Method development for amino acid characterization of fibroin in silk using RP-UHPLC-FLD - A quantitative determination of primary amino acid composition to analyse the degradation state of historical silk fibres

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Bachelor Thesis Scheikunde

Method development for amino acid characterization

of fibroin in silk using RP-UHPLC-FLD

A quantitative determination of primary amino acid composition to

analyse the degradation state of historical silk fibres

door

Indra Mellema

13 juli 2017

Studentnummer 10819282 Onderzoeksinstituut

Rijksdienst voor Cultureel Erfgoed Onderzoeksgroep

Conservatie en Restauratie

Verantwoordelijk docent Maarten van Bommel Begeleider

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Indra Mellema, 10819282, Amino acid characterization, July 2017

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Samenvatting

Er is in 2016 een grote hoeveelheid aan historisch zijde gevonden, waaronder de japon in afbeelding 1. Dit zijde heeft zo’n 350 jaar onder water gelegen. Ondanks dit is het zijde in goede staat gevonden. Nooit eerder is er zo’n grote hoeveelheid zijde in deze staat gevonden. Daarbij is het zijde van erg goede kwaliteit en rijkelijk versierd. De japon was waarschijnlijk eigendom van een koninklijke of adellijke vrouw. Maar hoe is het zijde zo goed bewaard gebleven? Informatie over hoe zijde het best bewaard kan worden is van belang voor kunstcollecties. Musea kunnen met deze informatie

zijde objecten onder de juist omstandigheden tentoonstellen zonder dat het zijde erg beschadigt. Kunstobjecten zijn van maatschappelijk belang; ze laten zien hoe er vroeger geleefd werd (educatie) en de cultuur van een gebied, het bevordert de economie en wordt uiteraard vooral mooi gevonden. Zijde is een van de sterkere textielsoorten, maar veroudert ook langzaam. Degradatie, het verouderen van zijde, zorgt ervoor dat zijde breekbaar wordt, minder glanst en zijn kleur verliest. Het verval van de waardevolle objecten moet dus worden tegengegaan. Hiervoor is het belangrijk dat er onderzoek wordt gedaan naar de degradatie van zijde. Zijde degradeert onder invloed van vier hoofdfactoren: luchtvochtigheid, temperatuur, Uv-licht en het zuurstofgehalte van de omgeving. In dit onderzoek is op moleculair niveau onderzocht wat de invloed is van deze factoren op zijde. Er is gekeken naar de verandering van de structuur. Zijde is gemaakt van ketens opgebouwd uit aminozuren; eiwitten. Bij degradatie worden deze ketens verbroken en veranderen de aminozuren in andere moleculen. Door te kijken naar de verandering van aminozuren in het zijde, kan bepaald worden welke factoren het schadelijkst zijn voor het zijde.

Hiervoor is er een analysemethode opgezet waarbij bijna alle aminozuren uit het zijde gescheiden kunnen worden. Hierdoor kan de hoeveelheid voor elk aminozuur apart bepaald worden en kunnen de hoeveelheden vergeleken worden met de hoeveelheid aminozuren in nieuw zijde. Hoe meer aminozuren er verloren zijn gegaan, hoe meer het zijde gedegradeerd is.

De aminozuren worden gescheiden met een analysetechniek genaamd vloeistofchromatografie i.c.m. fluorescentie detector. Bij de scheiding worden de aminozuren gescheiden op grootte, polariteit (of het aminozuur een geladen kant heeft) en hydrofobiciteit (of het aminozuur wel of geen interactie heeft met water). De aminozuren worden opgelost in een loop vloeistof (mobiele fase) en dan gescheiden in een kolom met een vaste stof (stationaire fase). Grote moleculen hebben moeite om door de smalle kolom te gaan en komen laat uit de kolom. Een geladen aminozuur heeft interactie met de loopvloeistof en komt sneller uit de kolom dan een ongeladen aminozuur dat interactie heeft met de vaste stof in de kolom. Hydrofobe aminozuren hebben interactie met de hydrofobe stationaire fase en ondergaan zo vertraging en komen later uit de kolom. Een scheiding van aminozuren is het resultaat. De aminozuren worden na de kolom gedetecteerd door een fluorescentie detector. Omdat er 17 verschillende soorten aminozuren in zijde voorkomen zijn er verschillende loopvloeistoffen gecombineerd om alle 17 aminozuren te kunnen scheiden.

Uit het onderzoek is gebleken dat zuurstof het degradatieproces stimuleert. Er is geen verband gevonden voor de relatieve luchtvochtigheid. Wel is er ontdekt dat gekleurd zijde minder snel degradeert dan ongeverfd zijde en de verf dus als het ware de zijde beschermt.

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Abstract

With the historical silk found in 2016, which was in relatively good state, questions were raised how this silk was conserved that well. The silk was found in the sea, dark, cold and in a low-oxygen environment. In this project different environmental degradation factors of silk; air composition (N2, O2 & air), dyes, relative humidity (0% & 100%), temperature and light exposure were analysed. This will give information on the best environment for silk in storage and exhibition. To analyse this, research was done on a method to characterize the amino acid composition of fibroin of historical silk. An RP-UHPLC-FLR method was successfully developed to detect all amino acids of fibroin, except the secondary amino acid proline. The precolumn derivatization reagent ο-phthaldialdehyde that is used, is unstable and forms very unstable derivatives with amino acids. Therefore, the amino acid derivatives should be kept cool and injected directly after derivatization. With this method different silk samples were analysed on amino acid composition. Cochineal dyed fibres showed less degradation and thus it is stated that this dye has a conservative effect. Washing historical silk with demi-water shows least degradation. No correlation was found for the different humidity levels of the environment of the silk fibres. Samples exposed to oxygen showed most degradation and therefore it is assumed that oxygen increases silk degradation. Further research should analyse more samples with stable conditions in order to find this correlation and the best way to conserve silk.

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Index

Samenvatting... 1 Abstract ... 2 Index ... 3 Introduction ... 5 Theory ... 7 Silk ... 7 Degradation ... 8 Oxidation... 9

Amino acid characterization ... 10

Hydrolysis ... 10

Derivatization ... 11

RP-UHPLC-FLD analysis... 12

Degradation indicators ... 13

Experimental ... 15

Chemicals and reagents ... 15

Equipment ... 15

Chromatographic condition ... 16

OPA reagent ... 16

Amino acid stock solution (standards) ... 17

Samples and sample preparation (hydrolysis & derivatization) ... 17

Validation ... 18

Calibration curve ... 18

Results ... 20

Ratio OPA:AA ... 20

Stability of the OPA and OPA-AA derivatives ... 21

Stability of samples ... 22

Amino acid standards ... 23

Calibration curve ... 24

Limit of Detection (LOD) ... 25

Xenotest samples ... 25 Oven samples ... 27 Historical samples ... 29 Texel textiles ... 30 Discussion ... 32 Ratio OPA:AA ... 32

Stability of the OPA and OPA-AA derivatives ... 32

Stability of the samples ... 32

Amino acid standards ... 33

Calibration curve ... 33 LOD ... 33 Xenotest samples ... 33 Oven samples ... 34 Historical samples ... 34 Texel textiles ... 35

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Conclusion ... 36

Acknowledgements ... 37

References ... 38

Attachments ... 40

I. Artificially aged samples ... 40

Xenotest samples ... 40

Oven samples ... 41

II. Historical aged samples ... 42

III. Texel Textiles ... 43

IV. Final diluted concentrations ... 45

V. Integration method ... 46

VI. Amino acid Library for UHPLC-FLR ... 47

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Introduction

In 2016 a rare discovery was done in Texel, the Netherlands. A silk dress, most likely of the seventeenth century, was found together with other textile fragments, in the Waddenzee in a shipwreck (van Bommel, 2017). It is a unique finding for several reasons. First because of the origin of the dress and the number of objects, but also the state of the finding was notable; the textile was in a relatively good condition after all these years of exposure to the environment. The dress is made of high quality materials, mostly silk. Textiles like this dress were normally reused afterwards, adapted or recycled into new costumes because silk was, and still is, an expensive textile. This is why not many intact dresses are found nowadays. Textiles are normally found in small fragments and that makes this finding of a complete original dress special.

The dress was found buried in the sea for about 350 years, so the fragments have been in a cold, dark and low-oxygen environment. Not much is known about the influences of these conditions on the textiles, which makes it hard for conservators to keep the silk in the relatively good state that it is now. It is known that silk degrades under certain circumstances. The main degradation factors are relative humidity, temperature, light exposure and lastly the oxygen-level (van Bommel, 1991).

In this research, several silk samples will be analysed on the amino acid composition of their fibres. These samples are obtained at several artificially ageing conditions (relative humidity, temperature, UV-light exposure, dyed/undyed and oxygen level) to understand the influence on the amino acid composition. Afterwards historical samples and cleaned samples of the Texel collection will be analysed. The results should give information about the degradation factors and can be used to improve the conservation of silk objects in the future. The most suitable conservation conditions for preserving silk in museums or storage can be determined and this can help to slow down further degradation and keep the silk in the best condition. Nowadays there is not much known about the influence of oxygen on silk textiles. People have been doing research about the influence of oxygen on the degradation of colour, but not much about the chemical properties of the fibre itself. With results from this research conservators can decide, whether or not, it is desired to store silk fragments oxygen free.

There are several methods to test the degradation state of silk. Physical properties comprise the evaluation of colour fading and brittleness, while chemical properties include the difference in molar mass, crystallinity, or amino acid composition (Gohl & Vilensky, 1983). The method used in this project is amino acid characterization with reversed phase liquid chromatography and fluorescence detector. A method will be developed to get an elution between the amino acids of the silk fibre. Another research project will be done on the same samples (historical, Texel textiles and artificially aged) using X-ray Diffraction (XRD) and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR).*_{These techniques give information about the crystallinity of the} fibres (see Theory section) and the amount of oxidation. The results of that project combined with this project’s results should give a broad view on the influence of the degradation factors on the chemical properties of silk.

Silk is a protein that exists of threads of fibroin and a ‘glue’ keeping the threads together; sericin. Ninety per cent of fibroin is formed by 4 amino acids glycine, alanine, serine and tyrosine (Yanagi, Kondo & Hirabayashi, 2000; Van Den Berghe, 2012). Degradation causes changes in the amino acid composition by breaking of peptide bonds and decrease/increase of some amino acids

*_{Researchproject done by Jennifer van der Schaft for RCE “De kristalliniteitbepaling in relatie tot de degradatie}

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concentrations. The changes in amino acid composition gives information about the degradation state of the protein on molecular level. Any change in relative amounts of amino acids in fibroin can be indicative for degradation (Wouters et al., 2000).

To get respectable results a separation of the amino acids by RP-UHPLC is required. The separation of amino acids (in the mobile phase) by UHPLC relies on the difference in affinity with the stationary phase. Because amino acids are different in polarity and thus affinity, an isocratic gradient is not the best option. In this project, a method will be developed to get a good separation for all amino acids by optimization of the mobile phase. The hypothesis is that the gradient should go from polar to nonpolar in time, to get both the polar and nonpolar amino acids through the column. This method will then be used to analyse the influence of oxygen, dyes, relative humidity, temperature, and UV-light exposure on the amino acid composition of fibroin.

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Theory

This research project will be focussed on the fibroin in silk fibres of historical textiles, like the fragments found in Texel. The amino acid composition of the fibroin threads will be analysed by RP-UHPLC-FLD, reversed phase ultra-performance liquid chromatography with fluorescence detection.

Silk

Silk is made from an animal fibre produced by worms, most common the Bombyx Mori. The worm produces a double protein thread of fibroin which hardens when exposed to air (Zhang et al., 2011). The threads are encased with sericin, another protein produced by the worm. Sericin needs to be removed to dye the silk, so that the dyes attach better to the fibres. Degumming, the removal of sericin, not only improves the colour but also the sheen, the texture and handle. (Qian, 2005). But in fact, the sericin could also be a good factor, because it prevents damage of fibroin from light-exposure (Becker et al). The silk samples that will be analysed are all degummed silk, and will only have the fibroin filaments.

Fibroin is a protein composed of 17 different amino acids (AA) but mainly glycine (gly), alanine (ala), serine (ser) and tyrosine (tyr), table 1. The material is highly orientated in alternating amorphous and crystalline regions (Vilaplana et al., 2015; Li et al., 2013). When polymers are orientated well, the chains have inter-polymer interactions and the fibre will be stronger. The longer the polymer the stronger the fibre will be.

Table 1 Composition of amino acids in silk fibroin (Yanagi, Y., Kondo, Y., & Hirabayashi, K., 2000)

Amino acid Mol % Weight %

Glycine, GLY 43.68 34.58

Alanine, ALA 30.34 28.51

serine, SER 9.93 11.01

Tyrosine, TYR 5.21 9.97

Other 13 amino acids

(aspartic acid (ASP), threonine (THR), serine (SER), glutamic acid (GLU), proline (PRO), cysteine (CYS), valine (VAL), methionine (MET), isoleucine (ILE), leucine (LEU),

phenylalanine (PHE), histidine (HIS), arginine (ARG))

10.84 15.93

The crystalline region contains mainly glycine, alanine, serine and tyrosine in repeating motifs. Dipeptides of glycine and the other three main amino acids form hexapeptides as shown in figure 1. These hexapeptides account 70 % of the total crystalline region (Zhou Confalonieri 2001). The crystalline region makes the polymer strong. The crystalline part is stiffer and more durable. Because of the well-orientated structure, it is difficult for molecules to enter and settle in the fibre what makes dying harder, but it also makes the degradation slower (Gohl & Vilensky, 1983).

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Figure 1 Hexapeptides in 70% of crystalline region consist of gly-X dipeptides where X is ala (65%), ser (23%) or tyr(9%), Zhou Confalonieri 2001

The peptides fold into antiparallel β-sheets motifs. These sequences are separated by amorphous sequences which contain mostly tyrosine, amino acids that are absent in crystalline regions and amino acids that have bulky or polar side chains (Yanagi, Kondo, & Hirabayashi, 2000). While the crystalline regions are characterized by an antiparallel β-sheet secondary structure, the amorphous regions distorts this structure and the polymers are aligned at random (Asakura, Yao, Yamane 2002; Gohl & Vilensky, 1983; Bommel, 1991). These amorphous regions have distorted β-sheets, distorted β-turns and 31-helices, figure 2 (Li et al., 2013). These structures cause the elastic property of silk. Because of the distortion in orientation, the amorphous fibres are weaker, easier dyed, and more pliable than crystalline fibres (Gohl & Vilensky, 1983). This also causes a difference in degradation of the regions. The resistance of fibroin fibre against degradation factors are thus determined by its structure.

Figure 2 Well-ordered crystalline regions and disordered amorphous regions (exaggerated and simplified) (Gohl & Vilensky, 1983).

Because most silk is dyed, some samples analysed were also dyed. The details about the dyes are listed in Attachment I. Literature states that the dyes have a preserving effect on the fibres in the degradation process except from black dyes. This suggests that the way of dyeing and the composition of the dyes influence their preserving ability (Van Den Berghe, 2012). This is mostly because dyes can absorb a part of the UV-light and the silk is then exposed to less UV-light (Cristea & Vilarem, 2006).

Degradation

The silk fibre is relatively more sensitive for degradation factors than other natural fibres. This is caused by the lack of cross-linkages, covalent or ionic bond between the polypeptides, which make fibres rigid (Gohl & Vilensky, 1983). The silk loses its strength what makes it hard to conserve (Vilaplana et al., 2015). The degradation reactions are mostly breaking of the peptide bonds (Van Oosten, 1991). This hydrolysis can be caused by enzymes, acids or base. Strong acid hydrolysis causes polypeptides to completely divide into separate amino acids. Weak acid hydrolysis results in

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di-, tri-, and tetrapeptides (Van Oosten, 1991). The peptide bonds of serine, threonine and aspartic acid are most sensitive for hydrolysis and break easily (Truter, 1973).

Oxidation

Another path of degradation of fibroin is through oxidation: by air, temperature (thermo-oxidation) or UV-light (photo-(thermo-oxidation). This can occur in 3 ways: oxidation of an amino acid sidechain, oxidation of the terminal amino acid of a chain and oxidation of a peptide bond in the main chain. The oxidation of amino acids results in new components: derivatives of the amino acids. This will result in a decrease in the amount of original amino acid. But they can also transform in other amino acids. Tyrosine can turn into alanine by losing a phenol group for example. The tyr/ala ratio is used to give a value about the transformation of tyrosine into alanine. This ratio is decreasing during the degradation process (Vilaplana et al., 2015). The amount of tyrosine is a marker for oxidation of silk (Vilaplana et al., 2015). Research of Yanagi, Y., Kondo, Y., & Hirabayashi, K. in 2000 also assumes that amino acid decomposition is affected by oxygen. Oxygen causes a loss of tyrosine in the fibroin due to photo and thermo oxidation (Li, et al., 2013; Becker and Tuross, 1994; Becker et al., 1995). Tyrosine is the main component of the amorphous regions, so tyrosine loss causes a decrease in amorphous material and relatively more crystallinity which affects the mechanical properties of silk making it less elongated and more brittle (Yanagi, Kondo & Hirabayashi, 2000). The amorphous regions are degraded before the crystalline regions because they are less rigid and therefore easier to break. Also, because the amorphous part consists of bulky amino acids they are more accessible for degradation reagents. The longer the polymers the longer ‘the path of break’, because the longitude packing is harder to get through, figures 3 & 4 (Gohl & Vilensky, 1983).

Figure 4 "A strong fiber because it has long polymers which are well aligned or oriented, giving it a long 'path of break'. (Gohl & Vilensky, 1983)

Figure 3 "A weak fiber because it has short polymers; although they are well oriented, the shortness of its path of break tend to make it weak."( Gohl & Vilensky, 1983)

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Yellowing

A physical observation of degradation is also the yellow colour of prior white silk. This is also caused by the degradation that decreases the relative amount of tyrosine, serine and phenylalanine (Becker & Tuross, 1994; Becker et al., 1995, 1997; Yanagi, Kondo & Hirabayashi, 2000). The ultraviolet radiation of sunlight causes the degradation. These sunrays cause the peptide and disulphide bonds in the fabric to break and that results in polymer degradation. The degradation products cause the fibres to absorb even more light and distribute the light further through the fibre (Gohl & Vilensky, 1983; Yanagi, Kondo & Hirabayashi, 2000). A decrease in tyrosine is also related to the yellowing of keratin in wool which composition is very alike with fibroin in silk (Van den Berghe, 2012). Further research analysed the change in tyrosine content under influence of UV-irradiation. As expected a loss of tyrosine caused the tensile strength to decrease and the yellowing to increase together with an increase of formed quinone-based tyrosine derivatives. Those derivatives are the result of tyrosine oxidation (Solazzo et al, 2012).

Thermo-oxidation & relative humidity

Rising temperature speeds up the degradation reaction. The peptide bonds, salt linkages and hydrogen bonds of the polymer break down when the temperature gets above 100°C (Gohl & Vilensky, 1983). The high temperature reduces the molecular weight and the fibre loses its strength (Vilaplana et al., 2015). Loss of serine in the crystalline region is characteristic for thermal degradation instead of oxidative degradation (Yanagi, Kondo & Hirabayashi, 2000).

Relative humidity also plays a role in the deterioration of silk. High and low levels accelerate the degradation (Vilaplana et al., 2015). When silk is wet it loses its strength, because the water molecules hydrolyse the hydrogen bonds between the silk polymers and weaken them (Gohl & Vilensky, 1983).

The degradation factors thus cause differences in amino acid composition, disordered structure, orientation and polypeptide length. Thereby degradation of the protein fibres causes a release of ammonia gas which reduces the molar mass (Becker & Tuross, 1994; Becker et al., 1995).

Amino acid characterization

As mentioned above the amino acid composition correlates with the visual properties like colour change & brightness, and mechanical properties of silk such as strength of the fibres. Any differences in the relative amounts of amino acids are indicative for degradation (Vilaplana et al., 2015). The amino acid characterization in this research includes 3 steps: hydrolysis, derivatization and analysis, which will be discussed below. The analysis will be done by RP-UHPLC and a fluorescence detector so that the alterations of the amino acid composition during degradation can be monitored. A fluorescence detector is more sensitive than a PDA detector and requires less sample. Therefor a fluorescence detector is more suitable for this research. Due to technical issues, first analyses were performed with PDA detector. These analyses include the amino acid standards and the stability tests. In a later stage of the project, the fluorescence detector was available and the samples could be analysed with the FLR detector.

Hydrolysis

The first step of the amino acid characterization is hydrolysis of the fibroin protein to get individual amino acids that can be characterized by UHPLC. This step is necessary because in the derivatization step every individual amino acid needs to react with the derivatization agent. The acid hydrolysis method is the most common method for amino acids, because it is more specific than basic

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hydrolysis. Where basic hydrolysis gives partly separated amino acids, acid hydrolysis results in completely separated amino acids. The acid hydrolysis reaction is shown in equation 1 for a dipeptide.

Equation 1 Acid hydrolysis of a dipeptide

Derivatization

Because amino acids are not detectable with a fluorescent detector by itself, a chromophore group, a molecule used for its fluorescent properties, should be inserted. This process is called derivatization of the amino acids. This will be done by using an ο-phthaldialdehyde-reagent, because it forms a highly fluorescent adduct and reacts with all primary amino acids except cysteine (Dai et al., 2014). The derivatization reaction is shown in equation 2, reaction time: 7 min (Molnàr-Perl & Vasanits, 1999). This short reaction time of the reagent is an advantage compared to other derivatization reagents. The ο-phthaldialdehyde-reagent contains ο-phthaldialdehyde(OPA), 3-mercaptopropionic acid (MPA) and a borate buffer (pH 9.5). The reaction results in the composition of a fluorescent adduct containing OPA, an amino and a SH-group. MPA is an iso-indole that forms the SH-group on the derivatives. MPA is used for the reaction with OPA because it provides relatively more stable iso-indoles than other SH-group containing alternatives. The separation could be performed at room temperature which is a practical advantage, but the derivatives are unstable which is a disadvantage. Therefor measurements will be done to define the instability. Lastly the derivatives of the OPA reagent have a stable chromatography baseline, what makes integration easier (Dai et al., 2014).

Proline is a secondary amino acid and will therefore not react with the OPA reagent. As stated before (see table 1), proline is not found in a large amount in silk. So, proline is neglected in the analysis.

Lysine, glycine and histidine can form more than one OPA-derivative (Kutlán, & Molnár-Perl, 2001). This makes them less stable than other amino acids.

Equation 2 Derivatization step with OPA-reagent

Stability of OPA reagent

The OPA reagent overall is an unstable solution which should be prepared fresh before derivatization and be stored in the fridge (~4 °C, dark) to stabilize the reagent (Molnàr-Perl & Vasanits, 1999; Molnàr-Perl, 2001). Some OPA amino acid derivatives, like the derivatives of glycine and lysine, are not very stable and can form more than one OPA derivative (Mengerink et al., 2002). Multiple peaks will appear in the chromatogram. The amount of primarily formed derivative is influenced by the concentration of reactant and the composition of the derivatization reagent. The most common ratio OPA/MPA = 1:3 and this ratio will be used in this analysis as well (Mengerink et

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al., 2002). The ratio of amino acid/OPA is supposed to be 1:20 according to literature, so that the OPA reagent is in excess (Molnàr-pearl & Vasanits, 1999; Molnàr-Perl, 2001). This ratio will be tested in this research.

Fluorescence

Ο-phthaldialdehyde (OPA) is a chromophore, because of its aromatic group and its ability to form another ring with the nitrogen of the amino acid, creating a sequence of alternating double and single bonds; conjugation (see equation 2). The final adduct contains many π-bonds. Conjugated double bonds are useful for chromophores because the π-bonds in the ring structures have small energy differences (ΔE) between excited and ground state orbitals. This difference is small enough for low-energy photons to excite the electrons. The more π orbitals in the conjugated system, the smaller the ΔE, so less energy is needed from the exciting light. And the longer the conjugated system, the longer the wavelength of the absorbed light (Lichtman, J. W., & Conchello, J. A. (2005). Fluorescence is the radiational transition of an excited electron (S1) falling back to the ground state (S0) emitting a photon (Harris, 2010). The fluorescence spectra (emission energy) have maxima lower in energy than absorption spectra (excitation energy), because molecules emit radiation at longer wavelengths than the radiation they absorb. In the emission spectrum, the emission intensity is plotted versus the emission wavelength (Harris, 2010). Perucho et al did research to determine the optimal excitation and emission wavelengths to analyse amino acids using OPA precolumn derivatization. The maximum signal of emission wavelength was detected between 445-454 nm. The optimal emission wavelength in order to perform OPA-amino acids analysis seems to be 450 nm, with λex=240 nm (Perucho et al., 2015).

RP-UHPLC-FLD analysis

In this research, the analytical technique reversed phase- ultrahigh performance liquid chromatography with fluorescence detection will be used. With HPLC analysis technique two phases are utilized. The liquid chromatography refers to the liquid mobile phase that is pushed through a column with high pressure. The column is part of the other phase, the stationary phase, a packing of particles in a column. The particles in a C18 column are made of silica with octadecyl-groups which make the column non-polar, figure 5. The differences in chemical properties, solubility, hydrophobicity and/or adsorption with the phases, will induce a separation of the amino acids. Because of the differences, the amino acids will go through the column at different speeds and will be detected at different times (retention times) (Dong, 2006). The retention time of the derivatives is also influenced by the column, the gradient, the mobile phase and the flow rate.

The OPA-amino acid-derivatives will be analysed with ultrahigh performance liquid chromatography(UHPLC). The difference with normal HPLC is that UHPLC is more improved and is resistant to higher pressures. The high pressure is caused by the smaller particles an UPLC column contains. Despite the high pressure, the smaller particles improve separation. The resolution increases, which decreases the detection limit. The advantage of using this technique is the small sample size that is needed. This is an important advantage for analysing historical samples, because you want to use as less sample as possible. Previous research on natural dyes supports that UHPLC gives more improved data than HPLC. Higher resolutions were obtained and the limit of detection (LOD) was decreased (Serrano, van Bommel & Hallet, 2013). Because the separation improves the running time will decrease as well, which is a practical advantage.

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Figure 5 Stationary and mobile phases of UHPLC

Mobile phases & gradient

The amino acids will be dissolved in the mobile phase. The mobile phase changes during a sample run (a gradient), so every amino acid can be separated. When the mobile phases are in a constant ratio the gradient is called isocratic. Silk is made of 17 different amino acids, all with different polarity and hydrophobic properties and will therefore all have different affinity with the mobile and stationary phases. A gradient elution is needed for the UHPLC analysis instead of an isocratic elution, because the amino acid derivatives will also have very different polarities. In this research, different gradient programs will be tested to develop the best one.

Reversed phase chromatography uses a mobile phase that is more polar than the stationary phase (non-polar C18 column). A combination of three mobile phases were used in the gradient elution. Solvent A: 10% methanol in water, solvent B: 100% methanol and solvent C: Sodium Acetate 1 M (pH 7). The amount of B will increase during the sample analysis, creating a less polar mobile phase. In the beginning the polar amino acids will have more affinity with the mobile phase and will elute fast. These amino acids will have a short retention time. Nonpolar or less polar amino acids will have more affinity with the stationary phase or the less polar mobile phase that is created later in the gradient. This results in a later elution, higher retention times, because they are retained. The retention times are specific for the derivatives (Perucho et al., 2015). Each amino acid has a different R group, but this difference can be minor like for glycine and alanine, figure 6. This results in amino acids that have the same affinity with the stationary phase. When both signals are detected at the same time this is called co-elution. The retention times will be close to each other and the signals will be hard to separate. Thus, a gradient elution program is not only necessary to separate the different amino acid derivatives because they have different polarities and affinities, but also to get a good separation of amino acids that are very alike.

Figure 6 Standard amino acid, glycine and alanine

Degradation indicators

The state of degradation can be determined by analysing the concentrations of the main amino acids in fibroin. Alanine, serine and glycine are the main components of the crystalline region while tyrosine is mainly present in amorphous regions. As mentioned before the amorphous region degrades sooner than the crystalline region. So, the assumption is that the amount of tyrosine will decrease sooner than the amount of alanine, serine and glycine. In a further state of degradation both the

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amorphous and crystalline region will degrade and the amount of alanine and glycine will also decrease along with the tyrosine concentration. The tyr/ala ratio will also be determined, to see if tyrosine molecules have degraded into alanine. Serine loss is also said to be causing yellowing of silk. Therefore, it is interesting to check if this is seen in the aged samples as well. The amount of alanine is affected by the loss of alanine by degradation but also by the gain of alanine by tyrosine degradation. This makes the alanine content a difficult degradation indicator.

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Experimental

Chemicals and reagents

Standard amino acid solutions of 2.5 mM were used to make an amino acid library. The amino acids were obtained from H. van Keulen. The amino acids were dissolved in HCl 35% from Acros Organics (Geel, Belgium) bottle opened in 2012. This HCl was diluted to 0.1 M (4.41 mL HCl filled to 500 mL with water). The 6 M HCl for hydrolysis of the silk was made by taking 53 mL HCl and added to final volume of 100 mL with water. A sodium acetate buffer 1M (pH 6.91) was made from 20.6 g sodium acetate trihydrate (Aldrich, now Sigma-Aldrich, Germany) dissolved in 100 mL water, 50 μl acetic acid (Merck, Darmstadt, Germany) and water added to final volume of 500 mL, filtered through 0.2 μm Nylon Millipore filter. A borate buffer 0.1M (pH 9.21) was made from 17 g sodium tetra borate decahydrate 99% (Sigma-Aldrich, Germany) and dissolved in 500 mL water, together with 3 g boric acid (JT Baker, USA). LC-MS-hyper grade methanol (Sigma-Aldrich, Germany) filtered through 0.2 μm Nylon Millipore filter was used for mobile phase and OPA reagent. The derivatization reagent was made from o-phthaldialdehyde (Sigma-Aldrich, Germany) and 3-mercaptopropionic acid (Sigma-Aldrich, Germany). Deionized water (Millipore Simplicity™ Simpak®_{2, 0.22-μm filter, MA, USA) was used from purification system.}

Equipment

UHPLC-FLD analyses were performed using a Waters AcquityTM_{H-class UHPLC system} (Waters Corporation, Milford, MA, U.S.A.) equipped with a quaternary solvent delivery system, a column oven, an autosampler, a PDA detector and a fluorescence detector (FLD). PDA data was recorded from 200 to 800 nm with a resolution of 1.2 nm (2 scan/s). The samples were analysed at a detection wavelength of 340 nm for the PDA. The equipment was controlled by Empower®_3.0 Chromatography Data Software from Waters Corporation. Peaks were integrated as shown in attachment V. Areas were calculated by software.

Analytical conditions were carried out using in a WatersAcquity®_{UHPLC BEH Shield RP18} 1.7 µm of 2.1 × 150 mm column, protected by a filter unit (0.2µm), with injection volume 0.2µl, a flow rate of 0.2 ml/min and a constant column temperature of 40 °C.

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Chromatographic condition

The gradient of the mobile phases was prepared by 3 solvents; methanol in water 10% (solvent A), methanol 100% (solvent B) and sodium acetate 0.1M pH=7 (solvent C). These solvents followed the elution program below in Table 2 and Figure 7. This gradient is developed based on literature (Perucho et al., 2015). The gradient is adapted to the system to create a shorter analysis time. The system pressure also played a role in creating the method. The system needed to stabilize between the separate samples, and not reach a pressure higher than 14 000.00 psi. Thereby the system pressure had to change smoothly during the measurement to prevent the column from damaging.

Table 2 Elution program used (flowrate: 0.2 mL/min)

Time (min)

% A %B %C

Figure 7 Visual representation of elution program

0 75 15 10 3 75 15 10 10 54 36 10 13 39 51 10 16 39 51 10 18 36 54 10 19 10 80 10 21 10 80 10 23 75 15 10 33 75 15 10

The excitation/emission wavelengths of the fluorescence detector used were respectively 240/450nm, based on previous research (Perucho et al., 2015).

OPA reagent

The OPA reagent was made by dissolving ±2.55 mg OPA in 0.5 mL methanol. Then 5 µl MPA and 2 mL borate buffer (0.1 M pH 9) were added to the solution (Perucho et al., 2015). The solution was put in fridge for 90 minutes at least to react. The reagent was freshly prepared every day and stored in the fridge (-4 °C). Ratio MPA/OPA in OPA reagent was 3:1.

The stability of the OPA reagent is tested in different ways to see if the chromatograms change. The stability of the OPA-AA-derivatives and the influence of the temperature at which the OPA-AA-derivatives are stored is tested by measuring samples from the derivatives daily. One half of OPA-AA-derivatives is kept in the autosampler between the measurements (27 °C) and the other half of the samples is kept in the fridge between analysis (Experiment 1). The stability of the OPA reagent is also tested by doing the derivatization of alanine with prepared OPA-reagent added on different days. This experiment was done with alanine because alanine is an amino acid that is widely present in silk and does not have multiple derivatives (Experiment 2).

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Amino acid stock solution (standards)

A 4 mL stock solution was made for the 17 amino acids in 0.1 M HCl, see table 3 (stored in fridge -4°C). The standard measurements were done by mixing the AA stock solution with the OPA reagent in ratio AA:OPA = 1/20. The overall ratio AA:(OPA/MPA) was 1/(20/60). The AA standards were measured with FLR to make a library of the AA and their retention times.

Table 3 Stock solutions amino acids

Amino acid Amount amino acid added

(mg) Concentration (mM) stock solution L-Glycine GLY 0.9757 3.25 L-Lysine LYS 1.5609 2.67 L-Serine SER 1.1627 2.77 L-Tyrosine TYR 1.8119 2.77 L-Alanine ALA 1.1482 3.22 L-Valine VAL 1.1948 2.55 L-Cysteine CYS 1.3521 2.79 L-Isoleucine ILE 1.2662 2.41 L-Leucine LEU 1.5154 2.89 L-Threonine THR 1.3004 2.73 L-Methionine MET 1.3560 2.27 L-Arginine ARG 1.9558 2.81 L-Phenylalanine PHE 1.7242 2.61 L-Proline PRO 1.4033 3.05 L-Histidine HIS 1.4772 2.38

L-Aspartic acid ASP 1.4269 2.68

L-Glutamic acid GLU 1.3776 2.34

Samples and sample preparation (hydrolysis & derivatization)

The artificially aged samples obtained from A. Serrano are listed in attachment 1 and these were aged at different conditions (relative humidity, temperature, UV-light exposure and oxygen-level.The historical samples were collected from 17th-century naturally-aged textiles (attachment 2) and from the 17th-century archaeological textiles found in Texel (attachment 3). The former naturally-aged samples belong to the RCE (Rijksdienst voor Cultureel Erfgoed, Cultural Heritage Agency of The Netherlands) reference collection and the latter archaeological Texel samples, to the Province of North-Holland. They comprise weaved fragments, which threads are called weft and warp. The weft

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forms the visible thread and the warp the skeleton of the textile. The weft is the thread that is inserted on the skeleton of warp threads (Burnham, 1980). For every sample, the weft and warp are analysed separately. For velvet samples, a third analysis is done of the upper layer, which is called pile. The unaged sample, S2, is documented in table 3.

From each sample 0.1-0.2 mg was weighted with an analytical balance and put in a heat protected glass vial. Then 100 µl 6 M HCl was added to hydrolyse the fibroin. The vial was closed and then put in an oven for 18 hours (105-110 °C). After 18 hours, the vials were taken out and the lid was removed. The HCl was evaporated by a gentle N2 flow. The residue was dissolved in 200 µl 0.1 M HCl and vortexed for 10 seconds to dissolve all the residues. An amount of 200 µl precolumn reagent of O-phthaldialdehyde, OPA, was added to 5 µl of the mixture in a small vial (OPA/AA=20/1). The reaction mixture was vortexed for 10 seconds and then placed in the UHPLC autosampler for injection.

Table 4 Not aged sample, S2, mass and silk information

Sample Silk Mass (mg)

S2 New silk (habotai) 0.1010

Validation

Besides the instability tests of OPA and the derivatives more tests are done for the validation of the analysis method.

Calibration curve

An amino acid stock solution was made for the 15 amino acid in table 5 in 1.0 mL HCl (0.1M). Two calibration mixtures were made. One stock solution for tyrosine and one solution for the other 15 amino acids, because tyrosine is hard to dissolve (solubility in water: 0.38 g/l, European Molecular Biology Laboratory, 2017). The 15 amino acids with 0.1 M concentration were mixed. Then the dilutions were made following table 6. An amount of 5 µl of the dilution mixture was reacted with 200 µl OPA reagent and then 0.2 µl of the mixture was injected (3 times). For dilution factors 500 and 1000 a stock solution 2, as mentioned in table 5, is used.

Table 5 AA stock solution for calibration curve

AA Concentration (mol/L) AA Concentration (mol/L) Ala 0.107 Leu 0.113 Arg 0.114 Lys 0.092 Asp 0.112 Met 0.120 Cys 0.124 Phe 0.100 Glu 0.103 Ser 0.112 Gly 0.096 Thr 0.114 His 0.117 Val 0.098 Ile 0.082 Tyr 0.00561

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19 Table 6 Dilution scheme of calibration curve measurements

Dilution Factor (DF) = Vf/Vi Stock solution AA (μl) HCl 0.1 M (μl)

1 15 0 1.2 15 3 1.5 15 7.5 2 15 15 3 15 30 5 15 60 10 15 135 20 15 285 40 15 585 80 15 1185 100 (stock solution 2) 15 1485

Dilution Factor (DF) = Vf/Vi Stock solution 2, DF=100 (μl) HCl 0.1 M (μl)

500 15 135

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Results

All samples and standards are measured three times, on the same day as the derivatization step and the OPA reagent is made freshly every day and kept in fridge as much as possible.

Ratio OPA:AA

The OPA reagent had a ratio of 1:3 (OPA:MPA), based on literature (Molnar-Perl, 2001). The ratio OPA:AA (20:1) was based on literature (Molnar-Perl, 2001) and the results of the ratio analyses, shown in figure 8 and 9.

Response with ratio 1:3 OPA/MPA ← no need to change ratio

Figure 9 FLR spectrum of amino acids. Blue= ratio 20:1, Red= ratio 40:1

Figure 8 Chromatograms of glycine extracted at 340 nm. Blue=ratio 20:1, OPA:AA. Black=ratio10:1, OPA:AA Note: this chromatogram is measured with a different method and therefor shows different retention times than used for sample analysis.

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Stability of the OPA and OPA-AA derivatives

Experiment 1: In Figure 10 the results of serine is shown. The biggest intensity was found when the sample was measured immediately after preparation. The room temperature sample is always measured after the fridge sample. This means that during the sample measurements of the cooled samples the room temperature samples degrade in the autosampler.

Figure 10 Bar plot of the peak area of the serine PDA chromatograms extracted at 340 nm. Samples were kept in fridge and at room temperature.

The OPA-AA-derivatives stability is also tested with alanine samples. These results are shown in Figure 11. It shows the influence of store temperature of the OPA-AA-derivatives on the PDA response.

Figure 11 Bar plot of the peak area of the alanine PDA chromatograms extracted at 340 nm. Samples were kept in fridge and at room temperature.

Leucine, an amino acid with multiple derivatives, also shows that in time the intensity of the multiple peaks change. From its chromatogram, it can be concluded that the main peak around 16.4 min decreases in time and the derivative peak around 14.7 gets bigger. Thereby a third peak appears on the

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3rd day of measurement. This assumes that more derivatives will appear in time, which is in line with literature (Kutlán & Molnár-Perl, 2001).

Figure 12 Chromatogram of leucine extracted at 340 nm. Samples stored in autosampler (RT) and in fridge (cooled).

In experiment 2 the stability of the OPA reagent was tested by making one stock of OPA reagent, keep that cooled and add the reagent to alanine solution on different days and measure the samples. The results are shown in figure 13.

Figure 13 Bar plot of the peak area of the alanine PDA chromatograms extracted at 340 nm. Samples were derivatizied ond different days with the same OPA stock solution (kept in fridge).

Stability of samples

As shown above both the OPA reagent and the OPA-AA-derivatives are unstable. This is also seen during the sample measurements. The amino acids with more than one derivative: glycine and lysine are very unstable. All samples are measured three times and the three measurements differ for the amino acids. This is represented in the figure 14 below, where the decrease in area in the three measurements is presented. The relative standard deviation (RSD) of the measurements also gives information about the stability of the derivatives. The RSD is represented in figure 15 for dilution factors 1 and 100.

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Amino acid standards

In figure 16 the chromatogram of a mixture of all amino acids is shown. The concentrations of all amino acids stock solutions (table 3) were around 2.5 mM. A library of the amino acids and their retention times is documented in attachment VI.

Figure 14 Decrease in area of 3 measurements. Samples were kept in autosampler (RT) between measurements. Time between the measurements was approximate 6 hours

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24 Figure 16 Chromatogram of amino acid mixture and a blanc

Calibration curve

Calibration curves were made for all 15 amino acids. For the 4 main components, the curves are shown below.

a.

b.

c.

d.

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Limit of Detection (LOD)

The lowest concentration detectable is different for every amino acid. Some amino acids give higher fluorescent response than others with the same concentration which is also seen in figure 16. The lowest concentration whereby all 15 amino acids could still be detected was DF=100. This responds to concentration of amino acids between 3.9-5.6 µM (attachment IV).

Xenotest samples

In total eleven samples aged in the Xenotest are analysed. Figures 18-20 show the amount of the 3 main components of silk, alanine, tyrosine and, in the samples. The reference sample is called S2 and is a silk sample that was covered in the Xenotest and therefore not exposed to the ageing factor. Sample 10C is a cochineal sample covered in the Xenotest.

Figure 18 Alanine content in silk samples aged in Xenotest. Green bars, samples in N2-pouches; red bars, samples in O2

-pouches; blue bars, samples in air--pouches; white bars, samples without pouches (white silk and C cochineal); grey, not aged (white silk S2 and C cochineal).

Figure 19 Tyrosine content in silk samples aged in Xenotest. Green bars, samples in N2-pouches; red bars, samples in O2

-pouches; blue bars, samples in air--pouches; white bars, samples without pouches (white silk S2 and C cochineal); grey bars, not aged(white silk and C cochineal).

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Figure 20 Serine content in silk samples aged in Xenotest. Green bars, samples in N2-pouches; red bars, samples in O2

-pouches; blue bars, samples in air--pouches; white bars, samples without pouches (white silk S2 and C cochineal); grey bars, not aged(white silk and C cochineal).

In figure 21 the tyrosine/alanine ratio is plotted for the samples. This ratio is the mole ratio in which the amino acids are found in the samples.

Figure 21 Tyrosine/alanine ratio in Xenotest samples. Green bars, samples in N2-pouches; red bars, samples in O2-pouches;

blue bars, samples in air-pouches; white bars, samples without pouches (white silk S2 and C cochineal); grey, not aged (white silk and C cochineal).

Figure 22 shows the difference in dyed and undyed silk fibre samples. The y-axis shows the decrease in amino acids content.

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Based on visual interpretation, samples X9 and X10, the samples exposed without pouch, seems most yellowed, figure 23.

Figure 23 Yellowing of sample X10. Left side exposed to UV-light in Xenotest, right-side covered.

Oven samples

Ten oven samples are analysed. The amino acid content of alanine, tyrosine and serine of the oven samples are plotted in figures 24-26.

Figure 24 Alanine content in silk samples aged in oven. Green bars, samples in N2-pouches; red bars, samples in O2

-pouches; blue bars, samples in air--pouches; grey, not aged.

Figure 22 The influence of cochineal dye on the decrease of tyrosine, alanine and serine content in silk and the tyr/ala ratio. Dark red are cochineal samples, and white bars are the new habotai sample.

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Figure 25 Tyrosine content in silk samples aged in oven. Green bars, samples in N2-pouches; red bars, samples in O2

-pouches; blue bars, samples in air--pouches; grey, not aged.

Figure 26 Serine content in silk samples aged in oven. Green bars, samples in N2-pouches; red bars, samples in O2-pouches;

blue bars, samples in air-pouches;grey, not aged.

In figure 27 the tyrosine/alanine ratio is plotted for the oven samples. This ratio is the mole ratio in which the amino acids are found in the samples.

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Figure 27 Tyrosine/alanine ratio in oven samples. Green bars, samples in N2-pouches; red bars, samples in O2-pouches;

blue bars, samples in air-pouches; white bars, samples without pouches; grey, not aged.

Historical samples

Three historical samples were obtained from the RCE historical collection. The details of the samples can be found in attachment II. Different fibres were taken as samples; warp, weft, and pile. Results are shown in figures 28-31.

Figure 28 Alanine content in historical aged samples

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30 Figure 30 Serine content in historical aged samples

Figure 31 Tyrosine/Alanine ratio in historical samples.

Texel textiles

In figure 32-35 the results of the Texel textiles are represented. The different cleaning methods are displayed with different colours. More details about the samples can be found in attachment III.

Figure 32 Alanine content in Texel samples. Light blue, washed with tap water; white, not washed; blue, washed with demi-water; orange, washed with ethanol; grey, silk sample not aged

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Figure 34 Serine content in Texel samples. Light blue, washed with tap water; white, not washed; blue, washed with demi-water; orange, washed with ethanol; grey, silk sample not aged

Figure 33 Tyrosine content in Texel samples. Light blue, washed with tap water; white, not washed; blue, washed with demi-water; orange, washed with ethanol; grey, silk sample not aged

Figure 35 Tyrosine/Alanine ratio in Texel samples. Light blue, washed with tap water; white, not washed; blue, washed with demi-water; orange, washed with ethanol; grey, silk sample not aged

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Discussion

Ratio OPA:AA

The results of the different ratios OPA & amino acids show the highest intensity of peaks for the ratio 20:1 (OPA:AA). This ratio is also suggested in literature. The ratio 10:1 shows lower intensity than ratio 20:1. The same result was found in FLR data of all amino acids for ratio 40:1. Therefor the sample measurements were done with this ratio.

Stability of the OPA and OPA-AA derivatives

From the results of serine, it can be concluded that storing samples after derivatization in the fridge improves stability. The serine peak of the room temperature sample was only found in the chromatogram on the first day, while the cooled samples could still be found after 4 days. This means that the OPA-AA-derivatives are more stable when kept cool in comparison to kept at room temperature. Still whenever the samples are cooled in the fridge degradation takes place due to the instability.

The trend seen for serine samples is also seen for the alanine samples. During the 4 days of measurement the intensity of the peaks decreases. Consistent with the literature we therefore may conclude that the derivatives are not stable and should be kept in the fridge to increase the stability.

The results of the leucine stability measurements, show that more peaks appear in time and the intensity of the main peak decreases.

In experiment 2, the stability of OPA was tested with alanine. The alanine peak differs in retention time and in height on the different days that the OPA was added. The area is almost equal in the first 3 days. The fourth day the area of the peak increases, but this difference is minor. The difference in retention time is also negligible. Since in this research both retention time and area are used for analysis, the OPA itself is considered as unstable too. The OPA is less unstable than the derivatives, but still not stable enough to use several days. To ensure the derivatization of the OPA reagent, the reagent was freshly made every day.

Stability of the samples

The instability was also seen in the sample measurements. The area decreases during the 3 measurements. This means that the fluorescence intensity decreases in time. The RSD of all amino acid derivatives show that some derivatives are more stable than others. The high RSD value of lysine, histidine and glycine can be caused by the multiple derivatives that they form. The total area is taken by adding all multiple derivative peak areas together, as suggested in literature (Kutlán & Molnár-Perl, 2001). Because they can switch to multiple derivatives these amino acids are more unstable. The amino acids with low RSD values are most stable, but as can be seen in Figure 7 the area of these peaks still differ throughout time.

To improve the stability of the derivatives in this method the sample should thus be kept cool. For further research, this information should be kept in mind. Also, the results show that the highest intensity is reached when the sample is measured directly after derivatization. The samples should thus be injected as soon as possible after derivatization. Another idea for further research would be to use an autosampler not only for the sampling but also for the derivatization step. The derivatives could then be injected immediately after derivatization when they are still stable. Lastly it is suggested to make the OPA reagent fresh every day.

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Amino acid standards

Sixteen amino acids could be found with the analytical method. Cysteine could only be detected in very high concentrations and according to literature not present in silk in high concentrations and is therefore eliminated from further measurements. Literature also states that OPA does not react with cysteine (Dai et al,, 2014). This is probably due the structure of cysteine that contains a SH-group like the MPA. This could cause a problem in the derivatization step.

Proline is a secondary amino acid and could therefore not react with the OPA reagent and form a chromophore. Proline could thus not be detected by FLR. Literature suggested to use FMOC, 9-fluoronylmethyl chloroformate, as reagent that could derivatize secondary amino acids (Buha et al., 2011). For further research, this could be used as derivatization reagent.

The retention times of the amino acids are as what can be expected from the theory. The polar amino acids serine, aspartic acid, glutamine acid, arginine, histidine and threonine all have relative short retention times because they have more affection with the polar mobile phase than the nonpolar column. In correlation the hydrophobic amino acids valine, leucine, isoleucine and phenylalanine have less affection with the mobile phase and have long retention times.

Calibration curve

As mentioned before the derivatives are not very stable. This is also seen in the calibration curves that have R² between 0.94-0.99 and the error bars. The calibration curves for glycine (and histidine & lysine) have a very low R² because of the multiple derivatives. Therefor glycine is not analysed further. The measurements are also affected by the instability in time. The dilution factors are made and measured on different days. About 4 dilutions were measured on 1 day. This means that the dilution factor measured first was more stable than the dilution factor measured last. This could have influenced the measurements. Because the higher concentrations differed from the linear trendlines, the 3 highest concentrations (DF=1, DF=1.2 & DF=1.5) were deleted from the calculations. Also, was taken in account that the concentrations of the silk samples would correspond to the higher dilution factors instead of the low dilution factors.

LOD

The lowest concentration detectable was different for the different amino acids. DF 100 was the highest dilution factor where all amino acid of the mixture could still be measured. For further research with more data, the LOD could be measured with signal to noise ratios.

Because of the low lineairty (R²) and the high RSD values due to instability of the derivatives, further development is needed to optimize the method. Despite that, with the current method samples have been analysed to find a trend for the different conditions they have been aged in. The RSD value shows that the results are not reliable, so only assumptions can be made.

Xenotest samples

Twelve samples were artificially aged by photo-oxidation in a Xenotest. All samples were exposed to UV-light. The samples were kept in different conditions. Most had pouches, a transparent bag covering the samples. The air compositions in those bags differed; air, oxygen and nitrogen. Samples X1, X4, X7 and X9 have been aged for one week and the others for two weeks. For more details see Attachment I.

For all samples the concentrations of the main components alanine, tyrosine and serine are calculated and the tyrosine/alanine ratio. The stability of the OPA-AA-derivatives could have influenced the results. Samples measured later after derivatization could show lower concentrations than samples measured first.

Sample X1 shows a high standard deviation and this measurement probably contains a wrong measurement, because X1, X2 and X3 have the same conditions and should thus have approximate same results. After the hydrolysis of the samples X1-X5, all HCl was already gone. This could mean that the vial was not properly closed and some HCl was evaporated. This could explain the results.

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The tyrosine content of the samples decreased during the ageing. If X1 is excluded, all samples show lower concentrations than the unaged S2 sample. There is no difference found between the different air conditions. For alanine and serine the aged samples also seem to have a lower content, but its not clear. The tyrosine/alanine plot shows a relatively high ratio for the oxygen-free condition (N2) and the most degradation for the samples without pouch. This suggests that oxygen accelerates the degradation and the pouches protect the samples from the UV-light.

The hypothesis is that, based on visual interpretation, X9 and X10 would have the least amount of serine and tyrosine. Serine and tyrosine decrease is said to be causing yellowing of silk. Samples X9 and X10 indeed belong to the samples with most tyrosine and serine loss, but no correlation can be found due to the low amount of samples.

For the open samples, dyed samples were also tested to investigate the influence of a cochineal dye on the amino acid composition. As seen in the barplot the percentual decrease in tyrosine and alanine content was more for the undyed samples. Also the tyrosine/alanine ratio decreases more for undyed samples. A decrease of this ratio means more degradation as written in the theory section. This thus supports what is suggested in literature that dyes have a preservence effect on degradation (Van Den Berghe, 2012).

Samples X1, X4, X7 and X9 were in aged for 1 week and the other samples aged for 2 weeks in the Xenotest. Yet, no constistent difference is seen.

Oven samples

Ten other silk samples were aged in a oven at 85 °C by thermal oxidation. The conditions of the pouches where the samples were in had different relative humidity levels (0% and 100%), and air compositions(nitrogen, oxygen and air). For further information see Attachment 1.

The oven samples show a more reliable result. The alanine, tyrosine and serine content decrease due to the thermal ageing. Despite there is a decrease in content of main amino acids, there is no correlation found for the relative humidity. This can be explained by literature (Vilaplana et al., 2015). Literature stated that both high and low levels of relative humidity (0% and 100%) stimulate degradation of silk.

The samples exposed to oxygen show lower amounts of serine, alanine and tyrosine. But because of the high standard error, this correlation should be tested again with more stable conditions and more samples. The correlation was not found in the tyrosine/alanine ratio.

If the bar plots of the oven samples are compared to the Xenotest samples, it is clear that the decrease in alanine is bigger for the oven samples. This means that the oven samples are further degraded. The tyrosine in the amorphous phase was first degraded and the alanine in the more stable crystalline phase is also degraded. In the Xenotest, the decrease in alanine is less noticeable and thus can be stated that the crystalline part in less degraded yet. Also the serine and tyrosine content is lower for the oven aged samples. The temperature of the oven was higher (85°) than in the Xenotest (55°C). The bigger decrease in serine content could be caused by thermal oxidation. In theory section, it was stated that serine loss is a sign for thermal oxidation, what refers to the oven conditions. Tyrosine loss in contrast refers to thermal and photo oxidation. The difference between the different ageing techniques can thus be caused by different types of oxidation, photo-oxidation and thermal, or different temperatures.

Historical samples

For the 3 historical samples, different fibres were taken as sample; weft, warp and pile. They all have different compositions. It was expected that the pile and warp, that are more exposed to the environment, would be more degraded, but no correlation was found. Thus, when analysing samples, the warp, weft and pile should be measured separately. The tyrosine/alanine ratio for the historical samples, all from 17th_{century, lies between 0.15-0.2. This is lower than the unaged sample (0.24), and} shows a degradation.

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Texel textiles

The samples taken from the Texel textiles are thought to be from the 17th century as well. The tyrosine/alanine ratio for the Texel textile lies between 0.1-0.15. This means the Texel textiles are more degraded than the historical samples. For the Texel samples washed with ethanol this ratio is the lowest. The samples washed with demi-water and tap water show ratio’s close to the untreated samples whereby demi-water has the highest ratio.

If the weft samples are compared on tyrosine content, the samples washed with demi-water again shows the highest content and thus is less degraded. The ethanol sample has the lowest tyrosine content and is therefore the most degraded sample. The warp samples seem to be the same for all conditions.

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Conclusion

In this research, a method was developed to analyse the amino acid composition of aged and unaged silk samples. The method developed using UHPLC-FLD had a gradient elution program with a mobile phase of methanol (10%), methanol and 0.1 M sodium acetate buffer (pH 6.9). The samples were derivatized with methanolic O-phthaldialdehyde and 3-mercapthopropionic acid in a borate buffer. Fifteen of the 17 amino acids in silk were successfully found with this method. The OPA reagent and its derivatives are not stable and should be kept in fridge. The samples should be measured soon after the derivatization step.

With this method the influence of oxygen, presence of a dye, relative humidity, washing method and ageing method on degradation was tested. The oven samples exposed to oxygen showed more degradation. The assumption can be made that in conditions with a high oxygen-level, silk will degrade more. No correlation was found for the different humidity levels tested. The cochineal dyed fibres showed less degradation, so it is stated that dyes have a conservative effect. The best washing method, based on the decrease in tyrosine content and the tyrosine/alanine ratio, is demi-water where ethanol gave the worst results. Those conclusions can only be interpreted as assumptions, because of the high RSD that makes the results unreliable.