Chromatography Forum

When quantitating samples use HPLC with a PDA detector, why would one use a fixed wavelength (for example, at the lambda max of the compound of interest) instead of the max plot. I always use a fixed wavelength and think this is correct, but it has been a matter of debate in our lab (we are not analytical chemists!) where some prefer one method over the other.

With HPLC - UV you have two sources of selectivity; the retention - different compounds elute at different times, and the wavelngth - different compounds absorb at different wavelengths.

If you use the lambda max you lose the wavelength selectivity. Which may or may not matter depending on what you are analysing.

Peter

You could also extract the maximum selectivity (not lambda max) of each analyte in separate traces, and base your quantification on the separate extracted channels. This is what I would do but unfortunately I don't have a PDA...

There are even times when you might want to select something that isn't the wavelength maximum, for example if you're trying to analyse two things simultaneously, one much more abundant than the other. In that case, you might deliberately select a wavelength at which the more abundant thing absorbs less. I believe Shimadzu have a software option that deliberately selects a different wavelength according to concentration, with the view to increasing the linear range over which you can measure something. I haven't seen it in action, and my only concern is how you address selectivity, given that it might look great at one wavelength, and then deteriorate horribly as the wavelength gets moved, and some near-coeluting compound starts to appear.