The present disclosure relates to the field of polarization spectroscopy. More specifically, it provides a means by which circular dichroism (CD) measurements may be performed in the vacuum ultraviolet (VUV). In one embodiment a highly efficient laboratory-scale VUV CD spectrometer is provided. As used herein, vacuum ultraviolet (VUV) light includes, generally, wavelengths of light that are about 190 nm and less wavelengths.
When light passes through a solution of optically active (e.g. chiral) molecules, left- and right-circularly polarized components traverse the solution with different speeds and are absorbed by the solution to different extents. The differing propagation speeds lead to the effect of optical rotation (OR), which can be probed by measuring the rotation of the polarization plane via optical rotatory dispersion (ORD). Near absorption bands, circular dichroism (CD) spectroscopy measures the differential absorption of left- and right-circularly polarized light by the substance. Both methods have been successfully used to characterize properties of solutions containing optically active molecules. In addition to being sensitive to inherent chirality, ORD and CD are also sensitive to different conformations of complex molecules. As a result, one of the most prolific applications of CD spectroscopy has been the study of the secondary structure of soluble proteins.
Optical rotation and circular dichroism have the same underlying cause, and are related to each other via Kramers-Kronig integration. Defining circular birefringence (CB) as twice the optical rotation, a complex general circular retardation can be defined by:C=CB−iCD  Eqn. 1where CB and CD are functions of wavelength. The form of Eqn. 1 and the Kramers-Kronig relationship it obeys are familiar from the (isotropic) optical dispersion of materials, n-ik, where n is the material's refractive index and k is the extinction coefficient. That n and k obey the Kramers-Kronig relation implies that the wavelength dependence of the index of refraction is determined by the material's absorption, and vice versa. Similarly, that CB and CD are related via Kramers-Kronig integration implies that complete knowledge of one over all wavelengths or energies determines the other.
Practical experiments, however, occur over finite wavelength ranges and ORD and CD spectroscopy have different advantages, depending on the wavelength range explored. While the ORD spectrum can be determined far from the absorption bands responsible for its effect, the CD spectrum directly probes the bands, and as such, is considered more sensitive and spectrally “compact”. In ORD spectroscopy the information associated with a given absorption band is typically spread out over a large energy range. As a result, the ORD spectrum in a given wavelength range contains overlapping contributions from multiple absorption bands. In contrast, with CD spectroscopy the information due to a single absorption band is localized within a smaller energy range, resulting in much less overlap between measured features. If the absorption region is accessible experimentally, CD spectroscopy is typically the preferred technique since it more directly probes the underlying “cause” of the optical activity of the substance being measured.
In the case of soluble proteins, the secondary structure of the molecules results in the optical activity of the solution, and different types of secondary structure, such as alpha-helix, beta sheet, polyproline-II helix, etc. give rise to distinct features in CD spectra. The electronic bands responsible for protein CD largely reside in the ultraviolet. The alpha-helical structures generally involve bands centered at ˜222, 208, 192, 175, 160, and 140 nm. Meanwhile, beta sheet structures have weaker bands at ˜215, 198, 175, and 168 nm. Variations in beta sheet geometry result in further modifications to CD spectra. The polyprolene-II helix has observed bands near 226 nm and 206 nm, and gives rise to spectra similar to those of proteins previously characterized as “un-ordered”. In all cases, other bands may exist but have yet to be observed and/or identified.
The ultraviolet wavelength region may be considered as consisting of two distinct segments: the near-UV region from ˜190-400 nm, and the “far-UV” or vacuum ultraviolet (VUV) region below 190 nm. Conventional CD spectrometers are limited in operation down to about 190 nm. The primary motivation to extend CD studies into the VUV lies in the existence of additional absorption bands present at shorter wavelengths. As a consequence of these supplementary features, VUV CD spectra inherently posses increased information content relative to their longer wavelength counterparts. While traditional CD instruments are often limited to determining the amount of alpha-helix structure present in a solution, VUV CD systems are capable of extracting numerous secondary component fractions. In addition, these powerful systems can also provide insight into conformational changes, such as fold state, independent of secondary structure. It is important to note that this information enhancement is not simply due to a “more data is better” argument; the improved capabilities are a direct consequence of the additional absorption bands present in the VUV.
Optical studies in the VUV are difficult to conduct due to the intrinsic absorption of most materials in this region. This phenomenon precludes the simple extension of, or modification to, traditional longer wavelength optical instrumentation to facilitate operation at these energies. To achieve efficient optical performance in the VUV, an instrument must be explicitly designed to do so. Specifically, conventional optical systems are designed to operate in atmospheric conditions and typically lack, among other things, the controlled environment required for operation at these shorter wavelengths. VUV radiation is strongly absorbed by both oxygen and moisture; hence, these species must be maintained at sufficiently low levels in order to permit transmission of VUV photons through instrument optical paths. Attempts to reach shorter wavelengths by simply purging with non-absorbing gases generally yield poor results. Furthermore, transmissive optical components that are otherwise suitable for near-UV or visible wavelength operation, routinely absorb strongly in the VUV. Consequently, reflective elements must instead be employed, greatly restricting design options. As a result, it is comparatively difficult to achieve high optical throughput in VUV optical instrumentation.
With the exception of early work by Johnson (Johnson W. C. (1971). “A circular dichroism spectrometer for the vacuum ultraviolet”. Rev. Sci. Instrum. 42(9): 1283-1286), progress towards development of a dedicated, highly efficient VUV-CD instrument has been limited. Today's commercial bench top systems are designed to operate at near-UV and longer wavelengths. Several of these systems offer simplistic purge functionality in an effort to extend capabilities into the VUV. Practically however, poor data quality restricts these instruments to operation at wavelengths above approximately 185 nm.
The most capable VUV CD systems in existence are those integrated into synchrotron radiation (SR) beam lines. The advent of such instruments in the 1980's and 1990's brought about tremendous enhancements in both CD data quality and information content. These improvements however, were largely the result of the remarkable intensity of SR sources, rather than fundamental advancements in instrument designs. As a consequence of these developments, interest in VUV-CD spectroscopy has grown considerably, resulting in the commissioning of several new beam lines and the identification of a myriad of applications for this technology. SR-CD systems are powerful, but the disadvantages are obvious: synchrotron facilities are huge and enormously expensive, making accessibility a severe limitation.
It follows that there would be great benefit in the development of a highly efficient, high throughput laboratory-scale VUV CD spectrometer, which does not require synchrotron radiation. Such an instrument would render high-throughput structural investigations of proteins widely available, thus creating new opportunities to accelerate discoveries in structural proteomics.