Chromatography Forum

Hi all,

I'm doing some chromatography using Caffeine. The caffeine peak elutes at ~1 min and at about 4 mins a negative peak appears. I tried changing mobile phase, column, diluent and even to a different HPLC but this negative peak always appears, I was wondering whether someone could explain the presence of this peak.

Mobile Phase: 10% ACN:Water 0.1% Triethylamine

Column: Prodigy ODS 30 x 4.6mm x 3 micron

Diluent: 10% ACN:Water


Any help is appreciated!

Thanks

I do not know what your negative peak is. However, have you measured the pH of your mobile phase and have you compared it to the manufacturers recommendations?

No I haven't measured the pH of the mobile phase and these are trial runs so there are no manufacturer specifications

ganna,

I'm with Uwe, in that I doubt it's a great idea to be using the mobile phase you describe since it will be damaging to most silica-based media. However, the negative peak you are observing is most likely due to hydroxide anion (assuming you are working at relatively low wavelengths). Since triethylamine is a fairly strong base, a significant fraction of it will be present in your mobile phase as triethylammonium hydroxide, causing elevation of your eluent background signal. Unless you're sample contains the same concentration of triethylamine, you will have a triethylamine vacancy peak along with a decrease in the associated ionized fraction of triethylammonium hydroxide and thus a decrease in the eluent background signal.

This is an unusual mobile phase for caffeine. Caffeine can be analyzed on almost any C-18 column using 20-30% methanol in water with 1% acetic acid and detection at 254 nm. Many of the chromatography vendors have application examples for caffeine.

Hi

My guess is that the negative peak arises from the lack of triethylamine in your diluent. If you use your mobile phase as a diluent, probably the negative peak will disappear...

/Peps

Peps,

Triethylamine is UV transparent. As I said earlier, hydroxide absorbs in the ultraviolet at low wavelengths and since a fraction of the triethylamine will be present as triethylamonium hydroxide, hydroxide anion is most likely be the cause of the negative peak (assuming Ganna is really using the free base in the mobile phase).

Chris

The mobile phase is damaging because it is rendered basic by the triethylamine? or some other reason?

Oliver: you have basic pH. Most C18 columns are not stable under these conditions.

Caffeine Separation with same column dimensions

http://www.silvertonesciences.com/modul ... TI067E.pdf