RAMAN Spectrometer, Dual Channel, Portable, MAS 6083
Abstract
Recently, Raman spectroscopy become a popular and potential analytical technique for the analysis of pharmaceuticals as a result of its advancement. The innovation of laser technology, Fourier Transform-Raman spectrometers with charge coupled device (CCD) detectors, ease of sample preparation and handling, mitigation of sub-sampling problems using different geometric laser irradiance patterns and invention of different optical components of Raman spectrometers are contributors of the advancement of Raman spectroscopy. Transmission Raman Spectroscopy is a useful tool in pharmaceutical analysis to address the problems related with sub-sampling in conventional Raman back scattering. More importantly, the development of surface-enhanced Raman scattering (SERS) has been a prominent advancement for Raman spectroscopy to be applied for pharmaceuticals analysis as it avoids the inherent insensitivity and fluorescence problems. As the active pharmaceutical ingredients (APIs) contain aromatic or conjugated domains with strong Raman scattering activity, Raman spectroscopy is an attractive alternative conventional analytical method for pharmaceuticals. Coupling of Raman spectroscopy with separation techniques is also another advancement applied to reduce or avoid possible spectral interferences. Therefore, in this review, transmission Raman spectroscopy, SERS, and SERS coupled with various separation techniques for pharmaceutical analysis are presented.
Introduction
Pharmaceutical products, like other industrial commercial products, undergo a lengthy process in order to prove that the product is suitable for the intended purpose. The pharmaceutical product authorized for commercialization should demonstrate that the product meets customer expectations of safety and efficacy [1]. This can be assured through analysis of drug raw materials, intermediates, products, formulations, impurities and degradation products, as well as biological samples containing the drugs and their metabolites for pharmacokinetic studies [2]. The analytical investigation, therefore, helps to understand the physical and chemical properties of the drug beginning from its development until marketing and post-marketing. Consequently, appropriate corrective action can be taken at any stage of the analytical investigative process to meet customer expectations.
For instance, the drug formulation needs to be scrutinized so that the manufactured product can meet the expected safety and efficacy requirements. Not only the physicochemical properties of the drug, but also the effect of excipients on the drug formulation have to be evaluated. The potential incompatibilities between the drug and excipients, the suitability of the excipients in facilitating drug administration and release of the active component in the body for optimum bioavailability, the ability of the excipients in stabilizing the drug against degradation from the environment should be realized for the optimum therapeutic efficacy and safety of the drugs [3].
Structural differences of pharmaceutical solids often have a significant effect on physicochemical properties such as dissolution rates, bioavailability, ease of manufacturing, and product stability [4,5]. Therefore, a critical investigation of the polymorphism of pharmaceutical solids is also a common procedure in the pharmaceutical research and drug development process. Analysis of some drugs and their metabolites in biological fluids are also important in monitoring factors such as the drug concentration, absorption and toxicity to enable corrective measures, if any, to be taken. To measure these factors, different analytical techniques such as volumetric, gravimetric, spectroscopic, electrochemical, kinetic techniques, x-ray diffraction, and different separation techniques have been employed.
Among these techniques Raman spectroscopy has, in recent years, received much interest and is considered the first choice in many applications in pharmaceutical analysis, mainly due to its noninvasiveness and specificity. Its potential application in the pharmaceutical industry, from production and quality control to shelf-life determination and the detection of counterfeits, is growing rapidly in recent times [6]. This review, therefore, focuses on the potential analytical applications of transmission Raman spectroscopy, SERS and hyphenated SERS techniques in pharmaceutical analysis. Moreover, qualitative and quantitative analysis of active pharmaceutical ingredients and related issues are addressed.
The phenomena of inelastic scattering light, known as Raman effect, was discovered in 1928 by C.V. Raman and K.S. Krishnan. After illuminating the samples with powerful incident light, they observed scattered light, later known as Raman scattering, exhibiting a different frequency from the original incident light [7]. This powerful light, usually from a laser, interacts with the sample of interest and two types of scattered light, namely elastic and inelastic, are produced. Elastic scattering (Rayleigh) occurs with no change in photon frequency whereas in inelastic scattering, the energy of the fraction of the photons scattered can either be lost (Stokes) with less energy than they had before or gained (anti-Stokes) with greater energy than those of the exciting radiation [8]. In Raman scattering processes, the incident photon distorts the electron clouds of the molecule resulting in the molecule elevates itself from lower energy state to the excited vibrational state. Inelastic scattering of photons by molecular bonds within the sample, therefore, provides information about molecular vibrations of molecules that interact with incident light. The difference in energy between the incident and scattered photon corresponds to the energy required to excite a particular molecular vibration that produces a fingerprint Raman spectrum for a molecule [9].
The Raman spectral features are well resolved and the bands are distinct with good sensitivity to the molecular structure, symmetry, electronic environment and bonding of the molecule, which assists with obtaining qualitative and quantitative information of a given molecule. The Raman spectrum shows intensity as a function of the difference in the frequency between the incident and the scattered light (Raman shift). For a given molecule, it consists of a series of peaks or bands each shifted by one of the characteristic vibrational frequencies of each specific chemical compound. Each molecule has its own unique spectrum and, in this way, a Raman spectrum can provide a “fingerprint” of a molecule. Furthermore, the intensity of a band is proportional to the concentration of the molecule from which the band arises.
While typical cross-sections for absorption in the ultraviolet and infrared regions are ca. 10−18 and 10−21 cm2per molecule, those for Raman scattering are quite low at ca. 10−29 cm2per molecule. This low efficiency of Raman scattering was a limiting factor for the use of Raman spectroscopy as a preferred analytical technique. Therefore, in order to record conventional Raman spectra, analytical concentrations greater than 0.01 M are usually required [10]. The innovation in laser technology and the different optical components of the Raman spectrometer has, however, advanced Raman spectroscopy to be one of the most important analytical tools for various chemical and biochemical analyses. The discovery of various other Raman phenomena, such as resonance Raman (RR), coherent anti-Stokes Raman scattering (CARS) and surface-enhanced Raman scattering (SERS), also helped improve the development of Raman as a very sensitive analytical tool [11]. Critical to the effective use of Raman spectroscopy as an analytical tool is the careful selection of a suitable laser source based on the nature of the sample and the geometry of the spectrometer. For example, samples with possible fluorescence interference require a longer excitation wavelength such as near-infrared (NIR) or red lasers [12].
The requirement for vibrational activity in Raman spectroscopy is not a change in dipole moment, as it is with IR spectroscopy, but a change in the polarizability of the molecule; i.e., there must be a change in the electronic polarizability of molecule with the vibrational mode. Therefore, molecules with delocalized electrons are susceptible to polarizability and have strong Raman scattering properties; aromatic conjugated systems are good examples of molecules with strong Raman scattering [13]. Moreover, unlike IR spectroscopy, Raman spectroscopy is not susceptible to interference from aqueous environments, due to its small cross-section for water, thereby providing it with an advantage over IR spectroscopy for the investigation of aqueous samples [14,15]. Another advantage of Raman spectroscopy as an analytical technique is the straightforward and quick sample preparation and handling. Depending on the effectiveness of the notch filtering, a large spectral range between 4000 cm−1 and 100 cm−1 can be covered in Raman spectroscopic measurements. All these advantages make Raman spectroscopy a potential analytical tool for pharmaceutical analysis.
A potential limitation of Raman spectroscopy has been its low sensitivity associated with the small Raman cross-section of many materials, thereby precluding the use of Raman spectroscopy as a method for ultrasensitive detection [16] of like biomedical and pharmaceuticals analysis. High levels of fluorescence, whether intrinsic or caused by impurities that overlay Raman bands, are also another problem encountered during Raman measurements as the fluorescence background is often many times more intense than the weak Raman signals.
Another limitation for Raman spectroscopy in pharmaceutical applications was due to sub-sampling [17]. This occurs as a result of the limited depth of light penetration such that representative sampling is difficult. For example, in a typical analysis of a single tablet, only a small fraction of the tablet could interact with the light and, therefore, the signal produced may not be fully representative of the content of the tablet.
These problems, however have been mitigated after the technological advancements of Raman spectroscopy through the following key inventions. The extension of the range of excitation wavelengths to the NIR region in the past few years reduces the background fluorescence and the photo-induced degradation of the sample [16]. In fact, the sample may decompose thermally if the laser intensities are too high and proper attention should be given in selecting the appropriate laser type and power. Problem associated with low sensitivity of Raman measurement has been reduced after invention of SERS. Implementing different geometric laser irradiance patterns has become a potential strategy to reduce sub-sampling problems.
As a result of these technological advancements, Raman spectroscopy has recently seen an upsurge in applications for pharmaceutical analysis. Several practical features make Raman spectroscopy attractive for pharmaceuticals analysis. Most APIs contain aromatic or conjugated domains with strong Raman scattering properties while aliphatic excipients, on the other hand, have poor Raman scattering properties. This provides an advantage for the use of Raman spectroscopy in the analysis of pharmaceuticals because spectral interferences from excipients are highly unlikely making it easier to profile APIs. The poor Raman scattering property of water also makes Raman spectroscopy more superior to mid- and near-IR spectroscopy for the analysis of APIs in formulations especially when the drug concentration is low [18]. Thus, application of Raman spectroscopy in pharmaceutical analysis including assessment and quantification of APIs, polymorphism study, investigation of drug stability, monitoring of drug metabolites in biofluids has been increasing tremendously.
The traditional back scattering Raman analysis acquires data from a small spot of analyte thereby failing to obtain fully representative spectra of the sample. Various approaches such as, sample rotation during spectral acquisition, temporal and spatial averaging of acquired data by scanning different regions of the sample, and simultaneous wide-angle illumination were implemented to avoid this sub-sampling problem [19,20]. Besides these approaches, it enables light to penetrate substantially deeper into the sample in particular for pharmaceuticals. In the transmission Raman geometry, the laser beam is incident on one side of the probed capsule and the Raman signal is collected from the opposite side [21] as illustrated in Fig. 1.
This geometry can be used for non-absorbing or weakly absorbing diffusely scattering samples [22]. For instance, layer interference from a capsule is highly reduced thereby providing detailed spectroscopic information on the interior composition of the drug and the bulk content of the probed turbid medium. The generation of Raman light and its subsequent behavior within deep layers of turbid media is governed chiefly by the ‘random walk’ of the laser photons migrating through the sample and producing Raman photons that in turn randomly migrate within the probed medium, referred to as Raman photon migration. In many applications Transmission Raman Spectroscopy has the added benefit of inherently suppressing inherently fluorescence emission that originates from the surface layers of the probed sample. On the contrary, with conventional backscattering Raman geometry, the fluorescence interfering signals are rather highly emphasized [23].
Though Raman spectroscopy provides rich information regarding the molecular structure of the sample, the problems associated with low sensitivity was remained a challenge until the discovery of SERS and its possible mechanisms [24]. The phenomenon was initially observed in 1974 when the Raman signal of pyridine adsorbed onto silver electrode was enhanced [23,25]. This enhancement of the Raman signal enables the detection of even a single molecule with high structural selectivity [26,27] and also overcomes the discrepancy between Raman-scattering cross sections as well as the fluorescence cross sections [28]. The phenomenon of a strongly increased Raman signal can be observed when Raman active molecules are attached or closed to metallic nanostructures called SERS-active substrates. These SERS-active substrates are metallic structures such as silver and gold with sizes in the order of tens of nanometers. The most common types of SERS-active substrates exhibiting the largest effects are colloidal silver or gold particles ranging in size from 10 to 150 nm, and silver or gold electrodes or evaporated films of these metals [16]. In recent times, the use of dynamic surface enhanced Raman spectroscopy for the ultra-sensitive detection of Raman active analytes has increased; in which the target analyte molecules are trapped in between the nanoparticles and a hot spot effect created in the process resulting in an extremely high SERS signal [29,30].
Recently, the application and development of SERS in chemical and biological analytes has increased at faster rate. The commercialization of SERS substrates produced by different companies such as Sigma-Aldrich [31,32], AtoID [33], Horiba Scientific [34], Ocean Optics [35], Silmeco [36] could be stated. This may show its maturity and potential for routine chemical analysis. Different techniques like lithography and chemical methods are used to fabricate these commercially available SERS substrates. Generally, the commercialization of SERS substrates ensures the potential of them in applying for routine analysis and quality control in pharmaceuticals.