Recent Advances in Raman and Surface Enhanced Raman Spectroscopy: Pharmaceutical, Forensic and Biomedical Applications

Raman spectroscopy is a potent analytical tool with an extraordinary and increasing number of applications. C.V. Raman in 1928 received the Nobel Prize in Physics for this discovery which was named after him, although the technique was first predicted by A. Smekal in 1923. In the last 35 years the pace of change for this technique has been rapid, as exemplified by the number of publications in the literature, where it has endured an extraordinary journey. Raman spectroscopy has advanced in recent years where its use in both industry and academia has increased significantly, and is redrawing the landscape in many fields such as biomedical and pharmaceutical R&D. The current excitement in this extremely active arena is clear to see by the explosion of publications in the last decade. This is largely a consequence of improvements in instrumentation, decreased cost, user-friendliness (one no longer needs to be a laser specialist to use this method) and employment of chemometrics (to assist in data analysis), as well as the development of Raman techniques such as SERS and TERS (surface-enhanced Raman scattering and tip-enhanced Raman scattering, respectively) and many other exciting variants of the ‘normal’ Raman technique which are continually emerging. Interest in applying Raman spectroscopy in the pharmaceutical community is growing and examples of such applications will be discussed. In addition, novel applications of variants of Raman spectroscopy (such as SERS), which have recently been reported, illustrates the diverse nature of this technique and its extraordinary ability to solve many biological problems of interest to the pharmaceutical industry, forensics and medicine. Raman spectroscopy is now well established as a complementary technique to much of the analytical instrumentation currently available, and Raman-based applications cover a broad range from drug discovery to manufacturing; including identifying polymorphs, monitoring real-time processes, imaging solid dosage formulations, imaging active pharmaceutical ingredients in cells, as well as diagnostic applications. Every compound has its own unique Raman spectrum which can be used for sample identification, detection and quantification. The differences in energy between the incident photons (usually provided by a laser) and the scattered photons correspond to vibrations in the molecule or crystal, and provide a “fingerprint” of the sample’s composition and molecular structure. There are several advantages of Raman spectroscopy, many of which are significant in the life sciences. These often include minimal sample preparation, the ability to observe the Raman spectra of analytes in aqueous solutions, as water is a poor Raman scatterer, leading to in situ analysis which is generally straightforward and desirable. Raman spectroscopy can also be used to investigate analytes through (e.g.) glass and fibre optics which can be employed for remote sampling, without the need to expose the instrument or operator to hazardous materials. Raman spectroscopy can be used for the analysis of organic and inorganic materials giving vibrational bands belonging to symmetrical vibrations (generally very weak in infrared spectroscopy). In addition, Raman bands of solids are usually sharper than infrared bands, making the spectra more information-rich and easier to interpret. Most of the disadvantages of Raman spectroscopic methods arise directly from the fact that Raman scattering is a rather weak effect and has to compete with any fluorescence contribution from the sample. Thus Raman tends to require intense laser excitation sources and sensitive (CCD) detectors which in turn has led to the relatively (and arguably) high costs of Raman instrumentation. The latter is one of the main obstacles to the widespread application of Raman spectroscopy for routine biological analysis. This situation is now changing as lasers and in particular detector costs have fallen (who doesn’t have a CCD in their mobile phone!) and instrument performance has steadily improved, with concomitant improvements in spectral resolution, and several vendors now offer portable easy-todeploy systems. When light is scattered from a molecule, the vast majority of photons are elastically scattered; this is termed “Rayleigh scattered light”. The scattered photons have the same energy (frequency) and, therefore, wavelength, as the incident photons. A small fraction of light (approximately 1 in 107 photons, although this is analyte specific) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process leading to this inelastic scatter is termed the Raman effect. Raman spectroscopy has evolved to include several variants of the normal dispersive technique; most notably two of these are SERS and the emerging technique of TERS-AFM (tip-enhanced Raman spectroscopyatomic force microscopy) (Fig.1). From the perspective of pharmaceutical analysis, Raman scattering has enabled the rapid non-invasive