Raman optical activity (ROA) is, like vibrational circular dichroism (VCD), a spectroscopic method that probes the chirality, or handedness, of molecular vibrations. ROA offers a novel alternative to X-ray crystallography, permitting absolute configuration determination for a neat liquid, oil or solution sample. ROA requires no derivatisation of the sample or growth of a pure single crystal.
Thanks to new developments in instrumentation, ROA has been applied to the absolute configuration of small chiral molecules when combining the application of ab initio methods to the analysis of experimental ROA spectra1. The calculations can also be carried out in commercial packages such as Gaussian09 (Gaussian Inc).
In addition, ROA holds great promise for the determination of the three-dimensional structure and conformational distribution of chiral molecules in unprecedented detail. ROA combines the structural specificity of Raman spectroscopy with the stereochemical sensitivity of chiroptical spectroscopy such as circular dichroism (CD).
Prof Laurence Barron recently stated in a review that: "The many structure-sensitive bands in the ROA spectra of aqueous solutions of biomolecules provide detailed structural information, in the case of proteins, not only secondary structure elements such as helix and sheet but also the tertiary fold. ROA studies of unfolded and partially folded proteins are providing new insight into the residual structure in denatured proteins and the aberrant behaviour of proteins responsible for misfolding diseases. It is even possible to measure the ROA spectra of intact viruses, from which information about the folds of the major coat proteins and the structure of the nucleic acid core may be obtained2."
ROA spectra are typically measured with 532 nm laser excitation, although 780 nm laser excitation is also commercially available, with a spectral window of 200 cm-1 to 2500 cm-1. The advances in the measurement of ROA over the past three decades benefit from the progress in general Raman instrumentation: multi-channel detection with charge coupled devices (CCD), holographic gratings and the advent of frequency doubled solid-state lasers. Additionally, ROA has experienced rapid development in recent years due to the implementation of an artifact reducing scheme based on the concept of the virtual enantiomer by Prof Werner Hug in 19993, that overcomes the ubiquitous offset problem in an ROA spectrometer.
Most recently, we have demonstrated the simultaneously acquisition of all four forms of ROA, named incident circular polarisation (ICP), scattered circular polarisation (SCP), in-phase dual circular polarisation (DCPI) and out-of-phase dual circular polarisation (DCPII)4. This new achievement allows the direct comparison of all four forms of ROA of molecules within or without resonance. Moreover, at present, a theoretical group at Rice University has been calculating DCPI ROA and predicted that DCPI has significant advantages over ICP or SCP for measuring much more reliable surface-enhanced Raman optical activities (SEROA) due to its inherent depolarization properties and its removal of large useless parent Raman radiation, which only contributes noise in ROA or SEROA measurements5. In this case, DCP-SEROA will improve the detection sensitivity by several thousand orders of magnitude compared to previous spontaneous ROA measurements and significantly decrease the amount of sample needed for down to only several thousands of molecules.
In addition to simultaneous acquisition of all four forms of ROA, all polarisation state (highly polarised, depolarised, unpolarised) measurements of parent Raman spectra can be obtained at the same time with the current BioTools ChiralRAMAN-2X spectrometer. In addition, the degree of circularity, which can be easily converted to depolarisation ratio, can also be acquired. In Figure 1, eight traces of Raman spectra of neat S-pinene have been shown. All traces were obtained for a single experimental setup. They are highly polarised (top spectrum, DCPII), depolarised (middle spectrum, DCPI), and unpolarized (lower spectrum, SCP or ICP) Raman in the top panel, and SCP, DCPI, ICP, DCPII ROA as labelled in the middle panel, and the degree of circularity spectrum in the lower panel.
The most recent development of the ROA spectrometer combines an ROA spectrometer (BioTools ChiralRAMAN-2X) to a multi-port inverted microscope (IX71/81 from Olympus) for Raman microscopy measurements. The attachment of a Raman microscope allows for micro analysis of trace samples, like particles of micron size, droplets, cells, spores, bacteria, etc. The addition of a microscope enables various traditional microscopic observations, like phase contract, relief contrast, differential interference contrast, FRET, TIRFM, to be applied to the same samples undergoing ROA measurement.
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Honggang Li is a Senior Research Scientist, BioTools Inc, Jupiter, Florida, USA. www.btools.com. Laurence A Nafie is a Chief Technology Officer, BioTools Inc and a Distinguished Professor, Department of Chemistry, Syracuse University, Syracuse, New York, USA. www.syr.ed
1. Haesler, J; Schindelholz, I; Riguet, E; Bochet, C G; Hug, W Nature 2007, 446 526-529;
2. Barron, L D; Hecht, L; McColl, I H; Blanch, E W. Molecular Phys. 2004, 102, 731-744;
3. Hug, W; Hangartner, G J Raman Spectrosc. 1999, 30, 841-852;
4. Li, H; Dukor, R K; Nafie, L A XXII-ICORS 2010, 1267, 159;
5. Lombardini, R; Acevedo, R; Halas, N J; Johnson, B R J Phys. Chem. C 2010, 114, 7390-7400.