Forensic analysis of dyed textile fibers

Transcription

1 Anal Bioanal Chem (2009) 394: DOI /s REVIEW Forensic analysis of dyed textile fibers John V. Goodpaster Elisa A. Liszewski Received: 17 April 2009 /Revised: 28 May 2009 /Accepted: 29 May 2009 /Published online: 20 June 2009 # Springer-Verlag 2009 Abstract Textile fibers are a key form of trace evidence, and the ability to reliably associate or discriminate them is crucial for forensic scientists worldwide. While microscopic and instrumental analysis can be used to determine the composition of the fiber itself, additional specificity is gained by examining fiber color. This is particularly important when the bulk composition of the fiber is relatively uninformative, as it is with cotton, wool, or other natural fibers. Such analyses pose several problems, including extremely small sample sizes, the desire for nondestructive techniques, and the vast complexity of modern dye compositions. This review will focus on more recent methods for comparing fiber color by using chromatography, spectroscopy, and mass spectrometry. The increasing use of multivariate statistics and other data analysis techniques for the differentiation of spectra from dyed fibers will also be discussed. Keywords Forensic science. Textile fibers. Fiber dyes. Trace evidence The forensic significance of textile fibers Trace evidence is a generic term that can be broadly defined as small, often microscopic fragments of various types of material that transfer between people, places and objects, and persist there for a time [1]. Given this definition, trace evidence can consist of a vast array of materials such as J. V. Goodpaster (*) : E. A. Liszewski Department of Chemistry and Chemical Biology, Forensic and Investigative Sciences Program, Indiana University Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA jvgoodpa@iupui.edu hair, fibers, paint, soil, glass, pollen, and dust. In the context of a criminal investigation, trace evidence is inherently associative. Its forensic significance stems from the fact that transfer of trace material can link suspects, victims, and crime scenes by implying contact between two individuals or between individuals and objects. Textile fibers are arguably one of the most important forms of trace evidence given that they have many classifications and subtypes, are physically and chemically differentiable, have various processing procedures, and are transferred easily. Various characteristics are evident in fibers, and certain ones play significant roles in fiber analysis. One of the most important characteristics for fiber comparisons is color, which reflects the dyes and pigments that were used on the fabric. In fact, the only characteristic of many fibers, such as cotton, that can be reliably used for discrimination of samples is its color as generated by a fiber dye. Perhaps the most important treatise in this area is Forensic Examination of Fibres, edited by James Robertson and Michael Grieve, the second edition of which was published in 1999 [2]. Forensic fiber examiners have also established several working groups such as the European Fibres Group (EFG), which is a part of the European Network of Forensic Science Institutes (ENFSI). The EFG published the Manual of Best Practice for the Forensic Examination of Fibres in 2001, and Wiggins has summarized this and other efforts of the EFG during its first ten years ( ) [3]. In the USA, the Scientific Working Group for Materials Analysis (SWGMAT) published the Forensic Fiber Examination Guidelines in 1999 [4]. Following review articles by Wong in 1989 [5], Rendle and Wiggins in 1995 [6], and Grieve and Wiggins in 2001 [7], there do not appear to be any recent reviews of the literature concerning the current analytical methods that may be applied to dyed fibers. Hence, it is the goal of this

2 2010 J.V. Goodpaster, E.A. Liszewski article to summarize recent developments in this area, with a particular focus on those that have been reported since the publication of Forensic Examination of Fibres in Fibers and fiber dyes Textile fibers are the basic unit of matter that form the components of fabrics and textiles. Although a multitude of classifications and subtypes exist, fibers can be broadly classified as either natural or man-made [8]. Natural fibers are further subdivided according to their source (animal, vegetable, or mineral). Man-made fibers are subdivided according to their base material (synthetic polymer, natural polymer, or other). There are over 1,500 manufactured fiber plants worldwide, according to 2007 data [9]. A majority of these plants are synthetic fiber producers, followed by cellulosic and glass fiber manufacturers. Regionally, Asia has the most manufactured fiber plants. Within the USA, 145 fiber manufacturing plants exist, the vast majority of which are synthetic manufacturing plants. Fibers can be a part of primary (direct) or secondary (indirect) transfers [10, 11]. Forensic science laboratories can then examine these transferred fibers and compare them to a known fiber in order to discover possible common origins. However, a significant factor in fiber examination is the passage of time. For example, studies of fiber persistence have demonstrated that up to 80% of transferred fibers are lost after the first 4 h [10]. Therefore, it is important for investigators to collect garments and any possible fibers quickly. Factors that can influence how fibers adhere to materials include the type of fiber transferred, the type of receiving material, and the extent to which the receiving material is used after transfer [10]. Dyeing is the process of imparting color to a textile material by interaction with a dye. A dye is a colored substance that is able to absorb and reflect certain visible wavelengths of light. Dyes must have affinity for the substrate on which they are being applied. In contrast, a pigment has no affinity for the substrate and is instead incorporated into the fiber during production or bonded to the surface. An important feature of fiber dyes is that very few textiles are colored with only one dye, and therefore many dye combinations exist. Furthermore, thousands of dyes are produced worldwide. Important reference works such as the Colour Index published by the American Association of Textile Chemists and Colorists allow forensic chemists to keep abreast of the chemical dyes that are available. A three-color dye system is what is most frequently used to give a textile its color, and how color is applied and absorbed on the fiber is an important comparison. Fiber dyes are classified in various ways, including their method of application, chemical class, or the type of fiber to which they are applied. The main classification scheme typically used by forensic chemists is based on the method of dye application. The major dye classes in this scheme are: acid dyes, basic dyes, azoic dyes, direct dyes, disperse dyes, metallized dyes, reactive dyes, sulfur dyes, and vat dyes. While readers should consult Wiggins [12] for a more detailed discussion of fiber dyes, the characteristics of the major dye classes are summarized in Table 1 and can be described as follows: Acid dyes are applied under acidic conditions so that basic functional groups (e.g., amino) on the substrate are protonated and positively charged. These groups form ionic bonds with deprotonated functional groups (e.g., sulfonate) on the dye molecule. Basic dyes are also applied under acidic conditions, but in this case protonated/positively charged functional groups (e.g., ammonium) on the dye form ionic bonds with negatively charged functional groups on the substrate. Table 1 Summary of dye classes and the typical fiber types to which they are applied [12] Dye class Description Typical fiber substrates Acid Water-soluble anionic compounds; ionic bond between dye molecule and polymer Wool, silk, polyamide, polypropylene Basic Water-soluble, applied in weakly acidic dye baths; very bright dyes; negatively charged fiber draws the dye cation Polyacrylonitrile, acrylic, occasionally polyester and polypropylene Direct Water-soluble, anionic compounds; applied directly to fiber from aqueous Cotton, rayon, other cellulosics medium that has an electrolyte; positively charged ion is attracted to negatively charged fiber surface and the dye is able to enter the fiber Disperse Not water-soluble; aqueous dispersion; hydrogen bonds and weak van der Waals Polyester, acetate forces hold dye molecule in fiber Reactive Water-soluble; form covalent bond with functional groups of fiber; similar in Cotton, wool, other celluolosics structure to acid dyes and application similar to direct dyes Sulfur Organic compounds containing sulfur or sodium sulfide; reduced using sodium Cellulosics sulfide or sodium hydrosulfite; dye enters fiber and is oxidized to original form Vat Oldest dyes; more chemically complex; water-insoluble; good colorfastness Cellulosics

3 Forensic analysis of dyed textile fibers 2011 Azoic dyes form a colored product via coupling between a diazo salt and a coupling component such as a naphthol. Direct dyes are directly incorporated into cellulosic fibers in the presence of heat and an electrolyte. Disperse dyes are also directly incorporated into synthetic fibers, associating with the substrate through weak van der Waals forces and some hydrogen bonding. Metallized dyes form colored metal complexes within the fiber through the reaction of a mordant (binding agent) such as chrome with a separate dye molecule. Reactive dyes form covalent bonds with functional groups on the fiber. These dyes are growing in popularity in part because they are less likely to be removed by washing. Sulfur dyes require a reducing agent to make them soluble, where it is applied to the fiber, then oxidized within the fiber back to its original insoluble form. Vat dyes also require a reducing agent to make them soluble and undergo oxidation within the fiber to create an insoluble dye. Analysis of dyed textile fibers The primary methods for the identification and comparison of textile fibers according to their type (e.g., synthetic polymer) and subtype (e.g., nylon) rely upon the principles of microscopy, spectroscopy, chromatography, and mass spectrometry. Microscopy has and will remain an important aspect of fiber examination [13]. For example, a stereomicroscope can record fiber characteristics such as size, crimp, color, and luster. Polarized light microscopy (PLM) is especially helpful with manufactured and synthetic dyed fibers because it reveals the polymer class based on the rotation of incident polarized light by the fiber. Infrared microspectroscopy is widely used in forensic laboratories to identify and compare single fibers [14]. Specific spectral features are used to discriminate between classes and subclasses of fiber. Given the polymeric nature of synthetic and natural fibers, pyrolysis coupled to gas chromatography (as well as mass spectrometry) is an informative and minimally destructive technique given its small sample sizes [15]. Finally, scanning electron microscopy coupled with energy dispersive spectrometry (SEM-EDS) can be used primarily to examine the elemental content of fibers, with inorganic materials arising from the residues of the manufacturing process, additives, or environmental contaminants [16]. Approaches for comparing fiber color and identifying fiber dyes have been developed alongside those for identifying fiber materials. Analytical techniques that have been applied to the analysis of dyed fibers and will be discussed in this review are shown in Fig. 1. Older methods of comparing fiber color involved comparing unknown shades to known hues and finding the best color match. Now, however, more reliable techniques are used to compare colors and dyes. Microscopic exams remain a key tool for color comparisons [13]. For example, simple observation of fiber color under the microscope can immediately eliminate two fibers as having come from the same source based on clear visible differences in hue. Polarized light microscopy can be used to detect dichroism (dependence of the observed color on the direction of the plane polarized light) in cases where the dye molecules are linear (e.g., Congo Red) and oriented along the fiber axis. Finally, the fluorescence of the dyes or optical brighteners added to fibers can be used as a distinguishing characteristic. For example, target fiber studies using cinema and car seats have shown that the number of apparent matches with green cotton target fibers using white light microscopy was reduced greatly by using fluorescence microscopy [17]. Beyond microscopy, however, are several techniques that have found broad usage and remain active areas of research. In some cases, extracting the dye from the fiber is Fig. 1 Analytical techniques that have been applied to the analysis of dyed fibers (key references are given in parenthesis)

4 2012 J.V. Goodpaster, E.A. Liszewski crucial to the success of these techniques. The best known methods for dye identification are based on determining the application class, which is the type of dye that was used (acid, direct, etc.) and the generic structure. The current status of these methods will be discussed below in the approximate order in which they would be applied to an unknown sample. UV visible microspectrophotometry (MSP) UV visible microspectrophotometry uses a microscope to gather light from the sample and transmit it to a UV visible spectrometer. This technique is ideal for objective observations of the color of many forms of trace evidence [18], and the application of MSP to fiber analysis has been discussed in detail [19]. The use of an instrumental technique such as MSP is significant because it is repeatable, nondestructive, and requires little sample preparation, unlike other methods that require extraction of the dye. MSP can distinguish between colored fibers that may appear visually similar because absorbance in the visible region is dependent on the molecular structure of the chromophore as well as the environment in which the chromophore is found. It is important to note that MSP is not capable of identifying particular dyes or mixtures of dyes, but rather identifying the spectral characteristics of a sample for the purposes of comparison. Typical MSP protocols call for several fibers to be examined per item (e.g., five for man-made fibers and ten for natural fibers). Upon examination using MSP, manmade fibers are usually found to have a homogenous chemical composition as the dye is bonded to a relatively constant chemical environment. Cotton and other natural fibers are composed of many different chemical components which are not homogeneously distributed throughout the fiber [19]. Hence, variation within a control fiber sample may occur due to uneven dye uptake. Other sources of variation are the presence of dye precursors, faulty dye bath cleaning, or by changing the dye bath conditions. The implications of these effects on spectra have been demonstrated by Grieve [20, 21]. In addition, fibers often fluoresce due to optical brightness or dyes applied to them. If measured in the UV region, dyes that are not colorfast may photobleach. MSP is also limited in cases where dyes have similar structures or where fibers are either very lightly or very darkly dyed [19]. Despite these limitations, MSP is currently the pivotal method in color comparisons of individual fibers. Modern UV visible NIR MSP instruments can measure transmittance, absorbance, reflectance, and fluorescence, as well as the polarization and luminescence of microscopic samples. Research into the use of MSP for the analysis of various dyed textile fibers has been ongoing for several decades with some degree of focus on natural fibers such as wool [22] and cotton [20, 21] given that color is the prime distinguishing characteristic for these fiber types. For example, MSP has been shown to discriminate between cotton fibers of similar color quite well, and the degree to which it discriminates is highest for blue followed by red cotton fibers [20, 21]. Although initial studies indicated that black fibers are difficult to discriminate (and potentially less valuable as evidence), more recent MSP measurements of numerous black cotton samples illustrated that this may not be the case, particularly when differentiating sulfur, reactive, and direct dyes [23]. Additional MSP studies have been made of other blocks of color (defined in [24] asa group of fiber samples of the same generic type having the same subjective color ). In these studies, subtypes of orange, green, blue, and red cotton fibers have been distinguished according to their spectral features, particularly in the UV range [24, 25]. The use of MSP together with thin-layer chromatography (TLC) is also a long-standing practice and subject of study [21, 26, 27]. MSP is generally held to be a complementary technique to TLC, and in some cases MSP can distinguish pairs of fibers that were not discriminated by use of TLC [19]. Recently, Wiggins compared the ability of comparison microscopy, visible-range microspectrophotometry, ultraviolet-range microspectrophotometry, and thin layer chromatography to differentiate reactive dyes in wool and cotton [28]. UV microspectrophotometry was identified as an important technique for differentiating red wool and blue cotton in particular, as well as for being nondestructive. A similar study using these techniques focused on detecting variation between dye batches, and microspectrophotometry was generally found to be insensitive to this effect [29]. Spectra obtained from MSP are often normalized and compared to each other visually. Some efforts to develop more sophisticated approaches have been reported. For example, the use of color theory (colorimetry) allows for quantitative descriptions of perceived color and its relationship to spectroscopic measurements. The application of colorimetry to forensic science is well established [18, 30] and Adolf and Dunlop present a detailed discussion of the use of colorimetry for fiber color measurements [19]. Originally, determination of color utilized visual comparison to known physical color standards; however, methods whereby instrumental methods are related to observed color are even more objective. For example, the trichromatic theory relies upon a model of color perception based on red, blue, and green receptors in the human eye. Colors can then be defined according to these three dimensions and expressed as relative amounts of each wavelength present in a given color spectrum. Therefore, transmittance or absorbance data can be converted to chromaticity coordinates (CC) or complementary

5 Forensic analysis of dyed textile fibers 2013 chromaticity coordinates (CCC), respectively [19]. The use of CCC to describe MSP results for single textile fibers has been demonstrated [20, 31, 32]. Other approaches to spectral comparison have used properties such as the difference in wavelength at corresponding peak maxima (λ max ), the sum of squares of the differences in absorbances, the sum of the absolute differences in gradient at corresponding data points, and the maximum difference between the absorbance distributions obtained by successive addition [22]. Most recently, Wiggins examined the utility of calculating the first derivative of the absorbance spectra [33]. The Savitzky Golay smoothing algorithm was used to remove noise that tends to be magnified in this approach. Overall, the first derivative provided more points of comparison when absorbance spectra were tightly grouped, broad, and lacking detail. However, differences between spectra from fibers that exhibited high intrasample variation were exaggerated, raising the possibility of false exclusions. Infrared and Raman spectroscopy While the use of infrared microscopy is quite common for determining the composition of fibers [14, 34], its use for identifying fiber dyes is not. This stems from the inherently low level of dye that is found in most textiles and the lack of sensitivity of infrared absorbance to components that represent less than approximately 5% of a sample. Although aromatic or azo functional groups can result in small but sharp peaks in the region between 1,500 and 1,600 cm 1, absorptions from the scissoring vibration of atmospheric water vapor can interfere in this region [14]. More specific contributions of known dyes to this region of the infrared spectra of acrylic fibers have been reported [35]. Although attenuated total reflectance (ATR) infrared microspectroscopy can be used to identify surface finishes [36], diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) has been more successful in characterizing fiber dyes. DRIFTS has the advantage of not following Beer s law, so reflectance measurements are more sensitive than transmission at low concentrations [37]. Both dye color and reactive dye state were discriminated on cotton [38, 39] by using DRIFTS. In these studies, the spectra were analyzed using chemometric methods, the applications of which will be discussed in more detail below. A thermospray interface for high-performance liquid chromatography (HPLC) that allows analysis of the dyes present in the effluent via DRIFTS has also been developed [40]. Raman spectroscopy has steadily become established as a technique for fiber analysis [14, 41] and it has been identified as a priority research area by the EFG [3]. While techniques such as MSP provide nondestructive ways to measure color, little information is gained about the specific dye. Raman is well suited for the analysis of colored fibers because this technique requires no sample preparation, is nondestructive, and requires only a small sample. Therefore, multiple tests can be performed on the same fiber. Raman microprobe spectroscopy has also been used to characterize dyes in both natural and synthetic fibers [42 44]. Both the dye state and dye concentrations could be discerned on cotton fabric based on the Raman spectra and multivariate statistical analysis. For synthetic fibers, it was determined that fibers of the same polymer type from different manufacturers have Raman spectra which are slightly different but could be distinguished by multivariate statistical techniques. In both cases, the Raman spectra of dyed fibers could be obtained and exhibit bands from both dye and fiber. The optimization of Raman for fiber analysis is ongoing. Studies of the effect of varying laser wavelength showed that excitation in the near-ir (785 and 830 nm) provided the best results in terms of spectral quality, sample degradation, and speed of acquisition [45]. Subsequently, a large-scale collaborative study has been carried out by the EFG using spectrometers from six different manufacturers with nine different laser sources [46]. Nine dyes and three test fibers (two red acrylic fibers and one red wool fiber) contained three different but known dyes. The results showed that the Raman spectra of identical dyes measured under the same excitation wavelength were consistent regardless of the instrument used. The excitation wavelength was a significant factor in the quality of the spectra obtained, with red lasers (633 and 685 nm) yielding the poorest spectra and blue (488 nm) and green (532 nm) lasers yielding the best quality spectra. However, the Raman spectra of identical dyes measured using different excitation wavelengths were noticeably different. The success of Raman for determining fiber dyes is due in large part to the high Raman cross section of dye molecules. Extraction of the dye and then spectral subtraction allows for the separate spectra of the polymer and the dye to be obtained. Therefore, fibers of similar color but different dyes will have different spectra and can be easily distinguished. While Raman spectroscopy can identify the main dye present in a fiber sample, the spectrum obtained must be of good quality (no fluorescence) and that the corresponding dye spectra must be available. Minor dyes, however, are difficult to detect. Fluorescence can be reduced, or removed, by mathematical postprocessing of the data, changing the laser wavelength, or using techniques (surface enhanced resonance Raman scattering or SERRS), which increase the signal and reduce the fluorescence. Thin-layer chromatography (TLC) Thin-layer chromatography is a long-standing tool for the separation of common classes of fiber dyes, with silica gel

6 2014 J.V. Goodpaster, E.A. Liszewski being the most typically used stationary phase [12]. Selecting an appropriate extraction and developing solvent can be difficult, however, and numerous solvent mixtures have been tabulated for these purposes [12]. Specific schemes for dye extraction and TLC have been published for dyes on cotton [47, 48], wool [27, 49 51], and various synthetics [47, 52, 53]. TLC has the advantage of being able to identify small amounts of shading colors or intermediates that can often be found in extracted dye mixtures. Most textile fibers contain multiple dyes to obtain the desired shade, so TLC of a few fibers may produce very complex dye patterns. However, no single solvent system exists that can separate all classes of dyes, so some amount of prescreening is necessary. The discriminating power of TLC is increased by running dyes on two different, noncorrelated solvent systems. Using multiple solvents maximizes the quantity of information and increases the significance of data that can be obtained. For example, multiple solvent systems have been used to separate dyes from several classes (disperse, acid, basic, reactive). The correlation between the solvent systems could be determined, and thus the selection of the most appropriate pair of systems for a particular dye class could be chosen [47, 53]. In this approach, linear correlation coefficients and critical values were calculated for the combinations of solvent systems. Scatter diagrams were created of R f values in one system against R f values in the second system in order to assist in detection of nonlinear correlation. Correlation coefficients close to + 1 or 1 were considered high and correlation coefficients near zero were considered low. Standard deviations, linear correlation data, and scatter diagrams were able to illustrate that the paired systems to be selected were those with a combination of large distribution of R f values and low correlation. TLC has been used with other techniques, such as microscopy and MSP, to compare fiber dye batches [29, 51]. Results have shown that variation was detected in more fiber types than not. Differences between dye batches can arise due to alterations in the dye components by the dyer due to cost or effectiveness, supply difficulties resulting in the need to change dyes, and dyeing over pale shades that were otherwise going to be waste. Overall, these studies claim that TLC may illustrate differences in dye batches that no other technique can. High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) As discussed by Griffin and Spears, HPLC has many advantages over TLC and MSP for the analysis of fiber dyes [54]. For example, MSP is limited when attempting to analyze highly absorbing (dark color) fibers. Furthermore, TLC requires relatively large quantities of dye and different eluent systems for various dye classes. HPLC, however, exhibits better chromatographic resolution, greater sensitivity, and it can be used for quantitation. There are several practical considerations when developing an HPLC system for fiber dye analysis, such as the general chemical nature of the dye (acidic, basic, or neutral), possible extraction solvents, and possible degradation of the sample. For example, organic solvents used in the extraction of the fiber dye can either negatively impact the separation or present interferences to a UV visible detector [54]. Some dye classes are also known to degrade, so the use of antioxidants, prompt analysis, and/or low extraction temperatures are necessary in these cases [54]. Reversed-phase HPLC has been used to separate anionic, cationic, nonionic, and ionic dyes, although anionic dyes have met with more success than cationic dyes. Basic dyes can be separated by employing a system based on the ionexchange properties of silica. Taken alone, HPLC only characterizes a dye based on retention time and hence a sensitive and specific detection method must also be used. The routine use of multiwavelength detectors allows for the recording of full UV visible spectra of each dye in a mixture, determination of peak purity, and the generation of spectral databases [54]. Capillary electrophoresis and its related techniques offer even greater separation efficiency than traditional chromatographic techniques and hold great promise for analytes ranging from small ionizable compounds to large nonpolar species. While the application of CE to dye analysis in the context of monitoring the dyeing process or its environmental consequences are well known, the use of CE as an analytical tool for fiber examiners remains limited. For example, early studies of the use of CE for fiber dye analysis focused on water-soluble dyes and conventional buffer systems [55]. Attempts to develop rigorous CE methods for fiber dyes were initially foiled by lack of reproducible migration times, poor sensitivity, and an inability to separate nonionizable dyes [55]. The introduction of micelles in the buffer (micellar electrokinetic chromatography or MEKC) now allows for the separation of water-insoluble dyes. For example, natural dyes such as flavonoids and anthraquinones were extracted from contemporary and historical wool samples and analyzed using MEKC [56]. A related technique called sample-induced isotachophoresis combined with MEKC has been demonstrated. In this case, relatively large volumes of dye extracts from various natural and synthetic fibers were introduced into a MEKC system. This dramatically increased sensitivity and brought sample sizes down to the single fiber level [57]. Lastly, although the technique has not yet been applied to fibers, nonaqueous capillary electrophoresis has shown promise for hydrophobic basic dyes [58].

7 Forensic analysis of dyed textile fibers 2015 Mass spectrometry Coupling the separation efficiency of HPLC and CE with the sensitivity and specificity of mass spectrometry (MS) is a natural combination of instrumental methods. The first report in this area described the use of thermospray high-performance liquid chromatography mass spectrometry (TSP-HPLC-MS) to identify and quantify dyes in various matrices [59]. Disperse and basic dyes were extracted from single fibers in capillary tubes and the extracts injected into the instrument. The mass spectra obtained from single fibers agreed with standard dyes as well as with previously published data. Only the identification of the major dye in the extract was of interest in this case, as determining any other components would require improved HPLC separation techniques. Since this time, several authors have discussed the analysis of fiber dyes by HPLC coupled to a mass spectrometer through an electrospray ionization (ESI) source [60 63]. LC-ESI-MS with in-line UV visible detection allowed for the analysis of textile dyes extracted from samples similar in size to what is seen in forensic laboratories. The UV visible absorption detector was a good monitor for the chromatographic elution of dyes of known color. Ultimately, LC-MS with electrospray ionization can make a distinction between sets of dyes that are otherwise indistinguishable by UV visible methods and also can introduce dye molecules into a mass spectrometer with minimal fragmentation [62]. Many commercial fiber dyes have minor structural differences, so several pairs of common dyestuffs were examined by UV visible spectroscopy and by LC-MS in order to differentiate between structurally related dyes. A set of ten red cotton items of similar color were used, and single fibers from these garments were indistinguishable based on appearance. In fact, two pairs of fibers were indistinguishable via MSP and were only able to be distinguished via differences in the separation and identification of the dyes by using LC-MS. The technique of LC-MS for fiber dye analysis has continued to mature and a method for the extraction, separation, and identification of 15 basic and 13 disperse dyes by LC-MS has been reported [63]. Capillary electrophoresis can also be coupled to a mass spectrometer and CE-MS has been successfully applied to the discrimination of dyed textile fibers [64]. In this research, four fiber types were used (acrylic, nylon, cotton, and polyester). Each fiber type was treated with different solvents to extract the dye. Anionic acid, direct, reactive, and vat dyes extracted from cotton and nylon by using CE analysis required a high ph buffer to make sure that the solutes were negatively charged. Vat dyes are insoluble and were extracted from cotton by using a reducing agent. Separation of the major and minor components of all dyes, however, was possible. Cationic basic dyes required a low ph buffer for CE analysis, and disperse dyes were poorly soluble in water. Diode array detection (DAD) allowed for the comparison of peak migration times and UV visible spectra of fiber dye extracts while the sensitivity of the mass spectrometer allowed fibers as small as 2 mm to be successfully analyzed [64]. A previously discussed method for the analysis of natural dyes by capillary electrophoresis [56] has been expanded to include mass spectrometry as a detection method and 11 natural dyes of plant and insect origin were rapidly separated with low detection limits [65]. Recent advances in mass spectrometry have been focused on less invasive ionization techniques that are nondestructive, yet preserve sensitivity and specificity. One such technique, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, has been successfully applied to single textile fibers [66]. Acidic and basic dyes could be immediately distinguished by gathering both positive and negative ion mass spectra. Furthermore, the use of a TOF mass spectrometer results in highly accurate mass determination. Chemometric analysis When trying to determine if known and unknown fiber samples could have a common source, forensic chemists often rely only upon visual comparisons of complex chromatograms and other spectra. Because of this, examiners do not have any statistical basis for determining the value of the evidence in question. This is a concern for forensic laboratories regarding the reliability of comparisons of dyed fibers, and the ability to compare samples in a quantitative way is desirable. A more recent approach to discriminating fiber colors is by using multivariate statistics. This statistical analysis consists of various mathematical techniques such as agglomerative hierarchical clustering (AHC), principal components analysis (PCA), and discriminant analysis (DA). These methods will remove as much noise as possible from the data, extract as much information as possible from the data, and allow accurate predictions to be made about the data. The application of multivariate statistics to analytical chemistry is well documented and several sources discuss chemometrics in detail [67 69]. The most relevant aspects of data preprocessing and the algorithms in use for fiber dye comparisons are summarized below. Preprocessing the data prior to performing multivariate statistical tests is typically required. Doing this can remove random noise and variation that might later confuse interpretation. Preprocessing can include normalization of the data, artifact removal and/or linearization, centering, smoothing, or scaling and weighting. Normalization of the data can eliminate variability arising from sample amount,

8 2016 J.V. Goodpaster, E.A. Liszewski concentration, and instrument response. Artifact removal and/or linearization of the data can include baseline correction of a spectrum. Baseline correction improves the accuracy of integrals and the appearance of the spectrum. Centering involves taking the mean spectrum from the entire data set and subtracting it from each spectrum. Smoothing the data can increase the signal-to-noise ratio if there is unnecessary noise. However, it can also cause distortions in peak height and width, as well as impair resolutions. Therefore, smoothing must be done cautiously. Scaling and weighting the data entails multiplying all of the spectra by a different scaling factor for each wavelength. By doing this, the influence of the calibration on each wavelength will be increased or decreased. Once the pretreatment of data is complete, statistical analysis can be performed on the data. AHC is a technique that analyzes data for groups of spectra that are similar to each other more so than others in that population without prior knowledge of sample groupings. PCA is another way of identifying patterns in data. It can be used to verify the clustering of data from AHC and also to filter out noise. PCA compresses the data by reducing the number of dimensions without significant loss of information. DA is often utilized as a follow-up to PCA. It constructs a new set of axes that best separates the data into groups. Together with soft independent modeling of class analogies (SIMCA), DA is an example of a supervised technique, meaning that knowledge of group relationships for each sample is required. While chemometric techniques have been applied to data generated from the base material of fibers, applications of chemometrics to spectra, chromatograms, etc. of fiber dyes has been somewhat limited. One example is the discrimination of cellulosic fabrics by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). In this approach, PCA and SIMCA were carried out on DRIFTS spectra obtained from dyed and undyed cellulosic fabrics [38, 39]. Chemometrics was used to match and discriminate different natural and synthetic cellulosic samples based on DRIFTS data. Overall, it was determined that samples could be discriminated based on fabric dye, fabric type, and textile processing. A specific example of the success of DRIFTS is seen with dyed poplin samples whose spectra were discriminated from other undyed fabric spectra but also from each other by using PCA. The spectrum of each colored sample shows peaks due to the cellulosic components of the fiber as well as peaks from the dye. Overall, these studies revealed that DRIFTS may be useful for matching and discrimination of reactive dyes on cotton even if the dye structure is unknown. Furthermore, DRIFTS is sensitive enough to distinguish between spectral features of dyed cotton fabrics without having to extract the dye. PCA and LDA have been used to discriminate between various yellow and red fibers by using UV Vis and fluorescence MSP [70, 71]. The fibers used were cotton, acrylic, nylon, and polyester that were similar in appearance. These results showed that fluorescence was more discriminating than absorbance. Chemometrics has also been applied to FT-Raman spectra from undyed poplin cotton fabric and from the same fabric differently dyed with a bifunctional reactive dye [42]. The four dyed samples each contained the dye in different states (unfixed, treated and unfixed, fixed, and treated and fixed). The spectra, consisting of the spectral range from 1,680 to 952 cm 1, were dominated by the dye but the different states of the dye could not be distinguished. Peaks in the 1,630 to 1,550 cm 1 region belonged to the dye. The 60 FT-Raman spectra of dyed cotton samples and five undyed cotton spectra were submitted to PCA. PCA was performed and the spectral groups of the four dye states were discriminated from each other and from the undyed cotton. Principal component (PC) 1 showed the separation of the undyed cotton but the other four groups could not be distinguished on this PC. The scores plot of PC2 vs. PC3 illustrated almost complete separation of the spectra of all five groups. PC3 separated the fixed from the unfixed dye spectra. An overall trend appeared in which the four groups were separated according to the chemical nature of the dye on the cotton. Conclusions The importance of fiber evidence in criminal investigations has spurred a significant amount of research into microscopic and instrumental methods for their analysis. While the identification of the generic chemical class of the fiber is of clear importance, additional information can be gained by examining the various dyes that are used to impart color to textile fibers. This is particularly true when the fiber is of natural origin (e.g., cotton or wool) given the lack of discriminating features of these materials. Methodologies for fiber dye analysis have progressed from simple visual comparison of an unknown to exemplars to the use of advanced instrumental techniques to specifically identify the dye compounds that may be present. Increased reliance on mathematical techniques for comparing spectroscopic data is also a clear trend in this area. The more traditional methods of comparison using techniques such as polarized light microscopy and microspectrophotometry remain of prime importance. However, the impact of more recent developments in instrumentation has not yet been fully realized. Acknowledgements The authors acknowledge the assistance of Robert Orr and Mark Ahonen from the Indiana State Police Microanalysis Unit in obtaining information about fibers and fiber dye analysis. The authors also acknowledge Dr. Jay Siegel for his assistance.

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