Raman spectroscopy is a vibrational spectroscopic technique that measures the inelastic scattering of monochromatic light, typically from a laser. It provides complementary information to infrared spectroscopy and is particularly valuable for analyzing aqueous samples, crystalline materials, and biological specimens.
Principle of Raman Scattering
When monochromatic light interacts with a molecule, most photons undergo Rayleigh scattering (elastic, no energy change), while a small fraction (approximately 1 in 10^7 photons) undergoes Raman scattering (inelastic) with a change in energy. In Stokes Raman scattering, the scattered photon has lower energy (longer wavelength) than the incident light, corresponding to molecular vibration excitation. In Anti-Stokes Raman scattering, the scattered photon has higher energy (shorter wavelength), occurring when the molecule starts in an excited vibrational state. The Raman shift (cm-1) is the energy difference between incident and scattered light, independent of the laser wavelength.
Raman vs. Infrared Spectroscopy
Raman and IR are complementary techniques governed by different selection rules. Raman activity requires a change in polarizability during vibration, while IR activity requires a change in dipole moment. Symmetric vibrations (e.g., C=C stretching in alkenes) are typically strong in Raman but weak in IR, whereas asymmetric vibrations are strong in IR but weak in Raman. Water is a weak Raman scatterer but a strong IR absorber, making Raman ideal for aqueous solutions and biological samples. Additionally, Raman spectra have less fluorescence interference than IR when using longer wavelength lasers (785 nm, 1064 nm).
Instrumentation
A Raman spectrometer includes a laser source with common wavelengths of 532 nm (green), 633 nm (red), 785 nm (near-IR), and 1064 nm (IR); longer wavelengths reduce fluorescence but follow the inverse fourth power law resulting in lower Raman intensity. The laser is focused onto the sample through a microscope objective (micro-Raman) or lens, enabling analysis of microgram quantities and spatial resolution down to 1 µm. A spectrograph with a diffraction grating separates the Raman-scattered light by wavelength, and notch or edge filters remove the intense Rayleigh line. Charge-coupled device (CCD) detectors offer high sensitivity for multichannel detection, while Fourier Transform (FT)-Raman uses an interferometer and single-channel detector with 1064 nm excitation.
Special Raman Techniques
Several advanced Raman techniques enhance its capabilities. Surface-Enhanced Raman Spectroscopy (SERS) uses adsorption of analyte molecules on roughened noble metal surfaces (Ag, Au) to enhance Raman signals by 10^6-10^10, enabling single-molecule detection. Resonance Raman Spectroscopy (RRS) matches the laser wavelength to an electronic absorption band of the analyte, enhancing specific vibrational modes by 10^3-10^5. Confocal Raman Microscopy uses a pinhole to reject out-of-focus light, enabling depth profiling and three-dimensional chemical imaging of heterogeneous samples. Tip-Enhanced Raman Spectroscopy (TERS) combines scanning probe microscopy with SERS for chemical imaging with nanometer spatial resolution.
Applications
Raman spectroscopy is used for identification of pigments in art and archeological artifacts through non-destructive analysis, pharmaceutical polymorph screening and counterfeit drug detection, diagnosis of diseases through Raman spectral fingerprinting of tissues and biofluids, quality control of semiconductors, polymers, and carbon materials (graphene, carbon nanotubes), and in vivo biomedical imaging and endoscopy using fiber-optic Raman probes.
