Chromatography Forum

Dear All, I may need advice and insights into the following problem.

I am trying to separate what I suspect to be dinitrophenolic isomers using HPLC.

I am using a C-18 column, and the mobile phases are methanol and water. I tried using gradient elution of all sorts using methanol/water mixture but they have all turned out unsuccessful; the peaks are not baseline resolved.

Does anyone have any experience on separation of such dinitrophenolic isomers?

Also, I noticed 2 things:

1. The baseline actually creeps higher with time, and I think this is due to the changing polarity of the solvent mixture. Is an upward drifting baseline acceptable in a chromatogram if a gradient elution mode is employed?

2. The chromatogram actually shows "troughs" which dips obviously below the baseline, instead of the usual peaks that are expected in chromatograms. What causes these? All these features make my chromatogram look "ugly" and "messy".

Please enlighten me, someone?

Dips can be caused by several factors, but the most common is a difference between Mobile phase and sample solvent. Is your sample solvent the same as the mobile phase at the start of the gradient? If not, that may be your problem. I have also obtained dips in methods that use triethylamine is the mobile phase.

It is normal for the baseline to go up during a gradient run. Methanol is more UV adsorbent than water, so the baseline rises as the gradient progresses. What is your detector wavelength? Unless the wavelength is high (over about 250) methanol is usually a poor choice for a gradient system. Can you use Acetonitrile? It absorbs less.

As far as your separation goes, you may want to try a phenyl column instead of a C18, this should be more selective for aromatic compounds.

Good luck!

Thank you Albany, you have answered many of my queries.

Yes, my samples are purely aqueous while the mobile phase used was a mixture of organic (methanol) and milliq water.

And yes, the baseline drifted upwards when the % methanol increased.

Thank you

Albany is correct - phenyl column should work better for that application:

http://www.imtakt.com/TecInfo/TI087E.pdf

Dionex has a complementary pair of columns designed for explosives analysis using methanol-water mobile phases. The Acclaim E2 has different selectivity than C18, and might do what you want. http://www1.dionex.com/en-us/columns_ac ... 35329.html Also, our Polar Advantage has very different selectivity for nitroaromatics than C18 does.

Your baseline trouble might be due to dirty methanol. What wavelength are you using? Also, some detectors have trouble with refractive index effects, and sharp gradients cause baseline disturbances.

Althernatively, you might want to use a porous graphitic carbon column (i.e. Hypercarb...) as it has much higher pi-pi interactions than phenyl columns. Generally speaking porous graphitic carbon columns provide better separation for phenolic isomers than phenyl columns...