Chromatography Forum

Hello,

I am currently trying to validate RP-HPLC isocratic method. I am getting negative peak interfering with my analyte peak. Negative peak appears because there is a difference in UV absorbance between sample solvent and the mobile phase.
Is there any method to integrate my analyte peaks without changing LC conditions (especially sample's solvent)?

Conditions are:
Eluent: Methanol-MiliQ water (60:40)
sample: wastewater
Sampe solvent: water
flow: 1 ml/min
Detection: PDA (220 nm)

Thank you in advance for your reply.

Dear Sebek, since your negativ peak interferes with the analyte it seems that your analyte is not retained. Did you consider to reduce the amount of organic in the mobile phase? Normally it helps. If not, than you should think about changing either the stationary phase or the organic part of the mobile phase.
Success

Before jumping to conclusions:
- what size column?
- what is the retention time of your analyte peak?

What kind of PDA?

If you have an Agilent, or any model that has a "reference" setting, you could have an interference that is absorbing in the reference wavelength range, causing the baseline to be negative.