Chromatography Forum

Hi,

I am working with olive oil waste water in RPLC and looking for phenolic compounds. I am using a gradient solvent (%3, acetic acid (Solvent A), %3 acetic acid, %25 aceto nitrile, Solvent B) and extracting the simple with %50 methanol before RPLC.
The problem is that standard mix's cromatogram is ok with the base line but my simple's cromatograms have baseline drifting in pozitive abs. as u see on the pic

Maybe my extraction method is not proper for the simple or something else... What is your suggestion ?

280 nm, 1 ml/s, 25 c, C18 column

Please keep your extracion as it is, but after extraction please cool the sample down for some minutes at -10°C and filter the sample through an syringe filter. That will remove the rest of the oil and you will see a much better baseline. Works also with fat creams.

Your advice seems so reasonable i will try what u said and inform you
Thanks you so much !

i have tried your suggestion but it didnt work for me.

Are you kidding me ?
A 4.5 HOUR run for one sample by HPLC ???
1 mAU drift over three hours.

i can get the peaks with in 50 min. so i dont run the whole precedure..

the drift is about 2 mAU. is it ok?

Should I assume that the chromatogram you provided was just to illustrate the drift over several hours and NOT your actual analysis ? 50 - 60 minutes still sounds too long of a run for Olive Oil phenols. It looks like your actual method runs for hours.

I have developed many HPLC and LC-MS methods over the years for edible oils and their constitutes. Most of these methods utilized detailed sample prep procedures and often a gradient with Water/ACN plus a less polar solvent (i.e. Methylene Chloride, IPA, Hexane and sometimes various mixtures of three solvents, depending on the detector used). There are many literature examples of HPLC methods for this so I would think about reviewing some of the existing literature to develop a better sample prep procedure and method. Here is an example of one method I found with just a quick search on the web: http://lib3.dss.go.th/fulltext/Journal/ ... 5B1%5D.pdf

As for your baseline drift... as I said in my previous post, 2 mAU in three hours is not a big deal. Esp over a 4.5 hour run !
Worry less about drift and more about injecting a clean sample combined with a good method.

There is an increased drift here during a peak (the baseline does not drop to where the drift ahead of peaks would predict). The only explanation I have for this is that he has incredible crud on the column which is even displaced partially by a peak. This is also indicated by the initial crud lasting one hour, one can expect a change of column properties with that much dirt on it.