Laser Raman spectroscopy (RAMAN)
Laser Raman spectroscopy (RAMAN)
Raman spectroscopy is the analysis method of studying the relationship between the scattering of scattered light, the energy difference of incident light, the vibration frequency and the rotation frequency of compound after the molecule is irradiated by light. Similar to infrared spectroscopy, Raman spectroscopy is a vibrational spectroscopy technique. The difference is that the former is related to the change of the dipole moment when the molecule vibrates, while the Raman effect is the result of the change of the molecular polarizability. The measured inelastic scattering radiation.
A certain wavelength of electromagnetic waves acting on the molecules of the material under study, causing the corresponding energy level transition, resulting in molecular absorption spectroscopy. The spectrum that causes the molecular electronic level transition is called the electron absorption spectrum, and its wavelength is located in the ultraviolet to visible region, so it is called the ultraviolet-visible spectrum. Electronic energy level transition accompanied by vibrational energy level and rotational energy level transition. The spectrum that causes the molecular vibrational level transition is called the vibrational spectrum, and the vibrational level transition is accompanied by the transition of the rotational energy level. Raman scattering spectra are vibrational-rotational spectra of molecules. When the molecule is irradiated with far-infrared light waves, it only causes the transition of the rotational energy level in the molecule and the pure rotation spectrum is obtained.
The advantage of Raman spectroscopy is that it is fast, accurate and usually does not destroy the sample (solid, semi-solid, liquid or gas) during the measurement, and the sample preparation is simple or even without sample preparation. Band signals are usually in the visible or near-infrared range and can be effectively used with optical fibers. This also means that the band signal can be obtained by encapsulating any laser-transparent medium, such as glass, plastic, or by dissolving the sample in water. Modern Raman spectrometers are easy to use, analyze fast (seconds to minutes), and depend on performance. Therefore, Raman spectroscopy in combination with other analytical techniques is in some ways easier than other spectroscopic techniques (univariate and multivariate methods and calibrations can be used.
Special Raman spectrum
In addition to the conventional Raman spectrum, there are some more specific Raman techniques. They are resonance Raman, surface-enhanced Raman spectroscopy, Raman polarimetry, related-anti-Stokes Raman spectroscopy, Raman gain or loss spectra, and super Raman spectroscopy. Among them, the relatively large number of drug analysis applications are resonance Raman and surface-enhanced Raman spectroscopy.
Resonance Raman spectroscopy
When the laser frequency close to or equal to the molecular electronic transition frequency, can cause strong absorption or resonance, resulting in sharp increase in the intensity of some Raman bands millions of times, which is the resonance Raman effect.

Surface Enhanced Raman Spectroscopy (SERS)
SERS phenomenon is mainly caused by the excitation of the metal surface matrix to enhance the local electromagnetic field. The strength of the effect depends on the size of the surface roughness corresponding to the wavelength of light and the degree of complex metal-dielectric interaction associated with the wavelength.
Qualitative and quantitative determination
Qualitative identification
Raman spectroscopy provides structural information on the functional groups in any molecule. It can be used to identify tests and structural analysis. Polymorphism can refer to the infrared processing.
Quantitative determination
The relationship between Raman bands intensity and analyte concentration follows Beer's law: IV = KLCI 0 where IV is the peak intensity at a given wavelength, K the instrument and sample parameters, L the optical path length, C the specific The molar concentration of the components, I 0 is the laser intensity. In practice, the optical path length is more accurately described as the sample volume, an instrument variable that describes the laser focus and acquisition optics. The above equation is the basis for quantitative Raman applications.
Influencing factors
The main disturbing factors are fluorescence, the thermal effects of the sample and the absorption of the matrix or sample itself. In Raman spectroscopy, fluorescence interference appears as a typical oblique broad background. Therefore, the impact of fluorescence on the quantitative mainly for baseline deviation and signal to noise ratio decline, the fluorescence wavelength and intensity depends on the type and concentration of fluorescent substances. Fluorescence is generally a quantum-more efficient process than Raman scattering, and even fluorescence of very small amounts of impure material can result in significant Raman signal reduction. Fluorescence can be significantly attenuated using longer wavelengths such as excitation at 785 nm or 1064 nm. However, the intensity of the Raman signal is proportional to λ-4 and λ is the excitation wavelength. The best signal-to-noise ratio is obtained by balancing fluorescence interference, signal strength and detector response. Before measuring the sample with a laser irradiation for a certain period of time, the fluorescence of the solid matter can also be weakened. This process, known as photo-bleaching, is achieved by degrading highly-absorbing substances. Photobleaching is not evident in liquids, either due to the fluidity of the liquid sample or the fluorescent material is not trace.
Sample heating causes a number of problems, such as changes in physical state (melting), transformation of the crystalline form, or burning of the sample. This is a problem that often occurs with tinted, small particles that have strong absorption or low heat conduction. The effects of sample heating are usually observable, manifesting the apparent change in Raman spectrum or sample over a period of time. In addition to reducing the laser flux, there are many ways to reduce the thermal effects, such as moving the sample or laser during the measurement or improving the thermal conductivity of the sample by thermal contact or liquid immersion. The matrix or sample itself can also absorb Raman signals. In the long-wave Fourier transform Raman system, the Raman signal can overlap with the near-infrared ubiquitous absorption. This effect is related to the system's optics and sample morphology. The variability of solid scattering caused by differences in packing and particle size is related to this effect. However, due to the limited penetration depth of the sample in the Raman spectrum and the relatively narrow wavelength range, none of these effects are of a size that is severe in the near-infrared spectrum.
Quantitative Raman spectroscopy, unlike many other spectroscopic techniques, is a single beam zero background measurement. Careful sample preparation and the use of well-designed instruments minimize this variability but do not eliminate them all. Therefore, the absolute Raman signal intensity is difficult to use directly for the quantification of the analyte. Potential sources of variation are the opacity of the sample and the inhomogeneity of the sample, changes in the laser power of the irradiated sample, and changes in optical geometry or sample position. These effects can be reduced by means of repeatable or representative sample handling.
Because of the absolute intensity of the Raman signal fluctuations, the use of internal standards is the most common and effective way to reduce variability. There are several variations of the internal standard method. An internal standard can be purposefully added that should have a unique band that does not interfere with the analyte for detection. In solution, unique bands of solvents can also be used because the solvent will be relatively constant from sample to sample. In addition, the peak of the excipient may be used in the formulation if the amount of excipient is significantly greater than the component to be tested. Assuming that changes in laser and sample positioning will equally affect the full spectrum, the full spectrum can also be used as a reference.
Important factors to consider in sample determination are spectral contamination. Raman is a weak effect that can be masked by many exogenous influences. Common sources of contamination include sample supports (containers or matrices) and ambient light. Often these problems can be reconciled by careful experimentation
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