Raman Spectroscopy in Analysis of Biomolecules
Authors: Niaura Gediminas 
Published: September 2006

Raman spectroscopy (RS) is a vibrational spectroscopy technique, based on the phenomenon of inelastic light scattering from matter (the Raman effect). It is an irreplaceable tool for the study of biological events at the molecular level and for identification of biomolecules. There are several reasons for this. First of all, RS measures the vibrations of atoms. This implies that the positions of the bands, widths and intensities are sensitive to the molecular structure, and minor perturbations induced by intramolecular or intermolecular interactions can easily be followed. Vibrations of some molecular groups are very characteristic (characteristic vibrations) and therefore can be used for the identification of certain groups or even whole molecules. In addition, work using isotope labeling, model compounds, site‐directed mutagenesis and normal coordinate analysis has led to the identification of so‐called marker bands, which serve as a bridge in establishing spectrum–structure correlations. Second, water causes weak Raman scattering, and consequently biomolecules can be studied in their natural environment without solvent interference. The third reason for the growing interest in RS is related to the considerable intensity enhancement (up to 106) observable in resonance Raman spectroscopy (RRS) and surface‐enhanced Raman spectroscopy (SERS). Important structural units, such as chromophoric active centers, aromatic amino acid residues, bases of nucleic acids, and peptide bonds, can be studied selectively by exploring the resonance Raman (RR) phenomenon and tuning the excitation wavelength (λex) into the electronic absorption band of certain structural elements. Such an approach allows the major problem in vibrational spectroscopy of biomolecules to be overcome – namely interpretation difficulties due to overlapping of numerous bands. Because the Raman effect is inherently weak, the future for analytical applications is closely connected with both RRS and SERS. The combination of these two methods might be especially productive. Fourthly, because the Raman effect is instantaneous (timescale in the order of 10−14 s), and due to the recent advances in laser technology, the time‐resolved action of biomolecules down to picosecond resolution can be followed, retaining the value of structural information. The ability to focus a laser beam down to the dimensions of λexis the fifth advantage of RS. Combined with RRS or SERS, this approach allows the composition of a single living cell to be analyzed.

To explore fully the advantages of RS, the limitations of the technique must also be considered. First of all, the probability of Raman scattering is very low, so the effect is weak and high concentrations of biomolecules are required. Secondly, in RR experiments excitation within the electronic absorption band often causes photodegradation of the active center in the biomolecule. To avoid such problems, low laser powers, moving samples and independent inspection of the sample integrity are often employed. The same problems are also present in the SERS field. Overheating by the laser beam could be an obstacle in Fourier transform Raman (FTR) studies. Finally, many active centers in biomolecules, or impurities in the sample, exhibit intense fluorescence (Fl), obscuring the Raman spectra.