Extensive tests to determine an optimal method for using a Bio-Rad FTS-60A spectrometer for measurement of vibrational circular dichroism (VCD) in both step-scan and rapid-scan modes are reported. In the latter case, results of “fast-scan” and “slow-scan” experiments are also presented for comparison. In step-scan mode, phase modulation (PM) during the polarization-modulation measurements is found to be detrimental to the signal-to-noise ratio (S/N), but is very useful for the normal IR measurements at slow step speeds. VCD S/N is improved by doing sequential spectral collection of the polarization-modulated spectrum without PM and the instrument response spectra with phase modulation. Systematic comparative studies are used to identify optimal parameter sets for doing the step-scan VCD measurements. These conditions use more rapid data collection with faster steps than previously suggested, which permits a more realistic evaluation of S/N and reproducibility of the spectra through comparison of spectra collected in blocks. Example VCD spectra are presented for small molecules in nonaqueous solution measured in short times and for proteins in aqueous solution measured over longer times scales. Near-IR FT-VCD measured in step-scan mode at both moderate resolution and high S/N are presented. At this stage, fast-scan, slow-scan, and step-scan techniques are shown to be virtually equivalent in the mid-IR, but the reduction in frequency dependence of the phase correction and the new capability for VCD measurement in the near-IR favor continued development of step-scan techniques.
Investigation of the Effects of Surface Chemistry and Solution Concentration on the Conformation of Adsorbed Proteins Using an Improved Circular Dichroism Method