Chirascan™ Circular Dichroism Spectrometer

Performance Information

Quality of measurement and its relation to light throughput

The high light throughput of Chirascan and Chirascan-plus, particularly at far-UV wavelengths, is the key reason for their superior sensitivity and speed. Shown below are calibrated radiometric scans of the light flux various bandwidths.

The photon flux for a 1nm bandwidth is in excess of 10¹³ per second for all UV wavelengths from 360nm to 180nm. This is many times as intense as the flux of any other commercially available circular dichroism (CD) spectrometer. Should more light be required, a bandwidth of up to 4nm can be maintained down to 178nm, a feature that is unique amongst prism-based CD spectrometers and derives directly from the innovative optical design of Chirascan. It means that for Chirascan, where the photon flux at 1nm is already superior to that of other CD spectrometers, there is more than a further order of magnitude of light flux in reserve.

The superior light throughput of Chirascan translates directly to superior quality of measurement for a given measurement time or, equivalently, a measurement of a given quality can be completed much more quickly. Chirascan-plus has still greater performance benefits due to its more sensitive photodiode detector. Here are a few examples of why this is important:

• In laboratories where there is high demand on the instrument, productivity is significantly increased without compromising data quality.
• In experiments that are demanding of sample, for example CD stopped-flow, the number of repeat measurements (and hence volume of sample) required to achieve acceptable quality is reduced. With ten times the light, less than one-third of the sample is required.
• More data can be generated in a single experiment - for example; far-UV wavelength-scanning in combination with continuous-ramp thermal melts generate a complete picture of protein secondary structure denaturation in approximately one hour.

Advantages of stepped scanning

Both Chirascan and Chirascan-plus acquire CD spectra using stepped scans. The CD signal is sampled at discrete wavelength points in the spectrum for a time defined by the user. Each measured point is the average of tens of thousands of samples and the number of points in the CD spectrum (or the wavelength increment) is determined by the user.

There are very good reasons for using stepped scans and they include:

• Each CD value in the spectrum is accurate because it is sampled at a single point with the monochromator held stationary
• Each CD measurement has an associated error and therefore has scientific validity
• No electronic filtering (time-constant filtering) is used to smooth and potentially distort the CD spectrum

Because Chirascan has very high light throughput (see above), the raw, unsmoothed spectrum is of high quality and in general will need no further treatment. However, post-acquisition smoothing tools can be used to remove random noise elements if required. Two key advantages of this method of smoothing are:

• A residual plot can be generated and inspection will show whether or not the spectrum has been distorted by the smoothing process
• The smoothing process is fully reversible and the original (raw) data are never lost

Of course data can be over-smoothed even when using post-acquisition filtering techniques (see below) but this is always evident and can be corrected.

It should be noted that for continuous scanning, none of the above is true: measurements are made with the monochromator in motion; time constants are applied which damp the signal and disguise the true quality of the spectrum; it is impossible to calculate the error in a measurement; smoothing is carried out during data acquisition and may distort the spectrum; electronic smoothing it is not reversible. Continuous scans can appear to have low noise but, because the data are smoothed during the acquisition, the user cannot assess the true quality of the CD spectrum or whether the smoothing process has distorted its shape. There is no valid reason for collecting data in this way: it disguises poor data quality and can distort spectral features.

When comparing the performance of different CD spectrometers, it is essential to compare the stepped scan data in order to make a valid comparison.

Post-acquisition smoothing

The CD spectrum shown above (left) was recorded on a Chirascan spectrometer and is raw (unsmoothed) data. In this example the spectrum is fairly noisy because of the small size of the CD signal and high scan speed used. If we now over-smooth this spectrum (above right), the resulting spectrum (blue trace) appears very smooth but the distortion is immediately obvious simply by comparing it with the raw spectrum (red trace). The distortion can also be seen from the residual spectrum (green trace) which is non-random about the x-axis.

A valid smooth of the same spectrum (above left) shows no distortion and, as can be seen from the residual trace, only random noise has been subtracted from the data.

The second example (below) is more typical. The unsmoothed spectrum was acquired on a Chirascan spectrometer in 38 seconds and is of high quality due to the high light throughput of Chirascan in the far-UV wavelength region. However some post-acquisition smoothing may be desirable to remove random noise elements.

In summary, continuous scans use electronic filters to generate smooth CD spectra but may also produce distortion of the CD spectra. Stepped-scans record unfiltered CD measurements and so are always undistorted. If required, post-acquisition tools can be applied to smooth these spectra in a controlled and reversible way.

Long Term Stability and Accuracy

Long term stability is of key importance for any CD spectrometer as it will be expected to be in service for many years. The spectra on the left show the CD spectra of Vitamin B12 recorded on two Chirascan instruments: a new instrument ready to leave the factory and an 18 month old instrument with a lamp that is nearing the end of its recommended life. The spectra shown are raw (unsmoothed) data collected back-to-back using the same sample and under the conditions tabulated below. The corresponding baseline for each instrument is overlaid (blue spectrum).

Following baseline subtraction, these CD spectra are overlaid for comparison in the figure (right). As can be seen, the spectra are virtually identical, underlining the long term accuracy and stability of Chirascan - and the long lifetime of the lamp.

Low Nitrogen Usage

It is essential that CD spectrometers are purged with a steady stream of nitrogen gas in order to:

• prevent generation of ozone by the xenon arc source
• remove oxygen from the light path (oxygen will absorb light at wavelengths in the far-UV)

Chirascan requires a total nitrogen flow of only 5 litres/min irrespective of the wavelength range being used.

Chirascan's sealed design ensures that even when it has been unused (and unpurged) for several days, the start-up time to achieve a good nitrogen environment for far-UV CD measurements is still rapid.

For the purpose of efficient nitrogen purging Chirascan has three separate nitrogen inlets, each with its own flow-meter:

• a sealed lamp housing (requiring 1 l/min)
• a sealed monochromator (requiring 3 l/min)
• a sealed light path within the sample chamber (requiring 1 l/min)

The sealed light path within the sample chamber ensures that the sample chamber can be opened, and the sample cell removed, without compromising the nitrogen environment in the region of the cell holder. Hence the user does not have to wait for the nitrogen environment to be re-established after changing the sample under investigation. This also enables the routine measurement of absorbance spectra with CD spectra because good absorbance measurements depend on a comparison of the detector voltage with and without the sample under identical nitrogen conditions.

Long Lamp Life

It is recommended that the xenon lamp is replaced every 1000 hours. The current lamp life is recorded automatically monitored and displayed by the lamp ignition switch.

Software Upgrades, Licences and Access

Relevant software upgrades are always available for the lifetime of the instrument and are free of charge.

The Pro-Data Viewer software (for data display, manipulation and analysis) can be installed on an unlimited number of PCs for off-line data inspection.

An emulator version of Pro-Data Chirascan (instrument control software) can also be installed on an unlimited number of PCs allowing users to gain familiarity with the instrument control at their desktop.

Chirascan - the new standard in circular dichroism