Colorant Degradation Analysis by LC and LC-MS

Bachelor Thesis Chemistry

Colorant degradation analysis by LC and LC-MS

A study on the degradation process of crystal violet, purpurin, alizarin and carminic acid

by

Nienke Meekel

28 June 2017

Student number 10719059

Research institute Supervisor Van ‘t Hoff Institute for Molecular Dhr. prof. dr. ir. P.J. (Peter) Schoenmakers Sciences

Research group Daily supervisor Analytical Chemistry Dhr. B.W.J. (Bob) Pirok MSc Abstract

Many coloured paintings and textiles fade over time under influence of light irradiation. Intramolecular bonds break due to the energy of the absorbed photons, the loss of side groups in the dye molecules causes changes in the conjugated system and therefore changes in the colouring properties of the molecule. In the worst case, no light is absorbed and no colour is observed anymore. The light degradation of dyestuffs is not fully understood yet, this project aimed to gain insight in the light degradation (365 nm and 254 nm) of crystal violet, alizarin, purpurin and carminic acid. Crystal violet is firstly demethylated and then di-demethylated, it is proposed that alizarin and purpurin lose a hydroxyl group first. The composition of the natural dye carminic acid was not in accordance with the literature, but degradation resulted in the formation of both more hydrophobic and hydrophilic compounds. For further separation and identification of degradation compounds, LC-MS and comprehensive 2D-LC-MS are required.

2 Samenvatting

In 2014 is het wrak van een 17e eeuws VOC-schip voor de kust van Texel onderzocht en werd een goed bewaarde jurk gevonden. De versieringen en de samenstelling van de kleuren op de jurk kunnen veel informatie geven over de herkomst en de tijd waarin de jurk gemaakt is. De kleuren hebben nu echter niet meer dezelfde intensiteit als ze vroeger hadden. De veroudering van kleuren gebeurt ook op andere objecten zoals tekeningen of schilderijen en wordt vaak veroorzaakt door blootstelling aan licht. Met name licht van korte golflengtes dat een hoge energie heeft, zoals UV-straling, heeft een sterk verouderend ofwel degraderend effect op de kleuren. In dit project werden de kleurstoffen kristalviolet, purpurine, alizarine en karmijnzuur voor verschillende tijden verouderd onder een UV-lamp met een golflengte van 254 nm of 365 nm. Na veroudering werden de degradatieproducten van de kleurstoffen gescheiden en gemeten met behulp van respectievelijk vloeistofchromatografie en massaspectrometrie. Vloeistofchromatografie is een scheidingsmethode waarbij mengsels door een kolom geperst worden, de kolom bestaat uit een stationaire fase waar sommige moleculen meer affiniteit mee hebben dan andere moleculen. Met behulp van vloeistofchromatografie kunnen complexe mengsels gescheiden worden op basis van verschillende molecuuleigenschappen zoals grootte, lading en hydrofobiciteit. De laatste wordt

‘reversed-phase N N N vloeistofchromatografie’ genoemd,

N N N N deze methode werd ook gebruikt in H H H N NH2 dit project om de verschillende O

N degradatieproducten en de kleurstof N zelf van elkaar te scheiden. Als de componenten van elkaar gescheiden N N N NH2 H zijn worden ze gemeten met een kristalviolet detector en wordt het signaal omgezet in een chromatogram zoals in Chromatogram van kristalviolet dat 4 uur gedegradeerd werd met 254 nm, de meest rechtse piek is kristalviolet. De andere pieken zijn degradatieproducten. het figuur te zien is. Door een massaspectrometer aan te sluiten op de kolom werden de gescheiden componenten gemeten in de massaspectrometer en kon zo de massa bepaald worden. In bovenstaand figuur is gedegradeerd kristalviolet te zien, alle pieken op het chromatogram zijn verschillende degradatieproducten. Een aantal van deze pieken kon worden geïdentificeerd met behulp van massaspectrometrie zoals te zien is in het figuur. Hieruit blijkt dat er onder andere methylgroepen afbreken, wat de eigenschappen van het molecuul en dus ook de zichtbare kleur kan veranderen. Op basis van de veranderingen in hydrofobiciteit wordt verwacht dat er een hydroxylgroep afbreekt van de moleculen alizarine en purpurine. De samenstelling van het monster van karmijnzuur leek niet overeen te komen met de waardes uit de literatuur, dus wellicht heeft de kleurstof gereageerd met het oplosmiddel of is het al deels gedegradeerd. Omdat de monsters in de praktijk veel complexer zijn, bijvoorbeeld als er gemengde kleuren van de 17e eeuwse jurk afkomen, moeten er in de toekomst geavanceerdere scheidingsmethodes gebruikt worden om de degradatiecomponenten te kunnen identificeren.

3 Table of Contents

1. Introduction ...... 5

2. Theory ...... 9 2.1 High performance liquid chromatography ...... 9 2.1.1 Ion-pair reversed-phase chromatography ...... 9 2.1.2 Solvent effect on reversed-phase analysis ...... 9 2.2 Mass spectrometry ...... 10 2.2.1 Ion source ...... 10 2.2.2 Mass analyser ...... 11 2.3 LC-MS ...... 11

3. Experimental ...... 12 3.1 Instrumental ...... 12 3.2 Chemicals ...... 13 3.3 Analytical conditions ...... 13 3.3.1 Sample preparation ...... 13 3.3.2 Methods ...... 13

4. Results & Discussion ...... 15 4.1 Crystal violet ...... 15 4.2 Purpurin ...... 19 4.3 Alizarin ...... 20 4.4 Carminic acid ...... 22

5. Conclusion ...... 24

6. Acknowledgements ...... 24

7. References ...... 25

Appendix I – LC-MS results crystal violet ...... 26

Appendix II – LC results system (3), purpurin ...... 28

Appendix III – LC results system (3), alizarin ...... 31

Appendix IV – LC results system (3), carminic acid ...... 33

4 1. Introduction

Humans have been using colour since the Palaeolithic age (stone age); cave paintings are found which are about 15,000 years old according to 14C-dating systems.1 Thus colours have played an important role in human history, they do often have specific meanings for people or whole societies; some colours are very rare and/or considered as sacred in several communities.2 Colour can be added to objects by the usage of pigments or dyes and each pigment or dye has its own origin and history of use.3 These natural dyes and pigments have been used since the prehistoric age and were retrieved from for example rocks, flowers, plants and insects.2 Nowadays many dyes and pigments are being synthesized instead of retrieved from natural sources. The beginning of the synthetic dye industry was marked by the discovery of aniline purple, later known as mauveine, by William Henry Perkin in 1856.4 Many synthetic dyes were developed, for example azo-, triphenylmethane-, phthalocyanine- and anthraquinone dyes.4,5 These classes are determined by the structure and properties of the dyes. It is important to note the difference between pigments and dyestuffs; pigments are non-soluble and a binder (such as oil or gum) is needed to bind them onto the surface of textile or paper. On the other hand, dyestuffs are water-soluble substances of which the colouring component comes into the textile, no binder is needed.3 The different dyes do all have different colours; these colours are caused by the absorbance of light through the dye molecules. A molecule absorbs light of certain wavelengths and the remaining wavelengths are transmitted or reflected. These reflected wavelengths are complementary to the absorbed ones, therefore is the observed colour complementary to the light that is absorbed. This absorbance can be facilitated by several mechanisms, but the absorbance which occurs in the synthetic dyes is due to molecular orbitals and charge transfer.4 To detect the colour with a human eye, the wavelengths absorbed must lie into the visible range which is between 380 nm and 760 nm as is shown in figure 1.

Figure 1 – Visible light range of the electromagnetic spectrum.6

5 The absorption of energy includes the excitation of an electron from an orbital to a higher unoccupied orbital, for example excitation from the highest occupied molecular orbital (HOMO) into the lowest unoccupied molecular orbital (LUMO).7 The smaller the difference between the HOMO and LUMO, the longer the wavelength that is absorbed. This difference between the two orbitals is decreased by increasing conjugation. Therefore, more conjugated molecules do absorb longer wavelengths than less conjugated molecules. The strength of the conjugated system is determined by the number of double bonds and the electron-withdrawing or –donating capacities of the side groups; all these factors do have an influence on the absorbed wavelengths and the transmitted colour. The colour generated by these dyes can lose its brightness or, worse, its colouring properties and fade over time. This is observed in various art objects, for example a menu written with the use of crystal violet in 1886 by Van Gogh which was still visible in 1958 whereas the ink was completely disappeared in 2001.8 The drawing ‘Montmajour’ of Van Gogh showed discoloration in areas that were exposed to light, whereas the areas that were protected from light still remained the characteristic blue colour of crystal violet. The compound crystal violet, which is a triphenylmethane dye (see figure 2), faded and lost its colouring properties. Previous research showed that the fading of these triphenylmethane dyes probably is caused by decomposition of the molecules by demethylation, de-amination and/or oxidation, for the latter oxygen must be present.8,9

N

N N

Figure 2 – Crystal violet, a synthetic triphenylmethane dye.

Another example of degradation of dyes is a very well preserved dress and some clothing from the 17th century, found in a ship wreck near the island Texel in The Netherlands in 2014. Probably natural dyes have been used in these clothes because synthetic dyes were not invented yet in the 17th century. Among others, traces of carminic acid (see figure 3) were found in these textiles. But apart from the dyes, other compounds were also found. The question is whether these compounds are different dyes or degradation compounds of the former dyes. The influence of the salty water and sand must be taken into account because the dress has been stored at the seabed for such a long time.

6 OH O HO OH

O OH HO OH O CH O HO OH 3 OH Figure 3 – Carminic acid, a natural anthraquinone dye.

The environment can influence the appearance of the dyes; humidity, oxygen (or other gases) and light do have degrading effects on the dyes. Light occurs at several wavelengths and is supported by the next equation. The wavelength (�) is inversely proportional to the energy (�) according to the following formula in which Planck’s constant (ℎ) and the speed of light (�) are involved:

ℎ� � = �

The photons in light of shorter wavelengths contain more energy than the photons emitted with longer wavelengths. Therefore, UV-light (10 < � < 400 nm) will have the highest energy and thus the strongest degrading effect on the dye molecule. Photons emitted on the dye molecule are absorbed by the chemical bonds in the dye molecule. The absorbed energy facilitates electron- excitation from the electronic ground state into the electronic excited state. As a result of this, the chemical bond can overcome its activation barrier which corresponds to the bond dissociation energy as is shown in figure 4, and the bond will break. The loss of side-groups will affect the absorbing capacities of the molecule and influence the strength of the conjugated system and thus the colour appearance of the molecule.

Figure 4 – The dissociation energy is the energy needed to break the internuclear bond, for the breaking of stronger bonds, more energy is required.10

7 In order to determine the original appearance of the object and the future behaviour of the dyes, conservation scientists would like to gain more insight in the kinetic processes by which the dye molecule degrade. Therefore, it is important to gain more insight in the degradation processes of these natural and synthetic dyes that were used since the 19th century and earlier. Liquid chromatography (LC) is a technique used for dye-analysis; the differences between side groups and sizes of the dyes determine their retention times which will be explained in the following section. Artificially degrading with UV-light will yield degradation compounds, these can be separated with LC. To determine the structures of the different compounds, mass spectrometry can be used. Analysis of the degradation products with mass spectrometry (MS) will give more insight on the degradation kinetics of the dyes. In this project, the degradation of crystal violet, alizarin, purpurin and carminic acid under the influence of UV-light, the identification of the different degradation products and their formation over time was examined.

8 2. Theory 2.1 High performance liquid chromatography High performance liquid chromatography (HPLC) is a separation method in which high pressure is used to push a solvent containing analyte through a packed column.11 The column is packed with a porous stationary phase that commonly consists of silica particles with certain side groups covalently attached. In this project, a UHPLC column was used (1.8 µm particle size) for HPLC analysis at ± 350 bar. Several types of HPLC are possible; for example, separation based on size, charge and hydrophobicity. The latter is called reversed-phase chromatography and is commonly used as separation method in analysis of dyestuffs because of the complexity, limited chemical stability and non-volatility of dyes.12 The separated compounds that come off the column are detected by a diode array detector (DAD) which can use different detection wavelengths.

2.1.1 Ion-pair reversed-phase chromatography In reversed-phase chromatography, the column consists of an apolar stationary phase with in this case octadecyl alkyl (C18) chains attached to silica particles.11 The polar mobile phase runs through the column and hydrophobic analytes are retained because they interact with the stationary phase. Hydrophilic analytes have no interaction with the stationary phase and elute rapidly. The gradient used is programmed to run from an aqueous buffer to an organic solvent, thus eventually all compounds will come off. The hydrophobicity and therewith the retention of the molecules are determined by several parameters; the size of the molecule (especially its carbon backbone), the properties of the side groups and the charge of the molecule. The first two can increase the separation, but this does not count for the latter. Some dyes are charged and contain negative side groups that reduce the hydrophobicity of the molecule. This is an undesired effect because it reduces the separation; even when molecules are large, they will elute early because of their charge. To reduce or even prevent this effect, the pH is lowered and an ion-pairing reagent is used. An ion- pairing reagent contains a charged functional group surrounded by carbon chains, this is added to the mobile phase buffer and will form complexes with the analytes. These analytes are then neutralized and their retention time increases and separation improves.12 Tetramethylammonium (TMA) seems to be a good ion-pairing agent but is not compatible with MS; it must be volatile. However, triethylamine (TEA) is compatible with MS and is also a suitable ion-pairing agent thus this is used.13

2.1.2 Solvent effect on reversed-phase analysis The solvent-type used in the mobile phase influences the separation. Acetonitrile as a solvent is good for reversed-phase analysis because it inhibits π-π interactions.14 On the contrary, the π-

9 electron poor solvent methanol does not have these properties. The influence of these π-π interactions on the retention times must be taken into account when methanol is used in the mobile phase for reversed-phase analysis because separation is not purely on hydrophobicity anymore.

2.2 Mass spectrometry The mass of the dye molecules and their degradation compounds can be determined by mass spectrometry. In a mass spectrum, the number of ions detected at each value of mass-to-charge ratio, m/z, is displayed.11 A mass spectrometer is composed of an ion source, mass analyser and detector. The first two components are discussed in the following sections.

2.2.1 Ion source When a sample enters the mass spectrometer, it is converted into ions via a specific ionization method. Electrospray ionization (ESI) was used in this project, ESI is an example of atmospheric pressure ionization (API).15 At API, samples are ionized at atmospheric pressure and then transferred into the mass spectrometer which is under high vacuum. Molecular ions are lost due to this transfer, but this effect is compensated by the higher yield of the ionization method as a result of fast thermal stabilization at atmospheric conditions. API methods are suitable for direct on-line coupling of LC.15 The reason for using ESI in this experiment, is its suitability for thermally unstable compounds and little fragmentation occurs. The sample enters the ionization chamber at low flow rate via a capillary to which a very high voltage is applied (2-6 kV).16 The ion polarity of the mass spectrometer is important because it influences the ionization of the dyes. At negative polarity, the solvent undergoes reduction reactions and the analyte is deprotonated. At positive ion mode, oxidation reactions occur in the solvent and the analyte is protonated.16,17 Negatively charged dyes, such as Eosin A (see figure 5) show no peak in the total ion current (TIC) chromatogram measured in positive mode. Whereas positively charged dyes such as diamond green B (see figure 5) and crystal violet show no peak in the TIC chromatogram measured in negative mode. Neutral and negatively charged dyes are most abundant, thus the negative mode can be used mostly.

N Br Br O O O

Br Br

O N O Figure 5 – Eosin A and Diamond Green B, a negatively and positively charged dye respectively.

10 For ionization, a strong electric field is applied under atmospheric pressure which induces a charge accumulation at the liquid surface at the end of the capillary. The surface breaks and highly charged droplets are formed.15 The flow of nitrogen gas that is applied assists the evaporation of the solvent in these droplets.16 The remaining molecular ions pass into the mass analyser.

2.2.2 Mass analyser The different types of ions are separated in a time-of-flight mass analyser (TOF), which separates on velocity.15 All ions gained the same kinetic energy due to the electric field that is applied in the previous part. They are accelerated and enter the flight tube in which no electric field is present. The molecular ions do all have a different velocity due to their different mass-to-charge ratios. After separation, the ions are detected by the detector which generates an electric current that is proportional to their abundance.

2.3 LC-MS As mentioned before, LC can be coupled to MS. Firstly, the sample is injected and separated by the use of HPLC. After the separation, the flow is split and one part goes to the DAD and the other part flows into the MS and gets ionized. The flow rate and concentration in which the sample enters the mass spectrometer is much smaller than the flow rate and concentration of the part that enters the DAD. A TIC chromatogram is measured by the MS which is comparable to the chromatograms obtained with the DAD. The parallel measurements are important because both chromatograms can be compared. The mass spectrum of each peak provides the m/z values of the compound(s) present in the corresponding LC peak. Due to the use of ESI, little fragmentation occurs and therefore corresponds the most abundant mass to the mass of the dye molecule or degradation compound which is present in that LC peak.

11 3. Experimental 3.1 Instrumental For the degradation of the dyes, the Spectrolinker XL-1500 UV Crosslinker – Spectronics Corporation (365 nm & 254 nm, ± 1-3 µW/cm2) was used. Three systems were used for analysis; the major part (LC and LC-MS analysis) was performed on an Agilent system (1), the minor part on a Shimadzu (2) and a Waters system (3). The overview of all three LC systems is shown in table 1. The column used on system (1) and (2) was an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT (959941-902, 50 x 4.6 mm, 1.8 µm particles) column. The column used on system (3) was a Waters UHPLC BEH Shield RP18 (2.1 mm x 150 mm, 1.7 µm particles) column.

Table 1 – Overview of systems used for LC analysis. Agilent (1) Shimadzu (2) Waters (3) Pump 1100 series G1311A LC-10AD VP Degasser 1260 Infinity G1322A DGU-20A5 Waters AcquityTM H-class Jet Weaver V35 Mixer FCV-10ALVP UHPLC system (Waters G4220-60006 Corporation, Milford, MA, 1290 Infinity G4212- Detector SPD-10A VP U.S.A) 60008 Autosampler 1100 series G1313A - Loop/Injection volume 10 µL 20 µL 5 µL Data recording 40 Hz and 80 Hz 2 Hz 2 Hz OpenLAB CDS LCsolution Empower 2.0 Chromatography Software ChemStation Edition Version 1.21 SP1 Data Software Rev. C.01.04 [35]

For the LC-MS analysis, system (1) was coupled to a Bruker mass spectrometer, the flow (1.850 mL/min) was split to the DAD and the MS. The LC system was coupled to a Bruker MicrOTOF- Q mass spectrometer, the settings for both positive and negative ion mode are shown in table 2.

Table 2 – Acquisition parameter Bruker MicrOTOF-Q in positive and negative mode. Positive ion mode Negative ion mode Source type ESI ESI Focus Not active Not active Scan begin 50 m/z 50 m/z Scan end 1200 m/z 1200 m/z Ion polarity Positive Negative Set capillary 4400 V 3800 V Set end plate offset -500 V -500 V Set collision cell RF 1600.0 Vpp 1600.0 Vpp Set nebulizer 0.7 Bar 0.7 Bar Set dry heater 200 ˚C 200 ˚C Set dry gas 6.0 L/min 6.0 L/min Set divert valve Source Source

12 3.2 Chemicals The following chemicals were used for LC-MS analysis; triethylamine (³ 99.5%, Sigma-Aldrich), formic acid (³ 96%, Sigma-Aldrich), deionised water (Baker analysed ultra LC/MSTM, Avantor) and acetonitrile (LC-MS grade, Biosolve). Triethylamine (³ 99.5%, Sigma-Aldrich), formic acid (³ 96%, Sigma-Aldrich), deionised water (Millipore, Q-POD®, Milli-Q, R=18.2 MΩcm) and acetonitrile (AR grade, Biosolve) were used for ion-pair reversed-phase LC analysis. For reversed-phase analysis only, deionised water (Millipore SimplicityTM Simpak® 2, R=18.2 MΩcm, U.S.A.), methanol (99.9%, Sigma-Aldrich) and formic acid (Sigma-Aldrich) were used. Samples of dyestuff were obtained from the reference collection of the Cultural Heritage Agency of the Netherlands (RCE, Amsterdam, The Netherlands). These were dissolved in dimethyl sulfoxide (SAFC) and acetonitrile (LC-MS grade, Biosolve).

3.3 Analytical conditions

3.3.1 Sample preparation The dye samples were dissolved in acetonitrile/dimethyl sulfoxide 1:1 (v/v), for each sample the dye (0.500 mL; 200 ppm/40 ppm) was added to a 1.5 mL vial with transparent glass. The vials were placed vertically on a wooden slat (see figure 6) which lied on a beaker into the UV cabinet. Thicker vials were used for the degradation of crystal violet, the other three dyes were degraded in thinner vials to make the degradation process go faster.

Figure 6 – Samples of carminic acid, purpurin and alizarin on the wooden slat used for degradation.

The samples were irradiated with UV-light at 254 nm or 365 nm for different duration times. After degradation, the samples were stored in darkness at -19 ˚C.

3.3.2 Methods 3.3.2.1 System (1) and (2) The following mobile phases were used for ion-pair reversed-phase chromatography: [A] – buffer/acetonitrile 95:5 (v/v) [B] – buffer/acetonitrile 5:95 (v/v) Buffer was prepared containing triethylamine (5 mM) in water and formic acid (pH 3). The mobile phases were delivered at different flow rates.

13 3.3.2.2 System (3) The following mobile phases were used for reversed-phase chromatography: [A] – water/methanol 90:10 (v/v) [B] – pure methanol [C] – water/formic acid 99:1 (v/v) The mobile phases were delivered at a flow rate of 0.2 mL/min using the gradient shown in table 3.

Table 3 – Gradient used for reversed-phase analysis on system (3). Time % [A] % [B] % [C] 0 80 10 10 1.33 80 10 10 2.33 74 16 10 5.33 55 35 10 9 55 35 10 14 30 60 10 25 5 85 10 26 0 100 0 30 0 100 0 32 80 10 10 40 80 10 10

14 4. Results & Discussion 4.1 Crystal violet Firstly, the effect of two different wavelengths (254 nm and 365 nm) on the degradation process was examined. The results are shown in figure 7; the peak corresponding to crystal violet at 9.87 min is present in all samples but decreases with longer degradation time. As can be seen from the graph, the 60 minutes’ degradation with 254 nm delivers the most degradation compounds and the longer the degradation time, the more degradation compounds are generated. As expected, the degradation compounds of crystal violet are less retained than crystal violet itself. This result is also proved by Favaro et al.9 The molecule is getting less hydrophobic due to its decreasing size and the loss of methyl groups.

Figure 7 – Comparison of crystal violet with samples degraded at 254 nm and 365 nm for respectively 30 minutes and 60 minutes. Flow rate 0.75 mL/min. The gradient used was 0-0.5 min, isocratic at 100% A; 0.5- 12.5 min, linear gradient to 100% B, maintained at B for 0.5 min; 13.00-14.50 min linear gradient to 100% A. DAD detection wavelength 254 nm.

Secondly, crystal violet and its degradation compounds were analysed with different detection wavelengths for the diode array detector (DAD); 201 nm, 214 nm, 230 nm, 260 nm, 254 nm, 500 nm and 590 nm. A sample of crystal violet (250 µL; 200 ppm) which was degraded at 254 nm for 4 hours was used for this experiment. The chromatograms measured at 201 nm, 214 nm and 230 nm showed a shift of the baseline and not all peaks were detected. The chromatograms detected at 254 nm, 260 nm, 500 nm and 590 nm are shown in figure 8. The most peaks are present in the chromatogram detected at 590 nm, thus this detection wavelength was used for the further examination of crystal violet.

15

Figure 8 – Crystal violet degraded at 254nm for 4 hours measured with different DAD wavelengths. Flow rate 1.850 mL/min, injection volume 20 µL. The gradient used was 0-0.25 min, isocratic at 100% A; 0.25-6.25 min, linear gradient to 100% B, maintained at B for 0.5 min; 8.75-9.5 min linear gradient to 100% A.

Crystal violet and its degradation compounds were analysed with mass spectrometry. All masses could be detected in positive ion mode (see figure A in appendix I). The increase of degradation compounds of crystal violet during 4 hours of degradation at 365 nm were measured with 20 minutes’ interval. The peaks were numbered and the expected corresponding compounds are shown in figure 9. The mass spectrum of each peak is shown in figure B in appendix I.

Figure 9 – LC chromatogram of crystal violet degraded at 365 nm for 4 hours. Flow 1.850 mL/min. Gradient used was 0-0.5 min, isocratic at 100% A; 0.25-1.1 min, linear gradient to 45% B; 1.1-5.1 min, linear gradient to 55% B; 5.1-6.1 linear gradient to 100% B, maintained at B for 1 min; 7.1-8.1 min linear gradient to 100% A. DAD detection wavelength 590 nm. The proposed structures are shown per peak.9

16 The mass and characteristic peak at the UV-Vis spectrum of peak 4 are the same as the values for crystal violet reported in literature.9 Peak 3 has a m/z value of 358.2 which corresponds to the demethylated form of crystal violet. At longer irradiation time, peak 1 and 2 are formed which have the same m/z value and are not fully separated. Their mass corresponds to the di- demethylated form of crystal violet, which has two isomers as is shown in figure 9. It is expected that peak 1 corresponds to the isomer with a primary amine and a tertiary amine because this compound will have lower retention due to the primary amine group. Peak 2 has more retention and is likely to correspond to the isomer with two secondary amine groups, one methyl group is lost at both sides of the molecule. The areas of the four main peaks were plotted against the degradation time (see figure 10). The UV lamp intensity appeared to decrease over time, this was tracked and is also shown in figure 10. The crystal violet peak (peak 1) is decreasing, while the other three degradation compounds increase during degradation. The reaction kinetics cannot be determined from this graph due to the decreasing lamp intensity which is also shown in the graph. However, according to this graph and the previous LC-MS analysis can be concluded that crystal violet firstly is degraded into its demethylated form and secondly into its di-demethylated form.

Figure 10 – Peak areas of crystal violet degradation over time. Flow 1.850 mL/min, injection volume 20 µL. Gradient used was 0-0.5 min, isocratic at 100% A; 0.25-1.1 min, linear gradient to 45% B; 1.1-5.1 min, linear gradient to 55% B; 5.1-6.1 linear gradient to 100% B, maintained at B for 1 min; 7.1-8.1 min linear gradient to 100% A. DAD detection wavelength 590 nm.

17 Finally, the crystal violet sample degraded for 4 hours at 254 nm was analysed with LC-MS. The LC results are shown in figure 11 and the MS results are shown in figure C in appendix I. A few compounds could be determined by comparing the m/z values with the exact mass and with the

9 help of λmax and the data found by Favaro et al.

Figure 11 – Crystal Violet degraded at 254 nm for 4 hours. Flow 1.850 mL/min, injection volume 20 µL. The gradient used was 0-0.25 min, isocratic at 100% A; 0.25-6.25 min, linear gradient to 100% B, maintained at B for 0.5 min; 8.75-9.5 min linear gradient to 100% A. DAD detection wavelength 590 nm.

Only a few peaks could be assigned because of the large amount of peaks that are not fully separated (in both LC and TIC chromatogram) and the low intensity in the TIC chromatogram. To improve the analysis of these degradation compounds, even for mixtures of degraded dyes; a more advanced separation method is required. Comprehensive two dimensional liquid chromatography coupled to MS could give a better insight in the degradation compounds. To determine the degradation kinetics of the dyes quantitatively, a more advanced degradation method should be used. The dyes have been degraded in solution, but are actually applied on textiles and other fibres. Moreover, a light source with constant intensity should be used and for increasing accuracy and reproducibility an in-line degradation method can be used. As mentioned before, some natural dyes were also degraded and measured with LC. These results will be discussed in the following sections.

18 4.2 Purpurin The anthraquinone dye purpurin was also degraded at both 365 nm and 254 nm and the dye appeared to degrade much slower than crystal violet, which was also expected according to the ISO Blue Wool Standards. These are used for comparing fading rates of dyes. The dye was degraded at 254 nm for different duration times, the graphs measured on system (1) are shown in figure 12.

Figure 12 – Comparison of purpurin with samples degraded at 254 nm for respectively 30, 60, 120 and 180 minutes. Flow rate 1.850 mL/min. The gradient used was 0-0.25 min, isocratic at 100% A; 0.25-6.25 min, linear gradient to 100% B, maintained at B for 2.5 min; 8.75-9.50 linear gradient to 100% A. DAD detection wavelength 254 nm.

The peaks at 1.15; 3.12; 3.33; 3.80; 6.04 and 7.20 minutes are also present in the (degraded) blank samples. The peak around 4.5 minutes corresponds to purpurin and decreases along irradiation time. However, no other peaks increase that much as the purpurin-peak decreases. It is possible that the degradation compounds of purpurin have a different extinction coefficient and absorb less light of the detector. In that case, the area of those peaks is not comparable to the area of the purpurin-peak because it is not representative for the concentration of the compound. Nevertheless, if the peaks do have comparable absorption spectra, it may be assumed that the extinction coefficient has not changed that much. The samples were also measured on system (3) and the chromatograms were screened at all wavelengths. The blank (ACN-DMSO) samples showed peaks at a detection wavelength of 275 nm (see figure D in appendix II). The compounds present in the (degraded) purpurin samples absorbed light with wavelengths of 350 nm and 500 nm (see figure E and F in appendix II). At an absorption wavelength of 350 nm, a peak eluting at 15.10 minutes is formed which first increases

19 and later decreases along degradation time. The PDA spectrum of this compound (see figure G in appendix II) shows absorption at 351.8 nm. Several small peaks are present having comparable PDA spectra, with a very low intensity. These peaks could indicate a broken ring because of their absorption at 350 nm. They can still absorb light because of the remaining conjugated system. At an absorption wavelength of 500 nm are two compounds visible; purpurin is located at 25.06 minutes and a purpurin equivalent is eluted at 27.99 minutes. The corresponding PDA spectra are shown in figure 13. These are nearly the same so it is possible that a hydroxyl group is released because this leads to a more hydrophobic compound and thus more retention. However, the expected hypsochromic shift is not visible. The position of the hydroxyl group that might be dissociated could not be determined. But it can be stated that it is not the bottom hydroxyl group showed in figure 14 which is not present in alizarin that elutes at 21.18 minutes as is mentioned in the following section.

Figure 13 – PDA spectra of Purpurin (RT = 25.057 min) and a Purpurin equivalent degradation compound (RT = 27.989 min) respectively. O OH O OH OH OH

O OH O Figure 14 – Purpurin (left) and alizarin (right). The dyestuff alizarin is comparable to purpurin and its degradation will be discussed in the next paragraph.

4.3 Alizarin Alizarin appeared to degrade slower than purpurin as can be seen in figure 15, these chromatograms have been measured on system (1). The samples were degraded at 254 nm for respectively 30; 60; 120 and 180 minutes. The chromatograms contained the same impurities as present in the purpurin chromatograms. The peak corresponding to alizarin at 4.11 minutes decreases along degradation time; the peaks eluting at 3.38 minutes and 5.33 minutes increase in the first hour of degradation

20 and disappear in the last two hours of degradation. This is also confirmed by the results of the reversed-phase analysis on system (3) as is shown in figure H in appendix III. Alizarin might lose a hydroxyl group because a hypsochromic shift occurs (see figure 16); the chromophore –OH increases the absorption wavelength of the molecule. The wavelength of absorption will decrease in case this group is lost whereas the molecule will be more retained because of the increasing hydrophobicity of the molecule. On top of that, the increase in hydrophobicity does not count for the peak eluting earlier than the alizarin peak; also a hypsochromic shift occurred, but the compound is more hydrophilic than alizarin itself. So LC-MS analysis is necessary to determine the corresponding structure formulas.

Figure 15 – Comparison of alizarin with samples degraded at 254 nm for respectively 30, 60, 120 and 180 minutes. Flow rate 1.850 mL/min. The gradient used was 0-0.25 min, isocratic at 100% A; 0.25-6.25 min, linear gradient to 100% B, maintained at B for 2.5 min; 8.75-9.50 linear gradient to 100% A. DAD detection wavelength 254 nm.

Figure 16 – Absorbance of the compounds measured at 430 nm on system (3); degradation compound (RT = 17.970 min), alizarin (RT = 21.180 min) and degradation compound (RT = 25.149 min) respectively.

21 At the absorption wavelength of 350 nm (see figure I and J in appendix III) some low intensity peaks similar to those present in degraded purpurin have been found.

4.4 Carminic acid The natural dye carminic acid has been degraded as well, the results of the analysis on system (1) are shown in figure 17. The appearance of the carminic acid peak is a striking point because it is composed of two unresolved peaks which is not corresponding with the data found in literature.18 The dyestuff carminic acid consists of several components such as dcII (flavokermesic acid glycoside), dcIV and dcVII (both isomers of carminic acid), kermesic acid, flavokermesic acid and carminic acid itself.18,19 These are present in several ratios depending on the origin of the dye, but the main component carminic acid must be present for 94-98%.3 This might indicate that the sample is already partially degraded, or has interacted with the solvent (ACN-DMSO).

As can be seen in figure 17; t0 increases along degradation time which indicates that very hydrophilic compounds are formed, probably the glucoside or OH-groups that broke off. This is also supported by the peak around 2.79 minutes which is more hydrophobic than carminic acid and increases along degradation time. This could be a carminic acid molecule that lost some hydrophilic groups.

Figure 17 - Comparison of carminic acid with samples degraded at 254 nm for respectively 30, 60, 120 and 180 minutes. Flow rate 1.850 mL/min. The gradient used was 0-0.25 min, isocratic at 100% A; 0.25-6.25 min, linear gradient to 100% B, maintained at B for 2.5 min; 8.75-9.50 linear gradient to 100% A. DAD detection wavelength 254 nm.

22 The analysis on system (3) showed almost the same results (see figure K in appendix IV), the two unresolved peaks have the same absorption spectrum (see figure L in appendix IV). Furthermore, a large amount (> 18) of side components are present in the dyestuff. The components visible at 500 nm, were all disappeared after 120 minutes of degradation. The chromatogram at 275 nm (see figure M in appendix IV) shows that degradation compounds are formed that absorb UV wavelengths (see figure N in appendix IV), these could be anthraquinones. Carminic acid was found to degrade relatively fast in this experiment, thus it should be degraded slower to examine the degradation process more accurately.

23 5. Conclusion

Crystal violet was degraded with light of 245 nm and 365 nm for several timespans, the degradation compounds were visible on LC and some were identified with LC-MS. It was concluded that the dye first is demethylated and second di-demethylated in which two isomers are formed. The degradation method was also applied on alizarin, purpurin and carminic acid and they were analysed as well. These dyes appeared to degrade slower than crystal violet. It is proposed that purpurin and alizarin lose –OH groups during degradation and that eventually a ring breaks and components are formed that absorb around 350 nm. The composition of the dye carminic acid did not correspond to the values found in literature, it may be partially degraded or have interacted with the solvent. The dye molecule appeared to degrade and both more hydrophilic and hydrophobic compounds were formed. LC-MS or even LC-MS-MS analysis is required for identification of these degradation compounds. Future research should be done on identification of the degradation compounds of these dyes. For more accurate analysis of the degradation kinetics, a lamp with constant and repeatable intensity should be used for sample degradation. To gain results that are representative for the dyes on clothing, textiles or paper, the dyes should be applied and degraded on these mediums. Even the effects of other degrading factors such as moisture should be taken into account. For analysis, a more advanced method with a higher peak capacity such as comprehensive 2D-LC-MS is required to resolve and identify all peaks.

6. Acknowledgements

First of all, I would like to thank Prof. Peter Schoenmakers for providing me the opportunity to do this project within the Analytical Chemistry group. Secondly, my thanks to Bob Pirok for supervising me these three months; I have learned a lot due to your helpful feedback. Also thanks to Sanne and Noor for helping me in the lab and giving useful feedback. Furthermore, I would like to thank Maarten van Bommel for helping me with the analysis at the RCE and sharing some of your knowledge about colours and dyestuffs. Finally, I would like to thank the Analytical Chemistry group for the great time I had during my project.

24 7. References

(1) Brunello, F. The Art of Dyeing in the History of Mankind, Bernard Hi.; Neri Pozza Editore: Vicenza, 1973. (2) Finlay, V. Kleur. Een reis door de geschiedenis; Snijders, M., Ed.; Ambo|Anthos: Amsterdam, 2003. (3) Hofenk de Graaff, J. H. The Colourful Past - Origins, Chemistry and Identification of Natural Dyestuffs; Abegg-Stiftung and Archetype Publications Ltd.: Riggisberg | London, 2004. (4) Christie, R. M. Colour Chemistry; Royal Society of Chemistry: Cambridge, 2001. (5) Allen, N. S. Polym. Degrad. Stab. 1994, 44 (3), 357–374. (6) Stratman, C. Light spectrum: visible light https://www.tes.com/lessons/viLnecVwyGloOg/visible- light-models (accessed May 12, 2017). (7) Clayden, J.; Greeves, N.; Warren, S. Organic Chemistry, Second Edi.; Oxford University Press: New York, 2012. (8) Confortin, D.; Neevel, H.; Brustolon, M.; Franco, L.; Kettelarij, A. J.; Williams, R. M.; van Bommel, M. R. J. Phys. Conf. Ser. 2010, 231 (1), 1–9. (9) Favaro, G.; Confortin, D.; Pastore, P.; Brustolon, M. J. Mass Spectrom. 2012, 47 (12), 1660–1670. (10) Atkins, P.; Jones, L.; Laverman, L. Chemical Principles - The quest for insight, 6th ed.; W. H. Freeman and Company: New York, 2013. (11) Harris, D. C.; Lucy, C. A. Quantitative Chemical Analysis, Ninth Edit.; W. H. Freeman and Company: New York, 2016. (12) Pirok, B. W. J.; Knip, J.; van Bommel, M. R.; Schoenmakers, P. J. J. Chromatogr. A 2016, 1436, 141– 146. (13) Berbers, S. Natural and Synthetic Dye Characterization by Comprehensive 2D Liquid Chromatography, University of Amsterdam, 2017. (14) Croes, K.; Steffens, A.; Marchand, D. H.; Snyder, L. R. J. Chromatogr. A 2005, 1098 (1–2), 123–130. (15) Hoffmann, E.; Stroobant, V. Mass Spectrometry Principles and Applications, Third Edit.; John Wiley & Sons, Ltd: Chichester, 2007. (16) Banerjee, S.; Mazumdar, S. Int. J. Anal. Chem. 2012, 2012, 1–40. (17) Wilm, M. Mol. Cell. Proteomics 2011, 10 (7), M111.009407. (18) Serrano, A.; Van Bommel, M.; Hallett, J. J. Chromatogr. A 2013, 1318, 102–111. (19) Stathopoulou, K.; Valianou, L.; Skaltsounis, A. L.; Karapanagiotis, I.; Magiatis, P. Anal. Chim. Acta 2013, 804, 264–272.

25 Appendix I – LC-MS results crystal violet

Figure A – TIC chromatogram of crystal violet and the mass spectrum of the crystal violet peak at 3.4 min.

3 4

1 2

Peak 1 Peak 2

Peak 3 Peak 4

Figure B – TIC chromatogram of crystal violet degraded at 365 nm for 4 hours and the corresponding mass spectra.

Figure C – TIC chromatogram and corresponding MS spectra of crystal violet degraded at 254 nm for 4 hours.

27 Appendix II – LC results system (3), purpurin

0.20

0.10

0.00 Absorption at 257nm at Absorption 0.010

0.005

0.000

-0.005 Absorption at 257nm at Absorption

0.020

0.010

0.000 Absorption at 257nm at Absorption

0.04

0.02

0.00 Absorption at 257nm at Absorption 0.06

0.04

0.02

0.00

Absorption at 257nm at Absorption 0.00 2.00 4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.00 Retention time (min.) Figure D – ACN-DMSO measured on system (3), absorption wavelength 275 nm. Top to bottom: ACN- DMSO undegraded; 30 minutes; 60 minutes; 120 minutes and 180 minutes degraded. Note the different scales.

28 0.000

-0.002

-0.004

Absorption at 257nm at Absorption 0.030

0.020

0.010

0.000

Absorption at 257nm at Absorption 0.002 0.000 -0.002 -0.004 Absorption at 257nm at Absorption

0.000

-0.002

-0.004 Absorption at 257nm at Absorption

0.000

-0.002

-0.004 Absorption at 257nm at Absorption

0.000

-0.002

-0.004

Absorption at 257nm at Absorption 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Retention time (min.) Figure E – Purpurin measured on system (3), absorption wavelength 350 nm. Top to bottom: ACN-DMSO 180 minutes degraded; purpurin; purpurin 30 minutes, 60 minutes, 120 minutes and 180 minutes degraded.

29 0.10

0.05

0.00 Absorption at 257nm at Absorption 0.030 0.020 0.010 0.000 Absorption at 257nm at Absorption 0.030 0.020 0.010 0.000 Absorption at 257nm at Absorption 0.030 0.020 0.010 0.000 Absorption at 257nm at Absorption 0.030 0.020 0.010 0.000

Absorption at 257nm at Absorption 0.00 2.00 4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.00 Retention time (min.) Figure F – Purpurin measured on system (3), absorption wavelength 500 nm. Top to bottom: purpurin; purpurin 30 minutes, 60 minutes, 120 minutes and 180 minutes degraded.

15.103 nm 300.00 400.00 500.00 600.00 700.00 246.3 15.10

351.8

308.1

679.1699.9 792.0 429.0442.3 507.8522.5 648.4

Figure G – PDA spectrum of the peak at 15.103 minutes at an absorption wavelength of 350 nm.

30 Appendix III – LC results system (3), alizarin

0.010

0.000 Absorption at 430 nm 430 at Absorption

0.04

0.02

0.00 Absorption at 430 nm 430 at Absorption 0.020

0.010

0.000 Absorption at 430 nm 430 at Absorption 0.010 0.005 0.000 -0.005

Absorption at 430 nm 430 at Absorption 0.004 0.002 0.000 -0.002 -0.004

Absorption at 430 nm 430 at Absorption 0.004 0.002 0.000 -0.002 -0.004

Absorption at 430 nm 430 at Absorption 2.00 4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.00 Retention time (min.) Figure H - Alizarin measured on system (3), absorption wavelength 430 nm. Top to bottom: alizarin; alizarin 30 minutes, 60 minutes, 120 minutes and 180 minutes degraded.

31 0.000

-0.002

-0.004 Absorption at 350 nm 350 at Absorption 0.06

0.04

0.02

0.00 Absorption at 350 nm 350 at Absorption

0.020

0.010

0.000 Absorption at 350 nm 350 at Absorption 0.015 0.010 0.005 0.000 -0.005 Absorption at 350 nm 350 at Absorption 0.002 0.000 -0.002 -0.004 Absorption at 350 nm 350 at Absorption

0.000

-0.002

-0.004

Absorption at 350 nm 350 at Absorption 0.00 2.00 4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.00 Retention time (min.) Figure I - Alizarin measured on system (3), absorption wavelength 350 nm. Top to bottom: ACN-DMSO 120 min degraded; alizarin; alizarin 30 minutes, 60 minutes, 120 minutes and 180 minutes degraded.

12.506 13.493 15.100 16.895 20.325 nm nm nm nm nm 400.00 400.00 400.00 400.00 400.00 600.00 600.00 600.00 600.00 600.00 248.7 12.51 240.4 13.49 245.2 15.10 16.90 273.6 20.32

353.0 354.2 278.4 348.2 302.2 299.8

643.5666.8 350.6 498.1 306.9 349.4 704.8 557.8661.9 495.7 681.5724.5 794.5 468.9670.5715.9 661.9 455.6529.8

Figure J – Absorption spectra of the compounds present at 350 nm in alizarin degraded for 120 minutes.

32 Appendix IV – LC results system (3), carminic acid

0.12 0.10 0.08 0.06 0.04 0.02 0.00 Absorption at 430 nm 430 at Absorption

0.020

0.010

0.000 Absorption at 430 nm 430 at Absorption

0.015

0.010

0.005

0.000

Absorption at 430 nm 430 at Absorption -0.005

0.015

0.010

0.005

0.000

Absorption at 430 nm 430 at Absorption -0.005

0.015

0.010

0.005

0.000

Absorption at 430 nm 430 at Absorption -0.005 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 Retention time (min.) Figure K – Carminic acid measured on system (3), absorption wavelength 500 nm. Top to bottom: carminic acid; carminic acid 30 minutes, 60 minutes, 120 minutes and 180 minutes degraded.

12.817 12.987 14.244 14.350 15.541 15.629 nm nm nm nm nm nm 400.00 400.00 400.00 400.00 400.00 400.00 600.00 600.00 600.00 600.00 600.00 600.00 274.8 12.82 274.8 12.98 274.8 14.24 274.8 14.35 273.6 15.54 273.6 15.63

470.2 312.9 311.7 494.5 494.5 477.4 496.9 493.2

661.9 661.9 661.9 665.6 661.9

Figure L – Absorption spectra of some peaks present in carminic acid degraded for 30 minutes.

33 0.24

0.22

0.20

0.18

0.16

0.14

0.12

0.10

Absorption at 275 nm 275 at Absorption 0.08

0.06

0.04

0.02

0.00

0.00 2.00 4.00 6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.00 Retention time (min.) Figure M – Carminic acid, measured on system (3), absorption wavelength 275 nm. Top to bottom: carminic acid degraded for 30 minutes (black line); ACN-DMSO degraded for 30 minutes (blue line).

1.787 1.879 2.715 7.791 nm nm nm nm 400.00 400.00 400.00 400.00 600.00 600.00 600.00 600.00 1.78 1.87 254.6 2.72 260.6 7.78

668.0 299.8 303.4 459.2 644.7663.1676.6

Figure N – Absorption spectra of UV absorbing components of carminic acid degraded for 30 minutes.

34