My laboratory has recently purchased a new UV-VIS detector for HPLC. It works with 2 wavelengths, working and reference. The rest of detectors we have work with air as reference. Neither the manual nor the vendor have given clear explanations about how calculations are made with the new detector. I understand that the instrument performs a subtraction or ratio of absorbances in order to compensate for lamp fluctuations and get more stable outputs. Am I right?
The detector has two lamps (one for UV 190-360 nm, another one for 361-700 nm). I assume that working and reference wavelengths have to be taken from the same lamp, otherwise compensation of possible fluctuations wouldn’t work.
Multicomponent chromatograms with different individual spectra may look quite different when changing the reference wavelength unless it is carefully chosen.
I’d like to get comments on that issue. Thank you very much in advance

I think the reference wavelength is used more to compensate for refractive index changes due to flucuations in temperature, and compostion during a gradient.

The reference wavelength should be picked such that the wavelength minus half of the bandwidth dosen't overlap with your sample spectra.

With multicomponent spectra you shouldn't overlap with any of your sample spectra. If you are running a gradient with multiple components with varying lambda max's and overlapping spectra that are precluding you from choosing a suitable reference wavelength you might be able to use an isobestic wavelength with a relatively narrow bandwidth. However, anytime you reference with a wavelength contained in your sample spectra you could impact linearity.

Your detector manual will probably have some good information on optimizing settings. Good Luck.