Raman difference spectroscopy as a probe of biological molecules

The application of Raman difference spectroscopy to biological molecules, briefly reported previously,¹ is described here in detail. With Raman difference instrumentation frequency differences of less than 0.1 cm⁻¹ in 10 cm⁻¹ wide lines may be reliably detected. The limitation in the ultimate sensitivity for the detection of small differences lies in the signal to noise ratio, the presence of multiple lines and line shape differences. Spectrometer resetability, spectral resolution and step increment size impose no severe restrictions on obtaining small frequency differences. Differences in Raman excitation profiles between two samples may also be examined with this technique, and by making a simple adaptation Raman polarization difference spectra may be studied as well.