The negative peak always appear in the blanks.
I have tried several approaches which didn’t seem to work.
I have cleaned the HPLC with isopropanol + acetonitrile + hexane + dichloromethane (50:25:15:10%, v/v) overnight at a flow rate of 0.5ml/min without the column.
I have also injected the same mobile phase as the blank but the negative peak is still there.
I have set the reference wavelength of detection at 190 - 800nm, 200 - 600nm, 200 - 500nm and 220 - 600nm but no difference.
The last thing I did was washing the column with 100% acetonitrile but it didn’t solve the problem of the negative peak.
The column that I am using is a Waters Spherisorb 3µm ODS2, 4.6 × 250 mm.
Your help will be fully appreciated.
Thanks
Hello,
The negative peak appears only when injecting? When you monitors the baseline the negative peak appears?
If you are injecting solvent, the negative peak could be a bubble. Did you try to purge your injection system, like the syringe, and its tubings?
Best,
Hi Rafael,
Yes the negative peak appear only when I injected blanks or sample between 1.8 to 2.8mins and the negative peak is at the baseline.
Thanks.
Dear Oliver,
The negative peak is at 2mins on the baseline.
The mobile phase is acetonitrile + methanol (65:35%, v/v) with added 0.065% triethylamine.
You may see the chromatogram posted.
Thanks.
Which HPLC are you using? We have an old Beckman that puts the wash solution in the injection loop. This would cause a difference in the refractive index and show as a peak (either positive of negative depending on the mobile phase and the wash solution) all along the diode array from 190 to 400 nm.
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