Chromatography Forum

Dear All,
I'm trying to assay a drug in plasma extracts by a HPLC system equipped with DAD. The analyte has the maximum absorption at 341 nm, and no absorption around 450 nm. So I set the DAD condition on 341 nm for detection and 450 nm as the reference wavelenght. On the other hand, I'm using an isocratic elution, so I'm not sure if I basically need to use a reference wavelength or not. Can I expect improvement of the results, or a detrimental effect?

::moved to the correct forum::

My guess is that it probably won't make much difference, but try it both ways and see what happens.

In general, the use of a reference wavelength is rarely useful and often detrimental.

No reason to use it for isocratic methods as my recollection is the S/N is better without a reference wavelength.

My first choice would always be to start without a reference wavelength. In a second step you may try if the S/N improves. Make sure to choose a wide reference bandwidth, something like 100 nm.