CD spectrometry – principles of operation

A CD spectrometer (also referred to as a spectrophotometer, spectropolarimeter or a circular dichrograph) is a highly specialized variation of an absorbance spectrophotometer. This spectrometer measures the difference in light absorbance between left- (L-CPL) and right-circularly polarized light (R-CPL) at multiple wavelengths when passing through a chiral molecule.

Chirascan systems utilize a Xenon arc lamp optically coupled, via a double Czerny-Turner prism monochromator, to a photoelastic modulator (PEM) to produce an intense measuring beam. Two quartz prisms enable selection of horizontally polarized light of defined wavelength and bandwidth. Horizontally polarized light is converted to circularly polarized light by the PEM. The PEM consists of a silica block attached to a piezoelectric element. Light passing through the PEM will remain horizontally polarized when the PEM is at rest. However, when an alternating drive voltage is applied to the PEM the polarization state of the transmitted light will alternate between L-CPL and R-CPL. The optimum drive frequency is wavelength dependent and the detector electronics are synchronized to the PEM drive frequency.

Maximizing sensitivity

The limit of detection of any spectrometer is determined by the signal-to-noise (S/N) characteristics: the better the S/N, the higher the sensitivity.

Factors contributing to the S/N can be written as:

S/N = (Q.I.t) ½, , where Q = quantum efficiency, I=light intensity, t=time

There are therefore three options to maximize S/N:

1.    increase intensity of the incident linearly-polarized light from the monochromator (I),
2.    increase quantum efficiency of the detector (Q)
3.    spend more time collecting and averaging data points (t)

Maximizing light intensity and detector quantum efficiency significantly reduces the time required to carry out a measurement: quality data is collected in less time even when working under time-limited conditions such as during analysis of photolabile samples.

Increasing light intensity (I)

Chirascan systems utilize a xenon arc lamp optically coupled, via a double Czerny-Turner prism monochromator, to a photoelastic modulator (PEM) to produce an intense measuring beam. Chirascan monochromators use two quartz prisms, each grown as a single crystal.  Prisms offer the advantage of improved light dispersion in the far UV and do not produce higher order diffraction artefacts associated with grating-based monochromators.  Crystalline quartz prisms, in contrast to the more common fused silica type, are birefringent (have a refractive index that depends on the polarization and propagation direction of light) so not only disperse light into the component wavelengths, but also disperse high purity vertical and horizontal linearly polarized components.  The monochromator selects the horizonal component for conversion to circularly polarized light by the PEM. The use of two polarizing prisms causes a large wavelength/polarization state dispersion which means that wider slit settings can be used to maximize throughput of required light while rejecting stray light.

Maximizing quantum efficiency (Q)

The quantum efficiency of a detector is its efficiency in turning an incident photon into an electronic signal. Photomultiplier tubes have been used for UV and visible light detection in spectroscopy for many years and still feature in the basic Chirascan system.

Advances in photodiode technology have resulted in new, high-gain, large area solid state detectors. Chirascan V100 and Chirascan Q100 now contain an avalanche photodiode detector to provide significant improvements in quantum efficiency in the ultra-violet, visible and near-infra-red regions.

Improvements in S/N that result in higher sensitivity are clear when comparing the photomultiplier-based Chirascan with a Chirascan V100 that utilizes an avalanche photodiode detector

Increased sensitivity –
improved signal:noise –
beneficial when sample is limited

Lysozyme tertiary structure near-UV – raw data, no smoothing, 10 mM phosphate pH7.4, 20°C, baseline corrected, 3 scans sample and buffer

Fast measurement –
rapid acquisition of CD spectra –
beneficial when sample is photolabile

Lysozyme tertiary structure near-UV – raw data, no smoothing, 10 mM phosphate pH7.4, 20°C, baseline corrected, 3 scans sample and buffer

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