Chromatography Forum

Hello guys,

I'm trying to analyze 4-nitrobenzoic acid in phosphate buffer solution (PBS, pH 7) with reversed C18 column (Macherey-Nagel CC 125/4 NUCLEOSIL 100-5 C18). The problem is the target compound eluted so fast (retention time around one minute) that the peak almost overlapped with solvent peak. I tried the eluent as an isocratic 30% acetonitrile 70% DI water as well as PBS instead of DI water. Also tried to increase the proportion of polar eluent (DI or PBS) from 70% to 90%. All attempts have failed to delay the peak retention time. Here's the question: If I use phosphoric acid which pH is less than pKa of 4-nitrobenzoic acid for an eluent, will the retention time be delayed? Any other suggestions would be more than welcomed.
Thanks,

Sung

Try a HILIC mode column.

Here's the question: If I use phosphoric acid which pH is less than pKa of 4-nitrobenzoic acid for an eluent, will the retention time be delayed?

Yes. Using 0.1% phosphoric acid (or a phosphate buffer in the pH-range of ~2-2.5) as aqueous part you should be able to protonate the acid and thus get decent retention on C18.
Is your sample dissolved in PBS pH7? Then you should keep the inkection volume small in order to prevent any solvent/eluent mismatch effects.
I'd use a scouting gradient (something like 10-70% organic) in order to get a hint for a useful aqueous/organic ratio if you want to go isocratic.

Thank you for the reply. I tried phosphoric acid (pH~2) instead of PBS and it eluted the compound with the reasonable retention time. May I ask more about the 'scouting gradient'? You mentioned that you can get a hint for an eleunt ratio from the scouting gradient, but I can't understand how it works. For example, if the organic eluent keeps increasing from 10% to 70% as you said for 40 minutes and the compound elutes around 10 minutes (when the organic eluent is around 30%), how would you interpret this result to guess an organic/aqueous ratio for isocratic method?

Use this:
viewtopic.php?f=31&t=19492

Thank you Tom. The spreadsheet is very straightforward and simple to use. I will use as a guideline. Thanks again.
Regards,
Sung

you can try something like this:

http://www.sielc.com/Compound-3-Nitrophthalic-Acid.html

retained by combination of RP and anion-exchange mechanisms.