Comparison of Vibrational Circular Dichroism Instruments: Development of a New Dispersive VCD

Abstract

A dispersive vibrational circular dichroism (VCD) instrument has been designed and optimized for the measurement of mid-infrared (MIR) bands such as the amide I and amide II vibrational modes of peptides and proteins. The major design considerations were to construct a compact VCD instrument for biological molecules, to increase signal-to-noise (S/N) ratio, to simultaneously collect and digitize the sample transmission and polarization modulation signals, and to digitally ratio them to yield a VCD spectrum. These were realized by assembling new components using design factors adapted from previous VCD instruments. A collection of spectra for peptides and proteins having different dominant secondary structures (alpha-helix, beta-sheet, and random coil) measured for identical samples under the same conditions showed that the new instrument had substantially improved S/N as compared with our previous dispersive VCD instrument. These instruments both provide protein VCD for the amide I that are comparable to or somewhat better than those measurable with commercial Fourier transform (FT) VCD instruments if just the amide I band in the spectra is obtained at modest resolution (8 cm(-1)) with the same total data collection time on each type of instrument.

Mini review: Instrumentation for vibrational circular dichroism spectroscopy, still a role for dispersive instruments

“…Ratioing Equation by Equation , for wavelengths ( λ ) having α B = (n + 1)π/2 (ie, where sin α B = 1 and cos α B = 0), and then dividing by 1.15, yields Δ A . Alternatively magnitudes can be determined by comparison with the VCD of a standard sample to provide a (quicker) secondary calibration . After averaging sequential scans, subtraction of baseline scans obtained under the same conditions and with the same sample cell (or equivalent) completes processing.…”

“…An updated dispersive instrument optimized for biopolymer studies has been constructed at the University of Illinois at Chicago designed around a 0.3‐m focal length, ~f/4 monochromator (Acton Research, SpectraPro2300i) operated in first order (by adding a long‐wave pass, ~5 μ , filter), and capable of interchanging multiple gratings. A water‐cooled, resistively heated (2500 K) carbon rod, matched to the slit size, provides high‐intensity IR radiation (substantially brighter than a glowbar) that is passed through a chopper to modulate the transmission signal at a position close to where it is imaged onto the monochromator entrance slit .…”

“…A, Schematic diagram of the optical components of the updated dispersive vibrational circular dichroism (VCD) spectrometer constructed at University of Illinois at Chicago (UIC) and optimized for biopolymer studies . S,C‐rod light source in cooled housing; M1, flat mirror; M2, focusing mirror; C, rotating wheel chopper (150 Hz); G, exchangeable monochromator grating (on turret, 150 or 200 mm/groove); F, optical long wave pass filter; P, wire gird polarizer (BaF 2 ); PEM, photoelastic modulator (ZnSe); SC, sample cell; L, lens, D, MCT detector.…”

“…This instrument is optimized for study of peptides and proteins in both the amide I and II regions, for which we have found that a 6 μ blaze, 150 g/mm grating offers the best trade‐off of resolution and throughput for this restricted region. [Our initial updated design used a higher‐density grating, but that did not adequately cover the amide II region .…”

“…While FT‐VCD has obvious advantages for wide‐range IR coverage, the relatively broad bands and the restriction to spectral windows of water for biopolymer systems in aqueous solution can offset the FTIR throughput and multiplex advantages. As a result, dispersive VCD is still useful in practice, because it can focus measurement time/effort on a single spectral band or narrow region of interest (especially for proteins) and can have very high throughput (by using larger slits at low resolution), thereby offsetting both main advantages of FTIR . Fourier transform infrared‐based VCD instruments are best used where the desired data must cover a wide spectral range, eg, for absolute stereochemistry studies of small molecules in organic solvents.…”