Performance of a circular dichroism spectrophotometer

The limit of detection of a circular dichroism (CD) spectrophotometer (or any spectrophotometer) is determined by its signal-to-noise (S/N) characteristics: the better the S/N, the better its limit of detection. The signal-to-noise ratio is limited by photon shot noise, which is the statistical variation about an average in the number of photons per unit time detected by the light detector. The quantised nature of photons and their random arrival at the detector means that although the average number of photons detected per second may be say 5, the number in any particular one-second interval may be 0, 2, 7 or some other number. Thus, a measurement must be made over a sufficiently long period of time to determine the true average and the time taken to determine the true average will be inversely proportional to S/N. It is therefore important to design a circular dichroism spectrophotometer to maximise its S/N characteristics.

A general relationship between the contributing factors to the signal-to-noise in an optical spectrometer can be written as:

Q = detector performance, I = light intensity, t = time scale of the measurement.

where Q = detector quantum efficiency, I = light intensity, t = time scale of the measurement.

From this it is apparent there are three ways to improve the signal-to-noise of a circular dichroism spectrophotometer: increase the intensity of the incident linearly-polarised monochromatic light, increase the quantum efficiency of the detector, or spend more time collecting and averaging data points.

The first two factors, light intensity and detector performance, are those that can be influenced by the design of a circular dichroism spectrophotometer and work together to lower the last factor, the time required to carry out a measurement. The higher the light throughput and better the detector efficiency, the less time it takes to collect quality data or, equally, the higher the quality of data that can be collected in time-limited experiments such as stopped-flow measurements.

Increasing the intensity of the incident light is the main avenue for increasing the performance of circular dichroism spectrophotometers and this finds its ultimate expression in the use of synchrotron light-sources for CD spectroscopy. Synchrotron facilities provide tremendous light intensity across a very wide spectral range of wavelengths but access to them is expensive and limited and their use is restricted to the more cutting-edge applications of circular dichroism. For the vast majority of CD experiments, a high-intensity bench-top source is the only practical option: Applied Photophysics Chirascan has been designed from the ground up to maximise the light throughput from its Xe arc-lamp source to the sample.

The Chirascan monochromator uses two synthetic, single-crystal quartz prisms instead of the diffraction gratings that most people are familiar with from normal absorbance spectrophotometers. Quartz prisms are more efficient than diffraction gratings for a very wide range of wavelengths, particularly in the UV. Quartz is also birefringent and the prisms not only disperse light into the component wavelengths but also, because of their birefringence, disperse the linearly polarised components, one of which is selected for conversion to circularly polarised light. A further advantage of prisms is they do not pass second-order multiples of the desired wavelength, which is a major source of stray-light in grating-based monochromators.

Unlike the dispersion of a grating, which is linear and highly customisable, the dispersion of a prism is non-linear and is set by the properties of the prism material. Consequently the optics and mechanics of a prism monochromator have to be more complex than a grating monochromator, with the need to constantly vary the slit-width as a function of wavelength to maintain a constant band-pass, and a complex relationship between prism movement and wavelength. However, the large wavelength dispersion in the UV means that wider slits can be used even at a small spectral band-pass, which means greater light collection efficiency throughout the UV region. The large majority of circular dichroism applications are carried out in the UV and it is at these wavelengths that the characteristics of a prism are a major advantage.

In addition to the high intensity to improve signal-to-noise, the light from the monochromator must have a very low stray-light content and a very high purity of linear polarisation to provide accurate measurements. All of these three key elements have been optimised in the Chirascan circular dichroism spectrophotometer and have been achieved by key design features of the Chirascan monochromator. These design features are outlined in the slide show below.

The second influence on the S/N is the quantum efficiency of the detector used. i.e. its efficiency in turning an incident photon into an electronic signal. In circular dichroism spectrophotometers, the detector of choice in the last few decades has been the photomultiplier tube. The quantum efficiency of these detectors, which are traditionally used in spectroscopy for UV and visible light detection, has remained fairly static over that period. Recently, advances in photodiode technology have resulted in new, high-gain, large area solid state detectors, which provide significant improvements in quantum efficiency in the ultra-violet, visible and near-infra-red regions when compared with photomultiplier tubes (see Figure 1). One of these new high-performance solid state detectors is featured in the new Chirascan Plus and it gives further S/N improvements over the photomultiplier-based Chirascan spectrometer. The quantum efficiency improvement outlined below results in significant signal-to-noise improvements over and above the already high performance of the standard Chirascan.

For a more information on the Chirascan and Chirascan Plus circular dichroism spectrophotometers, please refer to the products section or email your questions.

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Introduction to Circular Dichroism Understanding circular dichroism: the basics of polarisation Chiral molecules: the origin of optical activity Chirality and biology Principle of operation of a circular dichroism spectrometer Performance of a circular dichroism spectrometer Circular dichroism signatures of secondary structural elements CD units and conversions

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