Nitrogen Purge Efficiency in the J-815

All CD spectrometers operating at wavelengths below 190 nm require N2 purging. One reason is to prevent the generation of ozone gas due to the exposure of atmospheric O2 to far UV radiation. Another related reason is to allow the monochromator to transmit light into the vacuum UV region. This spectral region, below 190 nm, is of critical importance for biomolecules, particularly in protein secondary structure estimation.

The JASCO J-815 incorporates an engineered distribution system which is unsurpassed in its ability to provide a pure N2 environment for light penetration into the deep UV. The engineered flow design eliminates the need for energy depleting lamp house and sample chamber windows which are found in other CD spectrometers. Safe ozone free operation down to 185 nm is possible with as little as 3L/min flow. Moreover, the high efficiency flush design enables the operator to easily scan as low as 163 nm with very modest consumption.

Stray Light in CD Measurement

Stray light in a spectrometer is defined as light whose wavelength is outside the expected spectral bandwidth of the instrument at a designated wavelength. It is extraneous light which finds its way to the detector and adds an artifact to the result. For CD spectrometers, stray light is usually manifested as a reduction in observed CD
intensity; an effect which becomes much worse at higher optical densities (OD).

As experienced users know, particularly in biomolecular applications, high OD conditions are often a fact of life due to high sample and buffer absorbance especially at the low UV wavelengths so critical for protein structure analysis.

The result of 40 + years experience in designing CD monochromators, the JASCO J-815 has by far the lowest stray light figure of any available CD spectrometer. Using accepted ASTM methods, the J-815 system stray light is measured as 0.00002% over most of its spectral range. The J-815 is the best tool available to enable quality CD spectra even under challenging sample conditions.

Step Scan vs. Continuous Scan

In the modern CD Spectrometer, at least two methods of data acquisition may be employed: step scan and continuous scan. Only JASCO allows the spectroscopist to select either method. Although it has been asserted that step scan is inherently superior to continuous scan and therefore continuous scan need not be considered, this statement is mathematically inaccurate.

Step scan, which is the only method available in competing CD spectrometers, involves recording a user defined number of data at discrete wavelength points, and taking the average of each data set at each wavelength point to represent the spectra. Superficially, the step scan method has one major advantage: Since the monochromator is held still during data acquisition, the analyst can unequivocally say that each data point was collected only at one wavelength, within the constraints of the spectral bandwidth that is selected.

However, even if we accept this as an advantage over continuous scan (which it is not, under closer analysis), there are two major disadvantages: higher noise levels per spectral point and much longer acquisition times per spectra.
First, the continuous scan method, which reports data as a moving average of the wavelengths covered during one Digital Integration Time (DIT) interval, can be shown to enjoy a greater reduction in noise per unit DIT, especially in long DITs.

Second and most importantly, since the continuous scan method maintains a moving average over the DIT, the user can sample their average at any wavelength interval he chooses. However, with the step scan method, changing from a 2 nm interval to a 1 nm interval means doubling the total scan time. This data interval is critically important: if we want a 1 nm spectral bandwidth (SBW), we lose the information content inherent in that bandwidth unless we sample at least a 0.5 mm data interval (in some arguments an even smaller data interval should be used).

In the description of step scan, we acknowledged that it allows an easy assumption of wavelength and bandwidth accuracy. The question is, does continuous scan compromise these?
The answer to both is no.

In the JASCO system, the data acquired in the DIT interval is assigned to the wavelength occurring at the middle of the interval, thus insuring wavelength accuracy. Is the effective bandwidth changed with the moving average continuous scan? Of course, but in a predictable and clearly defined way. JASCO software constrains the scan speed to a level which will never compromise the data integrity for the combination of DIT (desired signal to noise) and SBW (desired spectral resolution) that the user selects.

Auto Step Scan Mode

Auto Step Scan Mode is a unique mode of data acquisition in which the response time and therefore the total acquisition time per data point is varied automatically by the software to suit the particular noise conditions at a given wavelength. When noise is higher (usually due to decreased light level) the acquisition time is increased to compensate. Therefore the total acquisition time can be minimized by applying longer response times only when they are required by the wavelength and sampling conditions.

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