Chromatography Forum

Hi,

Our lab is trying to separate melamine from other impurities using LC-DAD.

The conditions used are as follows:
Flow rate - 1ml/min
Mobile phase - 85:15 10 mM ammonium formate with 0.2% formic acid, 10 mM sodium octane sulfonate: ACN
Column - Varian C18
Injection volume - 20 uL
Wavelength - 190 to 300 nm

I understand that for ion pair chromatography, longer column equilibration times are required. How long a time is sufficient typically? We allowed the column to equilibrate for at least an hour.

We achieved separation, but the UV spectra of all the peaks are the same, so I'm puzzled. We also ran the std solutions of the impurities and their RT do not match.

Also, is temperature very critical in ion pair chromatography?

Thanks,
Wan

How long a time is sufficient typically? We allowed the column to equilibrate for at least an hour.

There is no global answer. Do a series of injections, and if your retentions are repeatable, then you are equilibrated.

but the UV spectra of all the peaks are the same

No great surprise. UV spectra in solution are notoriously information-poor, and if your compounds are related, it is quite possible that they have similar chromophores.

We also ran the std solutions of the impurities and their RT do not match.

That is more troubling. You may not have fully equilibrated your column (see my comment on that above). If you get consistent retention for the standards, and *different* retention times from a sample, there are a couple of possibilities:
1. The compounds you are seeing are *not* those you are expecting.
2. You have problems with your diluent or matrix.

If you haven't already done so, spike a sample with detectable quantities of your expected impurities. That should confirm or deny the first possibility.

Also, is temperature very critical in ion pair chromatography?

Yes!!

but do we need ion pair in the first place?

Hi,

Our lab is trying to separate melamine from other impurities using LC-DAD.

The conditions used are as follows:
Flow rate - 1ml/min
Mobile phase - 85:15 10 mM ammonium formate with 0.2% formic acid, 10 mM sodium octane sulfonate: ACN
Column - Varian C18
Injection volume - 20 uL
Wavelength - 190 to 300 nm

I understand that for ion pair chromatography, longer column equilibration times are required. How long a time is sufficient typically? We allowed the column to equilibrate for at least an hour.

We achieved separation, but the UV spectra of all the peaks are the same, so I'm puzzled. We also ran the std solutions of the impurities and their RT do not match.

Also, is temperature very critical in ion pair chromatography?

Thanks,
Wan