Negative peak in Gradient Blanks Across Entire UV Range

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I'm developing a HPLC method to retain some polar compounds that have weak UV absorption (only one acid/ester group and sometimes an amide group). The route I've chosen to pursue is using a 100% aqueous initial gradient on a suitable column (have tried both the Atlantis T3 and Synergy Hydro-RP) with phosphoric acid as the modifier as formate/acetate kill the sensitivity for these compounds.

For both columns and using either phosphoric acid or 10 mM ammonium phosphate, the issue always persists. It has happened on two HPLCs as well.

If I start at 100% aqueous mobile phase, with no hold time or a 2-5 min hold, the negative peak is there. If I start with 2% or 5% organic, then the peak is massively reduced, though I don't get the retention I need.

The thing that is stumping me is why there's such a sharp peak in gradient blanks (no injection). It seems to be correlated to when acetonitrile starts getting introduced into the gradient.

Relative to 0 mAU at t0, the peak is about the same magnitude in single wavelength chromatograms (~-60 to -70 mAU) across the entire UV range I collected on my DAD (190 - 400 nm). Below is an isoabsorbance plot, spectrum of this peak, and chromatogram at 210nm of a no-injection gradient blank. Note the negative dip (represented by black color):


Method info:
A: 0.1% H3PO4 in HPLC-grade water
B: HPLC-grade acetonitrile

0-4 min: 0%B
4-10 min: 0-80%B
10-12 min: 80%B
12.1 min: 0%B

Phenomenex Hydro-RP, 150 x 4.6mm, 4um, 1 mL/min

Any ideas? Thank you!
What you see is an refractive index issue especially at 210nm. If you run 100% ACN and add water to the mobile phase you would see the same sharp peak, but positive peak.
Gerhard Kratz,
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