Interlaboratory comparisons of circular dichroism (CD) spectra are useful for developing confidence in the measurements associated with optically active molecules. These measurements also help define the higher-order (secondary and tertiary) structure of biopolymers. Unfortunately, the extent of the validity of these measurements has been unclear. In this work, a method is described to extend CD validation over the entire observed wavelength range using what will be called spectral similarity plots. The method involves plotting, wavelength by wavelength, all measured spectral intensities of a sample at one concentration against the intensity values of the same material at a different concentration or pathlength. These spectral similarity plots validate the instrument in terms of spectral shape and whether the shape is shifted in intensity and/or in wavelength. This comparison tests the linearity of instrument’s signal, the balance of its left and right polarizations, its wavelengths, and its spectral intensity scales. When the process is applied to materials with accepted and archived intensity values, the method can be linked to older single-wavelength and double-wavelength calibration techniques. Further, spectral similarity testing of CD spectra from samples with different concentrations run in different labs suggests that improved interlaboratory validation of CD data is possible. Since a database of archival CD measurements is available online, spectral similarity comparisons could possibly provide the ability to compare linearity, polarization balance, wavelength, and spectral intensity between all current CD instruments. If the preliminary results published here prove robust and transferable, then comparisons of full-wavelength range spectra to archived data using spectral similarity plots should become part of the standard process to validate and calibrate the performance of CD instruments.
Steps Toward Full Wavelength Range Calibration for Circular Dichroism Spectroscopy
