Measurement of circular dichroism and related spectroscopies with conventional and synchrotron light sources: Theory and instrumentation

A linearly polarized optical beam that passes through a photoelastic modulator can be used to measure circular dichroism, linear dichroism, optical rotary dispersion, fluorescence detected circular dichroism and fluorescence polarization anisotropy. These parameters, along with the simultaneous measurement of absorption and fluorescence are possible with a conceptually simple single beam spectrometer, with only minor adaptations. Practical spectrometers and the components needed to build them using both conventional (Xenon arc) and synchrotron light sources are reviewed. The need to match the components throughout the instrument is discussed and methods of calibrating critical components are described. Potential artifacts associated with sample inhomogenity are discussed. An explanation for the observation that a linear dichroism signal can appear at the modulation frequency expected for circular dichroism is presented. Spectra demonstrating the ability of a synchrotron source spectrometer to extend the range of wavelengths for the circular dichroism of proteins are presented. Three general classes of practical spectrometers are described based on the components used and the performance achievable. Vendors of polarization-modulation spectrometers and specialized components required to build them are listed, as are existing and planned synchrotron based user facilities.