# Microplastic Pollution: Chemical Characterization and Impact on Wildlife
## 2 The Chemical and Physical Characterization of Microplastics
### Raman Microscopy  
Another powerful method of analysis for microplastic particles is Raman spectroscopy. Raman spectroscopy is another form of vibrational spectroscopy; however, in contrast to FTIR, Raman microscopy is more appropriate for small microplastics less than 20 \(\upmu\)m. Raman spectroscopy is based on the Raman effect, whereby the frequency of a small portion of dispersed or scattered radiation emanating from a sample differs from the frequency of monochromatic incident light. The basic principle is illustrated within Figure 6A below. Raman spectroscopy involves illuminating the sample with a monochromatic laser beam that interacts with the molecules in the sample and produces scattered light. Raman spectra are produced by the inelastic interaction of the sample molecules electrons and incident monochromatic energy. When monochromatic radiation impacts the sample, it interacts with the sample molecules and scatters in all directions [64]. A small fraction of scattered light experiences a shift in wavelength due to the inelastic nature of the process. Incoming excitation light interacts with a sample optically to produce scattered light, which is then reduced in energy by the vibrational modes of the specimen's chemical bonds. This process is known as Raman scattering [65]. Light is scattered inelastically by Raman-active materials' molecules due to their chemical vibrations. The fundamental benefit of Raman microscopy is that amorphous carbon can be found when the entire wavelength range is utilized. As a result, microplastics subjected to UV degradation do not significantly affect their Raman spectra. Since visible light is often used as the incident beam, Raman microscopy has a spatial resolution much better than FTIR--about 1 \(\upmu\)m. Particle shape and thickness have no bearing on the measurement either. Raman microscopy is potentially a more sensitive method than FTIR for identifying microplastics because of these benefits. Some materials, nevertheless, fluoresce, which masks the vibrational information contained within the inelastic scattering within a bright background. However, this can be reduced through choosing an incident beam of longer wavelength. Dyeing agents as well as microbiological, organic, and inorganic chemicals also may have a significant impact on the Raman signal [35]. The advantages of Raman spectroscopy include nondestructiveness, quick acquisition, reliability, single-point measurements with good spatial resolution, surface maps of large samples, and the use of spectra as sources of qualitative and quantitative data. The disadvantages include the signal's susceptibility to changes in measurement parameters and the difficulty of interpreting data, particularly when a complex matrix is present [66]. The micro-Raman technique, which combines collections of Raman spectral information with microscopy, can identify tiny microplastics down to 1 \(\upmu\)m, and this resolution cannot be attained by other techniques [57]. Figure 6B depicts a commercially available Raman microscope which can be used for this purpose. Raman spectroscopy provides data which is complementary to FTIR measurements since both use vibrational transitions to provide molecular speciation. In FTIR, the transmittance or absorption of light at a  
Figure 6: (**A**) Schematic of the fundamental principles of Raman spectroscopy, highlighting the main scattering and fluorescence excitations of a sample after it is excited using a monochromatic light source of frequency equal to \(\upnu_{0}\). The Raman stokes line experiences a frequency shift of D\(\upnu\) that corresponds to the difference between vibrational energy levels. (**B**) A benchtop, commercially available Raman microscope. (**A**) from Ref. [67] via Creative Commons license and (**B**) from Thermo Fisher Scientific. particular wavelength is assessed. In Raman spectroscopy, the data stream is a plot of scattered light intensity vs. the shift in wavelength as measured from the incident beam.