I am working on graident HPLC, coupled with a fluorescence detection(EX and EM = 490 and 520 nm). The mobile phase is A: 50 mM NaOH (pH12.3) with 0.1 % TFA and B: CH3CN with 0.1 % TFA. Gradient is %(A/B) 100/0 to 50/50 for 30 minutes. Since the anlytes have fluorescence only under >7, the basic mobile phase is necessary. Accordingly, the column is polymer-based column to prevent damage at higher pH.
Initially, the base-line is stable, but at 8 minutes following injection, the base-line is suddenly dropped far below a detection limit of the integrator (< -1000mcV). Do you have any idea what's going on in my analysis? I would appreciate, if you kindly give me some inputs about this. I am not analytical scientist.

Thanks,

Has this been a consistent observation, or has this only been observed once?

This message is from a questioner regarding above.

This is consistent following vehicle and analyte/vehicle (w/ diferent concentrations)injections.

One additional information:
If the mobile phase started from 80/20 to 50/50 for 30 minutes, this drop never observed, but instead the retention time of analyte became too short.

Apprecate your sugestion.
Thanks

Are the mobile phases you use:
A: 50 mM NaOH (pH12.3) with 0.1 % TFA
B: CH3CN with 0.1 % TFA.

I would suggest you use 50mM NaOH (surely the TFA is pointless here?) and 50/50 50mM NaOH/ACN, then run the gradient from 100% A to 100% B, not forgetting to leave enough time to re-equilibrate in 100%A again at the end of your run!

I think the wavelengths you are using may be too close together unless you have a very narrow band pass on your detector.

This thing is puzzling me. Bryan may have a good point: the change in refractive index of the mobile phase could be the reason that some of the excitation light is passing through the emission filter at the beginning of the gradient. As the gradient progresses, the refractive of the mobile phase may be changing enough to make this disappear, and the baseline is taking the described dive. Good point, Bryan!

Do you have a UV detector with which you can determine whether there is a corresponding (to the drop in baseline) rise in absorption? If you absorb more light you get less scattering, the latter may produce your initial baseline´s position.

Thanks all for messages above.
It appears that this sudden drop was observed, when the mobile phase exceeded roughly 15-20 % AcCN. I tried Bryan's suggested mobile phase. However, the drop was still observed.

Adding TFA delayed a retention time a bit. As a result, the resolution from impurity peaks became better. I am not sure why. Any idea would be appreciated.

Thanks so much, anyway.

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.