Graduation report
The extraction and analysis of fibre dyes
using LC-PDA-Orbitrap-MS
Graduation report
The extraction and analysis of fibre dyes using
LC-PDA-Orbitrap-MS
Student:
Tom Schotman tg.schotman@student.avans.nl
Internship mentor Netherlands Forensic Institute:
Dr. Ir. Jaap van der Weerd j. van.der.weerd@nfi.minvenj.nl
Internship institution:
Netherlands Forensic Institute Department of Microtraces Paint, Fibres and Textiles Group Laan van Ypenburg 6
2497 GB, The Hague
Internship mentor Avans University:
Dr. Henk Haarman hf.haarman@avans.nl
Second mentor Avans University:
Ir. Hans van Amelsvoort jaga.vanamelsvoort@avans.nl
Internship period:
Periods 4.3 and 4.4; 28-01-2013 to 14-06-2013
Institute of eductation:
Forensic Science
School of Life Sciences and Environmental Technology Avans University of Applied Sciences, Breda
i
Preface
This report is written as an overview of the work carried out during my graduation project for applying my Bachelor at the Avans University of Applied Sciences with a major in Forensic Science. All work has been carried out at the Netherlands Forensic Institute (NFI) for the fibres and textiles group in collaboration with the chemical identification group from the NFI.
I would like to thank Xiaoma Xu for all his help with the LC-MS system and the insight to high resolution mass spectrometry. Also Nicole van Rodewijk is well acknowledged for her help with the LC-MS system and for preparing a set of, to me, unknown dye samples. I would like to thank Jaap van der Weerd as well for the opportunity to do this project and for the programming of one of the databases. Iris van der Ouden for the good and nice cooperation during the laboratory work.
Furthermore I would like to thank everybody from the fibres and textiles section from the NFI for their help during this period and Rene from the chemical identification group. Also Jorrit his advise on the application of derivatisation reactions is much appreciated. Maarten for the nice discussions and staying late.
Niels Blok is well acknowledged for his advise regarding organic chemistry and in general for the friendship and fun during my time at the NFI. May we encounter more Saabs in the future.
All structures in this report are drawn using Accelrys Draw 4.1 unless stated otherwise.
References are based on the standard form from the journal Science & Justice since I feel that this journal represents the forensic fibre society best.
ii
Abstract
At the Netherlands Forensic Institute (NFI), there has been an intensive study into the analysis of fibre dyes using one single method with liquid chromatography mass spectrometry (LC-MS) for all dye classes. This project has accomplished many goals so far apart from the analysis of some dye classes and the choice of a suitable database. Two of these dye classes are vat and sulphur dyes. This are very non-polar fibre dyes applied to cotton fibres by reducing the dyes to an
ionised form. For vat dyes this is achieved by reducing a ketone and for sulphur dyes a disulphide bond. From the measurements there will be a UV/Visible spectrum, a retention time and an accurate mass of the dye molecule. Ideally these three factors will be combined in one database. Since this is not readily available software of different databases has to be tested to compare and find the most suitable database. This comparison is done for disperse, direct, acid and basic dyes. Derivatisation of the ketones was achieved using semicarbazide and ammonium hydroxyl
chloride for conversion to an imine form. Also the Lawesson reagent was used to derivatise the oxygen atom to a sulphur atom. This sulphur atom is oxidised using Oxone making it more polar. For the sulphur dyes a oxidation is carried out using nitric acid to created sulfonate groups at the disulphide bond.
For the database a group of 6 to 8 dyes in each tested dye class is completely processed according to the database preferences. A group of prepared fibre dyes is run in this database to read out which dyes might be present. This group is prepared by another person to remove any form of confirmation bias. ToxID and a Matlab programmed database were compared with each other. Results show that the conversion of the imine form of the vat dyes do not influence or decrease the solubility of the dyes in the fibres. This negative effect is the opposite of the goal of this project so these derivatisation reactions should not be operated. The use of the Lawesson reagent however showed increased extraction and different MS signal than expected with the normal dye. The extraction of the sulphur dyes with nitric acid completely discoloured the fibre but also degraded the fibre. This degradation of the fibre makes it impossible for good analysis using LC-MS making this method unfavourable. A conversion with chloroactetic acid is suggested to make a good derivatisation but some practicalities have yet to be overcome to use this method.
The comparison of the ToxID and Matlab database showed some differences. From the ToxID database only 1 of the 17 (6%) dyes was not identified in contrast to 4 of the 17 (24%) dyes using the Matlab database. With the Matlab database one of these 4 was identified wrongly giving a false positive result. Using the paired t-test to compare whether or not these differences are significant no true significant difference was measured. However the fact that one false positive is given using the Matlab database might not correctly be represented in this test.
The results show that derivatisation of the vat dyes is very difficult but can be achieved using the Lawesson reagent. This increases the extraction of the vat dyes and chromatographic separation appears to be different compared to the dyes in their normal state. Optimisation of the extraction is still necessary. The extraction of sulphur dyes with nitric acid has not proven to be feasible for the LC-MS analysis. Future work must prove however if a chloroacetic acid acidification gives reasonable results and if this method can be successfully applied using the LC-MS analysis. Database comparison showed that ToxID is the most suitable database for the dye identification if no adaptations are made to the Matlab database. Hence the ToxID database is the database of choice for future database processing.
Contents
Preface ... i
Abstract ... ii
1 Introduction ... 1
2 Forensic trace evidence ... 2
2.1 Fibre evidence ... 2
2.2 Dye and fibre chemistry ... 2
2.2.1 Dyes for cellulose fibres ... 5
2.2.2 Dyes for polyester fibres ... 7
2.2.3 Dyes for polyamide fibres ... 8
2.2.4 Dyes for polyacrylic fibres ... 9
2.3 Fibre and dye analysis ... 10
2.3.1 Microscopy ... 10 2.3.2 Micro-infrared spectroscopy ... 10 2.3.3 Microspectrophotometry ... 11 2.3.4 Dye extraction ... 11 2.3.5 Liquid chromatography ... 13 2.3.6 Mass spectrometry ... 13
3 Material and methods ... 15
3.1 Chemicals and equipment ... 15
3.2 Dye extraction ... 15 3.3 Chromatographic separation ... 16 3.4 Database comparison ... 16 4 Results ... 18 4.1 Vat dyes ... 18 4.2 Sulphur dyes ... 21 4.3 Database comparison ... 21 5 Discussion ... 23 6 Conclusion ... 27 7 Recommendations ... 27 References ... 28 Appendix I Selected dyes for the databases ... XXXI Appendix II Lay outs for the different databases ... XXXII
1
1 Introduction
At the fibres and textiles department of the Netherlands Forensic Institute (NFI) there is a project focused on the analysis of fibre dyes for several years. The focus of this project is on all
commonly used fibre dyes, concerning dye extraction, dye analysis by chromatography and identification by high resolution mass spectrometry. For most dye classes the extraction and analysis is validated as well as the identification of these dyes. For all these different dye classes a single chromatographic separation method has been created in the past. Though most dye classes have been validated, the extraction of vat and sulphur dyes for chromatographic
separation has not yet been successful. Vat dyes are highly aromatic, non-polar compounds with only ketones as function groups which have only be successfully extracted using reduction methods. The same is true for sulphur dyes which are polymeric substances linked by disulphide bonds. It is thus important to find methods which can extract these dyes followed by analysis using, preferably with the same separation method as is used for other dye classes. The other factor of this project is the use of databases for automatic, or more rapid, dye identification. Two different databases are proposed, both operation from different angles, making it important to compare both databases.
The goal of this project is to achieve extractable vat and sulphur dyes suitable for analysis using the same method as used for other dye classes. It is also a goal to compare the two databases in order to select the most suitable database for dye identification. For the extraction of vat and sulphur dyes methods are employed to try to make the dyes more water soluble. This would make them more easy to extract and separation using the standard method can then be employed. For vat dyes this is done by derivatisation of the ketones to a more polar functional group. For the sulphur dyes this is done by breaking the disulphide bond followed by derivatisation on these places. After successful extraction, the dyes are analysed with the standard method and the derivatisation is confirmed based on the mass spectra of the eluting compounds.
For the comparison of the databases a set of known disperse, basic, acid and direct dyes is analysed and processed into the databases. A set of dyes, unknown to the database analyst, are analysed as well and processed into the databases. This in order to determine the number of (false) positives and negatives. The identification and comparison is based on the absorption spectra, retention times and the mass spectra of the different eluting compounds.
In order to understand the process of fibre and dye analysis detailed chemistry and other relevant theoretical topics are outlined in chapter two. The materials used and methods applied during this project are outlined in chapter three followed by the obtained results in chapter four. A discussion is formed to relate the results to the methods used in chapter five followed by a conclusion in chapter six. For future prospects several recommendations are made in chapter seven.
2
2 Forensic trace evidence
In forensic science many disciplines are involved in providing evidence to solve crimes. This can range from technical evidence from mobile devices and computers to chemical and biological evidence such as drugs, fire accelerants, fingermarks and DNA evidence [1]. Among the other types of forensic evidence is trace evidence, often referred to as physical evidence, which is the evidence of, often microscopic, small particles. These particles are for example paint, pollen grains, soil, hairs, glass, and most commonly fibres [1-8]. Trace evidence particles are easily transferred during contact between two objects obeying the Locard's Exchange Principle: 'Whenever two objects come into contact, there is an exchange of matter' [5]. For example in a car crash between two cars, paint can be transferred from the cars onto the other car, just as windshield glass can and the plastic covers of the vehicle lights. This provides information about the incident and can trace back the vehicles involved might one flee the scene of the incident [3]. The analysis of these particles are often based on microscopic analysis followed by other physical and chemical analysis [1-8]. The focus in this report is on fibre evidence and will be further discussed below.
2.1 Fibre evidence
Fibre evidence is one of the most important forms of trace evidence in forensic science, since fibres are transferred in most cases where textiles are involved [2]. Fibres are described as thin, flexible objects having a high length to cross-sectional shape diameter ratio [1]. This includes many items such as hairs, electric wires but in principle textile fibres are meant in this research area. Since fibres are processed in textile items, the fibres can transfer during contact of the textile with other items. The number of fibres being transferred is dependent on the nature of the textile, the contact and other factors attributing to transfer [2].
After transfer the fibres persist to the recipient item, the item to which the fibres transferred. This persistence is not infinite and the number of fibres remaining on the recipient item will decrease by time. The speed of this decrease is dependent on the transfer mechanisms and the movements of the recipient item after transfer [2].
For forensic evidence this means that in a certain timeframe transferred fibres can be recovered from the recipient item and analysed [2]. This makes fibre evidence very strong to give
information about the activities during the transfer of the fibres [2]. There are some limitations involved with fibre evidence, fibres are mass produced items making their evidential value lower than with for example DNA evidence [1]. In order to assess the evidential value as much as possible points of comparison should be assessed in order to include or exclude reference
material [2]. Most trends in fibre comparison are well developed but more recently there has been a major interest in the analysis and identification of dyes based on the mass spectra. Since the dye structure is important in relation to the fibre types used to dye the fibres some fundamental topics are discussed below followed by the means of fibre and dye identification.
2.2 Dye and fibre chemistry
The analysis of dyes can be important in several fields of forensic science, this can be the analysis dyes in documents, the dyes used to colour illicit drugs and is also very important in fibre analysis [2]. Colour is a very important aspect in life and many decisions are based on the colour of a certain object. This argument is also very true in fashion and therefore many different colours of textiles exist dyed with a different group of dyes [9]. The classification of dyes can be based on either the chemical composition or the method of application [2, 9, 10]. The latter is
3 used by the Colour Index (C.I.) from the Society of Dyers and Colourists [2]. This organisation registers all dyes submitted for registration by dyers giving them a C.I. number and dye name. This is written as: C.I. <method of application> <colour> <number> [11]. These C.I. names are very important for the identification of dyes [2].
The chemical composition is divided by the main structure and not by the colour influencing groups. Functional groups influencing the colour (chromophores) are halogens, amides, amines, nitriles, ketones, alcohols and others [9, 10]. Also the number of double carbon-carbon bounds must be relatively high for the molecule to absorb light in the visual spectrum [9]. In order to make the molecules more soluble, sulfonate groups are attached to the dye molecules [10]. The most important dye classes are outlined below.
Azo dyes
Azo dyes are the most important group of dye molecules and are characterised by their azo bond attached to aromatic rings as shown in figure 1 [10]. To these aromatic rings more chromophores can be attached including more azo bonds giving rise to diazo, triazo dyes and further. Often nitro, amines and hydroxyl groups are present as the chromophores [9]. Azo dyes represent all colours in the visible spectrum due to the many chromophore synthesize spaces however in this dye class the brilliant colour shades are missing [10].
Figure 1: Base of an azo dye with the azo bond connected to two aromatic rings.
Anthraquinone dyes
Anthraquinone dyes are based on the anthraquinone base structure (figure 2), a colourless natural compound historically derived from plant material [10]. Since the base structure leaves 8
synthesis places the number of shades that can be achieved is relatively low compared to azo dyes [10]. The main chromophores for anthraquinone dyes are amines, hydroxyl groups and amine linked aromatic groups [9]. The main advantage of the anthraquinone dyes is that these give a range of brilliant colours [10].
4
Indigoid Dyes
Indigoid dyes are based on the indigo molecule (see figure 3) which is far the most important of this dye group [10]. Indigo is a blue dye used for the dyeing of blue denim trousers and is derived from natural plant material before it was being synthesised in 1898 [10]. Some other
commercially used related structures are those in which the nitrogen is converted to sulphur [10]. These thioindigoid dyes have chromophores such as chlorine and methyl groups but are not that commonly used [9, 10].
Figure 3: Structure of the blue indigo dye.
Polycyclic aromatic carbonyl dyes
Polycyclic aromatic carbonyl dyes are molecules which are highly aromatic with few other functional groups other than ketones. In the structures often an anthraquinone related base is present such as shown for CI Vat Orange 9 in figure 4 [10]. The structures can me even more aromatic and sometimes chromophores as halogens are added to influence the shades [9, 10]. Furthermore some amide and amine linkages can be made between the anthraquinoid structures. The dyes represent mostly the blues and other dark shades [10].
Figure 4: Structure of CI Vat Orange 9.
Sulphur dyes
Sulphur dyes are classified as dyes that are synthesised by the heating of aromatic amines, phenols or nitro containing compounds. These compounds do contain alkali polysulfides and can be reduced using a sodium sulphide solution [10]. Since the structures are unknown of these dyes it is difficult to relate to the chromophores of the dyes and dyes with the same dye name can differ in structure due to synthesis conditions [10]. It is known that the dyes possess a polymeric like structure caused by disulphide bonds between the different molecules [9, 10].
5
Polymethine dyes
Polymethine dyes are derivatives from cyanine with the main important derivatives being the diazahemicyanines (base structure shown in figure 5) [9]. Polymethine dyes are known for their positive charge making them basic dyes (see section 2.2.4). Polymethine dyes have a wide range of chromophores and are well presented as red, blue and yellow brilliant colour shades [, 10].
Figure 5: Base structure of diazahemicyanines. 2.2.1 Dyes for cellulose fibres
From all cellulosic fibres, cotton is considered the most used fibre type and is natural of origin [2, 12]. The polymer chain of cotton exists primarily of cellulose which is a polymer from sugar monomers (see figure 6). Other common cellulosic fibres are viscose/Rayon and Lyocell which are all made of regenerated cellulose [12]. Microscopically cotton is easily recognised but has further no other distinguishable features when observing the fibre other than colour and dye analysis [2]. Fortunately cotton has the most dye classes used making it an important feature to analyse the dyes present on the fibres [2]. Cotton and other common cellulosic fibres are predominantly used in clothing and upholstery [12].
Figure 6: Repeating monomer units (2) of cellulose, the main component of cotton fibres.
Direct dyes
Direct dyes are, as the name would suggest, directly applied to cellulosic fibres in an aqueous solution. In order to achieve good dyeings an electrolyte is added to make the solution positively charged neutralizing the negatively charged fibre surface [2, 10]. This makes it more easy for the dye to penetrate the fibre just as increasing the temperature does [10]. To solute the dye in the aqueous medium several sulfonate groups are brought onto the molecule to increase solubility [9]. An example of a diazo direct dye is shown in figure 7, having 4 sulfonate groups to increase solubility.
6 Figure 7: Chemical structure of C.I. Direct Blue 80.
Reactive dyes
Reactive dyes react with the methyl hydroxyl group of cellulose forming a covalent bond with the fibre [10]. This reaction is achieved by the addition of nucleophilic groups onto the dye molecule such as chlorine atoms connected to a triazinic ring (C3N3) or alkyl ether sulfonates [10]. An
example of a reactive anthraquinone dye is shown in figure 8 having a alky ether sulfonate group as the reactive group. The sulfonate is a leaving group attaching the hydroxyl group of the cellulose to the carbon attached to the ether [10].
Figure 8: Chemical structure of C.I. Reactive Blue 19.
Vat dyes
Vat dyes belong to a class of highly non-polar chemical compounds from which indigo is the most important (figure 3) [2]. The dyes all have ketone functional groups which is can be reduced
7 using a strong reduction compound such as sodium dithionite or sodium sulphide. This reaction, as shown in reaction 1, makes the compound ionised with a sodium counter ion [10]. This form is water soluble and has good affinity with the cellulose of the fibre making it possible to penetrate the fibre. Oxidation converts the ion back to ketone bringing back the original colour and
trapping the dye molecule into the fibre [9, 10]. This process is known as the vatting process, hence the name vat dyes. Due to the very non-polar nature of the vat dyes this class proves relatively difficult to the dyer and the chemical analyst [10].
Na2S2O4 O – R R O R R Air (1) Sulphur dyes
The chemistry of sulphur dyes is discussed above. The application of sulphur dyes to a substrate goes by a comparable process as that of the vatting process [10]. The difference is that disulphide bond of the dyes is broken by a reduction compound leaving a sulphur ion with a sodium counter ion [10]. Commercially speaking sulphur black 1 is a very important compound. This accounts for about 80% of the sulphur dye usage. Since sulphur dyes are relatively cheap these dyes are very often in t-shirts and mixed with other, more expensive, dye classes [2, 9].
2.2.2 Dyes for polyester fibres
Polyester fibres are considered the most important synthetic fibre type based on world production and application areas [12]. The main polyester type is polyethylene therphtalate (PET) which structure is shown in figure 9 [2]. The fibre has a high crystallinity and is considered highly hydrophobic [2]. Textiles made from polyester are used in many kind of application areas such as sporting goods, upholstery, car interior, cotton blends, gloves, ropes and much other applications [12]. It is also the most commonly encountered synthetic fibre in forensic cases [2].
Figure 9: Chemical structure of the repeating monomer units of the polymer polyethylene therphtalate (PET).
The dyes used to dye polyester fibres are disperse dyes. These dyes are brought into a dispersion to make them soluble in a polar medium [2]. Disperse dyes were originally developed for dye cellulose acetate fibres which are also hydrophobic but the development of polyester made this dye class bigger [10]. Since polyester is highly crystalline, the fibre needs to be swollen in order for the dyes to penetrate the fibres pores. This achieved by dyeing under high pressure at elevated temperatures [10]. Disperse dyes mainly comprise out of anthraquinone related dye structures
8 because of their non-polar nature, however some azo dyes are used as well [9, 10]. An example of a commonly used anthraquinone disperse dye is given in figure 10.
Figure 10: Chemical structure of C.I. Disperse Blue 60.
2.2.3 Dyes for polyamide fibres
Polyamide fibres are either protein fibres such as silk and wool or synthetic fibres such as nylon 6 and nylon 6.6 (figure 11) [10, 12]. These fibres are characterised by their amide bond within the polymer structure. The protein fibres have a somewhat low crystallinity compared to the higher crystallinity of the synthetic fibres. This influences the dyeing behaviour of the dyes [10]. Silk is often used in high end suits and is very uncommon in forensic case work. Wool and nylon fibres are often used in carpets and clothing [12].
Figure 11: Chemical structure of the repeating monomer units of the polymer nylon 6.6.
Polyamide dyes are predominantly dyed with acid dyes and metal complex acid dyes [10]. Acid dyes are applied under acidic conditions making the amino groups in polyamide fibres positively charged. The negatively charged dye molecules attract to the polymer with a ionic bond. In some cases a salt linkage can bond the dye with the fibre [12]. Due to the differences in crystallinity, silk and wool can be dyed with relatively deep shades due to better dye penetration compared to nylon fibres [10]. For example a negatively charged acid azo dye is shown in figure 12. The metal complex dyes recently used are premetallised 1:1 and 1:2 metal complexes. A 1:1 metal complex dye has the metal (often chromium, copper, cobalt or iron) within the dye structure. This in contrast to 1:2 metal complexes in which two dye molecules are linked with a metal [2, 9, 10]. Polyamide fibres can also be dyed using reactive dyes but this is less common [2, 10].
9 Figure 12: Chemical structure of C.I. Acid Red 266.
2.2.4 Dyes for polyacrylic fibres
Polyacrylic fibres are synthetic fibres existing out of acrylonitrile units and can be divided in two groups. Acrylic and modified acrylic. The former exists for more than 85% out of acrylonitrile (for example figure 13) and the latter between 35% and 85% out of acrylontrile units [12]. The latter are mostly copolymers with, for example, vinyl chloride [2]. The main end use of acrylic fibres are clothing and interior textiles and are often regarded as substitutes for wool fibres [12].
Figure 13: Chemical structure of the repeating monomer units of the polymer polyacrylonitrile (PAN).
For the colouration of acrylic fibres on most occasions basic dyes are used [2]. Basic dyes are applied under acidic conditions, leaving a positive charge to the dye molecule and rendering the polymer negatively charged. The dye bath is heated for better uptake of the dye into the fibre. To prevent uneven dyeing, dye retarders are added which occupy the dye sites until the dye replaces the retarder [2, 10]. An example of a basic azo dye is shown in figure 14. The dye has positively charged nitrogen atom with a methanesulfonic acid counter ion. Other counter ions are often chlorine atoms (figure 5) [9, 10].
10 Figure 14: Chemical structure of C.I. Basic Blue 41.
2.3 Fibre and dye analysis
2.3.1 MicroscopyFibre evidence is dominantly recovered using tape lifting [13]. This is a technique in which transparent tape is applied to the recipient item in order to adhere the transferred debris onto the adhesive of the tape. In order to make use of the debris on the tape, reference material is
necessary from which similar items have to be searched on the tape lifts [2]. For this a
stereomicroscope is used. The stereomicroscope exists out of two separate lens systems, each towards one eye, giving a stereoscopic view. The lenses are constructed to give a high working distance between the object table and lens in order to analyse and prepare items without
interference of the lens system. The range of magnification for stereomicroscopes is most commonly 10x to [14]. The lighting of a stereomicroscope can be both oblique and transmitted making it possible to work with transparent and non-transparent specimen. Selected fibres can be cut out of the tapes and prepared for high power microscopy [2].
The high power microscopes are transmitted light microscopes with polarised light capabilities and oblique fluorescent light capabilities [2, 14-18]. For best comparison between reference materials and the unknown materials two microscopes are connected via an optical bridge giving a view of both the fibres in plain view [2]. This gives a means of directly comparing the materials and is the first exclusion point in the analysis. The technique is especially strong in visual
comparison and for the determination of the fibre's general class (e.g. polyester or cotton) using polarised light [18]. It is also a means of qualitatively determining the microscopical appearance of the fibres as well as the colour and serves as rapid technique [2]. Though the microscope has many strengths it appears often difficult to determine fibre's polymer sub-class and to quantify the colour of the fibre by eye [2].
2.3.2 Micro-infrared spectroscopy
In order to determine the general and sub-class of the fibre composition infrared spectroscopy coupled to a microscope is used [2]. Infrared (IR) spectroscopy is the study of bond vibration and bond bending by molecules when illuminated by infrared light [19]. The amount of vibration or bending and the wavelength where vibration/bending happens is dependent the type of bond. The vibration or bending happens in the presence of a dipole moment between the bonds [2, 19]. The wavelength of the absorption of the infrared light is related to the type of bonds present in the molecule. This makes it possible to determine the bonds present in a molecule, and this can often lead to identification [2, 19]. Hence IR spectroscopy is a strong tool for determining the structure of the compounds. Because the amount of dye present in a fibre, the in situ analysis of the fibres
11 does not provide much information about the dyes. Raman spectroscopy shows some potential for this but it does not work in all cases, dependent on the dye and fibre's polymer composition [2, 19, 20].
2.3.3 Microspectrophotometry
Microspectrophotometry (MSP) is a technique used for the quantification of colour based on the absorption of light in the UV and visible spectrum. A UV-Vis detector is used and this type of detection system is based on absorption of UV and visible light due to chromophores in
compounds [2, 21, 22]. For a compound the absorption can take place in both the visible and UV spectrum or solely in one of those. Dependant on which frequencies the molecules absorb the light a certain colour is observed (the exact opposite in the colour spectrum). The detector measures the amount of light being absorbed in contrast to conditions were no compounds are present [2]. Molecules absorb according to Beer’s law: E = c*l*ε, in which E is extinction, c is concentration, l is the path length and ε is the molar extinction coefficient. Since dyes are mostly present as a mixture the technique is not possible to determine the concentration of a certain dye but the colour is clear from the spectrum. For the recording of the spectra a diode array detector (DAD) is used. A DAD detector measures several light wave frequencies at the same time by splitting up the light in a broad range of wavelengths which are all analysed by an detector array [2]. In this way data is quickly acquired. Even though some molar extinction coefficients are known, MSP is by no means a method for dye identification. In order to identify the dyes, the fibres need to be extracted, separated from each other and identified by a suitable chemical identification technique.
2.3.4 Dye extraction
In order to analyse the dyes used in textile fibre dyeing, the dyes need to be extracted out of the fibre. Several methods are available for this and are dependent dye class based on method of application.
Traditionally for direct, disperse and acid dyes were extracted using a pyridine - water solution during heating [2, 23-25]. The idea of this process is to swell the fibre making it possible for the dye molecules to get out of the pores of the fibre molecule. Using a solvent which has more affinity with the dye molecules than the fibre self, the dyes get efficiently extracted [2]. The downside is that this mixture is not volatile enough and can pollute mass spectrometric detection systems used for dye identification. DMSO appears to have the same extraction efficiency for the dye molecules and is suitable for the used detection systems.
With basic dyes the strong ionic bond needs to be destroyed which can be done by an acid [23]. The acid of choice for this is formic acid, destroying the cationic bond between the dye and the fibre. The dye has more affinity for the acid and is extracted into the solution. Since acid solutions can destroy chromatographic columns the acid can be evaporated and replaced by a suitable solution such as DMSO.
Reactive dyes are not able to be extracted from the fibre due to the covalent bond between the dye and the fibre [26]. However the fibre itself can be destroyed using a cellulase solution which digest the polymer bonds of the fibre. In order to achieve this the cotton fibre needs to be swollen using a strong alkali like sodium hydroxide before adding the cellulase solution. The resultant
12 molecule is the dye molecule with several glucose molecules from the cellulose attached to the covalent bond places. In practice this will mean that several glucose molecules will be present representing two are three molecules per covalent bond [2, 26]. The mixture thus often has 3 to 4 dye molecules having a difference of 1 or 2 glucose molecules from the other.
Though some of the described extraction procedures can be relatively difficult these are well developed and validated for chromatographic analysis. This is not the case for the vat and sulphur dyes which do not only represent difficulty in dyeing but also in extraction [2]. As described in section 2.2.1 the vat dyes are reduced to an leuco form using a strong reducing agent. This makes the affinity for the cotton fibre quite high so in order to extract the dye out of the fibre a solution is needed with more affinity for the dye molecule than the fibre. Traditionally this has been polyvinylpyrrolidone (PVP) which is a polymeric substance. PVP is unsuitable for
chromatographic analysis since the polymer will cloth the column [2, 24, 25]. Due to auto oxidation by air the fibre reduces back to the non-polar compound which expresses also a major problem. The non-polar molecules can have such a high affinity to the non-polar column that no elution will take place. Either a polar column can be used but this will present difficulties with the separation of other dye classes requesting different methods based on the dye [24]. Or a derivatisation of the dye molecule should take place leaving few options. The only constant functional group of the vat dyes are the ketones [9, 10]. In general this are difficult groups for reactions since these are not very reactive. However a method using the Lawesson reagent is presented, converting the ketone into an sulphide bond (see reaction 2) [27]. The sulphide bond is further converted to an sulfonate group using Oxone (see reaction 3). This should make the molecule more water soluble and make it possible for extraction of the dye using a solvent like DMSO. Methods using semicarbizide [28] and hydroxylammonium chloride [29] to make the dye molecules more soluble in polar solvents have also been tried.
Laweson reagent
(2)
Oxone
(3)
For sulphur dyes the same method of reduction is used as for vat dyes, however instead of hydroxyl group a thiol appears [2, 24, 25]. This request a somewhat different approach. In the literature it is described how water soluble sulphur dyes are made using chloroacetic acid (see reaction 4) [30]. This method proved successful using a dye powder and will hence be tried using the matrix of a fibre.
13 Na2S2O4 2 ClCH2CO2Na 2 (4) 2.3.5 Liquid chromatography
In order to separate dye different dyes present in an extraction liquid, liquid chromatography is the method of choice. Liquid chromatography is a chromatic separation technique based on the affinity of compounds with a stationary and a mobile phase. Each compound will have a certain equilibrium between both phases. The amount of affinity with both phases is different for most compounds and this concept is fundamental for the application of chromatography [31]. The stationary phase, as its name suggests, is a non moving component in the chromatography
system. This phase is either polar or non polar and the length of the phase can also be varied. The mobile phase is, in liquid chromatography, a liquid migrating through the stationary phase. The compounds will thus interact with both phases but the amount of interaction determines the way the compounds migrate with the mobile phase. This migration is the retention of the compounds at a given system. The retention time or factor can give information about the structure of a compound relating to the polarity of the compound [31]. There are two main types of liquid chromatography; Thin Layer Chromatography (TLC) and High Performance Liquid
Chromatography (HPLC). TLC is a technique were the stationary phase is a thin layer plate of, most commonly, silica (a polar phase) and the mobile phase is a liquid which can be any type of polarity. The mobile phase migrates due to the capillary action and interacts with the compounds added on the plate. The height the compounds migrate with the liquid compared to the height the liquid migrates is the retention factor of the compounds. TLC is rapid technique bus does only provides qualitative information [2].
HPLC is a technique with a somewhat different mechanism. The mobile phase is also a liquid but the stationary phase is column with a fatty bonded phase of high non polarity (in most cases). Also the liquid does not migrate to a certain level but migrates through the column at a constant rate resulting in all compounds leaving the column at a certain point (elution of the compounds). This is achieved by a high pressure onto the column caused by the liquid. The liquid is processed further to an advanced detection system such as a UV-Vis detector [31] (see section 2.3.3) followed by a Mass Selective Detector (MSD, commonly Mass Spectrometer, MS). This detection system makes it possible to accurately and quantitatively register a signal from the eluting compounds at the time the compounds are eluting [2, 32-35].
2.3.6 Mass spectrometry
For the identification of dyes several methods can be applied, however in combination of HPLC, mass spectrometry (MS) is the most promising technique for qualitative identification. MS is the analysis of ionisable compounds based on their mass (m) divided by the charge of the molecules (z) expressed as m/z. This makes atomic composition possible which often relates to certain identification of the analysed compound [36].
In order to obtain a mass spectrum an ionisation source is needed to created ionised compounds. The ions need to be separated according to their m/z values using a mass analyser and the ions need to be detected using a detection system [36].
The used ionisation source is an electron spray ionisation (ESI) source which is connected to the HPLC system. It sprays the mobile phase, and the compounds present in it, out a capillary into an aerosol while evaporating the mobile phase. To the tip of the capillary a high voltage is applied
14 giving a better dispersion of the aerosol and charging the droplets. A flow of hot nitrogen assists in evaporation of the mobile phase and helps direct the charged compounds direct towards the analyser. Before entering the analyser the analytes enter a heated capillary to complete
dissolvation of the analytes [36].
The analyser used is an Orbitrap mass analyser which is a design by Makarov supported by Thermo Fisher Scientific [37, 38]. This analyser is unique in its design as an ion trapping device and has high resolution mass spec capabilities. A schematic representation of the Orbitrap is shown in figure 15. Starting from the ESI source, ions are focused into the analyser with focussing beams and forwarded through a quadrupole which can remove ions if needed. Then there is a linear ion trap (LIT) in which ions can be stored before injection into to Orbitrap [36-28]. The LIT can also inject ions in a separate detector giving both low and high resolution mass spectra simultaneously. In the C-trap ions can be directed towards the collision cell before entering the Orbitrap to give MS² fragments. When entering the Orbitrap (collided or not) the ions will harmonise around the Orbitrap [38]. During this harmonisation ions get separated based on their m/z values and these values get recorded using an Fourier Transform algorithm allowing for fast recording of a wide range of m/z values. Detailed reviews of the Orbitrap mass
spectrometer can be found elsewhere [37, 38 ].
Figure 15: Schematic overview of the Orbitrap mass analyser [39].
The MS analysis of dyes follows several trends. Firstly sulfonate groups can be easily ionisated due to loss of a hydrogen atom. This can give several negative charges, dependant on the number of sulfonate groups, but is mostly limited to a single or double negative charge. This is often the case for acid, reactive and direct dyes. Due to the high resolution of the Orbitrap small m/z differences can be measured making it possible to analyse carbon thirteen isotopes, indicating the number of charges on the molecule. Furthermore several adduct ions can be formed such as acetate (M+60), sodium (M+23) and water (M+18). This is especially the case for acid dyes and to a lesser extend for disperse dyes. Disperse dyes have mainly the loss of one hydrogen atom but can also form fragments due to loss of ethyl groups on a tertiary amine. Reactive dyes have, as described, several glucose molecules attached to the dye molecule. This can result in high masses, but the measured values are lower due to double or triple negative charges.
Different from the other molecules are metal complex dyes and basic these. Due to the negative charge in the metal complex and the positively charge in the basic dye normally nothing is lost. This will result in molecular ions rather than M-H or other adduct or fragment formation.
15
3 Material and methods
3.1 Chemicals and equipment
Sodiumacetate trihydrate was obtained from ITBaker. Hydroxylammonium chloride,
semicarbazide hydrochloride, ammonium acetate, sodium hydroxide (25%), toluene (analytical grade) and dimethyl sulfoxide (DMSO) were obtained from Merck. Methanol (HPLC grade) and formic acid were obtained from Acros Organics. Nitric acid (65%, HNO3) was obtained from
Ensure and acetonitrile (ACN, HPLC grade) was obtained from Rathburn. Oxone (monopersulfate) and Lawesson reagent (97%) were obtained from Sigma Aldrich.
For dye extraction Waters glass screw neck vials were used, covered with PTFE coated silicone septa. Vials from the same type with sharp ends were used for HPLC injection.
For chromatographic separation a Finnigan Surveyor LC system was used coupled to a Finnigan Surveyor PDA Plus detector (Thermo Fisher Scientific) (200-800 nm). Full loop injections were carried out using a Finnigan Surveyor Autosampler Plus with the injection liquid being methanol. A reversed phase Grom-sil 120 ODS-5 ST column (150×2.0mm i.d., 3μm, Grace Davison
Discovery Sciences) was used with an AJO-4286 and Guard cartridge holder KJ10-4282 pre-column. The PDA detector was directly connected to a LTQ-Orbitrap (Thermo fisher Scientific) Mass Spectrometer with an ESI ionisation source. The electron spray voltage was set to 5 kV with the sheat gas being pumped at 60 arbitrary units. The capillary inlet was heated to 275°C set at a voltage of -35 kV. Full scans were operated from m/z 150 to m/z 2000 with an resolution of 1000.000 for the negative ionisation mode with 30 ms acquisition time using the FTMS analyser. From the most intense ions MS/MS scans were acquired using the collision cell. For the same m/z range the ion trap was used to analyse positive ions. For basic dyes only positive FTMS was used with the same settings as for negative FTMS analysis. All measurements were processed using Xcalibur Qual Browser by Thermo Fisher Scientific.
3.2 Dye extraction
Acid, direct and disperse dyes
For the extraction of acid, direct and disperse dyes from single fibres (<1 cm) 20 μl of DMSO was used at 100°C for 15 to 60 minutes. Time range per dye and fibre type and each 15 minutes the fibre was checked for discolouration.
Basic dyes
The extraction of basic dyes was done by using 20 μl of formic acid at 60°C for 20 minutes until the fibre discoloured. The liquid was brought into a clean vial and evaporated until dryness. The solid extract was then reconstituted in 20 μl DMSO.
Vat dyes
Vat dyes were tried to extract using different solvent systems. A 1M semicarbazide hydrochloride solution in water at low (1.6) and high (8) pH values. A 1M semicarbazide hydrochloride with 1.2 M sodium acetate in water at low (1.3) and high (12) pH values. The pH was adjusted using a 3 M NaOH solution.
Solutions of 1M hydroxylammonium choride in methanol water 1:1 with and without 1M sodium acetate were made.
16 All the above solutions were tried on small tufts of fibres for different vat dyes (see chapter 4) at 60 and 100°C. Following the reaction the tufts were placed in a clean vial and DMSO was added to see if there was increased extraction at 100°C compared to the un-derivatised dyes in fibres. A 10 mM solution of Lawesson reagent was made in DMSO together with a separate solution of 10 mM Oxone in DMSO. Tufts of fibres were first places in the Lawesson solution and kept for 3 hours at 70°C. The tufts were removed and placed in a clean vial and left to dry for 30 minutes at 70°C with no cap on top of the vial. After this an equal volume of Oxone solution was placed in the vial and kept for 3 hours at 70°C. The solution was then placed in a HPLC vial and used for injection.
Sulphur dyes
Sulphur dyes were extracted from tufts of fibres using a nitric acid solution of ranging
concentrations at 65°C. After extraction the extract was transferred into a clean vial and the acid solution was evaporated followed by reconstituted in DMSO. Separate samples were extracted using DMSO at 100°C to compare to the standard method.
Dye extraction performance was assessed using a 0 to 5 scale with 0 being no visual dye extraction and 5 being complete discolouration of the fibres [24].
3.3 Chromatographic separation
A single elution HPLC method has been developed in the past for the separation of acid, reactive, disperse, direct and basic dyes. It is the goal to achieve a soluble form of vat and sulphur dyes to fit in this method. The method exists of a gradient from 1:1 methanol – ACN with 25 mM ammonium acetate (solvent A) and 95:5 water – methanol with 10 mM ammonium acetate (solvent B). After 1 minute of eluting with 100% solvent B a 52 minute gradient to 100% solvent A was operated. This was held for 14 minutes and directly changed to 100% solvent B for
equilibrating the column for a next run. The flow was held at 0.2 ml/min with a total run time of 67 minutes.
3.4 Database comparison
For the processing of analysis data two different sets of databases are used. One set is maintained using Mass Frontier version 7.0 which can process all MS information from the measurements. This data can be exported to ToxID by Thermo Fisher Scientific which can automatically process the data files of measurements. For all Fourier Transform MS measurements a 5 ppm range was selected for detection and for all Ion Trap MS measurements 500 ppm. ToxID however does not process PDA data and for this a NIST MS database is used. From the Qual Browser, PDA spectra can automatically be searched in this database. An overview of the databases can be found in table 1.
The second database set is one programmed at the NFI, using Matlab R2011b. In this software Microsoft Excel files are imported with information from the PDA and the MS at a given
retention time. From the MS spectrum at a given retention time the 10 most abundant peaks from the Total Ion Current chromatogram are used. Comparison is based on the presence of one or several of these MS peaks in both the unknown and reference files. This is combined using the PDA spectra and the retention time.
17 For the comparison of both databases a set of 24 dyes was used to build the databases, for this disperse, acid, direct and basic dyes were used. In appendix I the complete list of dyes present in the database is shown. A range of blue, black and red dyes was chosen since these represent the majority of fibre colours encountered as forensic evidence. A set of 10 unknown dyes or dye mixtures was prepared by another analyst, removing confirmation bias. The identification of the dyes present in these unknowns was done by both databases making it possible the compare the databases.
Table 1: Overview of software used for the running and processing of the LC-MS system and data
Software Application of software
Xcalibur Software which arranges the LC-system, it's sequences and which stores data which can be read using Qual Browser.
Qual Browser Software to process analysed data, which are stored as RAW files (contains all information about PDA, RT and MS).
Mass Frontier Software used to store information related to the mass spectra of the eluted components, Mass Frontier can process RAW files with all its MS information, main database used to export to ToxID.
NIST Software mainly used for storage and read out of mass spectra however, in this case used to store and search PDA spectra directly out of the Qual browser.
ToxID Software used to search in RAW files for masses present in the database to automatically identify compounds present in the sample run. Gives a full read out of the peaks and its intensity.
Matlab DB Software to program and process data, in this case a database is designed by Dr. Ir. J. van der Weerd to store information related to mass spectra and PDA spectra at a given retention time and to search for any of these compounds after manually extraction of the data from a sample run.
18
4 Results
4.1 Vat dyes
Extraction of the Vat dyes appeared quite difficult. In table 2 the extraction scores are shown using different extraction procedures. Only 6 of the dyes were successfully extracted using DMSO (extraction higher than 4). These appeared to be dyes with either halogen atoms or ethyl groups. Also small molecules, with an indigo related structure, were extracted relatively
successful. However in many cases no extraction was successful so another approach was needed.
From the dyes that extracted, the 4 with an extraction score of 5 were analysed using the standard chromatographic method. From these only one dye eluted within the timeframe of the method, at the very end. This is shown in figure 16a on the PDA spectrum from the measurement. The structure of vat green 1 is confirmed with mass spectrum [M]- as shown in figure 16b.
Derivatisation with semicarbazide decreased the extraction efficiency. Also no colour change was observed, which would be expected when moderating the ketone group of the vat dyes.
Furthermore there were insoluble crystals formed making liquid chromatography impossible. Since it was a study to find a means of extraction of the vat dyes no further research was done into the reaction between the semicarbazide.
The reaction with hydroxyl ammonium chloride did not appear to alter the extraction of the dyes so no further research has been done in this reaction.
A selected group of dyes was extracted using the Lawesson reagent and it proved to be extracting the dyes to some extent. In any case better than the DMSO extraction. The extracts where
analysed by LC-MS and this was also done for Vat green 1 which could be analysed by the method. Additional peaks were analysed using the method but it also included the main peak which was analysed using the underivatised vat green 1. The other peaks that eluted did not appear to absorb in the visible region indicating that they lack a main chromophore. This can be due to the lack of oxygen at the ketones but the structures have not yet been identified as
19 Table 2: Extraction efficiency scores (0-5) for Vat dyes using different procedures
Extraction/derivatisation with:
Dye Molecular
formulae
DMSO Semicarbazide Hydroxyl ammonium chloride Lawesson Reagent Vat Yellow 2 C28H14N2O2S2 0 0 0 - Vat Yellow 33 C54H32N4O6 0 0 0 - Vat Orange 1 C24H10Br2O2 5 1† 5 - Vat Orange 2 C30H12Br2O2 0 0 0 - Vat Orange 9 C30H14O2 1 * 0 1* - Vat Orange 11 C42H18N2O6 0 0 0 - Vat Red 1 C18H10Cl2O2S2 5 2† 5 - Vat Red 13 C32H22N4O2 0 0 0 1 Vat Red 29 C38H22N2O6 4 0 4 - Vat Violet 1 C34H14Cl2O2 2 * 0 2* - Vat Blue 4 C28H14N2O4 0 0 0 - Vat Blue 6 C28H12Cl2N2O4 0 0 0 - Vat Blue 18 C34H13Cl3O2 5 1† 5 - Vat Blue 20 C34H16O2 0 0 0 3 Vat Blue 43 C34H15O2 4 0 4 - Vat Green 1 C36H20O4 5 1† 5 Vat Green 2 C36H18Br2O4 0 0 0 2/3 Vat Green 3 C31H15NO3 0 0 0 - Vat Green 13 C45H21ClN2O5 0 0 0 - Vat Brown 1 C42H18N2O6 0 0 0 - Vat Brown 3 C42H23N3O6 0 0 0 - Vat Black 9 C126H58N4O10 0 0 0 - Vat Black 25 C45H22N2O5 0 0 0 - Vat Black 27 C42H23N3O6 0 0 0 1 *
Extraction solution appears to be saturated with the dye molecules
† Insoluble dye crystals have formed
20 a)
b)
Figure 16: (a) PDA spectrum of the chromatographic run with Vat Green 1 after DMSO
21
4.2 Sulphur dyes
The extraction of sulphur dyes showed some different trends than for vat dyes. In table 3 the extraction scores are shown using different extraction procedures. The extraction with DMSO resulted in decolourisation of the fibres but not of colourisation of the DMSO self. This can either mean that the dye decolourises but remains in the fibre our gets extracted out of the fibre without being coloured. Hence the chromatographic method was applied to the extract but no peaks eluted within the given retention range of the method. Temporary adaptations to the method, using only the organic solvent, did not result in dye peaks eluting. Hence it is unknown if any extracts were present. Air oxidation did not return the dye colour to the fibre however. Extraction with nitric acid did completely decolourised the fibre and turned the solution into a brownish colour. This would indicate that some form of extraction took place. After evaporation of the extraction liquid the residue dissolved in DMSO. The nitric acid concentration was not very crucial, hence the lowest concentration possible was chosen (0.1 M) to eliminate dissolving of the cotton and lowering the pH if some acid residue remained. But just as with DMSO, no dye peaks eluted, even when the same adaptations were made as for DMSO. This would indicate either that the dye is still too non-polar or that the dye molecules precipitated in the eluent giving it a lack of retention in the column.
Table 3: Extraction efficiency scores (0-5) for sulphur dyes using different procedures Extraction/derivatisation with:
Dye DMSO HNO3
Sulphur Yellow 1 2 5 Sulphur Orange 1 1 5 Sulphur Brown 10 3 5 Sulphur Brown 15:1 0 5 Sulphur Brown 14:1 0 5 Sulphur Brown 12 1 5 Sulphur Blue 11 2 5 Sluphur Black 1 2 5
Solubilized Sulphur Blue 11 4 5 Solubilized Sulphur Black 1 3 5
4.3 Database comparison
For the comparison of the two databases (Matlab DataBase (DB) and ToxID) 10 samples were analysed with a total of 18 dyes. Since Disperse Black 056 is most likely not present in the
extract a total of 17 dyes is actually present. Basic Red 018 is not present in the database files and can hence not be identified as such. An overview of the results is shown in table 4. The database output results can be seen in appendix II.
For the Matlab DB a total 4 dyes are not identified and in one of these cases a false positive is given. Here Basic Blue 022 is given instead of Basic Red 029 (sample 5). Even when combining the PDA spectrum. Sample 1 has only the first blue compound recognised, but not the Basic Blue 022. In one sample (sample 8), only one PDA peak is detected making it impossible to extract information from the second compound manually. Hence Acid Red 111 is not detected using the
22 Matlab DB. Also in sample 8 Basic Red 014 is not identified, this is wrongly identified as
Disperse Red 152. In total 4 of the 17 (24%) dyes are incorrectly identified or not identified at all using the Matlab DB.
For the ToxID database a total of 1 dye is not identified but no false positive is given in this case. This is the case for sample 6 in which Direct Black 080 is not identified. It is uncertain why this is the case and it could not be resolved by manual data analysis at this point. In total 1 of the 17 (6%) dyes is not identified using the ToxID DB.
Table 4: Results of the database comparison compared to the true values Sample Dyes present in the
unknown samples
Remarks Identified with
Matlab DB
Identified with ToxID
Sample 1 Basic Blue 003 Two fibres in vial Yes Yes
Basic Blue 022 No‡ Yes
Sample 2 Basic Red 018 Dye not in database! No‡ No‡
Sample 3 Disperse Blue 073 Normal length Yes Yes
Sample 4 Basic Red 014 Short fibre Yes Yes
Sample 5 Basic Red 029 Extraction liquids mixed No† Yes
Direct Black 056 No+ No+
Disperse Blue 073 Yes Yes
Sample 6 Direct Blue 071 Three fibres in vial Yes Yes
Direct Red 023 Yes Yes
Direct Black 080 Yes No‡
Sample 7 Acid Blue 025 Normal length Yes Yes
Sample 8 Basic Red 014 Extraction liquids mixed No‡ Yes
Acid Red 111 No* Yes
Sample 9 Disperse Red 073 Extra short Yes Yes
Sample 10 Direct Red 079 Extraction liquids mixed Yes Yes
Acid Red 111 Yes Yes
Disperse Red 054 Yes Yes
‡No False Positive †False Positive given +
Dye most likely not extracted and thus not present
*
No PDA signal so no visual detection
In order to state whether or not these differences are significant the paired t-test is used: t = dmean×√n/sd [40]
In which dmean is the mean difference when subtracting all values of ToxID from Matlab DB and
sd is the standard deviation of the subtracted values.
The values are obtained by replacing a positive identification (yes) for the value one and negative identification (no) for the value zero. For the false positive the value minus one is used. A
significant difference is obtained when the calculated value for t lays above the value in the t table for n-1. Using this test no true significant differences are calculated, even when removing sample 2 and Direct Black 056 from sample 5. However it is uncertain how to deal with the false positive and if this test certainly is fit for this purpose.
23
5 Discussion
The extraction of vat dyes using DMSO showed in only limited cases successful, the only dye molecules which extracted where small, relatively polar molecules. This is in line with the extraction of disperse dyes. These molecules are small and are more polar than vat dyes. In limited cases the extraction of vat dyes was successful as well with the classic pyridine : water solvent used [24]. The chromatographic separation of these dyes also demonstrate the need for a more polar form since only one of the DMSO extracted dyes eluted within the timeframe of 66 minutes using a non polar column.
Also the ionisation of the dye is an important feature when using mass spectrometry. It was shown, using vat green one, that the molecule did ionise but the signal was relatively low
compared to the background ions. This means that derivatisation does not only needs to make the molecule more polar, but also increase the ionisability of the molecule. Fortunately this is mostly achieved when introducing polar groups to a compound.
Since using the vatting process the dyes auto oxidize back to the non polar ketone form (reaction 1), after reduction, a more stable derivatisation was needed. This derivatisation could only be achieved the ketone functional group, which is a very difficult group to react with. The last step in organic synthesis of many oxygen containing compounds is the formation of a ketone. This makes the step of derivatisation quite difficult. Imine formation has been suggested using semicarbazide [41] to build a more polar compound as shown in reaction 5. With this reaction however insoluble dye crystals formed. This can be true since it is often used for melting point determination of ketones [27] but a higher melting point often means higher polarity when dealing with organic compounds. Since no success was achieved no further structure formation was studied because that was beyond the scope of this research. It can be that the structures are too large form polar crystals but so far no satisfactory explanation has been found for this.
(5)
Another imine formation was suggested by Morgan et al [29] using hydroxyl ammonium chloride as shown in reaction 6. As in line with their findings no increased extraction was found to occur with all studied vat dyes. Even when PVP was used to bind the dye to the polymer only limited success was achieved in their study. However using PVP, chromatography will be out of the question making it an unwanted procedure. It might be that complete reduction of the ketone makes it only polar enough for extraction out of the fibre using a medium with more affinity for the dye than the fibre itself.
24 (6)
The use of the Lawesson reagent has just very recently been applied for the vat dyes at the NFI, this means that the reaction is not fully studied yet. Because of this, not all structural information is derived at this point making a complete study and result for this reaction difficult. The
improved reaction however showed support for the use of this reagent since no extraction for these dyes appeared in any other way so far. The lack of complete discolouration can be due the fact that the concentration of ketones is higher than the concentration of the Lawesson reagent. This requests a higher concentration of the Lawesson reagent to derivatise all ketones. Another explanation could be that the matrix (a cotton fibre) makes it difficult to extract all the dye from the fibre. This might be overcome by, for example, swelling the fibre as is done for the cellulase extraction method for reactive dyes on cotton, using aqueous sodium hydroxide [26]. Apart from these factors, the temperature can be of influence and the reaction temperature. In order to demonstrate the reaction the other eluting peaks should be investigated using the mass spectral information to find more information about the reaction.
The extraction of sulphur dyes using DMSO clearly discoloured parts of the fibres but the solvent did not took up the colour. Since no peaks were found using the LC-MS system it might be that the dye is insoluble in the eluent and has no retention time or that nothing was present in the DMSO. This most likely means that DMSO has influence on the colour of the sulphur dyes. However no known mechanisms involving the breaking or influencing of the disulphide bond using DMSO are known. This means that it might influence the chromophores of the dyes which makes it difficult to predict a reaction with the DMSO. The fibres and the liquid was exposed to air, which normally oxidises reduced dyes, to see whether or not the colour returned to its original form.
The reaction of HNO3 at the boil as shown in reaction 7 would make the sulphur bonds of sulphur
dyes more stable and polar. This reaction shown complete extraction with colouration of the solvent which would indicate a reaction of some sort. Chromatographic elution did no shown any dye peaks eluting in the time frame of several separation methods. Which makes it impossible to determine any form of reaction. Even when dye peaks eluted, monitoring of the reaction would, of course, be impossible to lack of structural knowledge of sulphur dyes, but would at least indicate a polar component from the dye. Another problem with this reaction is that a thiol is needed before the reaction will take place. In normal situations for sulphur dyes a disulphide bond is present and would need reduction for a thiol to be present. It is unsure if the reaction did occur with the disulphide bond present, indicating that nitric acid is strong enough for this, or that a completely different reaction did occur. First reducing the dye would make it unsafe since the reducing agent must present when introducing the acid to prevent oxidation by air.
It is however though that the dye molecules are insoluble with the mobile phases giving them lack of retention time and possible making them unionisable with the ESI interface. Another downside of this method is that it degrades the cotton polymer to some extend giving rise to
25 macromolecules being introduced into the column. This can pollute the column and the mass spectrometer making the method unfavourable in any way.
HNO3 (boil)
(7)
The method described by Wang et al [30] theoretically seems very promising in the breaking of the disulphide bond followed by derivatisation of the thiol (reaction 4). However this method is not yet tested for dyes in fibres. Some difficulties in their protocol apply for the dyes in the fibres. They titrate the reducing agent in parts to their sample whilst for single fibre dye analysis this is on a too small scale to carry this out. Also a one pot derivatisation is most favourable for this situation. In the future it will be tested if a mixture of the chloroacetic acid and reducing agent a high enough extraction for dye analysis from single fibres.
When successful dye extraction is obtained for both vat and sulphur dye classes, the analytical challenge just starts. In order to validate the extraction process using analytical parameters some aspects need to be taken into consideration. The limit of detection after extraction must be quite low. Since in most fibre cases only limited amount of fibres is available, the limit of detection must be below 10 mm of fibre. Preferably below 1 mm since in many cases 10 mm is actually quite long. This must be both for the PDA spectrum and for the MS signal in order to have successful identification. The repeatability of the method must be demonstrated using the same analytical conditions over different periods of time. This should be done using a standard mixture with compounds eluting at a retention time within 2,5% of the mean retention time. This must be accompanied with the right PDA and MS results. The robustness of the method should be
demonstrated for both the extraction and the sample run. This by using different prepared
extraction solvents and eluents comprising for small differences in concentrations and pH values. The method should show a robust limit of detection and repeatability under these circumstances to make it a robust method. Only than the extraction has shown to be successive.
The comparison of the two databases has shown some good trends, both databases came up with right identities for most dyes with only one false positive (type 2 error) [40]. However trends did show that the Matlab database performed less ideal than the ToxID database. This will most likely be due to the fact that the mass spectra is reduced to the ten most abundant peaks which will not always be the most important set of peaks. This is especially true for small lengths of fibres and dyes with low ionisabillity. This makes the Matlab database weak for analyses were the background ions are the most abundant. This is also the cause of the false positive in the dataset, in which the mass spectra was very similar based on the most intense peaks. This is really a downside of the database. The ToxID in contrast searches in the measurement files for exact masses which are in the database. This proved much stronger since it is automatic and it can also find peaks which are only visible in the mass spectrum and not in the PDA spectrum. The
maintaining of the ToxID database is more time consuming and the PDA database is not connected to it but this did not appear to be a big problem.
26 The paired t-test used for the comparison of the results obtained with the dataset proved however that no significant difference is between the databases. This might be true using the test in this way but it might not provide complete information. For example the false positive is given a score -1 which is based on it being at the other side of a true positive. The strength of the false positive might not be measured in such a way. Furthermore the test is normally designed to calculate the difference of analytical data such as concentrations. The values might be too strong to account for an accurate comparison. However this is the only test that compares in such a way the data obtained using the results. The contrast from 6% for the ToxID database against the 24% of the Matlab database is still quite high. It might be that additional comparison samples might be needed or that a bigger database set might influence the results. The results favour the ToxID database when no adaptations are made for the Matlab database.
27
6 Conclusion
The extraction of vat dyes using DMSO, semicarbazide and hydroxylammonium chloride did not subsequent aid in the extraction of most vat dyes and the analysis using the standard LC method. The lawesson reagent however proved to increase the extraction of the vat dyes making this a possible reagent for the extraction and analysis of vat dyes. No ideal reaction conditions have been found so far.
Extraction of sulphur dyes using DMSO and nitric acid did extract or decolourise some or all of the dye stuff from the fibre. However both with DMSO extraction and nitric acid extraction no suitable chromatographic separation and dye identification could be done. Because of this, alternative reaction should be investigated.
Based on the comparison of the ToxID database and the Matlab database it appeared that the ToxID database proved to be more successful in identification of fibre dyes. Both the automatic datafile readout for the dyes and the clear report proved to be more satisfactory for the ToxID database. Also the lack of false positives and the lower number of unidentified dyes makes this database the better database. Though no significant differences were calculated using the paired t-test, it will be chosen to use the ToxID database for future dye identification.
7 Recommendations
It is recommended to optimise the conditions for the Lawesson reagent and to further explore the reaction with mass spectrometry. This can be achieved by different extraction times and/or temperatures and using different Lawesson concentrations. If the extraction is optimised the analysis should be validated for the limit of detection, repeatability and robustness.
For the sulphur dyes it is necessary to develop an extraction method using chloroacetic acid to make the dye suitable for chromatographic analysis. To accomplish this, several concentrations of reducing agent, added to the acid, should be tried and different reaction temperatures and/or times should be tested. If this extraction method is optimised the same validation should be carried out as for vat dyes.
