthesise condensed tannins although these compounds were found in
small percentages [40].
In Scandinavia, barks from Salix arenaria and Salix resseliana containing 7–11% of condensed
tannins were traditionally used to produce leather for gloves [20].
Tannins from the bark of Picea abies (Norway spruce) were identified as combinations of
(epi)catechin and (epi)gallocatechin units with a polymerization degree of up to 13 units [41].
During the late 18th and 19th century there existed a great demand on tanning vegetable
material. To overcome the problem, barks from what was called exotic trees were imported from
South America, South Africa, Australia and New Zealand. One of the most imported material was
the bark of Acacia mearnsii, black wattle, endemic in Australia and New Zealand and cultivated in
South Africa. This material is still very used today in Europe by leather industry. Barks were
collected from August to October. The outer part was discarded, and the inner part was milled. It
could be used as a powder or boiled to produce liquor that was evaporated to dryness and traded for
all Europe [20,42]. Acacia bark contains 22–48% of condensed tannins. This source of tannins is a very
effective tanning agent and is used to produce different types of leathers, with a light red colour
when new becoming darker with time.
Wattle tannins consist of about 9% monomers, 42% dimers, 40% trimers, 9% tetramers and 1%
pentamers and higher oligomers by mass. The starter unit is either catechin or gallocatechin and the
extender units, fisetinidol or robinetinidol (Figure 3a). The second extender unit is always linked to
the starter unit to give angular trimers (Figure 3b). The predominance of trimers in the vegetable
material is essential for leather tanning since high molecular weight oligomers would not be able to
penetrate skin collagen fibres. A. mearnsii proanthocyanidin has recently been investigated. The
percentage of the different monomers in the tannin of mimosa are around 15% of catechin, 65–70% of
robinetidinol and 15–20% of fisetinidol. It can be considered as a mixture of procyanidin or
profisetidin and prorobinetidin or prodelphinidin substructures (Table 2) most probably as a
mixture of procyanidin and prorobinetidin or prodelphinidin since the proportion of OH/H
substitution at position 5 is 6.8:1 and the proportion of OH/H substitution at position 5′ is 3:2 [43,44].

(a) (b)
Figure 3. Structure of flavan-3-ol monomers (a) and mimosa condensed tannin (b).
Molecules 2018, 23, 1081 8 of 20

2.2. Wood
Wood was mainly used in furniture and buildings, so its use in tanning was scarce until the
beginning of the 19th century when an urge need for new tanning materials appeared. Both chestnut
and oak wood began to be used as ground or aqueous extracts [45]. The wood of these species, like
the barks, are also rich in ellagitannins. Nowadays, chestnut extracts are still used in industry being
mainly produced in Italy, Central Europe and Baltic region. Research of chestnut heartwood
indicated that has a polyphenolic constitution similar to oak wood. Monomeric vescalagin and
castalagin predominate in oak wood representing 40–60% of the ellagitannins. Dimers (roburin A
and roburin D, (Figure 4a) and other compounds were other sugar unit like lyxose/xylose
derivatives grandinin, roburin B, roburin C, and roburin E (Figure 4a,b), were also identified in oak
woods [46]. Some of the previous roburins like roburin A and roburin E were also identified in small
amounts in oak bark [39].

(a)

(b)

Figure 4. Structure of ellagitanins present in oak wood: (a) grandinin and roburin D; (b) roburins A,
B, C and D.
Molecules 2018, 23, 1081 9 of 20

During the last decades of the 19th century Europe began to import an exotic wood, quebracho,
Schinopsis lorentzii and S. balasae, from south America [18,47]. This tree has hard and reddish wood
rich in condensed tannins, about 14–26% weight of the heartwood. Quebracho tannins extracts
contains oligomers based on catechin as starter unit linked to one, two, three, etc. ent-fisetinidol
extender units (Table 2, Figure 5). The trimer is angular with one fisetinidol linked to the C-8
position and the other to the C-6-position of catechin. Analysis of the tannin indicates that is
composed by about 33% dimers, 37% trimers, 21% tetramers, 8% pentamers, and 1% heptamers.
Compounds with higher degree of polymerisation, if present, exist in low concentration [1,48,49].

Figure 5. Structure of quebracho tannin.

2.3. Leaves
In Europe, until the 20th century, sumac (Rhus coriaria) leaves, together with oak bark, were one
of the most important materials used to produce leathers. Sumac has been used since Antiquity and
it is even mentioned in Pedanius Dioscorides book “De Materia Medica” written between 50–70 AD.
During the Middle Age it was a valuable material used to produce delicate, light colored and great
durability leathers. It was also used in the dying of leathers and textiles. In Iberian Peninsula it was
used to prepare the famous cordovan, leather prepared with goat skins, and basils. The word sumac
refers to several species belonging to the genera Rhus of the Anacardean family. It also refers to the
powder obtained milling the leaves and small branches of the shrub collected from July to
September. Good quality sumac contains 25–35% of gallotannins. Rhus coriaria grew spontaneously
or cultivated in several regions of the South Europe, namely Portugal, Spain and Sicilia, produced a
light yellow to green tanning powder.
During the 17th and 18th centuries there was a great demand for Portuguese sumac produced
in the north of the country and it is described in the firsts encyclopedias as having a superior quality.
It was exported to France and British Islands to be used by the textile and leather manufactures of
Northern Europe. However, during the 20th century Spanish and Sicilian sumac surpassed the
demand for Portuguese sumac [18,33,50].
Molecules 2018, 23, 1081 10 of 20

Table 2. Proanthocyanidins monomers.

Tannins Name
Procyanidin Profisetidin Prorobinetidin Prodelphinidin

Chemical structure of the building block

Coupling position 4–8 4–6 4–6 4–8

Molecules 2018, 23, 1081 11 of 20

In Turkey and in the Oriental Mediterranean another specie of sumac was used, named Turkish
or Venezian sumac, Rhus cotinus (syn. Cotinus coggygria) used in the Mediterranean region but
containing less amount of tannins. Both sumacs tanning materials produces pale and soft leathers,
contrasting with other tanning agents that originate brown coloured leathers. Pale leathers were
much appreciated since they could be dyed in light and bright colours without dark background
interference. There are however some drawbacks: sumac infusions ferment and hydrolyse easily and
the resulting material has no tanning properties. They were used in vat or bag tanning.
Besides sumac leaves other plants leaves were also used for leather production, such as myrtle,
mastic and rédoul. The last one is of French provenance, very used in the French regions of
Languedoc and Provence and also in Catalonia where was called redon. It was also known as French
or fake sumac and it was poorer tanning material, with only about 15% of tannins [7,20,51].
As stated above, sumac is a source of gallotannins. The simplest gallotannin is pentagalloyl
glucose, β-1,2,3,4,6-pentagalloyl-O-D-glucopyranose (PGG). This compound has the five free
hydroxyls of glucose esterified with a galloyl residues. The α anomer is not usual in nature. More
galloyl residues can be attached to PGG through a meta- or para depside bond (Figure 6) [26].
Tannic acid is considered the tanning agent in sumac. Commercial tannic acid has a molecular
weight of 1701.206 g/mol and a molecular formula of C 76H52O46, corresponding to a decagalloyl
glucose, sumac leaves contain a mixture of gallotannins, from penta to decagalloyl-glucoside [52–
54].
In Spain, true sumac was many times forged with mastic (Pistacia lentiscus) leaves. However this
plant contains condensed tannins and leathers thus obtained were darker and got a reddish colour
with light.
Leaves of Myrtus communis were widely used for leather production in Italic Peninsula. This
plant contains the following ellagitannins: oenothein B, eugeniflorin D2, and tellimagrandins I and II
[55].

Figure 6. Structure of gallic acid and pentagalloyl glucose and tannic acid
Molecules 2018, 23, 1081 12 of 20

2.4. Fruits
A few fruits were also used as tanning materials, the most important being valonia and
divi-divi. Valonia is the acorns of Quercus aegilops, a tree also known as Turkish oak, which grows
abundantly in Turkey, Greece and adjacent countries. Acorns are usually picked up in August and
then dried out from the domes. It has been widely used in the production of leather in Austria,
Germany and France and allows producing faster, harder, firmer, heavier and quite impermeable to
water leather. Valonia is a source of ellagitannins, whose main constituents are castalagin,
vescalagin and pentagalloylglucose (Figures 1 and 6) [56]. This material was often mixed with
ground oak bark to produce soles that were considered of excellent quality and durability [18,20].
The divi-divi is a pod of a shrub, Caesalpinia coriaria, native to South America. It became to be
imported and used in English tanneries in the 19th century. This material, mainly a source of
gallotannins containing a small amount of condensed tannins, produces very porous leathers of
brown or reddish-brown colour [20,57].

2.5. Galls
Galls are pathological excrescences formed in the branches, leaves or domes of plants as a
response to the bites of certain insects or other parasites. They are anomalous grow of plant tissues
and have different size, shape and composition depending on the plant and the agent that causes
them. Galls used in tanning have a spherical (globose) shape, are smooth or have a crown and are
characterized by a high content of tannins, 40% to 70% of gallotannins. The most important are the
so-called Turkish galls, Aleppo or Levant galls, and are produced by the insect Cynips gallae tinctoriae
in the branches of the Quercus infectoria (syn Q. lusitanica) growing mainly in Asia Minor. These galls
have a greenish or brownish colour, are rigid, compact, very astringent, bitter to taste and are one of
the raw materials used to obtain tannic acid [33].
The so-called white galls, brownish-yellow, lighter, less astringent and with bitter taste, having
less tannin content than greenish galls, are another type of galls produced by Q. infectoria [19,20].
Since Ancient Greece that both have commercial importance and, in addition to tanneries, were
widely used in traditional medicine, dyeing and to make writing ink (iron gallic ink).
Another important gall is known as knoppern, or acorn gall, which is caused by the insect C.
quercus-calycis and found in many Central European Quercus (Q. robur, Q. pyrenaica) (Austria,
Hungary, Serbia, Slovenia) and Greece [20].

3. Detection and Characterization of Tannins in Historic Vegetable Tanned Leathers

Vegetable tanned leathers, i.e., leathers produced with plant materials rich in tannins, are the
commonest European cultural heritage leathers.
In heritage conservation, identification of leather making process is important to comprehend
leather technology, degradation susceptibility and condition.
The analysis of tannins is a relatively common procedure in historic leather conservation and
heritage science studies. Tannins characterisation allows to elucidate aspects on historic leathers
composition, technology, and condition, but also to evaluate the suitability of new vegetable tanned
leathers for conservation purposes [9,58].
Tannins characterisation in leathers can be based on spot tests, formerly the ferric test and
vanillin test and more recently the rhodanine, nitrous acid and the acid butanol tests. These chemical
tests are employed for a fast-preliminary material characterisation, especially by leather
conservators [58–63].
Some studies using chromatographic techniques, namely high-performance liquid
chromatography (HPLC) have been also used, but more costly and time consuming and only allows
to distinguish the broad classes of condensed versus hydrolysable tannins [64].
Molecules 2018, 23, 1081 13 of 20

3.1. Colorimetric Tests

Different colorimetric tests developed in the field of phytochemistry [26] have been adapted for
tannins detection in historic and archaeological leathers. These tests, particularly ferrous and
vanillin test, have been performed as spot test directly in fibres collected from leathers [61,62,65].
Recently, more specific chemical tests have been adapted to analyse both leather fibres and extracts
prepared from those fibres. The analysis of fibres with these tests is done visually. However, the
identification of tannins can be difficult to interpret in coloured or aged leathers samples due to the
presence of colorants or degradation products of the leather itself. To overcome these limitations,
chemical analysis of historic leathers can be performed in leather fibres extracts and analysed
spectrophotometrically [66].
Colorimetric tests can be used as a global and fast tool to detect and classify vegetable tannins.
When specific tests for the detection of condensed tannins (HCl/butanol test), ellagitannins (nitrous
acid test) and gallic acid/gallotannins (rhodanine test) are performed, a combined evaluation of
results is possible allowing a more specific detection of the chemical nature of tannins used in the
manufacture of the leather (Figure 7) [66].

Figure 7. Key-guide for the identification of tannins by colorimetric tests.

3.1.1. Ferric Test

This type of test is specific for phenolic compounds and not only tannins. It is based on the
principle that phenolics react with iron salts forming a bluish or greenish black (or grey) product
[58,64]. Iron salts such as iron sulfate (III) (Fe2(SO4)3) or iron chloride (FeCl3) are the commonest
reagents used in ferric test.
Some literature refers that this test can be used to distinguish hydrolysable from condensed
tannins. Hydrolysable tannins, namely tannic acid, form blue black products while condensed
tannins form green black products. This subtle distinction is very difficult to observe in leather
fibres, particularly in aged dark leathers or when mixtures of different types of tannins are present
and if diverse coloured fibres are to be compared [61,62].
Molecules 2018, 23, 1081 14 of 20

3.1.2. Acidified Vanillin Test

Vanillin test has been widely used to detect condensed tannins. In acid conditions, vanillin
reacts specifically with meta-substitued flavanols, not only condensed tannins, to form a red
product. Widely distributed flavanols such as catechin and epicatechin, condensed tannins
monomers, also react with the disadvantage they give higher colour yield than the condensed
tannins. Furthermore, this test is very sensitive to the presence of water that quenchs colour yield. In
addition, quebracho proanthocyanidins do not produce an intense colour with this reagent [26].
Reactivity with vanillin cannot be considered sufficient evidence for the presence of condensed
tannins [23,67]. Furthermore, vanillin reacts reluctantly with aged leathers, becoming very difficult
to undoubtedly identify the presence of condensed tannins [59,61,62,68].

3.1.3. Acidified Butanol Test

Acid butanol test is a depolymerization method specific for condensed tannins detection. With
this method it is promoted an oxidative cleavage of interflavanoid bond of proanthocyanidins in hot
acidified alcohol solutions to yield correspondent red anthocyanidins with absorbance maxima
around 550 nm. The red colour can vary significantly depending on the anthocyanidin formed and
on the position of interflavan links (C4 → C6 were found to be more resistant to cleavage than C4 →
C8) [23,67].
This test, specific to condensed tannins, is more sensitive than vanillin test and results are
unequivocally [62].

3.1.4. Nitrous Acid Test

Ellagitannins react with nitrous acid, obtained by dissolving sodium nitrite in a diluted acid,
forming a red or pink colour, which slowly changes to purple or blue [23,67,69].
Nitrous acid test can also be used to detect ellagic acid, an ellagitannins degradation product.
With this reagent ellagic acid forms a red chromophore product while other phenolics can form
yellow or orange products.
Ellagic acid can be present in aged leather due to acid hydrolysis promoted by moisture and
acidic pollutants. The red colour formed in tested historic samples indicates ellagic acid may be
present [62].

3.1.5. Rhodanine Test

Rhodanine test is specific for gallic acid detection and was developed by Inoue and Hagerman
for gallotannins determination in plant materials. In basic solution, this reagent
(2-thio-4-ketothiazolidine) reacts with the vicinal hydroxyl groups of free gallic acid producing a red
complex, with a maximum absorbance around 520 nm. This red colour is not formed with galloyl
esters of gallotannins, ellagic acid, ellagitannins or condensed tannins. However, this method can be
used indirectly to detect gallotannins presence performing the test before and after hydrolysis of
tannins [70].
Rhodanine test was adapted to detect free gallic acid in fibres and, indirectly, to estimate
gallotannins, without hydrolysing samples, by comparing results with acid butanol and nitrous acid
tests. The presence of free gallic acid in historic leathers may be due not only to gallotannins
degradation (hydrolysis), but also to ellagitannins, complex tannins and even condensed tannins
degradation. Therefore, an unequivocal assumption of a leather tanning with a vegetable source of
gallotannins is only possible if ellagitannins and condensed tannins are not detected (Figure 7)
[62,66].
Molecules 2018, 23, 1081 15 of 20

3.2. Spectroscopic Techniques

3.2.1. UV-Vis
Ultraviolet-visible (UV-Vis) spectroscopy has been used to analyse tannins extracted from
European historic leathers, such as bookbinding and upholstery leathers, principally as a
complementary technique to other spectroscopic research studies.
It is well established that the different classes of tannins from different vegetable sources
present characteristic UV absorption bands. Briefly, and considering hydrolysable tannins, the
wavelength of the maximum absorbance (λmax) and the respective inflection point (λmin) are as
follows: while gallotannins show two characteristic absorption maximums, λ max1 around 212 nm
and λmax2 around 275 nm, with distinctive inflection point around 242 nm; ellagitannins present
strong absorption near 200 nm and a shoulder around 277 nm. Regarding condensed tannins, they
present a strong absorption around 200 nm, an inflection point (λmin) between 258–259 nm and λmax
between 279–281 nm [71,72].
This distinctive spectral data has been found when analysing leather fibers extracts gathered
from leathers of different periods and geographical origins. Studies demonstrate that UV spectra
can clearly indicate if gallotannins or condensed tannins are present in extracts obtained from
leathers tanned exclusively 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 [66].
Other authors [73] present similar values for bookbinding and upholstery leathers.

3.2.2. FTIR
Fourier transform infra-red (FTIR) spectroscopy is a very useful and common analytical
technique in heritage science studies and in the last decade different studies have been published
describing this technique to analyse tannins in historic leathers [36,66,73–77].
Tannins from different vegetable sources present characteristic absorption bands in mid-
infrared region of spectrum. The 1750–700 cm−1 region was considered the most informative. All
tannins FTIR spectra exhibit four strong bands, two of them at 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. Tannins also present another weak band at 1518–1507
cm−1 due to skeletal vibration of the aromatic rings.
And it is also demonstrated that the fingerprint region (1800–650 cm−1) of hydrolysable tannins
presents an absorption pattern distinct from condensed tannins. Hydrolysable tannins presented
bands at 1731–1704 and 1325–1317 cm−1. The gallotannins sub class present three distinctive bands
at 1088–1082, 872–870 and 763–758 cm−1.
Vegetable tanning materials classified as condensed tannins, showed 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 not found in the spectra of gallo- and ellagitannins. The 1288–1283 cm−1
indicates a characteristic feature for the flavonoid-based tannins. This band can be assigned to the
ethereal C–O asymmetric stretching vibration arising from the pyran-derived ring structure of this
class of tannins [78,79].
The tanning materials in historical leathers can be analysed by FTIR technique after extraction
with aqueous acetone, followed by lyophilization. The presence of the four strong bands at 1615–
1030 cm−1, as referred above, is a strong indication that the material had been tanned with vegetable
tannins. Further characterization is possible looking for the marker bands of each class (Table 3).
Ellagitannins can only be identified if marker bands for hydrolysable tannins are present and
marker bands for condensed and gallotannins are missing. Table 3 presents data obtained with ATR
device. If other technique is used like KBr pellet or diffuse reflectance identical or very close values
are obtained.
Usually historic leather tanned with sumac like morocco leather are easily identified since the
characteristic bands of gallotannins are well defined. However, if leathers were tanned with oak,
Molecules 2018, 23, 1081 16 of 20

which contains a mixture of ellagi- and condensed tannins, or if the leather was tanned with more
than one type of tannin, FTIR spectra present the characteristic bands of hydrolysable and
condensed tannins, do not allowing the differentiation between these two situations.
When spectra display intense bands around 1650 and 1550 cm−1 corresponding to the amide I
and II bands of collagen, respectively, is an indication that leather had suffered a considerable
degradation of the proteinaceous material.

Table 3. Main ATR-FTIR bands of vegetable tanning materials and their assignment [36].

Bands (cm−1) Assignment Tannin Identification

ν C=O phenolic esters lactones
1731–1704 (m-s) hydrolysable tannins
ν C=O phenolic esters
1615–1606 (m-vs) ν C=C aromatic ring present in all classes of tannins
1518–1507 (w-m) ν C=C skeletal ring present in all classes of tannins
1452–1446 (m-s) ν C=C aromatic ring present in all classes of tannins
1325–1317 (m-s) ν C-O lactones and O-H deformation hydrolysable tannins
1288–1282 (ms-vs) ν C-O pyran ring, flavonoids condensed tannins
1211–1196 (m-vs) ν aromatic C-OH present in all classes of tannins
1162–1155 (s) ν, asymmetric, C-O-C cyclic ether condensed tannins
1116–1110 (s-vs) ν, asymmetric, C-O-C cyclic ether condensed tannins
ν, symmetric, C-O-C aryl phenolic
gallotannins
1088–1082 (m) ester
1043–1030 (m-vs) β = C-H deformation present in all classes of tannins
976 (w) condensed tannins
844–842 (w) γ tetrasubsituted aromatic C-H condensed tannins
γ OH and γ tetrasubsituted aromatic
gallotannins
872–870 (w) C-H
ν, symmetric skeletal (sugar ring,
gallotannins
763–758 (w-m) breathing vibration)
ν stretching, β in plane, γ out-of plane; vs: very strong, s: strong, m: medium, w: weak.

3.2.3. Other Spectroscopic Techniques: Fluorescence Spectroscopy and Solid State 13C-NMR
There are few reports than the above mentioned of other spectroscopic techniques used to
characterize vegetable tannins in historical leathers.
Emission fluorescence spectra of five tannins, oak, valonea, chestnut, quebracho and mimosa,
were recorded between 200 and 800 nm after excitation at 220 nm and 250. Results were used by the
authors to confirm the conclusions obtained by FTIR and UV spectroscopy [73].
Solid state 13C-NMR was used to distinguish between leather tanned with vegetable material
from leather tanned with mineral tanning agents. Special attention was paid to the spectra between
about 165–171 ppm. In this region peaks of vegetable tannins are very important while those from
collagen are small and scarce. The technique was applied only to new tanned leathers and requires
the use of small amount of material, 1–2 mm, which were freeze in liquid nitrogen and then milled to
a powder. Besides the distinction between hydrolysable and condensed tannins the authors also to
distinguish leather tanned with mimosa from the leather tanned with quebracho [80].

Funding: This research was funded by FCT [Portuguese Foundation for Science and Technology], project
number [UID/MULTI/00612/2013]

Acknowledgments: The authors acknowledge the Fundação para a Ciência e Tecnologia (FCT) (Portuguese
Foundation for Science and Technology) for funding project UID/MULTI/00612/2013. Lina Falcão
acknowledges FCT for PhD grant SFRH/BD/62704/2009.

Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 1081 17 of 20

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