Vibrational Spectroscopy: Recent Developments to ... - ACS Publications

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Vibrational Spectroscopy: Recent Developments to Revolutionize Forensic Science Claire K. Muro, Kyle C. Doty, Justin Bueno, Lenka Halámková, and Igor K. Lednev*

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Chemistry Department, University at Albany, Albany, New York 12222, United States

CONTENTS

Chemometrics Trace Evidence Hair Analysis Fibers Paint Analysis Ink Analysis Forensic Biology and Anthropology Body Fluids Forensic Anthropology Gunshot Residue Controlled Substances Illicit Drugs Pharmaceuticals Counterterrorism and Homeland Security Explosives Chemical Agents Bioagents Emerging Technologies Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References

model techniques according to the requirements outlined by the National Academy of Sciences. Here we present a critical review of forensic developments made in the field of vibrational spectroscopy since 2012. During the past 2 years, many significant advances have been made and, of the studies reviewed here, there are a few that are particularly noteworthy. Nondestructive, rapid methods for detection and identification of biological stains, with on field potential, have been reported. Through their use of multidimensional Raman spectroscopic signatures the Lednev research group in Albany, NY, has developed methods to differentiate and identify body fluids.4,5 The van Leeuwen research group in Amsterdam, The Netherlands, created a method to estimate the age of a bloodstain based on nearinfrared (NIR) spectroscopy.6 Similarly impressive results have been obtained in gunshot residue research. The Lednev laboratory and the Garcı ́a-Ruiz research group in Madrid, Spain reported independently on a new method to identify ammunition using Raman spectroscopy.7,8 An IR imaging procedure to automatically detect gunshot residue particles was also developed.9 Edward Suzuki, supervisor of the Materials Analysis Unit at the Washington State Patrol Crime Laboratory Division, has used IR spectroscopy to identify pigments used in automotive paint.10 Jürgen Popp and co-workers in Jena, Germany, have used Raman spectroscopic techniques for detecting pathogens, which is an extremely important concern for biosafety disciplines.11−14 Modern vibrational spectroscopy includes a wide variety of techniques based on two fundamentally different phenomena, IR absorption and Raman scattering. Raman spectroscopy involves the inelastic scattering of light by a gas, liquid, or solid sample. Upon irradiation, molecules change their vibrational state resulting in a corresponding change in the energy of scattered photons, referred to as the Raman shift. Only normal vibrational modes exhibiting a change in molecular polarizability are Raman active. The portion of photons scattered by a particular normal vibrational mode depends on the scattering efficiency, or Raman cross section, and the abundance of the chemical groups. The efficiency of normal Raman spectroscopy is typically low, but it can be significantly increased due to resonance and (metal) surface enhancement. Conversely, IR spectroscopy measures the absorbance of infrared radiation. When the frequency of the light irradiating a compound is the same as the energy of the normal vibrational mode, the radiation is absorbed. However, a vibrational mode will only be

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orensic science is intimately involved in judicial systems, and as such it must be completely objective and reliable. Because forensics is so diverse and extensive, it can be difficult to hold the entire field to this standard. The National Academy of Sciences published a report outlining the current state of forensic science in the U.S., including issues being faced and necessary changes.1 The committee described that, given the nature of forensic science and its implications on the criminal justice system, there are specific features that methods must possess and others that must be avoided. In order to prevent bias from an investigator, analyst, or expert witness, methods should be quantitative and have an associated statistical confidence, so that the likelihood of error can be objectively estimated. It would also be ideal for analyses to be automated and cost-effective to maximize efficiency. Raman and infrared (IR) spectroscopy are becoming increasingly more popular in forensic science. Both methods are nondestructive, rapid, quantitative, and confirmatory. Raman spectroscopy, in particular, is known for its intrinsically selective nature.2 It has also been suggested that it is “suited to be the process control star of the next century.”3 These qualities, along with their automated capabilities, make Raman and IR spectroscopy © 2014 American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2015 Published: November 10, 2014 306

dx.doi.org/10.1021/ac504068a | Anal. Chem. 2015, 87, 306−327

Analytical Chemistry

Review

this property, through predictions, for unknown samples. There is a variety of multivariate calibration methods available to solve quantitative spectroscopic problems, such as principal component regression (PCR) and partial least-squares (PLS) regression analysis, among others. Their application and performance is often dependent on the particular data set being analyzed and the question being studied.

IR active if it is associated with a change in the dipole moment of the molecule. Thus, Raman scattering and IR absorption both report on molecular vibrational characteristics yet offer complementary information since different selection rules control these two phenomena. Spectral data collected during Raman spectroscopic and IR absorption measurements can be extremely complex. In order to analyze the spectroscopic data more effectively, advanced statistical analysis is widely used. In addition, statistical analysis provides a quantitative estimate of the confidence interval for the conclusions made, which is critical for forensic applications. Chemometrics is an excellent statistical tool to better understand the chemical data and recognize complex relationships that would otherwise be unknown. Chemometrics. Chemometrics is a discipline that uses mathematical and statistical methods to design optimal experimental procedures and extract information from complex analytical data. IR transmittance spectra are usually converted to absorbance before any statistical analysis, since absorbance of each component is proportional to its concentration in the sample.15 A review summarizing chemometric methods used in NIR spectroscopy has been published by Roggo et al., and several books on this topic are available that discuss this technique at length.16−19 Univariate statistical analyses may be used in some specific and simple analytical methods, for example, when the intensity value at only a single wavenumber is considered. However, Raman and IR spectra provide fingerprint-like signatures of samples, resulting in very complex spectra. Therefore, multivariate statistical analyses are required to process, quantify, or classify vibrational spectroscopic data. Out of a large number of multivariate chemometric techniques, only a small portion of them have experienced a broad acceptance. The most commonly used chemometric methods can be divided into three groups. The first group includes mathematical pretreatment methods used to enhance and organize the information related to the chemical variation. The second group of multivariate methods focuses on qualitative analyses that are applied to group samples into classes, according to similarities in the spectroscopic data. The last group includes calibration methods, which are applied for quantitative analysis. Calibration methods associate the spectral data with a quantifiable property of the sample, concentration, for example, and can be used for regression analysis. Classification methods can be further broken down into two groups: unsupervised and supervised. Unsupervised methods, such as principal component analysis (PCA) and cluster analysis, assign spectra to classes without any user-defined training classes. Supervised classification methods, such as soft independent modeling of class analogy (SIMCA), partial leastsquares-discriminant analysis (PLS-DA), and support vector machine discriminant analysis (SVMDA), require an initial class assignment of each spectrum to train the model for optimal performance. Then the model is validated by evaluating the prediction success (correct class assignment) of external data, which was omitted during the training process. Multivariate calibration methods in spectral analyses are all considered supervised techniques. In each case, the training data set includes the quantitative characteristic of interest, like concentration, for each spectrum. Spectral features, such as frequency and intensity, from many calibration samples are compared to each other and related to the known quantifiable property of the sample. These relationships are used to estimate

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TRACE EVIDENCE According to Edmond Locard’s exchange principle,20 which essentially states that any interaction between two objects results in a transfer of material between them, trace evidence includes some of the most common forms of evidence found at crime scenes. Therefore, almost any piece of evidence could be considered as trace evidence. However, according to the Scientific Working Group on Materials Analysis (SWGMAT), there are five main types of samples considered to be trace evidence including paint, fibers, hair, glass, and tape. Although not every branch of forensic science is completely standardized, there are many guidelines in place for trace evidence. In 2000, the Federal Bureau of Investigation created an updated set of detailed guidelines for trace evidence, titled “Trace Evidence Quality Assurance Guidelines”, which is directly related to the SWGMAT. In addition, various documents have been created by the American Society for Testing and Materials (ASTM) which supplies specific instructions for testing procedures. Some of these ASTM standards are applicable to all areas of forensic science,21 some to trace evidence as a whole, and others are specific to analyses performed for individual types of trace evidence. The most relevant standards for different types of trace evidence are listed below in Table 1. In general there are a few fundamental principles that should be considered for all areas of trace evidence analysis. First and foremost, during the collection of trace evidence the utmost care should be taken to not damage the evidence; if a piece of a wall needs to be cut or a large object be removed or disassembled to preserve the evidence then it should be done. Second, during evidence collection, tape lifting should be avoided whenever possible. The evidence may stick to the tape making it difficult to analyze later and the interaction with adhesive chemicals from the tape may affect the evidence and, subsequently, its analysis. The properties of all known samples of trace evidence being compared (i.e., color, size, shape, etc.) to the questioned sample should be as similar as possible to those of the questioned sample in order to conclude that they came from the same source. Furthermore, since this type of evidence can be very small, many of the properties cannot be identified without the use of microscopy; and, if known and unknown samples were not first matched visually, subsequent analyses may not be as significant. Hair Analysis. Hair is ubiquitous in nature and an extremely common form of trace evidence discovered at crime scenes. Therefore, determining the origin of a hair sample can prove to be very complicated. In all analyses, the matching of a known hair to an unknown should be done with the utmost care, taking into consideration the length, color, damage, and somatic (body) region characteristics. Typically in forensic hair analysis there is a list of characteristics which need to be determined about the evidence in question. Some of these attributes include whether the sample is in fact hair; if it is human or animal; if human, the somatic region, color, race, growth phase, chemical alteration, and so forth; if it is suitable for DNA analysis; and if it is similar to other evidence or a 307



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color and structure, which relate to the pigmentation, treatment, artifacts, and abnormalities observed. These details are what allow an examiner to differentiate between species, races, and the somatic origin (i.e., scalp, pubic, facial, limb, and body, etc.). It is known that animal hairs and human hairs can be differentiated microscopically.39,40 However, even for an experienced forensic hair examiner, the best conclusion that can be made is that two hairs could have come from the same source or that two hairs are consistent with one another based upon all physical attributes described. DNA analysis can be performed on hair samples, but this can be problematic if the root or cellular material is absent. Although it has been shown that mitochondrial DNA analyses can be performed on hair samples which do not contain a root or skin cells, this testing procedure is not as definitive for matching a hair to an individual since it only takes into account the DNA from a person’s mother.41 Most recently there have been only a few published studies pertaining to spectroscopic forensic hair analysis. Raman spectroscopy is far less commonly used than IR spectroscopy due to the high fluorescence interference from melanin granules which give hair its color.42 Since 2012 there have not been any major forensic studies published using Raman spectroscopy to analyze hair samples. However, there have been a few groups utilizing IR spectroscopy for hair analysis. One study used attenuated total reflectance-Fourier transform-infrared (ATRFT-IR) spectroscopy to determine that bleaching hair samples with hydrogen peroxide had an effect on the amount of ethyl glucuronide (the marker used for identifying alcohol abuse) present.43 In a different study, a new approach was used to analyze hairs by a single combined atomic force microscopy-IR instrument, with submicrometer spatial resolution. This specifically focused on identifying locations of structural lipids in the cortex and cuticle regions of the hair. It was determined that the middle cortex, outer cuticle, and inner medulla regions of the hair differ in their IR absorbance intensities of long-chain methylene-containing functional groups.44 Since 2012, there has not been much research published about forensic hair analysis by spectroscopic methods. Many studies have been carried out prior to 2012; however those are not the focus of this review. Forensic hair evidence, although very common, is actually one of the more difficult items of trace evidence to analyze and obtain a confirmatory result. The reason for this is that the current microscopic techniques may not hold up in court and are inherently subjective. By using spectroscopy, additional information based on the inherent chemistry of hair can be obtained. This information can be highly reliable and helpful when attempting to match a questioned hair to a known hair sample, potentially corroborating results for use in court. Fibers. Fibers from a crime scene may be found anywhere, including under a person’s fingernails, on clothing, in hair, in a vehicle, and many other locations. They can be natural or synthetic and originate from carpets, clothing, upholstery, plants, or other sources. Many times fiber evidence can link a suspect to a victim or the scene of a crime. Although this is important, it is just as important to invalidate a suspect’s involvement with a particular crime. In 2009 John V. Goodpaster and Elisa A. Liszewski published a review article which included studies of fibers using Raman, IR, and ultraviolet−visible (UV−vis) spectroscopy.45 One of the main conclusions stated in this article was that dyes in fibers should

Table 1. Most Relevant ASTM and SWGMAT Standards for Various Types of Trace Evidence22−34 evidence type

ASTM standard no. E 1492

all N/A N/A

E 131-10 fibers

E 1421-99

N/A

E 1610-14 D 16-12 D 1535-13 E 2808-11 paint E 2809-13 E 2937-13 E 308-13

description Standard Practice for Receiving, Documenting, Storing, and Retrieving Evidence in a Forensic Science Laboratory SWGMAT Quality Assurance Guidelines SWGMAT Trace Evidence Handling Guidelines

Standard Terminology Relating to Molecular Spectroscopy Standard Practice for Describing and Measuring Performance of Fourier Transform Infrared (FTIR) Spectrometers: Level Zero and Level One Tests SWGMAT Forensic Fiber Examination Guidelines

Standard Guide for Forensic Paint Analysis and Comparison Standard Terminology for Paint, Related Coatings, Materials, and Applications Standard Practice for Specifying Color by the Munsell System Standard Guide for Microspectrophotometry and Color Measurement in Forensic Paint Analysis Standard Guide for Using Scanning Electron Microscopy/X-ray Spectrometry in Forensic Paint Examinations Standard Guide for Using Infrared Spectroscopy in Forensic Paint Examinations Standard Practice for Computing the Colors of Objects by Using the CIE System

known hair. Although microscopic analysis can be utilized in determining specific properties about hair evidence, it has inherent flaws. Forensic hair examiners may be biased in their conclusions based on information they could have received prior to or during their analysis and, in general, microscopic examination is subjective. However, well-trained and experienced analysts should arrive at the same conclusion about a known and unknown sample (i.e., the two hairs are or are not consistent with each other). By having specific guidelines and protocols for examiners to follow, which are repeatable and reliable, the trained analysts can provide credible results.35 DNA analysis can help eliminate the inherent examiner bias but many times hairs recovered from a crime scene do not contain a root or the cellular material necessary to obtain a DNA profile. Proper hair analysis is extremely beneficial to an investigation for a variety of reasons including helping to exonerate an innocent person or convict a guilty criminal. Hair evidence can assist investigators in narrowing a search for a suspect or help corroborate testimony and link a suspect to a crime. Pet hair has even been shown to help solve crimes by linking a suspect to a crime or leading investigators to suspects or specific locations.36−38 Furthermore, spectroscopic analyses provide more usable information about a hair sample’s chemistry which microscopy cannot provide. This information can assist in corroborating an examiner’s microscopic conclusions thereby forming a more solid case. Currently, forensic hair analysis begins with microscopic examination and identification. In hair comparisons there are various physical attributes which are described, in the context of 308



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different side groups attached to PET fiber chains or to minor chemical modifications on the polyester chains.52 Raman and IR spectroscopy have been used to distinguish between poly(butylene adipate-co-terephthalate) and PET, two classes of polymers which are not easily differentiated using other techniques.53 Fiber evidence is not always pristine and may contain defects due to environmental conditions and mechanical alterations. In one study microspectroscopic and microscopic analyses of fibers collected at a crime scene where gunshot residue was found were shown to be extremely valuable in helping to solve the crime.54 The types of fibers found at a crime scene can vary in several aspects, such as length, color, thickness, and generic class. These variations add to the complexity of forensic fiber analysis. Although microscopic analysis is necessary in this field of forensics, it cannot always be relied upon. Other techniques, namely, spectroscopic methods, can dramatically enhance the amount of information obtained from fiber evidence. Spectroscopic techniques can corroborate a type of fiber in question as well as allow for discrimination between different classes of fibers and fibers of the same color based on differences in dye components, even when the amount of dye present is minute. Paint Analysis. Paint samples in forensic analysis can be very useful in a variety of contexts. Two generic classes of paints are maintenance and architectural paints which are found in a wide range of crimes.55 One of the most important crimes in which paint samples are of key evidentiary value is hit and run incidents. Many times paint from a suspect’s car will be found on a victim’s clothing or property which can be substantial to helping find the vehicle involved. Paint evidence can also be important in burglaries where paint chips from a door or window may be found on a suspect or the tools used to break in. Paint samples can be found in a variety of shapes and sizes and typically originate from some type of force used to transfer the paint from one object to another. Although the list is somewhat endless, paint evidence can be found on a variety of substrates including pieces of glass, vehicles, roadways, buildings, bridges, walls, floors, tools, hair, clothing, and fingernails. Known paint samples can come from spray paint cans, containers of paint, a suspect’s car, and more. When making conclusions for a paint examination the analyst should determine if there are “significant differences” between a known and questioned sample in order to conclude whether the two samples “could have a common origin.”56 Correct paint analysis can help to prove or disprove a suspect’s involvement in a crime as well as determine if a piece of artwork is an original or a forgery. For current forensic paint analysis there is a list of documents developed by the ASTM which should be followed (see Table 1). At present, according to ASTM E 1610-14, there are various methods which are used to analyze forensic paint samples. These include light microscopy, SEM-EDS, microspectrophotometry, PGC, microchemical tests, X-ray fluorescence (XRF), X-ray diffraction (XRD), as well as IR and Raman spectroscopy. Stereomicroscopy is typically used for physical (i.e., color, layers, surface, etc.) matching of paint samples. Polarized light microscopy may be used to obtain more detailed information about particles in a paint sample while SEM-EDS can determine the elemental composition and morphology. Microspectrophotometry has its own set of specific guidelines (ASTM E 2808-11) and is used to obtain objective information about a paint sample’s color with transmittance or reflectance

be analyzed frequently to assist in discrimination between two similar fibers. Currently, the methods used in forensic fiber analysis include microscopic examination and identification. Typically there are three types of microscopes at an analyst’s disposal including a stereomicroscope, a compound (polarized) light microscope, and a comparison microscope. For this analysis, fibers are placed on a glass slide in a mounting medium where different filters, magnifications, and illumination sources may be used to determine the diameter, color, refractive index, cross-sectional shape, and ultimately, the polymer type. Other tests which are used include solubility testing, heating, and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS). Also, visible microspectrophotometric analysis can be used with a minimum spectral range of 400−700 nm. For the analysis of dyes in fibers, thin layer chromatography (TLC) can be used. When performing TLC tests for dyes there are many important things to consider including the type of eluent and which dye extractant to use. Pyrolysis gas chromatography (PGC) may be utilized to identify the generic type of an unknown fiber and possibly the subclass within the generic class; however, PGC is a destructive technique. Lastly, IR spectroscopy, in the mid-IR region (400−4000 cm−1), can be used via an IR microscope where identification is made by comparing an unknown spectrum to reference spectra. Generally fibers are flattened prior to IR analysis, but this is destructive to the morphology and can result in minor differences in peak intensities and frequencies. Therefore, an analyst must be cautious when assigning a match for the type of fiber. Overall, in forensic fiber examination all properties and characteristics of the unknown and known fiber(s) should be compared in order to best determine if a known sample is consistent with the questioned sample. Fiber evidence typically starts with microscopic examination which requires that a fiber is fixed to a glass slide using a mounting medium. A common mounting medium is Entellan new, and one group recently reported that this substance had no effect on the Raman spectra of fibers.46 However, if multiple fibers from the same source are recovered, then some can be analyzed microscopically while others can be analyzed via other techniques without the need to worry about any possible interference from mounting mediums. Raman spectroscopy has also been used to show that the ability to differentiate between various types of the same class of fiber47 as well as dyed fibers depends on the laser wavelength, color, and type of fiber.48 Raman spectroscopy has been used to provide more information about a sample than attainable by microscopic analysis. In one study, two sets of cotton fibers with dye concentrations ranging from 0.5 to 0.005% (w/w) were analyzed.49 This group was able to determine that dye concentrations below the detection limit of microspectrophotometry and light microscopy could still be detected by Raman spectroscopy. A different study demonstrated that comparisons of the polarization ratios from the 1614 cm−1 Raman band alone could be used for the discrimination between fibers which have different diameters and degrees of orientation and crystallinity.50 Raman and IR spectroscopy can be used to differentiate various classes of fibers, including ramie, cotton, and viscose fibers, but differentiating between the same class of fibers can prove to be difficult.51 However, by using smart internal reflection spectroscopy it has been shown that dissimilarities in polyethylene terephthalate (PET) fibers are most likely due to 309



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identify the pigment bismuth vanadate using FT-IR and dispersive Raman spectroscopy as well as XRF spectrometry.66 As mentioned previously, paint samples do not only come from vehicles; sometimes crimes are committed which involve the use of spray paint. To understand evidence from these types of crimes, Buzzini and co-workers analyzed the effect of shaking time for various spray paints on Raman and IR spectra demonstrating noticeable differences up to 3 min.67 Figure 1

measurements. Both PGC and microchemical tests allow for analysis of the paint binder but are destructive to the sample and should be avoided if possible. XRF and XRD are nondestructive techniques which can provide elemental analysis of multiple layers (XRF) and identify the crystal form of fillers, extenders, and pigments (XRD). IR and Raman spectroscopy are also nondestructive techniques and are used to obtain information about both the organic and inorganic components in a paint sample. These two spectroscopic methods are advantageous since specific chemical information can be obtained allowing for the identification of specific pigments present. Although microscopic examination is helpful, it has limitations and cannot provide information about the specific chemical properties of the questioned and known samples. In 2007, a review of Raman spectroscopy for the analysis of art was published which incorporated topics such as pigment analysis, corrosion, biomolecules, and chemometrics.57 More recently, a review was published that discusses the applications of Raman and IR spectroscopy in forensic art analysis, which covered many studies on paint.58 However, that review does not cover many of the most recent studies on the spectroscopic analysis of paint. Raman spectroscopy has been a helpful technique in various types of forensic paint analysis. In one study, 66 blue automotive paint samples were analyzed with three different lasers (514, 633, and 785 nm) to differentiate between metallic and solid paint samples with discrimination powers of 99% and 97%, respectively.59 Pigments in an oil painting were also analyzed by Raman spectroscopy, and one study determined that all pigments except for one, chrome yellow, (thought to have come from 19th century retouching) were from the Renaissance period.60 This showed the importance of using historical research to assist with determining the origin of paint pigments. Lateral scanning Raman spectroscopy has been utilized to discriminate between various multilayer white paint chips, where some of these samples were optically indistinguishable.61 Spatially offset Raman spectroscopy (SORS) has also been used to analyze multiple layers of paint samples.62 A known disadvantage of Raman spectroscopy is the fluorescence interference commonly encountered, which can be prominent with paint evidence. However, one study was able to identify pigments by reducing fluorescence interference in 90% of their examined blue paint samples without altering Raman scattering.63 These studies demonstrate the versatility and advantage of using Raman spectroscopy in paint evidence analysis. IR spectroscopy can also be used for a variety of applications in forensic paint analysis. Synchrotron FT-IR microspectroscopy and PCA has been used to differentiate between primer surfacer coats of vehicle paint as well as determine the year and details about the make and manufacturer of the vehicle.64 A similar study analyzed 3 μm thick slices of vehicle paint chips by Raman spectroscopy with different excitation sources to show an advantage over IR analysis for pigment identification.65 These researchers also demonstrated the ability to identify the vehicle involved in an accident or establish the color and make of the vehicle, when no comparative samples were available. Specific pigments in automotive finishes including silver/white mica pearlescent and bismuth oxychloride pigments, as well as some cadmium pigments (which produce similar IR absorptions to the former two), have been identified using SEM-EDS analysis, FT-IR spectroscopy, and XRF spectrometry.10 Automotive finishes have also been analyzed in situ to

Figure 1. Influence of the shaking time on the forensic analysis of Raman spectra of spray paints. The Raman spectra of an acrylic red spray paint, containing the pigment Naphthol red, after 0, 1, 2, 3, 4, and 5 min of shaking are shown. The peak intensities decrease over time. The inset shows maximum counts calculated for the peak at 1350 cm−1 for the 15 replicates of each condition plotted using boxplots. The time point of 1 min shows a higher pigment concentration as indicated by the highest boxplot values. Reprinted from Muehlethaler, C.; et al. Influence of the shaking time on the forensic analysis of FTIR and Raman spectra of spray paints. Forensic Science International, 2014, 237, 78−85 (ref 67). Copyright 2014, with permission from Elsevier.

shows Raman spectra of one spray paint sample after being shaken for 0 to 5 min, corroborating that the most significant changes occur up to 3 min, and the highest pigment concentration was at 1 min. These results help to show that specific care should be taken when comparing a known and unknown sample for crimes involving spray paints. The spectroscopic analysis of forensic paint evidence has been shown to be highly useful. Although fluorescence can be an issue in Raman spectroscopic paint evidence analysis, work toward correcting for this is being undertaken. Many groups have demonstrated that by using IR and/or Raman spectroscopy more information about a piece of evidence can be obtained, all in a nondestructive manner. These techniques can help to discriminate between vehicle paints and ascertain the make and model of a vehicle as well as proving the authenticity of a piece of art. From this overview it can be concluded that spectroscopic methods for paint evidence analysis are highly versatile while providing reliable and valuable results. 310



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INK ANALYSIS

Forensic ink analysis is directly related to the branch of questioned documents. This type of evidence can be found in many forms for various crimes including ransom or hate crime letters, forgeries, and more. Ink analysis can be used to help link a suspect to a crime or determine the authenticity of a questioned document. One important fact to consider when analyzing ink from a questioned document is that slight chemical interactions may occur between the paper and the ink. Although ink analysis is part of the questioned documents branch of forensic science, this review will only focus on the analysis of inks and not on any other forms of analyses related to questioned documents. Currently, the methods used in forensic ink analysis include microscopy, alternative light sources, liquid chromatography, TLC, and IR spectroscopy. Although there are general ASTM standards for questioned document analysis, ASTM 1789-04 (Standard Guide for Writing Ink Identification) and ASTM E 1422-05 (Standard Guide for Test Methods for Forensic Writing Ink Comparison) were withdrawn in January 2013 and 2014, respectively, with no replacement. However, since ink analysis falls within the category of questioned document analysis, ASTM E 444 (Standard Guide for Scope of Work of Forensic Document Examiners) could be referenced. In 2013 Braz et al. published a review article on the use of Raman spectroscopy for the analysis of inks on questioned documents.68 They discussed studies which utilized various laser excitation wavelengths, which can help to provide compounding information, ultimately resulting in a chemical signature for a specific ink. However, fluorescence is still a shortcoming of Raman spectroscopy and paper can be highly fluorescent. In attempts to avoid this problem, surface enhanced resonance Raman spectroscopy (SERRS) and near infrared (NIR) methods have been utilized in the analysis of inks; however, SERRS can be considered to be destructive to the sample. That review also mentioned studies completed to distinguish between intersecting lines of writing, to determine which was written first, but this research is still quite limited. Because of its selectivity, Raman spectroscopy has been a reliable technique in differentiating between various types of inks, both printed and written. In one study Raman spectroscopy and capillary electrophoresis (CE) were used to analyze 23 printer ink samples, obtaining discriminating powers of 94.0% (Raman) and 95.6% (CE) for all samples analyzed.69 Another study analyzed 10 different ink cartridges, both black and color, from various manufacturers using Raman spectroscopy, laser desorption mass spectrometry, and matrix-assisted laser desorption ionization-mass spectrometry.70 It was discovered that the excitation wavelength has an effect on the spectral information obtained from colored inks as shown in Figure 2. These researchers demonstrated, for the first time, the ability to differentiate between black-printed documents from the same and different manufacturers. Ink evidence can also come from an ink pad, and one study used high-performance thin layer chromatography (HPTLC) and Raman spectroscopy to analyze nine different blue stamping inks from India.71 They determined that HPTLC and Raman spectroscopy are complementary techniques which can be used for differentiating inks based on their chemical composition. They also looked at intersecting lines drawn with different red and black pen inks, blue stamp pad ink, laser printer toner, and pencil to determine the order of writing, but were not successful in all

Figure 2. Determining the best excitation wavelength for Raman spectroscopy for the forensic analysis of black and color inkjet printed documents. Comparison of Raman spectra obtained for yellow Canon and Hewlett-Packard sample A (HP A) with 785 nm (red dotted line) and 458 nm (blue continuous line) excitation wavelengths. Differences between the two spectra from the same ink are explained by a resonance effect. Reprinted from Heudt, L.; et al. Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents. Forensic Science International, 2012, 219, 64−75. (ref 70). Copyright 2012, with permission from Elsevier.

cases. Since fluorescence can be an issue with Raman spectroscopy, one group used surface enhanced Raman spectroscopy (SERS) with silver-doped agarose gel disks to quench fluorescence when analyzing rhodamine 6G and crystal violet dyes.72 IR spectroscopy is a common technique in the analysis of inks from questioned documents and can be useful in helping to solve crimes. In one study ATR-FT-IR was used to prove a case of fraud by demonstrating that the red ink from two different seals (used as a signature) were not of the same origin or source, therefore discrediting the prosecutorial complaint.73 FT-IR spectroscopy was combined with Raman spectroscopy in one study to obtain individual identification of 70 different red ink entries.74 FT-IR microspectroscopy has been used in combination with SEM-EDS mapping to successfully determine the sequence of intersecting lines of red seal ink and laser toner.75 Black toners from 18 manufacturers in Portugal have also been analyzed and identified (and a spectral library developed) using a nondestructive diamond cell FT-IR spectroscopic approach, showing high repeatability and 100% correct identification.76 Inks from ballpoint pens have also been studied by a number of different groups. In particular, one group analyzed inks from 10 blue ballpoint pens using UV−vis and IR spectroscopy and HPTLC coupled with PCA to estimate the time at which a document was written.77 In a different study blue gel, ballpoint, and roller ball pens from different brands were analyzed on three types of paper using Universal ATR-FT-IR and linear discriminant analysis methods (including genetic algorithm, stepwise formulation, and successive projections algorithm) to successfully differentiate all pen brands on each type of paper; correct classification rates ranging from 91.3 to 100% were achieved.78 One group constructed a spectral database and library-searching program using analyzed signatures from 63 black ballpoint pens commercially available in Korea.79 They were able to identify the model, blend, and manufacturer of each pen as well as determine the sequence of line intersections 311



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Samples analyzed spectroscopically are preserved for further analysis, like DNA testing. By retaining the advantages offered by current techniques, and overcoming their limitations, spectroscopic methods exhibit the potential to be invaluable to forensic investigators. Our laboratory has been working for several years on the application of Raman spectroscopy to identify body fluid traces. We have introduced the concept of multidimensional spectroscopic signatures to account for the intrinsic heterogeneity and possible variations between donors of dried body fluids. Multidimensional Raman spectroscopic signatures have been created for several human body fluids including peripheral blood, sweat, saliva, semen, and vaginal fluid.4,5,88,89 In 2011, Sikirzhytski et al. published a comprehensive self-review which covered current methods used in the field as well as new methods developed by our research group.90 More recently, McLaughlin et al. published a modified blood signature to account for the dependence on the excitation laser power.91 The ability to discriminate peripheral and menstrual blood has also been demonstrated.92 In 2014 Rinke-Kneapler and Sigman published a review on current Raman analysis methods of body fluids.93 Besides characterizing a variety of human body fluids, Raman spectroscopy can also be used to differentiate blood from human and animal origin. McLaughlin et al. measured the Raman spectra of blood from human donors and 11 animal species and found that the two groups could be differentiated with 100% accuracy, as shown in Figure 3.94 A PLS-DA model built using a calibration data set was tested via a blind study and

for two signatures. Various statistical analyses have been used to assist with forensic ink analysis. Specifically, PLS-DA has been used in the differentiation of blue ballpoint pen ink.80 Cluster analysis, PCA, and discriminant analysis have been used to discriminate between black ballpoint pen ink varieties and brands.81,82 Blue, red, black, and green ballpoint pen inks from various sources have been analyzed by UV−vis-NIR, FT-IR, Raman spectroscopy, and SERRS.83 The ability to differentiate inks was demonstrated for some of the samples analyzed using chromatic analysis with the CIE-L*a*b* color system. More recently this group analyzed red and black ballpoint pen inks by TLC, XRF, and UV−vis-NIR spectroscopy (absorbance and diffuse reflectance modes), again utilizing chromatic analysis in the CIE-L*a*b* color system, to differentiate between ballpoint inks within the same coloristic palette.84 Within the questioned documents discipline of forensic science, ink analysis is one of the most important methods to obtain useful information from the evidence, besides DNA or fingerprint identification. A common struggle in questioned document analysis is the intersection of two lines where knowing which was written first can be extremely helpful in forgeries or in determining a match. This problem has been addressed with varying success using spectroscopy. Although the colors of inks can vary tremendously, it is those ink colors which are more common (i.e., black, blue, and red) that are important to study. It has been found that ink can be differentiated based on the pen brand as well as the origin of printed documents. Altogether, these studies show that spectroscopic analyses of ink can be of significant assistance in solving crimes that involve questioned documents.

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FORENSIC BIOLOGY AND ANTHROPOLOGY Body Fluids. Body fluids are commonly found at several types of crime scenes. Some of the techniques that are currently being used in forensic investigations to identify body fluids include luminol, Hemastix, the starch-iodine test, and Christmas tree staining.85 Most of these methods are destructive to the sample, and they only identify one body fluid. As such, a single piece of evidence may have to be submitted to several destructive tests before being identified. The ideal tool for a forensic investigator would be one that could be brought to a crime scene and identify all body fluid samples discovered, without destroying or compromising the evidence. The current methods used by forensic investigators to examine and identify body fluids fall into one of two categories: presumptive or confirmatory tests.86 Presumptive tests are typically simple to use sensitive tests that quickly provide results. As such, a positive result only indicates the body fluid in question may be present. While presumptive tests are rapid, they can result in false positives. For example, an acid phosphatase assay can be used to presumptively identify semen. However, vaginal fluid also contains acid phosphatase and will provide a false positive result.86 Presumptive tests can be followed by confirmatory assays. Confirmatory methods are far more selective, so they have the ability to conclusively identify a body fluid. Unfortunately, confirmatory tests are destructive.87 In criminal investigations, preserving forensic evidence is of the utmost importance so this poses a significant problem. Spectroscopic methods have several advantages over the traditional chemical assays. Spectroscopic methods are rapid and selective and, most importantly, they are nondestructive.

Figure 3. Discrimination of human and animal blood traces via Raman spectroscopy. (A) Comparison of the preprocessed averaged Raman spectra from all human (n = 10) spectra and all animal (n = 100) spectra. (B) Cross-validated prediction scores for human class using the binary PLS-DA model. Red line represents the default classification threshold. 312



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This information could include race, gender, age, or other physical characteristics. Ultimately, Raman and IR spectroscopy could serve as model forensic techniques for body fluid investigations. Their nondestructive, rapid, and confirmatory natures make them perfect for crime scene analysis. Sophisticated techniques, such as SERS, help to overcome sensitivity and sample size limitations but could be destructive to the evidence. Advanced statistics and chemometrics allow researchers to interpret complex spectral data sets and extract useful information which allows for more comprehensive and accurate results. Forensic Anthropology. The current methods for recovering, analyzing, and interpreting human remains varies depending on their condition. Usually a forensic pathologist can perform a post-mortem autopsy. However, forensic anthropologists are typically sought if the remains have skeletonized, decomposed, putrefied, or become otherwise unrecognizable. Forensic anthropologists can use information from both the skeleton and soft tissue to determine the individual’s sex, age, cause of death, and time since death. Unfortunately, many of the techniques used in forensic anthropology are qualitative and prone to investigator bias. Spectroscopic techniques could greatly improve the accuracy and precision of the field of forensic anthropology. Currently, many of the analyses performed by forensic anthropologists to determine gender, age, or time since death are carried out by gross examination.104 Standard procedures include making observations of the skeletal remains and tissue present, qualitative descriptions, and measurements of bones or osteological features. The size and morphology of bones can be used to predict an individual’s sex. Age is determined by epiphyseal union, dental development, and bone measurements. Often times, population specific information is required to make any determinations based on these observations.104 The post-mortem interval (PMI), or time since death, can be estimated based on observations of any soft tissue present, while taking into consideration recent weather conditions. Some researchers have found chemical methods to estimate PMI, but many of these methods are not reliable enough to be used by forensic practitioners.87 There is a clear and definite need for new forensic anthropological techniques which are accurate and reliable and do not require population or weather data for analysis. In 2013 Zapico et al. published a review of current techniques to estimate age at death.104 They reported on a group that used ultraviolet resonance Raman spectroscopy to detect age related changes in the amide I band of cortical bone and teeth. In a review published in 2012, Ozek et al. reported on the use of both Raman and FT-IR spectroscopy to determine PMI.87 The Raman and IR spectra of bone show peaks from both the organic and inorganic phases of the tissue. The organic phase is primarily collagen, while carbonated hydroxyapatite mineral, or bioapatite, comprises the inorganic phase. Some studies have used soft tissue, such as brain, liver, or kidney, to estimate PMI.87 Traditional Raman spectroscopy can be used to identify the different phases of bone tissue. Pestle et al. studied collagen in archeological bone with hand-held vibrational spectrometers and distinguished between the spectra of bone, collagen, and hydroxyapatite.105 Karampas et al. has developed a quantitative method to not only identify but determine the amount of bioapatite and collagen in bone using Raman spectroscopy.106

external validation. During the blind study, 10 unknown samples were all successfully identified as either human or animal. In total, 10 spectra taken from cow blood, a species which was not included in the calibration data set, were also identified as animal. This external validation demonstrated the model’s ability to correctly classify blood from a species of animal which was not initially accounted for, indicating its robustness. Body fluids discovered at a crime scene are seldom found in their pure form. Contaminants from the surrounding environment or other body fluids may be present. Raman spectroscopic mapping is capable of identifying mixtures of blood and semen.95 Several mixtures of the two body fluids were prepared in varying concentrations. Mixtures could be distinguished from pure body fluids with high sensitivity and specificity. Additionally, Raman spectroscopy with mapping can detect blood even in samples contaminated with sand, dust, or soil.96 Substrate interference can also hinder analysis. McLaughlin et al. explored this and found that the Raman signal arising from the substrate could be identified and subtracted from experimental spectra, leaving the signal due to blood.97 Substrate interference could also be reduced by altering the excitation laser wavelength. The application of advanced statistical modeling can greatly enhance the information contained within spectra. Edelman et al. developed a technique to identify bloodstains on colored backgrounds and estimate their age using NIR spectroscopy.6 Bloodstains on colored fabric were identified with 100% sensitivity and specificity. Furthermore, PLS regression was used to estimate their age, up to 1 month, with an 8.9% root mean squared error. While Raman spectra can be complex and difficult to interpret at times, Lemler et al. showed that traditional Raman spectra collected from dried blood stains excited at 785 nm are exclusively attributed to hemoglobin.98 By using advanced techniques, such as SERS, researchers are able to observe additional components of body fluids. Premasiri et al. analyzed bloodstains by SERS and found that the resulting spectra were due to plasma, not hemoglobin.99 SERS can also be more useful than traditional Raman spectroscopy because of its increased sensitivity. While traditional Raman spectroscopy can only detect bloodstains diluted up to 1:250, SERS amplifies the signal from blood so much that stains can be detected after a dilution of 1:100 000.100 Hu et al. used SERS to analyze whole human tears with silver nanoparticles and found a strong spectral contribution from hypoxanthine and uric acid.101 Bonnier et al. found that while Raman and FT-IR spectroscopy are both being used more commonly on body fluids in biomedical applications, both possess disadvantages.102 When body fluids are still in their whole, or liquid, form their FT-IR spectra are dominated by interference from water. Raman spectroscopy is not inhibited by water, but it does require a suitably concentrated analyte. Allowing a fluid to dry concentrates it but results in a more heterogeneous sample, thus decreasing reproducibility. However, IR spectroscopy still can be used for body fluid analysis. Ollesch et al. found that FTIR spectroscopy could differentiate between blood taken from patients with urinary bladder cancer and those with bladder inflammation.103 Recent studies have shown the ability to estimate the age of a bloodstain at a crime scene and suggest that a similar approach could be used for other body fluids. This needs to be explored further to confirm its potential. Additionally, it would be extremely helpful if spectroscopic techniques could characterize the donor of the body fluid beyond human or animal origin. 313



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In a novel study, surface enhanced spatially offset Raman spectroscopy (SESORS) was used to penetrate the surface of bone.107 This allows for the characterization of tissue or material beneath bone. This could suggest that if tissue under bone, such as the brain, needs to be characterized for PMI estimation, it may be possible that SESORS could be used to do so without damaging the bone. While being able to determine the PMI is crucial in death investigations, on a more basic level it is also important to be able to distinguish between recent and archeological remains. If a human skeleton is discovered, determining whether the remains are two versus two thousand years old will greatly influence whether or not a forensic investigation is necessary. Patonai et al. developed a method using FT-IR spectroscopy to analyze the crystallinity and carbonate-phosphate indices to differentiate between recent and ancient remains.108 When buried, several environmental factors can influence the collagen and bioapatite in bone, such as soil type, acidity, and temperature. In a controlled study, Howes et al. buried bone samples and varied soil type, moisture, pH, temperature, bone condition, and species and found that the amount of organic and carbonate content, calculated by FT-IR spectra, was strongly correlated with burial time.109 In a study of the ATRFT-IR spectra from rat brain tissue, Ke et al. found a very strong relationship between several different peak intensities and PMI, shown in Figure 4.110 Seven pairs of IR peaks were used to create regression equations to estimate PMI, with correlation coefficients between 93 and 97%. Both Raman and IR spectroscopy are useful in forensic anthropology. Their ability to quantitatively analyze human tissue, bone, and teeth allow investigators to estimate age and PMI. Characterizing bone and tissue beneath it by Raman spectroscopy can be greatly enhanced by SORS. Additionally, ATR-FT-IR spectroscopy can be used to gather more detailed information than traditional IR spectroscopy. Future forensic research will only improve the applicability and reliability of the methods developed thus far. Additionally, advancements can be made through medical research studies, which exhibit forensic relevance. It would be advantageous if spectroscopic techniques could accurately estimate PMI and age. Researchers are working toward this, but it is still not yet reliable enough to be implemented in the field. Moreover, spectroscopy could eventually replace current methods used to estimate gender and race, with more accuracy and precision, and a quantifiable associated error.

Figure 4. Brain tissue from rats was analyzed by FT-IR spectroscopy to estimate time since death. FT-IR spectra shown were acquired from brain tissue 0−144 h post mortem. Peak intensities at several frequencies were used to create calibration curves and estimate the post-mortem interval with 93−97% accuracy. Reprinted from Ke, Y.; et al. The Changes of Fourier Transform Infrared Spectrum in Rat Brain. Journal of Forensic Sciences, 2012, 57, 794−798 (ref 110). Copyright 2012, with permission from Wiley.

GUNSHOT RESIDUE Forensic investigators attempt to link suspects to particular shooting incidents by determining if recovered ballistic evidence originated from a particular firearm. This technique is based on tool mark examinations which compare striations on discharged projectiles or cartridge cases as being unique to a specific firearm. Unfortunately, tool mark comparisons require the opinion of an expert examiner, which introduces the potential for bias and variations between examiners. As noted by the 2009 National Academy report on forensic science, these types of analyses have “unarticulated standards with no statistical foundation for estimation of error rates.”1 Because of the drawbacks associated with tool mark examinations, other avenues must be explored for shooting incident reconstruction. Recent advances in analytical methodology may increase the value of gunshot residue (GSR) for shooting incident reconstruction. GSR is composed of the

burnt and partially unburnt byproducts resulting from the firearm discharge process, specifically trace metallic components from the discharged firearm, spent cartridge cases and projectiles, as well as chemical components from the ammunition propellant and primer.111 Particles originating from the primer and metallic components of the firearm are often labeled as inorganic GSR (IGSR), while particles from the propellant are commonly labeled as organic GSR (OGSR). OGSR is mainly composed of nitrate ester explosive materials such as nitrocellulose or nitroglycerin. When a firearm is discharged, GSR particles are deposited around the crime scene, most importantly on the body or clothing of the shooter. Thus, detecting GSR on a suspect may provide a way to link him or her to the crime scene. The most common and only standardized procedure for GSR analysis is SEM-EDS. SEM-EDS has a high affinity for the

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Figure 5. ATR-FT-IR imaging for detection of microscopic GSR. (a) Visual image of the mapped tape substrate area. (b−d) ATR images of mapped area, each pixel represents one raw ATR-FT-IR spectrum. The pixels are colored by intensities of transmitted light at (b) 1415 (chemical marker for IGSR), (c) 1646 (a chemical marker for OGSR), and (d) 1728 cm−1 (chemical marker for tape substrate), respectively. (e) Color scale determining intensities of detected chemical signal. Blue colored areas indicate strong absorption (low % T) by the analyte, at that specific frequency while red colored areas indicate little to no absorption (high % T) by the analyte, at that specific frequency. (f) FT-IR spectra of organic GSR, inorganic GSR, cotton fiber, and tape, showing the unique peaks used during mapping to identify samples.

(i.e., flameless atomic absorption), vibrational spectroscopy is capable of correlating the analytical data to a single GSR particle, greatly reducing the risk of false positives. Additionally, vibrational spectroscopy is nondestructive, rapid and automated Raman and IR microscopes are commercially available. Identifying a particle as GSR is the first step in many shooting incident investigations. As previously described, the most common approach for this application is SEM-EDS. Unfortunately this approach is only suitable for IGSR. Moreover, the technique is not ideal for forensic laboratories due to the complexity, time, and monetary investments required for the procedures. Although advances to the SEMEDS approach via the addition of voltammetry have been reported by O’ Mahony et al., vibrational spectroscopy is an emerging approach which offers innovative solutions for GSR detection.115 Vibrational spectroscopic mapping was targeted as an alternative approach for GSR detection. Microscopic-ATR imaging combines the ease of analysis of traditional ATR-FT-IR spectroscopy (no sample preparation required, nondestructive, rapid, and selective), with the high selectivity associated with vibrational spectroscopy, for the automated scanning of microscopic samples. Microscopic-ATR imaging combined with tape lifting was reported for the collection and detection of OGSR and IGSR.9 Common double sided pressure sensitive adhesive tape was used to collect GSR from a cloth discharge surface. This procedure was designed to mimic the collection of GSR from a suspect’s clothing. ATR-FT-IR maps were generated in which individual spectra were collected every 1.56 μm over a 0.5 mm2 range and are shown in Figure 5. The vibrational signatures from the GSR particles and tape collection substrate were unique, and the spatial resolution of the technique was able to discern GSR particles 4.7 μm in size and larger from the collection substrate.

analysis of the heavy metals Pb, Ba, and Sb whose presence in a spherical particle is considered unique to IGSR.112 Unfortunately, this technique is not applicable for the analysis of propellant residues (OGSR) or GSR samples originating from heavy metal free ammunition. Heavy metal free ammunition is growing in popularity as a result of its less harmful impact on the environment and on manufacturing practices.113 GSR particles generated from heavy metal free ammunition are composed of elements which are not targeted by the current SEM-EDS standardized procedures. Furthermore, particles originating from some manufacturing trades represent false positive threats to SEM-EDS analysis of GSR. Studies have determined that particles originating from automotive brake pads and tires may be composed of Pb, Ba, and Sb and in these scenarios could not be distinguished from IGSR via SEM-EDS analysis.114 Furthermore, even the successful identification of IGSR with SEM-EDS offers relatively limited forensic value. The most common conclusions are limited to estimating the shooting distance, confirming that a shooting incident occurred and/or that a suspect discharged or was within the proximity of a discharging firearm. Because of the drawbacks with tool mark examinations, the ability to link a GSR sample to a specific firearm or ammunition would be a novel and impactful advancement for the forensic community. Several researchers have investigated this approach through vibrational spectroscopic analysis of GSR. Preliminary results indicate that a fully developed method could mimic tool mark comparisons for the ability to link a GSR sample to a particular source. A comprehensive review of GSR analysis methods was provided by Dalby et al. in 2010.111 Since that time, a dramatic increase in the use of vibrational spectroscopy for GSR characterization and detection has occurred. Vibrational spectroscopy offers suitable spatial resolution for the analysis of individual GSR particles. As compared to bulk GSR analyses 315



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require a library of Raman spectra from numerous firearmammunition combinations, a long-term goal of the project. Similarly, ATR-FT-IR spectroscopy was used to differentiate GSR particles from different firearm-ammunition combinations.118 ATR-FT-IR offers rapid analysis of individual particles. ATR-FT-IR allowed for the analysis of additional discharge samples which could not be analyzed by Raman spectroscopy. Dark colored GSR particles whose Raman signal was obscured by fluorescence provided informative ATR-FT-IR spectra. A blind test via PLS-DA was utilized to achieve an external validation for the approach. GSR particles from a discharge not included in the training set were assigned as originating from the correct firearm-ammunition combination. Finally, GSR samples from different firearm-ammunition combinations were discriminated through the use of combining Raman and IR spectroscopy.119 The same GSR particles were analyzed with both Raman and IR spectroscopy, and the resulting spectra from each sample were combined into one spectrum. The approach of combining spectra from two different techniques to enhance statistical discrimination is relatively novel. Pallipurath et al. first reported this approach by combining Raman and fiber-optic reflectance spectroscopies for the differentiation of medieval paint samples.120 The combined approach was shown to extract additional vibrational data from each GSR particle and resulted in improved statistical classification as compared to the two individual methods. The effect of weapon memory on the resulting GSR sample is another key discharge parameter that was investigated by Raman microscopy.121 The effect of weapon memory can be described as residues from a previous firearm discharge (which have been stored within the firearm) being expelled with a subsequent discharge. Since different ammunition could be used between subsequent discharges, GSR particles from a previous discharge may “contaminate” the GSR particles originating from the currently discharged ammunition. LópezLópez and co-workers discharged the same firearm with two different types of ammunition without cleaning the weapon between discharges. A total of 20 discharge samples were collected and the two types of ammunition were rotated throughout the 20 discharges. The two samples of ammunition possessed propellants with different stabilizers (the initial discharge contained ethyl centralite while the subsequent ammunition contained diphenylamine). A Raman band at 1342 cm−1 (characteristic of a diphenylamine decomposition component) was used to determine from which ammunition individual GSR particles originated. Visual investigations of the spectra based upon the presence or absence of this peak determined that, on average, 4.5% of the GSR particles analyzed were from the subsequent discharge. The authors concluded that an appropriate number of GSR particles should be collected and analyzed in order to overcome the potential pitfall of weapon memory, especially when attempting to link or identify the GSR sample as originating from a specific ammunition type. GSR is becoming an increasingly important form of evidence for crime scene reconstruction. Applied spectroscopic research within the last 2 years has illustrated that GSR samples from different sources can be differentiated. This preliminary research may indicate the ability to link an unknown GSR sample to a specific forensically relevant parameter (caliber size, ammunition brand, etc.). In the future, false positive sources for OGSR should be investigated with vibrational spectroscopy.

Raman microspectroscopic mapping for the detection of GSR originating from both the propellant and primer of the ammunition was reported by Bueno et al.116 This approach, utilizing a Raman microscope equipped with an automated stage and autofocusing feature, is similar to the ATR-FT-IR imaging technique described above. Training data sets of Raman spectra from IGSR, OGSR, and the tape substrate were generated. These training sets were used as a predictive tool to classify unknown Raman spectra collected from tape lifted samples, via PLS-DA. Low error in the classification rates illustrated the proof of concept for this technique as a rapid and inexpensive alternative to GSR detection. Once a GSR sample has been identified, analysis can be performed to extract forensically relevant information. Discharge parameters (caliber, type or manufacturer of the ammunition and/or firearm) which may assist in crime scene reconstruction would be targeted with this approach. This concept is based on the hypothesis that, due to the numerous complex interactions involved in the firearm discharge process, GSR samples from different origins are unique. The following research investigates this hypothesis by attempting to differentiate GSR samples from different sources. The end goal for these projects resembles the goals of tool mark comparisons by attempting to link GSR samples to a particular source. López-López et al. applied Raman spectroscopy for the analysis of GSR particles from several types of ammunition.7 The ammunition possessed chemically different propellant, varying in presence or absence of specific additives. Manufacturers use different additives to achieve different goals within an ammunition (stabilizers, plasticizers, etc.). Raman spectra collected from the GSR samples were compared to the unburnt ammunition propellant powder. Two different propellant mixtures, one containing the stabilizer diphenylamine and the other utilizing ethyl centralite, were discharged. Chemical identification of the stabilizers was achieved in Raman spectroscopic analysis of the resulting GSR particles. Raman spectra were visually differentiated based upon the presence or absence of peaks associated with these additives. The authors concluded that the technique is applicable as a rapid screening tool for GSR analysis and discrimination based upon stabilizer composition of the discharged ammunition. Further characterization of unburnt ammunition propellant was performed with both Raman and IR spectroscopy.117 Discriminant analysis successfully differentiated spectra collected from single (containing mostly nitrocellulose) and double (containing nitrocellulose and nitroglycerin) based gunpowder. Propellant mixtures both containing and void of dinitrotoluene and diphenylamine additives were also differentiated. Similarly, the technique may be useful for the identification of unknown unburnt propellant mixtures. Bueno et al. investigated the ability to discriminate GSR samples based upon different discharge parameters.8 Specifically, GSR particles originating from different firearmammunition combinations (varying by caliber size) were probed with Raman microscopy combined with chemometrics. Several multivariate analyses were used to differentiate the Raman spectra with classification rates above 95%. The authors stated that the most appropriate application for this research is on a “case by case” basis for the exclusion of a particular firearm-ammunition combination as generating a crime scene sample. Conversely, the ability to link a GSR sample to an unknown firearm or ammunition with this approach would 316



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concentration of cocaine in the mixtures.131 Figure 6 shows the Raman spectra collected from the four binary mixtures, with varying concentrations for each. Portable Raman spectrometers can be used in the field and at crime scenes. This allows forensic investigators to obtain

CONTROLLED SUBSTANCES Illicit Drugs. Illicit drugs, those which are not prescribed by doctors, are encountered by almost all levels of law enforcement. From municipal police officers executing a search warrant, to state police officers carrying out a traffic stop, to federal agents at U.S. borders, drugs can be found in innumerable cases. While a concealed bag containing pills or a white powder may appear to be illicit drugs, the identity and quantity of the substance must be determined before drawing any conclusions. Forensic scientists use several different methods to sample and identify illicit drugs. Presumptive tests, like color tests, can be used at crime scenes, followed by confirmatory analyses in a laboratory, such as gas chromatography/mass spectrometry (GC/MS). However, too often color tests result in false positives.122 Additionally, GC/MS requires sample preparation, standards for validation, and sample-specific instrumental parameters. Conversely, vibrational spectroscopy, which is nondestructive so evidence is preserved for further analysis, can be used in situ. For these reasons, Raman and IR spectroscopy have become common techniques used in forensic drug analysis. Bell et al. published a comprehensive review of current methods used to analyze illicit drugs with Raman spectroscopy in 2012.123 This included identifying specific drugs, even in the presence of excipients, cutting agents, or other drugs. The authors also explained that while Raman spectroscopy is often used for quantitative analysis of pharmaceuticals, it is much more difficult with illicit drugs because they are often not found in their pure form. However, this variation between samples can be advantageous because it allows investigators to attribute multiple samples to a common source. Hargreaves reviewed both hand-held Raman and FT-IR spectrometer techniques in 2012.124 The portability of Raman and FT-IR instruments is one of many reasons these methods are favored in law enforcement. Hargreaves describes that while FT-IR, like Raman spectroscopy, is nondestructive, rapid, and confirmatory, it does require direct contact with the sample for analysis. Buckley et al. and Faulds et al. published reviews of SORS and SERS methods in 2012.125,126 Buckley et al. described that SORS can be used to analyze samples contained within packaging, such as plastic, paper, or fabric. Faulds et al. explained that SERS allows investigators to detect incredibly small amounts of narcotics with the use of different substrates. This is valuable when the concentration of the drug is very low, such as in body fluids. In the past 2 years, several new approaches have been developed to identify, characterize, and even quantify illegal drugs using Raman and IR spectroscopy. Raman spectroscopy is rapid and nondestructive in nature. It can be used to quickly characterize samples as a screening test, before performing more time-consuming analyses.127 Traditional Raman spectroscopy can be used to detect drugs collected at crime scenes with tape or contaminants present in illegally produced alcoholic beverages.128,129 It is also being used to characterize new synthetic drugs, often referred to as “legal highs” because their manufacturers create these new compounds by slightly modifying the structures of known drugs in order to circumvent current laws.130 Quantitative analysis can also be performed to estimate an active ingredient’s concentration in a mixture. De Oliveira Penido et al. used both Raman and FT-IR spectra collected from binary mixtures of cocaine and four common adulterants with PCR to quantify the

Figure 6. Quantifying cocaine in binary mixtures with adulterants. Raman spectra collected from binary mixtures of cocaine with (A) sodium carbonate, (B) caffeine, (C) benzocaine, and (D) lidocaine, each prepared in several concentrations. Quantitative analysis of spectral data resulted in calibration curves that could be used to estimate the concentration of cocaine. Reprinted from de Oliveira Penido, C. A. F.; et al. Quantification of binary mixtures of cocaine and adulterants using dispersive Raman and FT-IR spectroscopy and principle component regression. Instrumentation Science and Technology, 2012, 40, 441−456 (ref 131). Copyright 2012, with permission from Taylor & Francis Ltd. 317



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medicine produced by pharmaceutical companies which properly follow government regulations. As such, patients ingesting counterfeit pharmaceuticals may be subjected to unexpected side effects, allergic reactions, or toxic adulterants. Forensic investigations of pharmaceuticals are charged with differentiating genuine and counterfeit drugs and identifying potential toxic adulterants in counterfeits. This information may be forwarded to enforcement, regulatory, and/or police agencies as evidence of criminal activity and to help elucidate the source of the counterfeit drug. Identification of the counterfeit source may provide investigators with the ability to trace the drug to a point of diversion (i.e., from a legitimate source where the drugs were lost or to a particular clandestine lab), to contain the threat and assist in the prosecution.144 However, several pitfalls exist which make these determinations increasingly difficult. Foremost, the gap between counterfeit and legitimate pharmaceuticals is shrinking, due to the emergence of so-called “high quality counterfeits.”145 Prominent methods for quality assurance of pharmaceuticals include colorimetric testing and TLC. The German Pharma Health Fund “Minilab” combines both of these methods in a contained unit for use in developing countries.146 Unfortunately, color reactions possess narrow specificity, and TLC is a relatively elementary technique which requires confirmation through more robust analytical studies.147 Chromatography and mass spectrometry are well investigated as confirmatory counterfeit detection methods.148 Specifically, high-performance liquid chromatography (HPLC) has been coupled with various detection methods for anticounterfeiting.148 However, HPLC requires relatively excessive sample preparation and analysis times. Chemical fingerprinting via CE has been applied to characterize the trace components in bulk pharmaceuticals. This method catalogs the ingredients of a pharmaceutical and is capable of distinguishing manufacturers and can differentiate between counterfeit and genuine pharmaceuticals. These fingerprints may be used to generate a library for comparative purposes. However, the approach is not ideal for finished products as excipients or matrix materials may interfere with the analysis.149 As discussed in the introductory section, vibrational and laser spectroscopies offer several of the attributes which are required to combat the growing industry of counterfeit pharmaceuticals. Specifically, there is a clear need for an analytical method for counterfeit identification which offers on-site, rapid, and highly specific identification of potential counterfeit pharmaceuticals. A review of the spectroscopic applications for anticounterfeiting prior to 2012 was provided by Bunaciu et al.150 Since that review, a trend to increase the emphasis on multivariate analysis to distinguish counterfeit from genuine pharmaceuticals has occurred. The speed and ease of analysis applied to anticounterfeiting is paramount. As such, Hajjou and co-workers investigated the use of the TruScan hand-held Raman device for the field identification of counterfeit and substandard medicine.151 TruScan offers the high specificity of Raman spectroscopy combined with the ease of operation and rapid analysis of a portable device. Analgesics of different strengths (different concentrations of the active pharmaceutical ingredient, API) and antimalarial drugs were investigated with the TruScan device. The authors concluded that the technology shows promise for identifying counterfeits both absent of the API or with an ingredient, which is different than the intended API. However, the method could not differentiate drugs with

immediate, quantitative, reliable, information in real-time. Studies have shown it can even be used to analyze drugs on complex substrates, such as clothing and textiles.132 SORS and SERS are two advanced applications of Raman spectroscopy that allow scientists to investigate smaller or concealed samples. SORS can be used to detect and quantify the concentration of drugs through opaque surfaces, such as plastic.133 SERS can be used to easily collect a Raman spectrum, even in conditions that typically produce significant interference from fluorescence.134 IR has many variations, each with several applications in forensic drug analysis. Spectral libraries of known drugs are often used to identify samples acquired by police. Researchers are constantly adding to these libraries as new drugs are encountered to keep the databases as current and helpful as possible.135 Tsujikawa et al. used a portable IR spectrometer to study 120 psychoactive drugs.136 Researchers optimized pretreatment and library searching algorithms to optimize search results. The developed method was then used to correctly identify 8 of 11 forensic samples. One of the most common forensic uses of IR spectroscopy is the detection and quantification of alcohol in breath.137 IR has also gained interest because it requires no solvents or sample preparation, making it a “green,” or environmentally conscious, method.138 Reflectance NIR spectroscopy is an inexpensive method that is still reproducible and suitable for forensic purposes. It can be used to analyze mixtures of drugs and create a concentration calibration curve. The results can be comparable to similar FTIR methods, while costing less.122 By combining ATR-FT-IR spectra with chemometrics, drugs can be differentiated based on their purity, or concentration, and form, such as hydrochloride or the free base form.139 Because Raman and IR spectroscopy are complementary, they are often studied together. Experimental spectra can be compared to spectra calculated using density functional theory to interpret observed peaks. By calculating the vibrational spectra of different conformations of the same molecule, more information can be elucidated and understood from its Raman and IR spectra.140 Raman and IR spectroscopy exhibit several advantages over competitive techniques. Portability allows analysis to be performed directly at the scene of the crime or wherever the evidence is collected. Additionally, the instrumental parameters do not need to be specifically tailored to different substances, which is the case with GC/MS. Spectral libraries can be used to identify substances without obtaining and analyzing a standard. Even complicated samples, with high fluorescence profiles or low Raman activity, can be characterized by advanced techniques, such as SERS and SORS. Pharmaceuticals. The World Health Organization defines a counterfeit medicine as “deliberately and/or fraudulently mislabeled with respect to identity and/or source.”141 These drugs may fall into one or more of the following categories: products without active ingredients, products with incorrect quantities of active ingredients, products with wrong ingredients, products with correct quantities of active ingredients but with fake packaging, copies of an original product, and products with high levels of impurities and contaminants.142 Counterfeit drugs consist of approximately 10% of the global medicine market, with rates considerably higher in developing countries which lack strong regulatory and law enforcement agencies.143 Counterfeit drugs are not subjected to the same quality control and clinical trials as 318



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multivariate analysis approaches for EMA detection.159 Raman spectra collected from the adulterated samples were compared to pure samples via the use of a spectral library and HQI. HQI values were used to determine if sorbitol samples were adulterated with ethylene glycol (EG) and diethylene glycol (DEG). Samples adulterated with EG and DEG were flagged as suspicious only at quantities containing more than 10% adulterants. However, when these same spectra were used to build SIMCA and PLS models, the rates for the limit of detection were lowered to ∼2% and ∼0.9%, respectively. A common pitfall associated with the analysis of intact pharmaceutical capsules or medication in sealed containers with backscattering Raman spectroscopy is the potential for fluorescence interference. SORS is able to suppress signals from containers and collect data from the inner contents of a sample due to its inherent diffusion approach, which utilizes a distance between excitation and collection locations. SORS has been applied to analyze bulk pharmaceutical materials and adulterants through different containers160 and within different capsules.161 Bloomfield et al. applied SORS as a noninvasive, portable technique for pharmaceutical materials characterization.160 Transmission Raman spectroscopy, a form of SORS in which the area of excitation and collection are on different sides of the sample, was used to analyze API/excipient mixtures within various colored and capsules and capsule thicknesses.162 Lee et al. developed a calibration data set from samples of API/excipients (ambroxol/lactose, respectively) of different concentrations in glass containers of various thicknesses. The authors used this calibration data set to estimate the concentration of ambroxol within capsules of varying color and thickness. Results indicated that the transmission approach could suppress fluorescence from the capsules, and spectral features from the ambroxol/lactose did not vary based upon capsule color. The authors additionally investigated the relationship between the spectral data and the concentration of ambroxol via PLS analysis. They reported that the accuracy of the PLS predication of Raman spectroscopic data is comparable to current HPLC methods and satisfies Korean Pharmacopoeia requirements. SEM-EDS was used in conjunction with Raman spectroscopy for the chemical mapping of pharmaceutical tablets.163 The authors utilized multivariate analysis to determine the pharmaceutical dosage for solid state samples. Raman spectroscopy was used to distinguish components not identifiable in the EDS maps. The different approaches provide complementary information which may enhance the probability of detecting a counterfeit sample. Several emerging Raman technologies have been applied to forensic pharmaceutical analysis. A new method for the detection of pharmaceutical ingredients was developed with the use of SERS labeled sensing.164 It was observed that the SERS sensor provided different Raman signatures in the presence or absence of the target analyte. For this study, the target analyte was the antibiotic erythromycin. The developing approach shows promise as a rapid method for the detection of counterfeit medicine, void of an API. Additionally, ultraviolet fiber enhanced resonance Raman spectroscopy has been applied to the analysis of several antimalarial drugs.165 A linear Raman signal was obtained in terms of analyte concentration. Therefore, the method may have applications for detection of substandard medicine with lower than expected concentrations of an API.

different concentrations of API or that consisted of a mixture of APIs (i.e., common antimalarial and HIV/AIDS treatments). The authors concluded that the technique may have applications for the rapid detection of counterfeits void of the API but should not be applied to “substandard medicine” which contains lower than expected concentrations of the API. To address some of the concerns associated with portable Raman spectroscopy, Feng et al. combined the portable Raman spectroscopic approach with multivariate analysis.152 Local straight-line screening and PCA models were built to distinguish Raman spectra collected from counterfeit and authentic pharmaceuticals. The method was capable of discerning three different types of counterfeit hypoglycemics from their authentic counterparts: (1) counterfeits without active ingredients, (2) counterfeits with an API which differs drastically in chemical structure, and (3) counterfeits with an API which differs slightly in chemical structure (so-called chemical analogues for the correct API). The authors reported that the method was capable of analyzing a sample in less than 1 min and reported sensitivity/specificity results of 96.8% and 97.5%, respectively. Detection of counterfeit erectile dysfunction medication through spectroscopic analysis has included Raman153,154 and ATR-FT-IR155,156 spectroscopy on their own and coupled with several multivariate methods, as well as a combined approach utilizing NIR, FT-IR, and Raman spectroscopy.157 Fraser et al. attempted to differentiate counterfeit and authentic Cialis with Raman spectroscopy combined with multivariate analysis.154 The authors also targeted the identification and quantitation of the API (tadalafil) in this study. Results were found to be dependent upon the multivariate analysis utilized. For example, PLS, support vector machines (SVM), and PCR were the most effective for distinguishing samples; however, SIMCA was the most accurate at classifying the samples as genuine or counterfeit. The fingerprint region of Raman spectra was utilized to quantitatively estimate the concentration of the API via PLS regression. The authors suggest this scheme may be utilized to estimate the concentration of the API in substandard medicine. Alternatively, Ortiz et al. investigated the use of ATRFT-IR spectroscopy combined with multivariate analysis for the examination of counterfeit Cialis and Viagra.155 PCA differentiated spectra collected from authentic and counterfeit samples. Because of clustering of spectra in the PCA space, the authors postulate that counterfeits from seizures across the globe originated from the same sources. The ability to correlate counterfeits from different parts of the world to the same source may provide investigators with enough evidence to prosecute or augment the level of the charge. The authors recommended generating a spectroscopic library for real world applications of the approach. Similarly, Quin et al. recommended the use of a spectral library and hit quality index (HQI) algorithm for the identification of expired pharmaceuticals.158 Gryniewicz-Ruzicka and co-workers reported a novel advancement to lower the detection limit of economically motivated adulterants (EMAs) in pharmaceuticals when using Raman spectroscopy.159 EMAs are chemical analogues of excipients or bulking agents that are commonly used in pharmaceuticals. EMAs are considerably cheaper but, often, toxic alternatives. Therefore, the detection of EMAs regularly indicates the presence of a counterfeit drug. The authors compared the use of spectroscopic libraries (a method currently employed to pass/fail potential counterfeit pharmaceuticals) to 319



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Figure 7. Discrimination of 14 explosive compounds using Raman spectroscopy and principal component analysis. Key below figure shows color coding of the explosives. (a) Three-dimensional scatter plot of 14 different explosives scored on the first three components showing separation. (b) Closer view of the three-dimensional feature space showing the separation of 10 of the explosives. Reprinted from Hwang, J.; et al. Fast and sensitive recognition of various explosive compounds using Raman spectroscopy and principal component analysis. Journal of Molecular Structure, 2013, 1039, 130−136. (ref 183). Copyright 2013, with permission from Elsevier.

destructive to the sample, which is detrimental to further forensic investigation. Additional problems associated with these analytical methods include a lack of selectivity, inability to detect substances in sealed containers, and limited portability of instruments and standoff capacity. Raman and IR spectroscopy are well established techniques for explosives analysis. Portable instruments have even been in use for several years.169 Lewis et al. organized a tremendous amount of information about current methods used to study explosives and precursors by Raman spectroscopy as a detection tool.170 In their review, Caygill et al. addressed almost all analytical techniques for explosives detection, such as spectroscopic and sensor techniques, olfactory-type sensors, nanotechnology, and so forth.171 Skvortsov reviewed laser spectroscopy methods for standoff detection of explosives in the form of particles on objects’ surfaces.166 A recent review on IR and Raman spectroscopy by López-López and Garcı ́a-Ruiz summarizes new trends and progress that have been achieved for explosive material identification up until the beginning of 2014.172 The review covers several topics such as forensics, homeland and international security, and environmental applications. Significant progress has been made in the development of standoff Raman spectrometers, but there are still technical challenges that remain unresolved.166,168 Raman spectroscopy does not satisfy the requirements for trace level detection of explosives at long-range distances, but identification of bulk explosives has recently been studied.168 Raman spectroscopy has also been used to quantify the concentration of ingredients in explosive mixtures.173 However, there are a limited number of studies which have identified explosives in trace amounts using portable Raman spectrometers in the past 2 years.174,175 IR spectroscopy has shown the potential to be used in explosives detection from a distance. One study used FT-IR spectroscopy for the identification of several explosive samples measured at a distance of 5 m.176

Because of the advent of high-quality counterfeit pharmaceuticals, new approaches must be developed to protect the public from the dangers associated with them. Conventional vibrational spectroscopic approaches for anticounterfeiting have recently emphasized the need for chemometric analysis of spectral data. Additionally, SEM-EDS-Raman spectroscopy has illustrated great potential for the future of anticounterfeiting.

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COUNTERTERRORISM AND HOMELAND SECURITY Explosives. Explosive material can be divided into two classes. The first class has a relatively high nitrogen content due to at least one nitro (−NO2) or nitrate (−NO3) group. The second class includes peroxides, perchlorates, and azides.166 The mid-IR range contains the fundamental vibrational− rotational transitions of almost all explosives, which makes IR spectroscopy very promising for their identification.166 However, these energetic materials used to construct explosive devices are still difficult to detect and remain the primary threat to military and civilian personnel. Detection is challenging also in part due to the wide range of chemical structures, inherently low vapor pressures, and possible sample degradation.167 There is a critical need for reliable technology that offers standoff detection of explosive compounds, including the ability to detect explosives in very small amounts, in liquid or solid form, and in different containers. There is a desire for a detection method with high sensitivity and selectivity that would not be affected by environmental conditions.168 There are several different analytical methods that can be considered for detecting explosive traces, such as desorption electrospray ionization mass spectrometry, laser electrospray mass spectrometry, various forms of chromatography, colorimetric indicators, electrophoresis, ion mobility spectrometry (IMS), antibody/antigen-based assays, and laser-based techniques like laser-induced breakdown spectroscopy (LIBS).167 However, most of these techniques require a laboratory equipped with the appropriate materials, infrastructure, and trained personnel. Furthermore, some of these approaches are 320



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Raman spectroscopic methods but is limited to nonmetallic containers. There has been significant progress in explosives research under laboratory conditions using these advanced techniques as well as traditional Raman and IR spectroscopy, which will help to improve the current analyses used. Chemical Agents. When it comes to the detection of dangerous chemical agents that threaten public safety, the size and speed of the detector is crucial but still must allow for very high accuracy and reproducibility. Several methods are used to detect chemical agents, such as IMS, GC/MS, GC/flame photometric detection, and surface acoustic wave sensors. Although conventional and molecular assays are the standard techniques allowing for identification of toxins, they are laborious and time-consuming.186 The detection and prevention of chemical agent attacks requires sensitive, reliable, rapid, portable, and noninvasive techniques. Mogilevsky et al. published a comprehensive review in 2012, covering the applicability, strengths and weaknesses of several Raman spectroscopy techniques, including SERS, SORS, temporally offset Raman spectroscopy (TORS), and resonance Raman spectroscopy.187 Raman spectroscopy is a nondestructive technique that keeps the sample intact, can identify substances in any physical state as well as through sealed containers. As the authors demonstrated, basic Raman spectroscopic techniques can be further enhanced by combining them with other complementary technologies. In 2014, Bhardwaj et al. published a review of the field, which reported on updates since 2012, including the potential of SERS for chemical and biological toxin detection.186 SERS has shown a great capacity for detection of released biological and chemical toxins, which can be extended to defense applications. Advantages include the availability of hand-held instruments, label-free analysis, and simultaneous detection of multiple chemicals with highly enhanced sensitivity. Over the past decade, development of new technologies for the detection of chemical agents has experienced a shift from conventional to advanced techniques.186 SERS is a portable, ultrasensitive tool for detecting chemical agents based on molecular fingerprinting which makes this spectroscopic technique a potentially better choice over other currently established detection methods. However, there are still limitations in direct detection by SERS. This technique has an intrinsic lack of selectivity. Real samples are very complex and include variability of naturally occurring SERS active species that bind to SERS active surfaces. Bioagents. Biological agents (bioagents) are a group of organisms that are pathogenic and can be used as biological weapons. Bioagents are a serious concern in part because of the ease with which they can be produced and transported. They are usually very effective, odorless, and there is a lag time between the attack and appearance of a victim’s symptoms during which the bioagents stay undetected. It is clear that rapid and accurate bioagent (viruses, fungi, bacteria, and bacterial spores) identification is a task of a great importance for defense and homeland security in order to prepare for potential attacks. The requirement for rapid analysis is not met by current methods, such as polymerase chain reaction and MS. Immunoassay kits can be relatively fast, but they have shown only moderate sensitivity.188 Vibrational spectroscopic methods could potentially fulfill the need for rapid, portable, easy to use instruments for on-site detection of bioagents and their toxic byproducts.

Detection of explosives and their precursors in trace amounts is in high demand by security and counterterrorism forces. Nuntawong et al. used SERS to evaluate trace amounts of explosive samples using a novel preparation technique and successfully demonstrated its highly enhanced sensitivities.175 Trace amounts of explosives and their precursors can also be found on clothing or other belongings of subjects under investigation. Confocal Raman microscopy was used to identify some common explosives and precursors deposited on fabric material.177 Portable Raman spectrometers were used for in situ analysis, and spectra of the explosive substances were identified without significant interference from the substrate. Identification of unknown and possibly hazardous substances from nonmetallic containers is an issue of major importance in many different fields. SORS suppresses fluorescence and Raman scattering from containers and allows users to obtain a Raman spectrum of its contents. Recent studies have demonstrated the feasibility and applicability of SORS in noninvasive screenings of concealed substances.178,179 In order to improve the signal-to-noise ratio in SORS measurements, time-resolved and spatially offset Raman spectroscopy (TR-SORS) has been used to identify chemical substances. TR-SORS can be used to acquire high-resolution spectra under ambient light conditions and identify a concealed substance with only one measurement by suppressing fluorescence from the container’s surface. In a pilot study, Cletus et al. used TR-SORS to identify explosives in plastic containers under sunlight, fluorescent, and incandescent light from a distance of 6 cm.180 More recently, these authors extended the application of TR-SORS for the standoff detection of explosive ingredients inside opaque containers from a distance of 15 m.181 Loeffen et al. has used SORS to study chemical and explosive materials concealed within four different containers while varying offsets.182 Samples that present analytical complexities, such as fluorescence or heterogeneity, can usually be corrected for with simple adjustments to the method. For example, fluorescence can be avoided by changing the excitation laser wavelength. Hwang et al. used three different excitation laser wavelengths to analyze explosive compounds by Raman spectroscopy.183 They found that the 514.5 nm laser achieved the best scattering and resulted in a better signal-to-noise ratio than the two longer wavelengths, 633 and 785 nm. Subsequently, they used PCA for feature extraction and spectral identification of the 14 selected explosives, shown in Figure 7. López-López et al. developed a new technique to characterize dynamite, a heterogeneous solid mixture of several explosive compounds.184 Raman mapping was used to probe the surface of the dynamite and identify several of its major and minor components. Gares et al. studied photodegradation of solid trinitrotoluene (TNT) and TNT prepared in solutions using deep ultraviolet resonance Raman (DUVRR) spectroscopy.185 This enabled DUVRR detection of solid TNT and its solutions, as well as its photoproducts, after degradation by sunlight. Overall, vibrational spectroscopic techniques appear attractive for the analysis of explosives because of low false positive rates, fast collection times, and their nondestructive nature. Raman and IR spectroscopy can suffer from low sensitivity, limiting their applications in the field. However, SERS can be used to overcome this constraint on Raman spectroscopy. SORS is extremely beneficial for detailed chemical analysis of samples in sealed containers not accessible by conventional 321



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Raman remote sensing.192 More recently, Chalmers et al. edited a book which provides in-depth information about forensic uses of both Raman and IR spectroscopy.193 Although there have been numerous reviews regarding the use of Raman and IR spectroscopy for specific forensic applications, these three published works offer more comprehensive overviews and suggest that they are both growing and useful techniques for the field of forensic science. Moreover, there are a variety of studies which have been completed showing the continued promise of using Raman and IR spectroscopy in forensic evidence analysis. However, as important as these applications have been, vibrational spectroscopic techniques are still not widely used in practice for all areas of forensic science. Within the past few years there have been a number of technologies that have shown significant advancement and success in applying Raman and IR spectroscopy to forensic science. Although all of these cannot be mentioned here, some of the more promising technologies are discussed. One technology that has shown significant advancement is a confirmatory identification of body fluid traces using Raman microspectroscopy. This single nondestructive method could be used for all main body fluids found at a crime scene and is advantageous over current biochemical tests, which are destructive and body-fluid specific.194 A new method for the detection of biological stains has been introduced by Dr. Stephen Morgan and co-workers at the University of South Carolina, who have patented the technique of thermal infrared imaging analysis.195 These two emerging technologies have great potential to significantly improve the detection and identification of biological stains at a crime scene and the efficiency of forensic serology, including the utilization of DNA evidence. Another emerging technology is in the area of gunshot residue (GSR) detection and analysis using vibrational spectroscopy. Dr. Lednev’s laboratory in Albany, New York, and Dr. Garcı ́a-Ruiz’s research group in Madrid, Spain, have independently initiated this novel methodology and made significant progress over the past 2 years. These investigations include the analysis of GSR particles from several types of ammunition, discriminating between organic and inorganic GSR samples, analyzing GSR originating from both the primer and propellant of the ammunition as well as different propellant (gunpowder) mixtures, analysis of GSR from different firearmammunition combinations, and analysis of spectra from subsequent discharges (weapon memory).7−9,116−119,121 This area of forensic science is very important in helping to link a suspect to a shooting incident. Current techniques for GSR analysis have limitations and disadvantages, which could be overcome by using vibrational spectroscopy to provide additional sample information. SORS, invented by Dr. Matousek of the Rutherford Appleton Laboratory, has been used as a noninvasive approach for a variety of forensic applications within the past few years. These applications include the analysis of drugs, explosives, and more. To acquire Raman spectra, this technique utilizes a spatially offset region between the point of laser beam excitation and collection. By increasing the offset, deeper penetration beneath the surface is afforded, ultimately resulting in obtaining spectra of contents within various types and thicknesses of nonmetallic containers. In 2008, Neil A. Macleod and Pavel Matousek wrote an expert review on emerging noninvasive Raman methods for both forensics and process control discussing transmission Raman spectroscopy and SORS.196 This technique has been

Pathogens raise serious concerns for human health and are an important challenge for scientists. Pathogens can present themselves as contaminants in the environment, or food,14 or as biological weapons used in an attack. They can be identified directly, in food, or even in human body fluids, such as blood or urine.189 The Popp research group at the Institute of Physical Chemistry, Friedrich Schiller University, Jena, has made several new contributions to pathogen detection in biomedical diagnostics, the food industry, the environment, and bioagent analysis. One recent innovative technique is capturing microorganisms from complex media using an antibody-modified aluminum substrate and Raman spectroscopic analysis.190 Rösch et al. reviewed vibrational spectroscopic methods used for bioagent identification in forensic applications.11 For Raman spectroscopy, the measurements were mainly performed with excitation laser wavelengths in the region of NIR. Two methods were used in order to enhance the Raman signal; one was the use of excitation laser wavelengths in the ultraviolet region. In this case the Raman signals of specific compounds in the mixture are selectively enhanced. The second approach to increase Raman sensitivity was to use SERS for bioagent identification. Recently it has been shown that combining a rapid extraction of dipicolinic acid from Bacillus cereus spores with SERS analysis, the spores can be identified in 2.5 min using a portable Raman analyzer.188 These authors also summarized their effort to develop a portable Raman analyzer for the rapid detection of Bacillus anthracis spores using SERS active capillaries. Anthrax remains the most likely threat of all biological agents and, as such, it is the subject of great interest in the scientific community. Researchers are constantly working to improve detection and transmission prevention methods. Stöckel et al. has identified anthrax endospores in the presence of other Bacillus species in powder form.12 Using microscopy and Raman spectroscopy they distinguished anthrax from the other Bacillus species with an overall accuracy of 96.8%. Bioagents can be characterized by IR and Raman spectroscopy on the basis of their spectra, which provide specific fingerprints that allow for their accurate identification. Also, the nondestructive character, rapidness, and portability of many Raman instruments suggest that the number of applications will increase, especially with other technical developments. Further research into bioagent identification should focus on detection limits, reproducibility, selectivity, and speed of spectroscopic analysis.

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EMERGING TECHNOLOGIES Both Raman and IR spectroscopy have been important developing techniques in the field of forensic science. Through their selective nature they provide a greater amount of detail, in terms of specific chemical information, than many of the current techniques used by forensic examiners. These two techniques are not new; they are both very well established and have been progressing extensively over the past decade and, more prominently, within the last 2 years. In 2002 Edward Bartick published a paper for the proceedings of the 16th meeting of the International Association of Forensic Sciences demonstrating that Raman Spectroscopy is an emerging technology in forensic science.191 Since then there have been numerous studies undertaken which use Raman spectroscopy in a forensic context. In 2010 Emad L. Izake published a review on the use of portable Raman spectroscopy for homeland security applications focusing mainly on SORS, SERS, and 322



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advantages of using vibrational spectroscopy in a forensic context.

used in the identification of concealed explosives in glass and opaque plastic containers.197−200 More recently, SORS has been used to analyze different layers of paint samples.62 These important findings demonstrate the progress of SORS and its use for various forensic applications. A much newer technique, tip-enhanced Raman spectroscopy (TERS) has only recently been applied for uses related to forensics. TERS is a novel technique which takes advantage of the combined chemical information obtained from Raman spectroscopy with the high spatial resolution afforded by scanning probe microscopy to obtain sensitivity on the single molecule scale. A study out of the Van Duyne research group by Kurouski et al. used TERS to analyze iron gall ink and indigo dye on rice paper.201 This technique was performed in situ on a handwritten document dated back to the 19th century and on a reference sample. Both inks were identified based on observed vibrational modes in the spectra which were consistent with components in the inks. It has also been demonstrated by Deckert and co-workers that TERS has great potential for determining the sequence of a single DNA strand.202 This type of analysis could possibly allow for DNA typing for forensic purposes based on a single DNA molecule recovered at a crime scene. These studies provide some insight that TERS could be used for future forensic applications including, but not limited to, ink analysis for questioned documents and DNA typing. Combining chemical spectroscopic information obtained from Raman spectroscopy with elemental information offered by other methods opens new exciting opportunities in analytical chemistry in general and forensic science in particular. Two techniques that offer this dualistic type of information are Raman-laser-induced breakdown spectroscopy (Raman-LIBS) and Raman-scanning electron microscopy-energy dispersive Xray spectroscopy (Raman-SEM-EDS). LIBS and SEM-EDS have been used extensively as separate techniques for forensic applications but have only recently been combined with Raman spectroscopy for similar types of studies. LIBS uses a high power laser source to impact a sample and produce evaporated material due to the high temperature. The light emission from the plasma (containing both excited and neutral species of the ablated matter) generated creates the analytical signal measured by LIBS.203 SEM-EDS combines the extremely high magnification powers of SEM with the high spatial resolution and elemental selectivity of EDS for very accurate analyses. The incorporation of Raman spectroscopy with these two elemental analysis techniques allows for simultaneously obtaining two vastly different, but corroborating, types of data. This approach provides advantages over current methods since one sample can be used for two different types of analyses. The combination of Raman-SEM-EDS is a very new concept and therefore has not been extensively used for forensic studies to date. Although these integrated techniques have not been mentioned frequently in the literature for forensic use, their advantages could be extremely useful in the future. Overall, spectroscopic methods are becoming increasingly more popular for forensic evidence analysis. It would be beneficial if these methods were to be used for real-world evidence analysis. The incorporation of some of these applications into practical use is a tangible possibility for the near future. They could extend existing capabilities and corroborate results of techniques currently being used, potentially allowing for a more thorough case in court. These emerging technologies only fortify the breadth and potential

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CONCLUSIONS Forensic science is in a unique position, compared to other scientific fields, because of its direct and substantial impact on society. For these reasons, it must be held to the highest scientific and ethical standards. As stated by the National Academy of Sciences, forensic techniques must be sound methods that can accurately interpret evidence without allowing for personal bias. Additionally, destructive techniques must be avoided so that evidence can be preserved for future analysis. Overall, forensic methods must be efficient and reliable. Vibrational spectroscopy methods, such as Raman and IR, are already useful tools for forensic scientists. Their quantitative and nondestructive nature ensures that analyses are objective and preserve the evidence. As expected, spectroscopic methods are becoming increasingly more popular for forensic evidence analysis. These new developments could have a significant impact on the field of forensic investigation. Portable Raman and IR spectrometers continue to become more affordable and accessible to crime laboratories. Raman and IR spectroscopy could potentially be used at the scene of a crime to analyze almost any form of physical evidence, without the risk of destruction. This review demonstrates how valuable vibrational spectroscopy is in the field of forensic science. Many techniques are still being developed while others already have countless applications and have proven their validity. As the need for more information from evidence arises, the development of new vibrational spectroscopic techniques will follow suit. These methods have shown their importance in forensic science thus far and will continue to do so for years to come.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Claire K. Muro graduated magna cum laude from the University at Albany in 2012 with a B.S. in Forensic Chemistry and B.A. in Anthropology. After graduating she started working on her doctorate and joined the Lednev Research Group, where she is currently a Ph.D. candidate. She is particularly interested in new forensic developments in body fluid analysis and osteology. Her research projects center around Raman spectroscopy as a forensic tool and the use of chemometrics to extract complex information from spectral data. Kyle C. Doty earned his B.A. in Chemistry and B.S. in Forensic Chemistry (2009) from SUNY Buffalo State College. From 2010 to 2012 he worked as a formulations scientist in the division of research & development at Bausch & Lomb, Inc. He is skilled in the area of vibrational spectroscopy and the application of chemometrics for data analysis. Currently he is a third year doctoral student in the Chemistry department at the University at Albany, working under the mentorship of Dr. Igor Lednev, where he focuses on research projects which target the development of novel methods for forensic purposes. Justin Bueno studied at the University at Albany where he received his B.S. in Chemistry with an emphasis in forensics in 2010. He continued his work at University at Albany and received his Ph.D. in Analytical 323



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Chemistry in 2013. His Ph.D. dissertation investigated a new method for gunshot residue detection and characterization via vibrational spectroscopy. His thesis was granted the Distinguished Doctoral Dissertation award from the University at Albany College of Arts and Sciences. Justin is continuing his career in research utilizing Raman spectroscopy. Lenka Halámková received her B.S. and M.S. in Biology and Ph.D. in Geology from the Masaryk University (Brno) in the Czech Republic. After completion of her degrees she was appointed as a Research Associate in the Academy of Sciences, the Czech Republic. She has over 4 years of experience in bioanalytical science and technology where her principal focus was on biofuel cells controlled by biochemical signals, biocomputing based on enzymes and logical gates, bioelectronics, optobioelectronics, and biosensors. She recently joined the group of Dr. Igor Lednev (University at Albany) as a research scientist working on the application of advanced statistics for spectroscopic data analysis. Igor K. Lednev is a professor at the University at Albany. He received his Ph.D. from the Moscow Institute of Physics and Technology, Russian Federation. Lednev’s expertise is in the development and application of novel laser spectroscopy for biomedical research and forensic purposes. Dr. Lednev served as an advisory member for the White House Subcommittee on Forensic Science and on editorial boards of four scientific journals including the Journal of Raman Spectroscopy. Dr. Lednev is a fellow of the Society for Applied Spectroscopy. He received the Research Innovation Award and the University President Award for Excellence in Research. He has coauthored over 160 peer-reviewed publications.

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ACKNOWLEDGMENTS

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REFERENCES

This project was supported by Award No. 2011-DN-BX-K551 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice (I.K.L.). The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the U.S. Department of Justice. We would also like to thank Jeremy Manheim and Ewelina Mistek for their assistance in the preparation of this manuscript.

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