Probabily column overloading

[alemaggot]

Hi guys.

Today I've use for the first time Kinetex PFP 2.6um 75 mm x 4,6 mm such as column, on perkin elmer HPLC system. Mobile phase were 100 % H2O.

I injected 5ul of a sample solution (water as solvent) with 0.5 mg/ml such as concentration. My compund give two principal peaks. The second peak is orrible. The peak have a very small height and big fronting. It seems that I'm in the case of colum overloading.

but I can't understand... With this volume and concentration, how can I overload a column?

In my experience I don't use PFP phase before today. It's possibile that this phase isn't compatible with my compund?

Can you help me?

Thank you

[tom jupille]

The obvious tests:

Inject only 1 microliter and see what happens. Then dilute your sample by a factor of 5 and inject 5 microliters and see what happens.

If the 5-fold dilution shows significantly better peak shape, then you were overloading. In my experience, overloading more commonly causes tailing. The exception is for ionizable compounds and insufficient buffer (you said your mobile phase was 100% water).

If the 5-fold dilution has the same problem, but the 1-microliter injection shows better peak shape, then your problem is volume overload. I would not expect to see that in this case, but you never know.

[Mattias]

(assuming that the analyte has acidic and/or basic groups):

I assume that the Kinetex column is fine to run with 100% aqueous, but using 100% water sounds like the problem.

1. You will generally get better peak shapes if the analysis is run below or above the pKa of the substance.

2. Having no ion strength at all in the mobile phase will give the analyte the chance to interact with the silanol groups.

3. It is possible that the retention that you see is not due the reversed-phase but due to the secondary interactions (like silanols). The capacity of these is much less than of the reversed-phase groups, giving over-loading effects. (The Kinetex column contain a lot of free silanols, no matter what the sales rep says).

You should have no problem to inject 5 µl of a 0.5 mg/ml solution on a 4.6 mm I.D column....

[alemaggot]

Thanks for your reply.

I've tried to inject only 1ul of solution. The peak shape were better, but it's more orrible. There is a big asymmetry factor.

If I diluite more the solution the area respound of my compounds drecrase too.
The components is: 4-acetamidobenzoic salt of N-N, dimetilisopropanolammina. You advise to use a buffer? In this mode I can also block the secondary interaction that Mattias says.

In water solution the pH is about 6,8. I think to try acetate buffer. It's ok?

thank you!

[Mattias]

My best guess is to run this analysis with (lets say) 0.1% TFA in water.

It will make the bensoic acid molecule protonated, and give it a good retention. It will also shut down the silanols and drastically improve the peak shape of the N-N, dimethylisopropanolamine. The latter seems to contain a tertiary amine (?), which are notorious for bad peak shapes.

Good luck!

[tom jupille]

Perhaps a more important question: for which compound are you analyzing? The 4-acetamidobenzoic acid or the N-N, dimethylisopropanolamine? In aqueous solution the salt *will* dissociate.

[alemaggot]

I must analyze 4-acetamido benzoic acid. I didn't Know nothing about dissociation. Thanks! I think, however, that isn't a problem...

N-N, dimetilisopropanolammina isn't see by Uv detector. Right?

[tom jupille]

N-N, dimetilisopropanolammina isn't see by Uv detector. Right?

Only at very short wavelength, if at all.

You *do* need a buffer at fairly low pH, or at least some acid in the mobile phase to suppress the ionization of the 4-acetamido benzoic acid.

[alemaggot]

My best guess is to run this analysis with (lets say) 0.1% TFA in water.

It will make the bensoic acid molecule protonated, and give it a good retention. It will also shut down the silanols and drastically improve the peak shape of the N-N, dimethylisopropanolamine. The latter seems to contain a tertiary amine (?), which are notorious for bad peak shapes.

Good luck!

Today I've try this way. The result were wonderful!4-acetamido and 4-amminobenzoic peaks are perfect! Great height, great respound, good retention! All is great!

In other post I'll ask another thing about my method development.

Thank you very much!

[tom jupille]

Mattias, you get a gold star!

[alemaggot]

Mattias, you get a gold star!

ahahah