Chromatography Forum

Hi all,

I have a problem with my chromatogram. Three of my peacks are negatives.
What is the reason? I use Agilent sistem 1200 equiped with UV-DAD detector.
My standarts are chlorogenic acid, p-cumaric acid, caffeec acid, rutin, miricetyn, quercetin and naringenin. Mobile phase is: A- 0.5 % acetic acid, B- MeOH. Flow rate is 0, 800 ml/min, UV-DAD 280 nm, column- Poroshel 120 ( 4.6 x 75 mm x 2.7 ).

Negative peacks in my chromatogram are rutin, miricetyn and quercetin.

What you're seeing is consistent with an inappropriate reference wavelength setting. If a compound absorbs more strongly at the reference wavelength than it does at the measurement wavelength, it will generate a negative peak. Try turning off the reference wavelength.

Thank you for the answer Tom!
When I turned off reference wavelength all was ok!

I am glad because you could overcome your challenge, Zanio. Now, since you have an UV-DAD I recommend select your further reference wavelengths based on each analyte DAD spectrum. All the best for your Team.