The detector measures absorbance simultaneously at wavelengths from 190 to 640 nm. A UV-lamp provides good sensitivity over the whole wavelength range.
If you know little about the analytes in your sample, store all spectra over the full wavelength range. This provides full information but fills up your disk space rather quickly. Spectra can be used to check a peak’s purity and identity. Spectral information is also useful to optimize wavelength settings for your chromatographic signal.
The detector can compute and store at run time up to 8 signals with these properties:
sample wavelength, the center of a wavelength band with the width of sample bandwidth (BW), and optionally
reference wavelength, the center of a wavelength band with the width of reference bandwidth.
The signals comprises a series of data points over time, with the average absorbance in the sample wavelength band minus the average absorbance of the reference wavelength band.
Signal A in the detector default method is set to sample 254.0/4, reference 360.0/100, that is, the average absorbance from 252 – 256 nm minus the average absorbance from 310 – 410 nm. As all analytes show higher absorbance at 252 – 256 nm than at 310 – 410 nm, this signal will show you virtually every compound which can be detected by UV absorbance.
Many compounds show absorbance bands in the spectrum. Optimization of wavelength setting shows the spectrum of anisic acid as an example. To optimize for lowest possible detectable concentrations of anisic acid, set the sample wavelength to the peak of the absorbance band (that is, 252 nm) and the sample bandwidth to the width of the absorbance band (that is, 30 nm). A reference of 360,100 is adequate. Anisic acid does not absorb in this range.
If you work with high concentrations, you may get better linearity above 1.5 AU by setting the sample wavelength to a valley in the spectrum, like 225 nm for anisic acid.
A wide bandwidth has the advantage of reducing noise by averaging over a wavelength range — compared to a 4 nm bandwidth, the baseline noise is reduced by a factor of approximately 2.5, whereas the signal is about 75 % of a 4 nm wide band. The signal-to-noise ratio for a 30 nm bandwidth is twice that for a 4 nm bandwidth in our example.
Because the detector averages absorbance values that are calculated for each wavelength, using a wide bandwidth does not negatively impact linearity.
The use of a reference wavelength is highly recommended to further reduce baseline drift and wander induced by room temperature fluctuations or refractive index changes during a gradient.
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