The role of tannin-based dyes in the degradation of historical textiles: a multiple analytical approach

UNIVERSITA’ DI PISA

Dipartimento di Chimica e Chimica Industriale “Scuola di Dottorato Galileo Galilei” PhD course in Chemical Science (CHIM/01)

XXVI cycle (201

The role of tannin

degradation of historical textiles:

a multiple analytical approach

Annalaura Restivo

Supervisors:

Dr. Erika Ribechini

Dr. Ilaria Degano

UNIVERSITA’ DI PISA

Dipartimento di Chimica e Chimica Industriale “Scuola di Dottorato Galileo Galilei” PhD course in Chemical Science (CHIM/01)

cycle (2011-2013)

The role of tannin-based dyes in the

degradation of historical textiles:

multiple analytical approach

Annalaura Restivo

External supervisor:

Prof. Josefina-Pérez Arantegui

“Sono fra coloro che pensano che la scienza abbia una

grande bellezza. Uno studioso nel suo laboratorio non è solo

un tecnico, è anche un bambino messo di fronte a fenomeni

naturali che lo impressionano come una fiaba.”

(Marie Curie)

A Gabriele i cui difetti mi fanno impazzire ma i cui pregi mi hanno fatto innamorare Sei tutto ciò che io non sono ma proprio per questo ci completiamo Sei l’unico che nei momenti bui riesce a farmi scorgere una luce E’ grazie a te se divento ogni giorno una persona migliore

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Table of contents

Introduction and aims of the thesis ...

Chapter 1 State of the art

1.1 The chemistry of wool fibre ... p. 13 1.2 Wool degradation ... p. 17 1.2.1 Lipid degradation ... p. 17 1.2.2 Keratin degradation ... p. 18 1.3 Analytical techniques used in the study of wool composition and

degradation ... p. 31 1.4 The chemistry of tannins ... p. 36 1.5 The degradation of textile-dyestuff complexes ... p. 44 1.6 Analytical techniques used to study tannins ... p. 46

References ... p. 51

Chapter 2 SEM-EDX to study wool degradation

2.1 Introduction ... p. 63 2.2 Materials and methods ... p. 64 2.2.1 Instruments and working conditions ... p. 64 2.2.2 Chemicals and materials ... p. 64 2.2.3 Reference specimens ... p. 65 2.2.4 Historical and archaeological textiles ... p. 67 2.2.5 Cross-sections... p. 68 2.3 Results and discussion ... p. 69 2.3.1 Reference specimens ... p. 69 2.3.2 Historical and archaeological textiles ... p. 73

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Chapter 3 Artificial ageing of amino acids by merocyanine

540-mediated photosensitization

3.1 Introduction ... p. 81 3.2 Materials and methods ... p. 83 3.2.1 Instruments and working conditions ... p. 83 3.2.2 Chemicals ... p. 83 3.2.3 Amino acid solutions ... p. 84 3.3 Results and discussion ... p. 84 3.4 Conclusions ... p. 94

References... p. 97

Chapter 4 GC-MS characterization of wool lipid components

4.1 Introduction ... p. 99 4.2 Materials and methods ... p. 100 4.2.1 Analytical procedure... p. 100 4.2.2 Instruments and working conditions ... p. 100 4.2.3 Chemicals ... p. 101 4.2.4 Reference specimens ... p. 102 4.2.5 Historical and archaeological textiles ... p. 103 4.2.6 Quantitative analysis and Principal Component Analysis (PCA) ... p. 104 4.3 Results and discussion ... p. 104 4.3.1 Optimisation of sample pre-treatment ... p. 104 4.3.2 Analysis of the reference materials ... p. 108 4.3.2.1 Lipid composition in unaged specimens... p. 108 4.3.2.2 Lipid composition in accelerated aged specimens ... p. 116 4.3.2.3 Principal Component Analysis ... p. 118 4.3.3 Analysis of samples collected from historical and archaeological

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Chapter 5 Synchrotron Radiation based spectroscopies to study

wool degradation

5.1 Introduction ... p. 131 5.2 Materials and methods ... p. 132 5.2.1 Instruments and working conditions ... p. 132 5.2.2 Chemicals ... p. 132 5.2.3 Iron (III) gallate ... p. 133 5.2.4 Reference specimens ... p. 134 5.2.5 Wool cross-sections ... p. 134 5.2.6 Wool pellets... p. 135 5.2.7 Historical and archaeological textiles ... p. 135 5.3 Results and discussion ... p. 135 5.3.1 XRF ... p. 135 5.3.2 XANES... p. 137 5.3.3 EXAFS ... p. 138 5.4 Conclusions ... p. 140

References ... p. 142

Chapter 6 Characterization of tannin raw materials by DE-MS

6.1 Introduction ... p. 145 6.2 Materials and methods ... p. 146 6.2.1 Instruments and working conditions ... p. 146 6.2.2 Chemicals ... p. 147 6.2.3 Sample preparation ... p. 147 6.3 Results and discussion ... p. 147 6.3.1 Galls ... p. 148 6.3.2 Walnut ... p. 152 6.3.3 Catechu ... p. 154

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6.3.6 Onion ... p. 161 6.4 Conclusions ... p. 163

References... p. 166

Chapter 7 Development of an HPLC-ESI-Q-ToF method for the

analysis of complex mixtures of phenols

7.1 Introduction ... p. 169 7.2 Materials and methods ... p. 170 7.2.1 Instruments and working conditions ... p. 170 7.2.2 Chemicals ... p. 172 7.2.3 Raw materials ... p. 173 7.2.4 Van’t Hoff analysis ... p. 173 7.2.5 Method validation ... p. 173 7.2.6 Sample pre-treatment ... p. 174 7.3 Results and discussion ... p. 174 7.3.1 Optimization of chromatographic separation ... p. 174 7.3.2 Van’t Hoff analysis for RP-Amide stationary phase ... p. 176 7.3.3 Optimization of detection with ESI-Q-ToF ... p. 178 7.3.4 Method validation ... p. 184 7.3.5 Application to complex matrices ... p. 187 7.4 Conclusions ... p. 188

References... p. 190

Chapter 8 Conclusions and future work ...

Appendix A Tannins biosynthesis and reactivity

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Introduction and aims of the thesis

Historical textiles are an important part of our cultural heritage. Such textiles are subject to several types of degradation that damage their integrity and readability. Historical textiles are affected by fading, yellowing and loss of material. Thus, their readability is often difficult and sometimes impossible.

In the last few years a number of studies on textile degradation and conservation have investigated the chemical processes causing fibre deterioration in order to propose durable conservation methods (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14).

Historical textiles can be damaged by the way the fabric was used, by the original dyeing procedure, and by the conservation conditions. Textile dyeing is one of the principal causes of fibre damage and thus the effects have been widely studied (1) (2) (3) (5) (6) (7) (8) (11) (12). Thus textile conservation is an extremely challenging task.

Of the widespread range of dyeing techniques and materials used to date, iron-tannin dyestuffs are known to cause specific and intense damage to textiles, highly affecting the fibre integrity (1) (3) (5) (8) (11). This specific class of black dyestuffs is classified as mordant dyestuffs, due to the fact that they are applied using metal salts, called mordants, to bound tannin molecules to the fibre. Iron salts are largely used as mordants to create brown-black complexes, but also other metals were used, such as aluminium and copper (3) (5).

The deterioration of black textiles is a widespread problem. Several examples of textiles dyed with this kind of colorant suffer from severe degradation effects, which in most cases are much stronger than those acting on fibres subjected to the same conservation conditions but dyed with other dyestuffs (1) (3) (5) (8) (11). Several studies have focused on the reasons for the weakness of the tannin dyed fibres, however the answers obtained are still insufficient to develop conservation or

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restoration methods to arrest degradation . Starting from the well-known mechanism of iron-gall ink corrosion, oxidation and hydrolysis reactions are presumed to act on the fibres. Unfortunately, there are fundamental differences between inks and dyestuffs in terms of the method of application and treatment. Inks were also applied mainly to polysaccharide matrices, while tannin dyestuffs were commonly used in dyeing of proteinaceous fibres, such as wool or silk (5).

This PhD research is part of a project, “VAT, The short life of tannins”, which is being funded by the regional administration in Tuscany, Italy (PAR FAS 2008-2013). The aim of the project is to understand the chemical processes acting on textiles dyed with tannin dyestuffs. This thesis focuses on the degradative effects of tannins on wool threads. The final goal of this work will be the creation of a predictive model for describing the ageing processes that affect fibres.

A multi-analytical approach will be used employing complementary techniques to investigate the tannin-fibre complex. Specifically, three different research lines will be followed:

• investigation of wool matrix degradation

• characterization of the composition of tannin dyestuffs

• investigation of mordant oxidation state and structure

Chromatographic, mass spectrometric and spectroscopic techniques will be used. Gas Chromatography-Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) will be used to investigate wool matrix degradation, Direct Exposure Mass Spectrometry (DE-MS) and High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) will be used to characterize dyestuffs composition, and Synchrotron based spectroscopies will be used to obtain information regarding iron mordants. Scanning Electron Microscopy combined with Energy Dispersive X-Ray Spectroscopy (SEM-EDX) will also be used to gain information on both the wool matrix and metal mordant.

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The results will be used to develop innovative analytical protocols and to create a database of chemical properties . The resulting ageing model will be validated by analyzing samples collected from several historical tapestries and fabrics.

GC-MS (lipid fraction)

HPLC-DAD (proteinaceous fraction)

Wool thread

μ-XRF, μ-XANES, μ-EXAFS

SEM-EDX

Mordant

HPLC-ESI-QToF

Dyestuff

DE-MS

SEM-EDX

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References

1. Towards an early warning system for oxidative degradation of protein fibres in

historical tapestries by means of calibrated amino acid analysis. Berghe, I. Vanden.

2012, Journal of Archaeological Science, Vol. 39, pp. 1349-1359.

2. Phototendering of wool sensitized by naturally occurring polyphenolic dyes. G. J.

Smith, I. J. Miller, V. Daniels. 2005, Journal of Photochemistry and Photobiology

A: Chemistry, Vol. 169, pp. 147-152.

3. Production and validation of model iron-tannate dyed textiles for use as historic

textile substitutes in stabilisation treatment studies. H. Wilson, C. Carr, M. Hacke.

2012, Chemistry Central Journal, Vol. 6.

4. Hacke, Anne-Marei. Investigation into the nature and ageing of tapestry materials.

Ph.D. Thesis. [University of Manchester]. 2006.

5. Hofenk de Graaff, J. H. The colourful past. [Abegg-Stiftung and Archatype Pubblication Ltd.]. 2004.

6. Protection against phototendering of wool by metal salts and mordanted dyes. I. J.

Miller, G. J. Smith. 1995, Journal of the Society of Dyers and Colourists, Vol. 111,

pp. 103-106.

7. I.Degano. A multiple-analytical approach for the characterization of organic dyes in textiles. Ph.D. Thesis. [University of Pisa]. 2009.

8. Investigation into the nature of historical tapestries using time of flight secondary

ion mass spectrometry (ToF-SIMS). J. Batcheller, A. M. Hacke, R. Mitchell, C. M.

Carr. 2006, Applied Surface Science, Vol. 252, pp. 7113-7116.

9. Investigating the photo-oxidation of wool using FT-Raman and FT-IR

spectroscopies. Jones, D. C. 1998, Textile Research Journal, Vol. 68, pp. 739-748.

10. Photodegradation of tryptophan in wool. K. Schaefer, D. Goddinger, H.

Hocker. 1997, Journal of the Society of Dyers and Colourists, Vol. 113, pp. 350-355.

11. Studies on woollen threads from historical tapestries. M. Odlyha, C.

Theodorakopoulos, R. Campana. 2007, Research Journal, Vol. 7, pp. 9-18.

12. Peggie, D. A. The development and application of analytical methods for the identification of dyes on historical textilesc. Ph.D. Thesis. [University of Edimburgh].

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13. Yellowing of wool keratine on exposure to ultraviolet radiation. R.S. Asquith, K.

E. Brooke. 1968, Journal of the Society of Dyers and Colourists.

14. Radical mechanism involved in the photobleaching and photoyellowing of wool.

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Chapter 1

State of the art

1.1 The chemistry of wool fibre

Wool is a keratin-base mammalian hair constituted by an extremely complex structure. Wool is constituted almost exclusively of keratin (97 %), with low percentages of lipids, polysaccharides, nucleic acids and mineral salts (3%) (1) (2) (3) (4) (5) .

Lipids constituents are located in the intercellular region and on the surface of the fibre (1). Intercellular region lipids have lamellar structure divided into two domains: orthorhombic domain and liquid crystal one. Orthorhombic domain is made of ceramides, cholesterol, glycerides and free fatty acid separated from each other. Liquid crystal domain is constituted by the same components mixed together (1) (6).

Wool surface is covered by two kinds of lipid materials. The external layer, called wool wax, is secreted by sebaceous glands of sheep and consists in more than 15 different chemical classes divided into three groups: high-polarity lipids, medium polarity lipids and low polarity lipids. High polarity lipids are constituted by ceramides, 2-hydroxyacids from C14 to C18, 2-hydroxyalcohols from C14 to C24, 1,2 dihydroxyacid and 1,2 diols with even carbon number from C14 to C30 and cholesterol oxidation products such as cholestanediols, cholesta-3,5-diene-7-one and 3-hydroxy-lanosta-8-ene-7-one. Medium polarity class contains free saturated and monounsaturated fatty acids from C7 to C31, fatty alcohols from C12 to C35, cholesterol, lanosterol and desmosterol, together with hydroxycholesteryl esters. Low-polarity lipids are constituted by triacylglycerols, aliphatic diesters, aliphatic and steroidal monoesters, steryl, cholesteryl, lanosteryl and dihydrolanosteryl esters (1) (4) (7) (8) (9) (10) (11) (12) (13). Diacid compounds (11) and low percentage of

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hydrocarbons ranging from C14 to C33 (14)are also present in lanoline composition. Lipid chains assume three different isomeric structures: linear, iso- ((ω-1) monomethyl substituted) and anteiso- ((ω-2) monomethyl substituted) configuration (1) (4) (5) (7) (9) (10) (11) (12) (13).

Second surface lipid layer is covalently bonded to keratin and it is principally constituted from 18-methyeicosanoic acid (18-MEA), approximately 50-60% of the total amount. Lipids are linked via ester bonds to specific protein residues: cysteine and, less commonly, serine and threonine (1) (3) (5) (7) (15) (16) (17) (18) (19) (20) (21). Fatty acids from C16 to C20 are also present together with low amounts of cholesterol, cholesterol sulphate and ceramides (1) (16) (17) (18) (19) (21). Further branched-chain fatty acids are present: 14-methylhexadecanoic and 16-methyloctadecanoic acid (16). 18-MEA is supposed to have a fundamental structural role in cell adhesion (5) (7) (16) (19).

Lipid layers covering wool surface determine its high hydrophobicity (1) (5) (7) (17) (19) (20) (21) (22) (23). Scouring treatments with non ionic surfactants partial remove non-covalently bound surface wool wax (15) (17) (18). Consequently 18-MEA is fundamental in maintaining fibre hydrophobicity (16) (21). Possible anti-oxidative and bactericide action of 18-MEA was also supposed (21).

Inorganic material, constituting almost 1 % in weight of fibre total amount, consists in potassium, sodium, calcium, aluminium, iron, silicon dioxide, sulphate, carbonate, chloride and phosphorus pentoxide ions (1).

Keratin consists of almost one hundred proteins whose structure and amino acid composition is quite diversified. These proteins are built up mainly by amino acids with large side groups, as glutamic acid, aspartic acid, arginine, leucine, valine and cystine. Keratin proteins can be divided into three main groups, present in different percentages depending of wool specie: low-sulphur proteins (LSP), high sulphur proteins (HSP) and high-tyrosine proteins (HTP). Principal amino acids constituting keratin are shown below (Figure 1.1). Not all these proteins have the same influence on wool microscopic and macroscopic features (2) (7) (15) (24).

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Figure 1.1 Principal keratin amino acids

Wool keratin proteins are organized in four morphological components: cuticle, cortex, cell membrane complex and medulla. The cuticle is made of overlapped cuticle cells forming an external protective layer of almost 1 µm, divided into three parts: epicuticle, exocuticle and endocuticle. In the outer part is the epicuticle, which is in contact with the inner amorphous exocuticle. The inner exocuticle, of almost 0.3 µm thickness, is constituted by two different layers: the “a layer” outside, very rich in sulphur, and the “b layer” inside. The b layer is in contact with the third part of the cuticle, named endocuticle, not very rich in sulphur and consequently easily subjected to degradation. The high sulphur content of cuticle, increasing from endocuticle to epicuticle, determines its amorphous structure. Sulphur containing residues can form

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disulfide bridges, fundamental structures that give chemical resistance and physical strength to fibres (1) (3) (15).

The cortex makes up the bulk of the fibre (90 % of the total amount). It is made of cortical cells arranged parallel to fibre axis and packed in a sulphur rich amorphous matrix. The cortex can be divided into two different regions: the orthocortex, where the matrix is distributed around a high number of crystalline fibrils twisted together, and the paracortex, constituted almost of amorphous matrix concentrated in specific regions between fibrils randomly distributed. The different structure of ortho- and para-cortex is responsible of curly shape of wool. The high crystallinity of the orthocortex, due to its low sulphur content and consequent lack of disulfide bonds, makes it extremely sensitive to hydrolysis and chemical attack. Cortical crystalline proteins are arranged into helices, which form structures called superhelices or protofibrils by twisting together three helix chains. Protofibrils are organized in helices forming a structure called microfibril. Microfibrils are arranged in macrostructures called macrofibrils, which constitute the cortical cell. Helical secondary structure of wool can take two different conformations: α-helix and γ-helix. Among these, the γ-helix organisation is the less stable due to the difficulties in the formation of secondary bonds between the residues (2) (3) (5) (7). α-helix conformation allows amino acids large side groups to locate in the external part of the structure, reducing steric effects. This structural organization makes residues of single amino acids having a key role in determining wool mechanical and chemical properties (2) (3) (7) (15) (24).

A continuous layer of intercellular material named cell membrane complex (CMC), composed of proteins, lipids, carbohydrates and minerals, is placed between cuticle and cortex. This layer strongly helps in resistance to water penetration. CMC amount is almost 4-6 % of fibre mass and it is the only continuous phase in wool. Consequently, it is fundamental in determining fibre properties and intercellular adhesion. CMC lacks in mechanical strength and it is sensitive to biaxial and torsional stresses, causing a phenomenon known as fibrillation. CMC proteins are constituted principally by glycine, tyrosine and phenylalanine. The low number of disulphide cross-links suggests low cystine content (1).

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Moreover, coarse fibres can present another structural element called medulla, constituted by specialized and vacuolated cells located along fibre axis (2) (3) (5) (7) (23).

Keratin secondary bonds are fundamental to determine wool properties. They can be due to different type of interactions: salt linkages, hydrophobic interaction, hydrogen bonds and isopeptide bonds strongly contribute to fibre strength and chemical solubility (1) (7). These interactions also allow the linkages among α-helices to be formed, obtaining protofibrils formation. The presence of sulphur rich proteins, which can form disulphide bonds, has a strong influence on wool physical and chemical properties. These kinds of bonds, creating cross-link between keratin chains, have an important role in wool mechanical and thermal properties and they are fundamental in maintaining wool three-dimensional structure (2) (7) (15) (24). Most of disulphide linkages are located in the amorphous non-helical keratin matrix (7).

1.2 Wool degradation

1.2.1 Lipid degradation

Wool is easily subjected to oxidation and subsequent removal of the external lipid layer (1). Previous studies about wool degradative processes highlighted that 18-MEA is extremely sensitive to photodegradation mechanisms (1) (18) (20). Bleaching of fibres via hydrogen peroxide cleaved linkages between 18-MEA and wool keratin residues through oxidative reactions (20). 18-MEA ester bonds were more resistant to degradation processes that thioester ones (1).

Lability of ester linkage between 18-MEA and fibre surface is probably related to the position of this lipid in wool fibre structure. 18-MEA is located in an accessible overlayer and consequently it is more sensitive to cleavage if compared with other lipids bounded in more hydrophobic and isolated positions (20). 18-MEA reduction influences membrane fluidity, causes rigidity and reduces cells adhesion (18) (19).

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Analysis of lipid wool composition on aged fibres highlighted the presence of some oxidation products of sterols (25). Sterols component are highly sensitive to oxidative damages (26). Moreover, strongly alkaline and acid treatments, such as those during of dyeing procedures, cause surface lipid loss and reduction in strength. Particularly, tannin dyeing with ferrous sulphate and copper sulphate mordanting has stronger deleterious effects on fibre strength (18) (27). Keratin lipids were sensitive both to UV and Visible radiations (28).

1.2.2 Keratin degradation

Wool is subjected to several kinds of degradation processes, such as changes in tensile stretch, loss of elasticity and yellowing due to UV-Vis exposure (23) (29). Wool is sensitive to the action of heat, light, acids, alkalis, reducing agents and microorganisms (7) (15) (23). Cuticle, rich in sulphur bridge, acts as first protection against external agents. In case of its detriment with consequent rupture of sulphur cross-link, wool fibre becomes extremely sensitive to degradation. Furthermore, reactions of disulphide bonds, which are detailed afterwards, easily lead to the formation of volatile sulphur compounds which have detrimental effects on organic materials (23). As a consequence, reactions involving cystine and cysteine residues have a fundamental role in wool degradation processes (15).

In the following subsections are described degradative processes affecting wool keratin.

Degradation by light exposure

Wool is sensitive to UV-Vis radiation, which cause several phenomena: photo-yellowing, bleaching and tendering. They consist in the light induced excitation of chromophores with consequent deactivation via several processes: internal vibrational relaxation, emission of photons, intersystem crossing from singlet to triplet excited state with subsequent energy transfers (1). Amino acid residues are not equally sensitive to UV-Vis radiation: tryptophan, histidine and cystine are the one more subjected to degradation, followed by tyrosine, phenylalanine and methionine.

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Glycine and alanine degradation is due to secondary reaction mechanisms (1) (3) (23) (30). Mechanisms hypothesized were studied both on wool fibres (29) (31) (32) (33) (34) and amino acids solutions (35) (36).

Wool photo-oxidation reactions can follow two different pathways. The photosensitizer can react with the substrate, with formation of free radicals that react with oxygen. Otherwise, there is an energy transfer between excited state photosensitizer and ground state molecular oxygen, leading to singlet oxygen formation (24). Reactivity of singlet oxygen decreases from histidine to tryptophan, tyrosine, methionine and cysteine. Relaxation times of singlet oxygen in water are very low, limiting the extent of reaction. In dry wool keratin, relaxation time increases, but oxygen diffusion is low and reactions still occur only on fibre surface (24).

Electromagnetic radiation causing wool degradation can be divided into three regions, on the basis of its effects: tendering, yellowing and bleaching (1) (3) (15) (37). Processes causing photo-tendering, photo-yellowing and photo-bleaching of wool involve several keratin residues.

Photo-tendering occurs below 300 nm. It is principally caused by cystine disulphide bond break resulting in tensile strength and abrasion resistance loss (1) (38). UV radiation cause photo-oxidative rupture of disulphide cross-links with formation of several products, depending of the irradiation wavelength used. Cystine S-monoxide and cystine S,S-dioxide, partially oxidized products, are generated with irradiation at 254 nm. On the contrary irradiation at 360 and 420 nm leads to the formation of cysteine sulphonate (1) (33) (34) (37). Particularly, cysteine S-sulphonate formation is not a consequence of direct cystine absorption (37). These oxidation products are unstable and hydrolyse to sulphonic acid groups (Figure 1.2) (1) (7).

The reactions described affect fibre stability, causing hydrolysis of peptide bonds in the presence of moisture and subsequent formation of cysteic acid (Figure 1.3 ) (3) (7) (30) (33) (34). Thus cysteic acid was used as a marker of oxidative modifications affecting wool during ageing (38) (39).

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Figure 1.2 Disulphide oxidation pathway: (I) disulphide, (II) cystine monoxide, (III) cysteine sulphenic acid, (IV) cysteine sulphenate, (V) cystine dioxide, (VI) cysteine sulphinic acid, (VII) cysteine sulphonate, (VIII) cysteine sulphonic acid

Figure 1.3 Cysteic acid formation

Wool radiation between 250 and 320 nm causes yellowing phenomena. Absorption at this wavelength leads to the formation of free radicals and new chromophores absorbing at longer wavelengths (causing yellowing), together with significant decrease in histidine, tyrosine and tryptophan content (1) (24) (38). Chromophores absorbing between 250 and 300 nm are the aromatic residues of tyrosine, tryptophan and phenylalanine, together with cystine disulphide bonds (1) (24) (37). Their maxima of absorption are at 275, 280, 257 and 250 nm respectively (1) (24). Particularly, irradiation of tyrosine, tryptophan and phenylalanine leads to the formation of oxyndolylalanine, ortho-benzoquinone and benzoic acid respectively, increasing the number of oxygenic groups in the fibre (40). Wool yellowing is strongly influenced by humidity: in dry conditions yellowing occurs ten times slower than in humid ones (15) (24) (37). Several mechanisms are involved in

R= -CH2CH(NH-(polypeptide chain))CO-polypeptide chain

RSSR (I) RSOSR (II) RSOH (III) RSSOH (IV) RSO₂SR (V) RSO₂H (VI) RSSO₃H (VII) RSO₃H (VIII)

Sulphonic acid side group

H

O

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yellowing process. Cysteyl residues can quench excited singlet state of tryptophan and tyrosine, breaking disulphide bond and forming thiol radicals: UV radiation absorbed by tyrosine is transmitted to cystine causing disulphide bridge breaking and sulphur radical formation (3) (7) (23) (24) (33) (37) (41) (42) . In the presence of moisture, sulphur radicals turn firstly to thiol and sulphenic acid, with subsequent elimination of sulphenic group and formation of cysteine and aldehyde side groups (Figure 1.4) (3) (7) (23) (24) (33) (37) (41). These reactions are associated with an initial increase in absorption signal at 600 nm, due to sulphur radical formation, followed by a decrease due to the formation of thiol and sulphenic radicals (24) (40).

Figure 1.4 Effect of light exposure: sulphur radical reaction

Quenching reaction involving tyrosine, together with tyrosine reaction with singlet oxygen, lead to the formation of phenoxyl radicals. These radicals can couple forming dityrosine or can react with oxygen forming dihydroxyphenylalanine (dopa) (Figure 1.5). This product can be subjected to further oxidation, forming ortoquinone and, via condensations reaction, melanine (3) (24) (32). Another reaction product identified is hydroxydityrosine, deriving both from dityrosine oxidation and tyrosine-DOPA coupling (Figure 1.5) (24) (32) (35). All such products are chromophores causing yellowing phenomenon (35) (39).

Tyr

Oxidation H2O H2O

+

Cystine linked

with tyrosine Sulphur radicals

Thiol and sulphenic acid side groups

Thiol side group (cystine) and aldehyd side group S CH2 CH2 S SH CH2 CH2 HOS SH CH2 COH CH₂ S S CH2 CH₂ O H

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Figure 1.5 Tyrosine photo-oxidation pathway

Tyrosine absorption leads to an energy transfer from tyrosine to tryptophan residues. Consequently, co-presence of tryptophan and tyrosine reduces tyrosine degradation. This process occurs only if the residues involved are close to each others. Photosensitisation of tryptophan occurs even in the presence of other aromatic residues, such as phenylalanine (3) (32). Excited tryptophan could lead to several processes. Previous studies indicated correlations between wool yellowing, tryptophan initial content and tryptophan destruction rate (3) (32). Tryptophan could be quenched by disulphides through a static short-range interaction not involving electron/charge transfers (24) (42). Electron transfer reaction with disulphide, leading to tryptophan free radical formation, is evidenced in aqueous solution (24). Tryptophan and tyrosine are also involved in cross-link reactions, resulting in dityrosine and tryptophan-histidine linkages formation (24) (43). Moreover,

NH O O H NH O O H OH NH O O H NH O OH [O] [O] Tyrosine Tyrosine NH O O H NH O OH OH Tyrosine Dityrosine Dopa Hydroxydityrosine

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tryptophan can react with α-keto acids deriving from irradiation at 320, resulting in β-carbolin formation (Figure 1.6) (1) (24) (43). Instead, tryptophan is quenched by oxygen in hydrophobic environment (24). Oxidation of the C-2 position of the indole ring results in the formation of alanine. Hydroxylation of oxindole-3-alanine leads to dioxindole-3-oxindole-3-alanine (Figure 1.6). Otherwise, reactions between tryptophan and singlet oxygen generate hydroperoxide species such as 3α-hydroperoxypyrrolidinoindole, which leads to the formation of 3α-hydroxypyrrolidinoindole (Figure 1.6) (3) (43) (44). Hydroperoxides decompose with oxidative opening of the indole ring generating N-formylkynurenine (NFK), a yellow product which can be reduced forming kynurenine (Figure 1.6). NFK is known to be a photosensitizer (3) (24) (30) (32) (37) (43). Moreover photo-oxidation of tryptophan and kynurenine leads to the formation of 7-hydroxytryptophan and 3-hydroxykynurenine (3-HK) respectively (Figure 1.6) (1) (3) (24) (32) (43). 3-HK can also be derived from 7-hydroxytryptophan indole ring rupture (Figure 1.6) (3) (30). Kynurenine is known to form cross-links with basic amino acids, such as lysine and histidine (32).

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Figure 1.6 Photo-oxidative pathway of tryptophan

Hydroperoxides can further react with following two main ways: homolytic cleavage due to light exposure and reductive cleavage via electron transfer involving metal ions. Hydroxy radicals resulting from these reactions can abstract hydrogen from α-C-H bonds of α-amino acids, N-H bonds of tryptophan and S-H bonds of cystine (3). For example, reaction between hydroxy radicals and cysteine thiol groups leads to the formation of new disulphide cross-links (Figure 1.7) (3) (7).

HOOC NH2 NH O H NH HOOC NH2 O Tryptophan N-formylkynurenine 7-hydroxytryptophan Kynurenine 3-hydroxykynurenine hν O2 hν − CO NH2 HOOC NH2 O _{hν O} 2 OH NH2 HOOC NH2 O O H HOOC NH2 NH hν O2 hν O2 O NH2 NH HOOC OH O HOOC NH2 NH Oxindole-3-alanine Dioxindole-3-alanine hν O2 R N H N β-carboline hν Ο2 COOH OOH N H NH 3α-hydroperoxypyrrolidinoindole RCO₂H COOH OH N H NH 3α-hydroxypyrrolidinoindole hν

25

Figure 1.7 Disulphide cross-link formation

Moreover, hydroxy radicals can hydroxylate aromatic residues of tyrosine, phenylalanine and tryptophan, generating phenoxyl radicals from phenols (3). Finally tryptophan irradiation below 300 nm generates fluorescent degradation products. Excitation of these substances above 320 nm generates fluorescence at 450-500 nm (3). Photo-oxidation processes involving tryptophan highlight that also this molecule can be used as a marker of wool yellowing (3).

Bleaching phenomenon occurs between 400 and 460 nm and it is extremely important in terms of tapestries ageing studies. Tapestries are often kept behind window glasses, which filter lower wavelength UV light (1) (24). Bleaching causes the degradation of photo-oxidative products of tryptophan and tyrosine, which absorb between 350 and 500 nm (1) (3). Moreover, bleaching processes make fibres more sensitive to yellowing (3).

Sulphur and carbon free radicals are both involved in photodegradation reactions (1) (3) (24) (40). Sulphur radicals formation, due to irradiation at 310 nm, is a consequence of the radical free process involving cystine and tyrosine previously described (1) (3) (24) (45). Previous studies suggested that these radicals formation is not a direct consequence of radiation absorption by disulphide bond with subsequently cleavage (24). Carbon radicals, which structure is still unknown, derivate from tryptophan, tyrosine, phenylalanine and histidine residues (1). Kinetic analysis showed at least three different carbon centred radicals deriving from keratin residues (24). Free radicals formation increases in the presence of iron and copper ions, which form complex with carboxyl-acid groups and sulphur containing groups. Radical formation is a consequence of energy transfers between these complexes and keratin residues (24) (46). Both sulphur and carbon radicals disappear in the presence

O H

+

+

Cystine side groups Disulphide cross-link

SH

CH₂

26

of oxygen, suggesting the incoming of photo-oxidative reactions (1) (24). Energy transfer leads to the formation of excited oxygen and, consequently, to amino acids photolysis (1).

Previous studies attested the incoming of ageing processes involving wool keratin which lead to the formation of 4-hydroxybenzoic acid. Reaction mechanisms are still unknown, but involving of aromatic amino acids has been hypothesized. 4-hydroxybenzoic acid is supposed to be the product of oxidation of tyrosine and tryptophan residues, known to be sensitive to photo-oxidative reactions (Figure 1.8) (15) (41) (47).

Figure 1.8 Hypothesized reaction leading to 4-hydroxybenzoic acid formation

Degradation by heat

Wool subjected to high temperature treatments suffers from several physical and chemical detriments. First of all, temperature above 100 °C causes desiccation and formation of new cross-links during condensation reaction between water-bonding carboxyl and amino groups (Figure 1.9). Moreover, if temperature above 180 °C is reached, elimination reactions lead to the formation of hydrogen sulphide, ammonia and yellow-brown substances (7). Dyeing processes commonly need relatively high temperature treatments, causing detrimental effects on textiles (27) (48).

Figure 1.9 Effect of heat

OH O O H OH O NH₂ O H O₂ hν

Tyrosine 4-hydroxybenzoic acid

+

Glutamic acid residue Lysine residue Peptide cross-link COOH (CH2)2 O N H CH C NH O C C H (CH2)4 N H2 O NH O C C H (CH2)4 NH C (CH2)2 O N H CH C

27

Degradation by acids

Most of the recipes describing wool dyeing methods concern the use of hot and quite acidic solutions. These treatments became fundamental in wool dyeing with natural mordanted dyestuffs, but they cause several problems: peptide bond hydrolysis, breakage of polypeptide chain, loss in crystallinity and decreasing in fibre cohesion. Consequently, fibres strength is strongly reduced (15) (27).

Amorphous region is sensitive to acid attack, which can act on keratin peptide bonds, salt linkages and secondary bonds. Acid attack can also cause yellowing phenomenon: hydrolysis of amino side group of glutamic acid and asparagine leading to the formation of dehydroalanine groups and ammonia (Figure 1.10), was hypothesized.

Moreover, as previously explained, when disulphide cross-links turn into sulphonic acid because of photo-oxidation reactions, a consequent hydrolysis of neighbouring peptide bond occurs with formation of cysteic acid and shorter peptide chains (7).

Figure 1.10 Effect of acids: formation of dehydroalanine

Degradation by alkalis

Degraded wool is more sensitive to alkali detriment than not degraded one. In fact, when radiation or chemical treatments cause fibres oxidation with breaking of peptide and disulphide linkages, wool structure can be modified even by mild alkali conditions. Generally, wool dissolves completely in 5% sodium hydroxide solution, while a 2 % sodium chloride solution protects wool from alkali degradation. On the contrary, alcoholic solution increments alkali damages (7). Wool dyeing with vat dyes

Amino side group Dehydroalanine

side group H+

+

28

needs mandatory alkali conditions to bond dyestuff molecules to textile fibres. Consequently, controlled conditions are required (15).

Alkaline treatment leads to the rupture of disulphide linkages, with formation of thiol and sulphenic acid side groups. Sulphenic acid side group is unstable and decomposes giving dehydroalanine, water and sulphur (Figure 1.11).

Figure 1.11 Effect of alkali: formation of sulphenic acid side groups

Reaction between sodium and sulphur leads to the formation of sodium sulphide, that breaks disulphide link through an autocatalytic process (7). Sulphenic acid decomposition leads also to the formation of aldehyde side group and hydrogen sulphide, via elimination reaction (Figure 1.12).

Figure 1.12 Effect of alkali: sulphenic acid decomposition

Disulphide cross-link Thiol side group

OH

-Sulphenic acid side group

+

Dehydroalanine side group

SOH CH₂ NH O C CH OH -CHO NH O C CH

₊

Sulphenic acid side group Aldehyde side group

Sulphenic acid side group

NaOH

+

₊

29

Both aldehyde and dehydroalanine side groups are chromophore molecules that cause yellow, grey and brown discoloration if part of conjugated groups. Moreover, hydrogen sulphide and sodium sulphite can deteriorate organic material and cause the corrosion of any metals in contact with wool (7). Dehydroalanine residues can react in turn with the thiol group formed during the described reaction or with the cysteine side groups of wool, leading to lanthionine formation. This cross-link is stable in acid condition but reacts in the presence of alkali and it is easily subjected to photolysis. Furthermore, dehydroalanine can form lysinoalanine crosslink with lysine residues of neighbour chains (Figure 1.13) (7).

Figure 1.13 Effect of alkali: dehydroalanine reactions

Degradation by reducing agents

Disulphide links are extremely sensitive to the action of reducing agents that reduce cystine residues to thiol groups (Figure 1.14). Reoxidation by air or chemicals leads to the formation of new disulphide bridges in different positions. Reoxidation is a technique commonly used to obtain artificial modification of wool shape, creating new waves or eliminating undesired ones (7).

CH2 NH O C C Dehydroalanine residue S H CH2 O NH C H C

+

Thiol side group

S CH2 O N H CH C NH O C C H CH₂ Lanthionine cross-link CH2 NH O C C Dehydroalanine residue N H₂ (CH₂)₄ O NH C H C

+

30

Figure 1.14 Effect of reducing agents

Degradation by biological attack

Wool is sensitive to several fungi and bacteria. These organisms use different processes, as enzymatic activity, catalytic oxidation and reduction and hydrolysis, to destroy, eat and soil wool fibres. These processes mainly result in disulphide and peptide bonds break. These phenomena happen principally in the amorphous cuticle, rich in disulphide bonds.

Moreover, wool is easily affected by detrimental action of beetles and moths. These insects use reducing secretions to break disulphide bonds, making keratin sensitive to the attack of protein-digesting enzymes (7).

Cystine Thiol side groups

CH₂ S S CH₂ SH CH₂ C H₂ SH

31

1.3 Analytical techniques used in the study of wool composition

and degradation

Wool ageing studies employ a wide range of analytical techniques to investigate its composition, state of conservation and damages.

• Physical tests: physical properties of wool fibres are studied to asses tapestry condition and structural integrity. Stress and strain tests are used to evaluate changes in mechanical properties (1) (29). Particularly, Smith et Al. (29) attested the active role of metal ions used in mordanting procedures, which take part in reaction mechanisms induced by light exposure.

• Colorimetry: this technique, which evaluate fibre colour on the basis of CIE tristimulus values, is commonly employed in ageing investigation on dyed and not dyed wool (15) (27) (49). Wilson et Al. evidenced significant differences in colour changes of dyed and not dyed wool subjected to the same ageing protocol (27). Nevertheless, colour changes of complex structures as tapestry are not suitable as index of wool detriment, being extremely dependent from dyestuff sources and dyeing procedure (15) (49) (50).

• Thermal analyses: Dynamic Mechanical Thermal Analysis (DMTA), Differential Scanning Calorimetry (DSC) and Thermogravimetry (TGA) are commonly used to study thermodynamic properties of wool. These techniques were able to evidence differences in mechanical properties and thermal stability incoming during ageing processes, relating them with dyeing damages (51) (52) (53). Particularly, Odlyha et Al. (51) used DMTA, DSC and TD to characterize model tapestries, and to quantify the effects of mordanting and dyeing processes. Moreover, thermal alteration due to black dyeing was highlighted.

• IR spectroscopy: spectrophotometric techniques such as Fourier Transform Infra Red (FT-IR) (5) (36) (50) (54) (55) (56), Raman (5) (33) (48) (55) (56) and Attenuated Total Reflectance (ATR) (33) (34) (38) (56) spectroscopies are widely employed to study wool degradation. These techniques were successfully used as superficial-non destructive analytical methods (34) (38) (48) (54) (55) (56).

32

Information about amino acid oxidation state, disulphide bond and cysteic acid were obtained on the basis of IR absorption bands (33) (34) (38) (48) (55) (56). In particular, Odlyha et Al. (34) used ATR spectroscopy to evaluate historical treads degradation. Fibre degradation was attested on the basis of cysteic acid amount, quantifying its signal at 1040 cm-1. Also oxidative changes were highlighted and semi-quantified on the basis of cysteine monoxide and dioxide signals (1075 and 1125 cm-1 respectively). Moreover, the influence of dyestuff sources and mordants on wool degradation was highlighted. Church et Al. quantified degradative changes affecting tryptophan and tyrosine on the basis of their Raman lines at 1002 cm-1 for tryptophan, 851 and 829 cm-1 for tyrosine, but no identification of degradation product was achieved (33).

• Fluorescence spectroscopy: this technique is also applied in the identification of wool degradation products: secondary products formed during wool yellowing and bleaching highlight specific fluorescent emission (3) (24) (42). Particularly, Davidson (3) studied degradation of tryptophan (excitation at 320 nm gives fluorescence at 450-500 nm) which leads to the formation of new fluorescent products absorbing at 350-500 nm. These unknown products were destroyed by radiation wavelengths between 380 and 500 nm.

• Scanning Electron Microscopy (SEM): SEM analysis is widely employed in wool morphological studies. Detailed images obtained from SEM analysis highlighted physical damages occurring on wool fibre after dyeing, mordanting, washing procedures and degradation (1) (23) (40) (50) (54) (57) (58) (59). Results obtained with SEM analysis are exclusively morphologic and topographic. SEM-EDX coupling is employed to obtain information about elemental composition of wool surface (1) (17) (27) (60). Particularly, Hacke (1) employed SEM analysis to study degradative effects of ageing of dyed and not dyed fibres, comparing results with historical threads. The author highlighted no detrimental effects of dyeing procedures on wool morphology. On the contrary, on accelerated aged fibres deep longitudinal cracks were attested. Longitudinal cracks were highlighted also in historical threads, together with transverse cracking and scale ablation, indicative of more widespread deterioration.

33

• X-Ray Photoelectron Spectroscopy (XPS): SEM instrumentation is commonly coupled with XPS technique. XPS analysis gives information about chemical composition and degradation state of wool fibre surface, being complementary to SEM technique. Particularly, XPS analysis gives qualitative and quantitative information about elemental composition of fibre surface (1) (55) (56), measures changes incoming with degradation processes and allows identification of atoms oxidation state to be performed on the basis of binding energies of photoelectron ejected from the samples (1) (22) (61). This technique is largely employed to investigate amino acid and lipid wool components (1) (22) (61) and to characterize intercellular matrix structure (6). In particular, Hacke (1) studied C/N and C/O elemental ratios of wool surface. These ratios were related to surface lipid content (C/N) and oxidative state (C/O) of wool, and their changes were used to study wool detriment. Particularly, C/N and C/O decrease was attested during ageing, suggesting the detriment of surface lipids and the incoming of oxidative reactions. Complete loss of surface lipid layer and intense oxidation state was attested for historical threads.

• Secondary Ions Mass Spectrometry (SIMS): this technique allows the characterization of amino acidic and lipid composition of the fibre to be obtained, providing elemental and molecular mass data of the first 1-2 mm of the surface (1) (16) (17) (18). In particular, SIMS instrumentation was employed to investigate ester linkages between external lipid layer and wool keratin (17) (20). Hacke (1) highlighted the increase of oxidised sulphur species during ageing. Particularly, in negative ion ToF-SIMS spectra of accelerated aged fibres was evidenced an increase of SO3

-

signal (m/z 80) together with a decrease in SH- (m/z 33) signal. Moreover, SIMS analysis highlighted partial reduction of lipid surface layer due to dyeing and ageing of fibres. Particularly positive ion ToF-SIMS analysis detected cholesterol fragment [M-OH]+ (m/z 369) in not aged and artificially aged wool, while this signal disappeared in historical threads. Moreover, studies of [C20H41COS

-] (m/z 431) and [C20H41COO

-] (m/z 325) fragments, belonging to 18-MEA bonded with thioester and ester bond respectively, suggested higher resistance to degradation of oxygen ester bounds.

34

• Electron Spin Resonance (ESR): with ESR technique it is possible to characterize radical produced in irradiated wool. This technique help in elucidation of photodegradative mechanisms acting on wool on the basis of the radicals identified (3) (24) (37) (45). Millington et Al. (37) highlighted formation of RSSR+· and RSSR+·as photolysis production of wool UVC exposure, suggesting that only the radical cation leads to the formation of partially oxidized cystine. Moreover this study supported the identification of [RS(SR)SR] structure as the dominant stable radical specie generated in wool keratin by UV exposure.

• Thin Layer Chromatography (TLC): this technique is widely employed to study wool lipid composition (62) (63). With TLC separation of wool lipid components is achieved. Combination between TLC and Flame Ionization Detector (FID) has the advantage of avoiding hydrolysis procedure giving information on the original composition of wool lipid components (4) (8) (62) (64). Jover et Al. (4) used TLC-FID for the analysis of several chemical classes of compounds: fatty acids, mono- and diesters, fatty alcohols, diols, hydroxy acids, sterols and ceramides were detected. This technique does not give structural information: identification of compound is achieved through comparison with standards.

• Gas chromatographic separation: analysis of wool lipid molecular composition is commonly achieved using Gas Chromatography Mass Spectrometry (GC-MS) (9) (12) (13) and Pyrolysis/Gas Chromatography Mass Spectrometry (PY/GC-MS) (10). These analytical techniques provide characterization of complex mixtures of analytes through structural identification. Supercritical fluid extraction with carbon dioxide followed by dissolution in ethylacetate/ciclohexane (1:1) and alkaline water extraction (NaOH) were commonly employed in samples pre-treatment (9) (12). GC-MS and Py/GC-MS can be employed only on high volatile analytes. As a consequence, derivatization procedures to increase lipids volatility are used. Methylation and silanization reactions are commonly employed (9) (10) (12) (13). Moldovan et Al. Used GC-MS for the characterization of wool free fatty acids and free fatty alcohols from C9 to C33, 2-hydroxy fatty acids and 2-hydroxy fatty alcohols from C14 to C24 in both normal-, iso- and anteiso- structures (9). Py/GC-MS, which avoid long sample pre-treatment, is widely used as fingerprint method

35

for the characterization of wool wax composition (10). Asperger et Al. (10) identified branched and unbranched alkanes belonging to iso- and anteiso- fatty acids, aliphatic aldehydes deriving from 2-hydroxycarboxilic acid, and several steroid such as 2,5-cholestadiene and 3,5-cholestadiene.

• High performance Liquid Chromatography (HPLC): this chromatographic instrumentation is widely used to achieve separation of wool amino acid components (32) (39) (41) (43) (65) (66). Particularly, with this technique qualitative and quantitative characterization of wool products is achieved. HPLC technique does not require high volatile analytes, reducing the employment of derivatization procedure and curtailing sample pre-treatment. Analytes separation can be performed using different kinds of columns: C18 reversed phase columns are widely employed (32) (39) (41) (43) (67). HPLC instrumentation can be coupled to several detectors, as UV-Vis spectrophotometers or Diode Array Detectors (HPLC-DAD) (32) (41) (43) (65), Fluorescence spectrophotometers (39) (43) (65) and Mass Spectrometers (HPLC-MS) (32) (41) (66). HPLC-DAD and HPLC-Fluorescence instrumentation are extremely suitable for the characterization of chromophores produced in wool fibre during photo-oxidative reactions on the basis of their absorption and fluorescence bands (32) (41). Particularly, Vanden Berghe (39) employed HPLC-Fluorescence amino acids analysis to investigate oxidative degradation of keratin. This study evidenced the decrease of tyrosine together with the increase of cysteic acid during ageing. Consequently, these analytes were identified as oxidative degradation markers. Moreover, the author determined a parameter indicative of fibre oxidation state, named Keratin Degradation Factor, based on amino acids more sensitive to oxidation reaction. Fluorescence detector allows high sensitivity and sensibility to be achieved, but the low number of fluorescent chromophores in wool limits the application of this technique in the study of textiles. To avoid this problem, pre or post-column derivatization is employed (47). HPLC-DAD was also employed to obtain a preliminary fractionation of yellow chromophores followed by their structural characterization with MS detectors (32).

36

1.4 The chemistry of tannins

Tannins belong to plant natural phenols, a class of chemical compounds extremely complex and widespread in plant kingdom. Natural phenols are classified as natural organic substances, featuring one or more phenolic groups in their structure. These aromatic compounds are the principal group of secondary metabolites and bioactive substances of plants, also widespread in microorganism kingdom (68) (69) (70) (71). High amounts of tannins are found in wood, bark, leaves, buds, floral parts, seeds and roots of plants (68) (69).

Tannins have essential roles in plant physiology and ecology, such as growth, photosynthesis and reproduction. Their protein binding capacity, leading to astringency phenomenon, acts as a defensive mechanism by reducing plant digestibility. They act as phytotoxic, antimicrobial, antimycotic, antiviral, allopathic, phytoalexins and signal molecules. Moreover, tannins act also as antioxidants, due to their reducing power and free radical scavenging activity. They limit the occurrence of cancer in humans and protect against oxidative processes acting on proteins or carbohydrates. Antioxidant activity principally depends on tannins stereochemistry and degree of hydroxylation (68) (72) (73) (74). Other processes using tannins are tanning of leather and weighting of silk. Mordanting in Turkish red process, black textile dyeing and production of black inks also employ tannin compounds (69).

Tannins are a heterogeneous group of polymeric phenols constituted by aromatic ring structure with several hydroxy substituents (molecular weight ranges between 500 and 20000 Da). Tannins reactivity is related to their hydroxy substituents, which can conjugate other biomolecules: tannins precipitate protein, enzymes, carbohydrates and alkaloids (68) (75).

This class of molecules is divided into two different groups: hydrolysable tannins (HT) and condensed tannins (CD), known also as proanthocyanidins. Hydrolysable tannins are soluble in water: hydrolytic cleavage of hydrolysable tannins leads to phenolic acids and sugars (47) (68) (76). Hydrolysable tannins are present in angiosperms and green algae, while condensed tannins are found in wood

37

components of higher plants. Plant roots are mainly constituted by hydrolysable tannins (68). The ratio between hydrolysable and condensed tannins changes during plant life: young plants contain high amount of hydrolysable tannins, while at the end of plant life condensed tannins content increases. Environmental factors also influence tannin composition of plants: nitrogen fertilizations and temperature increment cause a decrease in condensed tannins amount, while CO2 increment causes an increase in condensed tannins content (68).

Hydrolysable tannins are divided into two main groups: gallotannins and ellagitannins. Gallotannins are constituted by gallic acid units esterified by hydroxy groups of a central polyol. This central molecule is commonly glucose, but it could be also shikimic acid, quinic acid, glucinol and cyclitol. Central polyol of gallotannins can also be esterified by chains of galloyl residues linked together by deepside bonds at the C3 position (Figure 1.15) (68) (77).

Pentagalloyl glucose is the simplest gallotannin and possible precursor of more complex gallotannins and ellagitannins (Figure 1.16) (68) (77). Pentagalloyl glucose

oligomers were also identified (78)

Figure 1.15 Gallotannins general structure

O O O O O O O OH O H OH O O H OH O H O OH OH OH O O H OH OH O O H O OH O O OH OH O O H OH OH n O O H O H OH OH Gallic acid unit

38

Figure 1.16 Pentagalloyl glucose structure

Ellagitannins are constituted by esters of hexahydroxydiphenic acid. These tannins are the product of oxidative C-C coupling reaction between adjacent residues of gallotannins. Residues involved in C-C coupling are commonly in C4 /C6 or C2/ C3 position of glucopyranose core (Figure 1.17) (68) (72). Coupling between galloyl residues at C1/C6, C1/C3 and C3/C6 position is also possible (72). Hydrolysis of ellagitannins leads to the formation of ellagic acid (72) (79). Gallotannins and ellagitannins can be found together in some tannin sources as oak and chestnut (68).

Figure 1.17 Ellagitannins general structure

O O O O O O O OH O H OH O O H OH O H O OH OH OH O O H OH OH O O H OH OH O O O O O O O O H O H O H O O H O H O H O OH OH OH O O H OH O H O O H OH OH OH O OH O H O H O H O O H OH OH O O O H O H O O OH OH Hexahydroxydiphenic acid unit

39

Tannins extraction procedure causes some structural modifications, leading to formation of several new products. Castalagin and castalin, together with their positional isomers, vescalagin and vescalin, are an example of rearrangement products deriving from hydrolysis of pentagalloyl glucose (Figure 1.18) (78) (80).

The high variety in the structure of hydrolysable tannins is due to oxidative coupling (Figure 1.19) of neighbour gallic acid units and aromatic ring oxidation (Figure 1.20). Products of these reactions are highly complex structures (72) (77).

Figure 1.18 Castalagin and castalin structures

O O O H O H OH OH OH OH OH OH OH O O O O O H O H H O O H O O H O H O H O H O H O H Castalagin O O O H O H OH OH OH OH OH OH OH O O H O O H O H H H O O H Castalin

40

Figure 1.19 Oxidative coupling reactions of hydrolysable tannins

Figure 1.20 Aromatic ring oxidation of hydrolysable tannins

O O OH OH O H O O OH O H O H O O OH OH O H O O OH OH O H O O O OH O H O O OH O H O H O O OH OH O H O O OH OH OH O O OH O H O H O O OH OH O H O O OH OH O O O OH OH O H O O OH O H O H O O O OH O H O O OH O H O H O O OH O H O H O O OH O O H Galloyl- Hexahydroxydiphenoyl- Dehydrodigalloyl- Flavogallonyl- Sanguisorboyl- Valoneoyl- Tergalloyl-O-OC OH O H O H COO -OH OH O H O-OC OH O H O H COO -OH OH O H O-OC OH OH OH O-OC OH OH O H O-OC OH O H O H COO -O OH O H Hexahydroxydiphenoyl- Flavogallonyl- Valoneoyl-O-OC O O O COO -OH OH O H O-OC OH O H O H COO -OH OH O H O-OC OH COOH COOH O-OC OH O H O H COO -O O O H O O H COO - Dehydrohexahydroxydiphenoyl-

Trilloyl-41

Galloylation range and anomeric stereochemistry of the core further increase structural variability of hydrolysable tannins (72). Moreover, glucose core polymerisation is also possible (78) (81).

Condensed tannins are oligomers or polymers constituted by 5-40 monomer units of 15-carbon polyhydroxyflavan-3-ol. Flavan-3-ol structure is constituted by two types of phenolic rings, named A- and B-ring, having different reactivity. This structure has also two chiral centres, C2 and C3 (68) (73) (74) (82) (83) (84) (85). Condensed tannins have high structural variability, depending on hydroxylation pattern of the A- and B- ring of flavan-3-ol, stereochemistry of chiral centres C2 and C3 and stereochemistry of interflavanol bond. Galloylation at the C3 position further increase variability (68) (74) (82) (83) (84) (85) (86). Single monomers are linked together by labile C-C bond: C4/C8 bonds, present in higher amount, give linear structure while C4/C6 bonds result in branched globular structures (A-type dimer) (68). Also doubles links through C2-O-C7 or C2-O-C5 are attested (B-type dimer) (Figure 1.21) (74) (82) (84) (87).

Figure 1.21 Condensed tannins: A- and B-type dimers

Condensed tannins classification is made on the basis of hydroxylation pattern of A- and B-rings of monomers. They can be divided into five different groups, depending of the structure of their monomeric units: procyanidins, prodelphinidins,

OH OH O O H R3 R1 OH R2 OH O O R3 R1 OH R2 B-type dimer A-type dimer OH OH O O H R3 R1 OH R2 OH O O H R3 R1 OH R2

42

profisetinidins, prorobinetinidins, propelargonidins (Figure 1.22) (68) (73) (74) (82) (85) (86) (87) (88). Profisetinidins and prorobinetinidins monomers are linked through C4-C6 linkages, while procyanidins and prodelphinidins ones trough C4-C8 linkages (89).

Figure 1.22 Tannins classification

Tannin dyestuff contains other phenolic compounds apart from tannins molecules. One of these molecules is juglone. Juglone is present in several plants of Juglandaceae family, nut-producing specie widely cultivated in temperate zones of Northern hemisphere. All parts of the plants, such as leaves, roots, stem, branches and nuts, contain amounts of this molecule. Juglone (5 –hydroxy-1,4-naphtoquinone) (Figure 1.23) belongs to naphthoquinones, a group of phenols deriving from naphthalene. This molecule is soluble in hot water, alcohol and ether. Alkali solutions of juglone are purplish red coloured, while concentrated sulphuric acid solutions have blood-red colour. Juglone is toxic for several plants, while it has no deleterious effects on human organism. Juglone is employed as wool and hair dyestuff, and it is largely

Procyanidins: R1_{= H R}2_,R5_{= OH}

Catechin: R3_{= H R}4_{= OH}

Epicatechin: R3_{= OH R}4_{= H}

Catechin gallate: R3 _{= gallic acid ester R}4_{= H}

Epicatechin gallate: R3_{= H R}4 _{= gallic acid ester}

Prodelphinidins: R1_,R2_,R5_{= OH}

Gallocatechin: R3_{= H R}4_{= OH}

Epigallocatechin: R3_{= OH R}4_{= H}

Gallocatechin gallate: R3 _{= gallic acid ester R}4_{= H}

Epigallocatechin gallate: R3_{= H R}4 _{= gallic acid ester}

Propelargonidins: R1_,R2_{= H R}5_{= OH}

Afzelechin: R3_{= H R}4_{= OH}

Epiafzelechin: R3_{= OH R}4_{= H}

Afzelechin gallate: R3 _{= gallic acid ester R}4_{= H}

Epiafzelechin gallate: R3_{= H R}4 _{= gallic acid ester}

Prorobinetinidins: R1_,R2_{= OH R}5_{= H}

Robinetinidin: R3_{= H R}4_{= OH}

Epirobinetinidin: R3_{= OH R}4_{= H}

Robinetinidin gallate: R3 = gallic acid ester R4= H Epirobinetinidin gallate: R3_{= H R}4 _{= gallic acid ester}

Profisetinidin: R2_{= OH R}1_,R5_{= H}

Fisetinidin: R3_{= H R}4_{= OH}

Epifisetinidin: R3_{= OH R}4_{= H}

Fisetinidin gallate: R3 = gallic acid ester R4= H Epifisetinidin gallate: R3_{= H R}4 _{= gallic acid ester}

R4 R2 R1 O R5 O H R3 OH A B 8 2 3 4 5 6 7 1

43

used in medicine against viral, fungal and bacterial infections. Anti-cancer action of juglone is reported. Moreover, it is also used for fish poisoning (69) (90).

In appendix A tannin biosynthesis and reactivity are described.

Figure 1.23 Structure of juglone

Tannin dyeing process

Tannins are classified as mordant dyestuffs because the dyeing process involves the use of a metal ion, called mordant, to link the textile to the dyestuff. Several tannin dyestuffs sources are described in Appendix B. Tannin-textile bond is possible both with proteinaceous and cellulosic fibres, but reaction with proteinaceous ones is promoted (69). When proteinaceous fibres are dyed with this technique, a complex constituted of a central metal ion is formed, which acts as a bridge between keratin residues and dyestuff molecules (91).

During dyeing process, molecular hydrogen bonding and coordinative bonding occur. Amino acids involved in the reaction are the ones with hydroxy, carbonyl, carboxyl, amine, amide and thiol side groups. Complex formation with carboxyl and thiol group is supposed to be prevalent (1) (27) (29) (69) (91) (92). This process is divided into two different steps: application of the mordant and application on the dyestuff. On the basis of the order followed, dyeing mechanisms are classified as pre-mordanting and last-pre-mordanting procedures. Pre-pre-mordanting procedure consists in a first reaction between textile fibres and metal salt, followed by interaction with tannin molecules. Last-mordanting is obtained through the reaction between textile and tannin agent, followed by mordant addition (Figure 1.24) (69) (91) (92). Simultaneous adding of dyestuff and mordant is also reported (69) (91). Tannin

OH O