Synchrotron radiation circular dichroism spectroscopy, commonly referred to as SRCD and also known as VUV-circular dichroism or VUVCD spectroscopy, is a powerful extension to the technique of circular dichroism (CD) spectroscopy, often used to study structural properties of biological molecules such as proteins and nucleic acids. The physical principles of SRCD are essentially identical to those of CD, in that the technique measures the difference in absorption (ΔA) of left (AL) and right (AR) circularly polarized light (ΔA=AL-AR) by a sample in solution. To obtain a CD(SRCD) spectrum the sample must be innately optically active (chiral), or, in some way be induced to have chiral properties, as only then will there be an observable difference in absorption of the left and right circularly polarized light. The major advantages of SRCD over CD arise from the ability to measure data over an extended wavelength range into the vacuum ultra violet (VUV) end of the spectrum. As these measurements are utilizing a light source with a higher photon flux (quantity of light stricking a given surface area) than a bench-top CD machine it means data are more accurate at these extended wavelengths because there is a larger signal over the background noise (the signal-to-noise ratio) and, generally, less sample is needed when recording the spectra and there is more information content available in the data. Many beamlines now exist around the world to enable the measurement of SRCD data.
Origins Extending the wavelength range for CD experiments had been both considered and instigated as far back as 1970. Three research groups had created their own "in-house" CD machines, with specialist lamps as their light source, to enable measurements in this range. Synchrotron radiation (SR) had been proposed for use as the light source at a meeting in Brookhaven National Laboratory on Long Island in 1972, however, it took a few years more before this came to fruition. Two research papers in 1980 reported the collection of CD data using SR as the light source for the experiments. Specifically, spectra were obtained in wavelength regions into the VUV range, from ~100 nanometers (nm) to ~200 nm, largely unavailable to laboratory-based bench-top spectrophotometers. Sutherland et al. focussed on the development of a versatile spectrophotometer capable of measuring CD, amongst other properties, in the VUV region of the spectrum, while Snyder and Rowe collected CD data from a small organic compound in the wavelength range 130.5 nm to 205 nm.
Simplified overview of an SRCD beamline setup As shown in the diagram, a number of baffles are used throughout to remove possible stray light being reflected off the sides of the beamline tube. The use of only one mirror minimizes the loss of photon flux which is most important in the VUV region where reflectivity is poor relative to the visible wavelength range. The first constructed SRCD beamlines initially tried to utilize the intrinsic properties of the SR radiation produced, whereby there exists a "central" linearly-polarized component with, above and below this, equally opposing regions of circularly-polarized components. The premise for this was that the overall signal produced from a chiral sample would be enhanced by the absorption difference (the signal) derived from these circularly polarized features of the beam. In an ideal situation this approach would work; however, this setup was modified such that all beamlines now include a linear polarizer (as shown) to remove these circularly polarized components. This was because even the minutest of movements in beam position (beam drift) led to unequal matching of the contributions of the circularly polarized components striking the sample and this, in turn, meant the SRCD signal produced was inaccurate and unreliable; often being irreproducible as a result. Whereas cCD machines are purged throughout with nitrogen to minimize the absorption by oxygen of the light from the source xenon arc lamp, in an SRCD arrangement the beam passes through a calcium fluoride (CaF2), or similar "VUV-wavelengths transparent", window where everything before this point is in vacuum, and everything beyond is in nitrogen. The beam interacts with a photoelastic modulator (PEM) which consequently produces an alternating right- then left-circularly polarized beam and these now interact with the sample. The resultant absorption difference by the sample is measured and amplified by a photomultiplier tube (PMT) and from this the SRCD spectrum is recorded. The wavelength range that is utilized for SRCD studies is typically in the UV to VUV region and can go to below this; potentially from ~100 nm, up to the visible region, ~400 nm. The exact range over which data can be collected relies on the beamline set up, the sample preparation and the wavelength range of the PMT detector used. One of the primary factors limiting the lower wavelength cut off is the sample usually being in solution as a large water absorption band exists centred ~167 nm. This high absorption background swamps any possibility of measuring the very small CD difference signal, although use of deuterated water (D2O) as the solvent reduces the solvent absorption increasing the lower wavelength data collection range by ~10 nm. Removing the solvating water completely, creating a film as a result, means that data can be recorded to significantly lower wavelengths, down to around ~130 nm.
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