Discovered by Nobel laureate C. V. Raman in 1928, Raman spectroscopy is an analytical technique based on the scattering of light, and has proven to be of great use to forensic investigations. The Raman spectrometer consists of a number of principal components: a light source to produce the incident light, a prism or mirror and focusing lenses to direct the beam towards the sample, and a spectrometer for the detection of scattered light.
A beam of monochromatic light (light of a single colour) is introduced to the sample, typically using a laser as the source. When light impinges on a substance, the majority of it will be absorbed or pass through the sample. However a small fraction of this light is scattered, and it is the scattered light that is of interest in Raman spectroscopy. The light will be scattered in one of two ways; elastic scattering (also known as Rayleigh scattering) or inelastic scattering (known as Raman Scattering).
When Rayleigh scattering occurs, a photon interacts with a molecule in such a way that it is raised to a virtual energy state. This state is very short-lived and the molecule soon returns to its ground state, releasing a photon of equal energy to the initial photon. In this instance the light does not lose or gain energy during the scattering process and thus the scattered light has the same wavelength as the excitation source.
However Raman scattering, or inelastic scattering, involves some kind of change in energy and thus a change in wavelength. When light encounters molecules in the sample, they can interact in such a way that energy can be either lost of gained by the scattered photons. The shift in frequency corresponding to the energy difference between the incident photon and the scattered photon is the Raman shift. This can occur as either a Stokes shift (in which the emitted photon has lost energy) or an anti-Stokes shift (emitted photon has gained energy). These shifts are detected and transformed into a visual representation of the data gathered.
The result of this analysis is the production of a Raman spectrum, which plots the intensity of the shifted light versus the frequency (typically measured in a unit called the wavenumber). This spectrum provides an array of information relating to the molecular vibrations experienced by a sample, the correct interpretation of which can allow for sample identification, as different materials have different vibrational modes and thus characteristic spectra. Understandably the spectra produced can be complex, however spectral fingerprinting can be used to compare the overall pattern of spectra, including any similarities and differences in band intensities and band positions.
Although analytical techniques such as chromatography and mass spectrometry are most typically used in forensic analysis, Raman spectroscopy has a distinct advantage in that it is a non-destructive technique. A light source is simply directed over the sample during analysis, after which the sample remains undamaged and can be used for alternative analyses. This is an obvious benefit to a forensic investigation in which only a limited amount of sample may be available for such work.
Furthermore, this technique is significantly simpler in terms of sample preparation. Gas or liquid chromatography require a sample to be at the very least dissolved in some form of solvent, if not first taken through procedures of extraction or separation before being amenable to chromatographic analysis. However Raman spectroscopy requires little or no sample preparation, saving time whilst also reducing the number of opportunities for samples to become compromised in some way. In addition to this, the sample does not have to be analysed in a particular state, as solid, liquid or gaseous samples are amenable to Raman analysis, and samples can even be analysed through glass, plastic or quartz, eliminating the need to even remove a sample from its packaging.
A problem faced by infrared spectroscopy, a technique somewhat similar to Raman spectroscopy, is the absorption effects caused by water. However because Raman spectroscopy is based on the scattering of light rather than absorption, it does not suffer from these effects and thus there is no need to dry samples prior to analysis. This is obviously a major benefit, both in terms of time spent preparing samples but also when conducting in situ field work.
However Raman spectroscopy is not without its caveats. When incident light interacts with a sample, only a small amount of the light is scattered, and an even smaller proportion of that light experiences Raman scattering. As a result of this, signals pertaining to Raman scattering are comparatively weak, and thus can be easily masked by fluorescence and other interferences. Although this technique can be applied to a vast range of sample types in any state, it cannot be used for the analysis of metals or alloys.
Advances in Raman Spectroscopy
Portable Raman Spectrometer
Raman spectroscopy lends itself to the possibility of portable instrumentation. The invention of handheld Raman devices allows for in situ analysis of samples at incident scenes, enabling investigators to quickly determine the likely identity of a substance without being required to collect and send the sample to the lab first. Modern handheld spectrometers are even fitted with a simple “Match/No Match” feature (when configured with the appropriate libraries), making analysis by Raman spectroscopy simple and even suitable for unskilled operators. Furthermore, the ‘point-and’click’ nature of these handheld devices can be particularly beneficial when dealing with potentially dangerous samples, eliminating the need to hazardously handle and transport substances for analysis. Similarly, fiber-optic probes can even allow for analysis from a distance.
Surface-Enhanced Raman Spectroscopy (SERS)
In order to remedy the problems of low sensitivity of Raman scattering, a technique known as Surface-Enhanced Raman Spectroscopy, or SERS, has been developed. This involves using a metal surface (often gold or silver) with a nanoscale roughness, typically produced by electrochemical roughening or metallic coating, onto which analyte molecules are absorbed. The extent to which Raman signals are enhanced can be so great that it allows for the detection of even a single molecule. Not only is SERS highly sensitive, it is also surface selective, meaning the surface of a material can be specifically analysed. This could not previously be achieved using Raman spectroscopy, as any Raman signals from surface analytes would most likely be overwhelmed by signals resulting from the bulk of the sample.
The potential applications of Raman spectroscopy to forensic analysis are plentiful, with the technique being increasingly used to ascertain details of a range of sample types.
A particularly beneficial application of Raman spectroscopy in forensic science is in the analysis of ink samples on questioned documents (Braz et al, 2013). If the source or authenticity of a particular document is drawn into question, physical and chemical analysis of inks used can often be vital in establishing whether or not any additions or alterations have been made, whether two ink contributions are likely to be from the same source, and even the chronological sequence of ink contributions. Pen inks are complex liquids composed of multiple pigments, dyes, resins and solvents, resulting in a wide range of potential ink compositions. These compositions will undoubtedly vary between different pen manufacturers, allowing for the analysis of seemingly identical ink samples to prove whether or not they are actually likely to have originated from the same source.
In the study of questioned documents, it is often not ideal to destroy a writing sample in order to conduct analytical chemical testing, such as would be required when carrying out mass spectrometry. Raman spectroscopy allows for disputed writing samples to be chemically analysed without the need for sample preparation and without losing the document’s evidential value. This particular application of Raman spectroscopy is not limited to pen ink, but can also be utilised in the analysis of printer inks and paint samples.
The analysis of blood has interestingly been subjected to Raman analysis, investigating the possibility of using Raman techniques to identify whether a substance is blood and even attempt to age the blood sample (Boyd et al, 2011). The research concluded that Raman spectroscopy was able to identify the presence of blood, with peaks characteristic of blood components, particularly haemoglobin, being frequently detected. This was achieved even when blood samples were significantly diluted. Furthermore, the research has investigated the possibility of using Raman spectroscopy to determine the age of blood in a rudimentary fashion, based on differences in scattering peaks between fresh and dried blood samples. With the possibility of portable Raman technology, the ability to detect blood using this technique would allow for in situ analysis at crime scenes, enabling investigators to quickly ascertain whether a substance is actually blood.
Although substances of abuse are most commonly analysed using chromatographic techniques coupled with mass spectrometry, Raman spectroscopy has been successfully utilised in the detection of illicit substances (Moreno et al, 2014). Provided the investigator has access to an appropriate library of reference spectra for comparison purposes, it may be possible to identify drugs of abuse and their metabolites based on the characteristic spectra produced. The benefits of the application of Raman spectroscopy in this case include the possibility of in situ analysis to quickly identify potential controlled substances and the ability to analyse samples without sample preparation or destruction.
In addition, Raman spectroscopy has been successfully applied to the analysis of gunshot and explosive residues, geological samples, fibres and various bodily fluids.
Boyd, S. et al. Raman spectroscopy of blood samples for forensic applications. Forensic Sci Int, 208 (2011), pp. 124-128.
Braz, A. et al. Raman spectroscopy for forensic analysis of inks in questioned documents. Forensic Sci Int, 232 (2013), pp. 206-212.
Izake, E. L. Forensic and homeland security applications of modern portable Raman spectroscopy. Forensic Sci Int, 202 (2010), pp. 1-8.
Lepot, L. et al. Application of Raman spectroscopy to forensic fibre cases. Sci Justice, 48 (2008), pp. 109-117.
Moreno, V. M. et al. Raman identification of drugs of abuse particles collected with colored and transparent tapes. Sci Justice, 54 (2014), pp. 164-169.
University of St Andrews. Raman Spectroscopy. [online] Available: https://www.st-andrews.ac.uk/seeinglife/science/research/Raman/Raman.html