Introduction and theory

Figure 1. Jablonski energy level diagram illustrating possible transitions. Solid lines represent absorption processes and dotted lines represent scattering processes. A, electronic transition with non–radiative decay (heat, zig–zag line) or radiative decay (fluorescence, thick line); B, Rayleigh scattering; C, Stokes Raman transition; D, anti-Stokes Raman transition. S0 is the singlet ground state, S1 the lowest singlet excited state and v represents vibrational energy levels within each electronic state.

Figure 2. FT–Raman spectrum of paracetamol.
Instrumentation
Dispersive spectrometers
Interferometric spectrometers
Microscopy
Fibre optics
Data processing and presentation of results

Figure 5. Examples of digitally processed spectral files. A, original Raman spectrum; B, spectral result after calculating the second derivative of the original Raman spectrum; C, spectral result after resolution enhancement by Fourier deconvolution of the original Raman spectrum; D, spectral result after Fourier smoothing the original Raman spectrum by 80%.
System suitability tests

Figure 6. FT–Raman spectra of wavelength calibration standards cyclohexane and sulfur.
Sample preparation and sample presentation
Interpretation of spectra
- three co–ordinates are required to locate the molecule in space
- an additional three co–ordinates are required to describe the orientation of the molecule based upon the three co–ordinates that define the position of the molecule in space.

Figure 7. Bending vibrational motions associated with CH3 ((+) represents movement above the plane, (-) represents movement below the plane).

Figure 8. Bending vibrational motions associated with CH2 ((+) represents movement above the plane, (-) represents movement below the plane).
Qualitative analysis

Figure 9. FT –Raman spectra of the polymorphs of carbamazepine (top, polymorph I, bottom, polymorph III).
- ability to work with aqueous–based systems with little spectral interference from water
- utilisation of a fibre–optic probe for direct and/or remote sampling
- collection of the Raman spectrum directly through the glass vessel with little or no spectral interference
- ability to analyse the spectrum quantitatively.

Figure 10. Examples of defined point, line and area maps.

Figure 11. Peak area profiles (images) representing: A, mannitol; B, aspartame; C, cellulose; D, magnesium stearate; E, corn starch; F, monoammonium glycyrrhizinate.
Quantitative analysis
- system suitability – an overall test of system function
- specificity – the ability of Raman spectroscopy to differentiate the analyte from the matrix
- working range – the concentration range over which the method is validated
- linearity – demonstration of a direct relationship between a measured analytical response and concentration over the working range of the method
- precision – the repeatability with which a number can be represented
- accuracy – degree of conformity of a measurement to a standard or true value
- limit of detection – lowest concentration at which an analyte can be detected
- minimum quantifiable limit – lowest concentration at which an analyte can be quantified with acceptable accuracy and precision
- robustness – demonstration of the reliability of an analysis with respect to deliberate variations in method parameters.
Chemometrics
Partial least square
Discriminant analysis
Collections of data
- comparing the reference spectrum to the sample spectrum
- ensuring that the reference spectrum is named accurately in the library (no library is completely perfect)
- determining that the suggested hit is a logical component to expect in that particular sample.
Last modified: 20 May, 2017