By Anonymous on Wednesday, March 10, 2004 - 07:32 am:
I am working on graident HPLC, coupled with a fluorescence detection(EX and EM = 228 and 305 nm). The mobile phase is A: Hexane:Isopropanol (98:2)and B: Isopropanol:Water (98:2). Gradient is %(A/B) 97/3 to 25/75 for 20 minutes. The base-line increase as the gradient (~150mV). It is normal? I would appreciate your help, I am not chemist.
By HW Mueller on Thursday, March 11, 2004 - 12:00 am:
It wouldnīt surprise me if you got more Raleigh scattering as the H2O mixes in. Nevertheless, run some fluorescence spectra to rule out that you have a fluorescing impurity somewhere (if varying Ex gives the same Em itīs fluorescence, if Em varies as does Ex itīs scattering). Scattering is less at longer Ex, but, probably you can not increase Ex enough to matter.