Hello Everybody,
I got a problem. Iīve found that I canīt elute benzoic acid at pH upper than 5, in a rp- C18 column, at 45% AcN, KH2PO4 5mM buffer.
I was expecting that it elute at time o, bat i canīt detecty it by expectral comparison. I detect it in a diode array at 200 nm.

Could it be possible that the way of interaction with the stationary phase was other than the expected and that in that conditions a chemical bonding was formed? .Iīve seen that preassure increases after injection.

Thank you very much..
Celia.

Celia,

Perhaps it's eluting unretained and you're not able to detect it in the void due to the relatively high background of acetonitrile at this wavelength. If you use a lower pH where in the benzoic acid is protonated, it should behave itself quite nicely. It should be detected okay at 254 nm.

If I understand you correctly, you are trying to detect the benzoic acid peak via a comparison to the UV spectrum obtained at low pH. Note that the spectrum will change, since benzoic acid is in an ionic form at high pH, i.e. at pH higher than 5. Maybe this is the reason that you can not identify the peak any more. Please clarify if this interpretation is incorrect!

Thanks, and could you tell me any bibliography where i could find how pH and solvatation affects absortion spectrum...
Celia

Dear Celia!
I expect you`ve come across the problem of peak broadening in RPLC of ionic substances.
5 years ago I was interersted in it, and I carried out an experiment using benzoic acid as a test compound. At pH 3 it elutes as a sharp peak? t = (for example) 15 min, at hH 7 -- t = 3 min (but peat is rather broad). In 6-4 pH range chrom. of benzoic acid is "camel-like" with 2 or even more maximums, and the "peak" is lasting from 3 to 15 minutes.
Excuse me for bad english,
Constantine

Hello, Constantine,

Iīm interested in the possibility of adsortion to stationare phase...Do you know something about it?
Thanks, Celia

Yes? unendcapped C18 phases do adsorb benzoic acid when using unaq. eluents for example pure acetonitrile, but adding of water leads to its complete desorption.
Constantine.

Sychov,
what buffers did you use?

I used kH2PO4 25mM, and I tried pH 3, and 7, with acetonitrile...
I found a lot of problems with pH 7: the columns didnīt last too much.

I was analysing antibacterial, b-lactamic antibacterials.

Sorry for my English..and thank you.

Sychov,
How do you get 25mM KH2PO4 into acetonitrile, and how do you measure its pH in acetonitrile?

i prepared a kh2po4 bufer 25mM, and i got itīs pH with H3po4.
After, i mixed it( buffer and AcN ) in the system .

Why?

Celia