Chromatography Forum

Forgive me if this has been asked before or is a mundane question, but I need some clarification from the experienced chromatographers here. In my experience with Normal Phase chromatography, i've only seen highly organic mobile phases and diluents.

Would someone have a reason to use say, a phosphate buffer as a sample diluent in normal phase seperations? (Specifically on an NH2 column with an 85% ACN/15% H2O Mobile Phase). Or does this not work at all?

My initial thoughts are that it won't work because in normal phase you have a polar column. If the sample is in buffer, it won't seperate because it is also polar?

Thanks,
Tim

In every case where your sample diluent elution power is too high for the mode of separation you use, then you will have problems (no retention, split peaks etc). In this case, as you correctly figured it out, an aqueous phosphate buffer as sample diluent won't work for your conditions.

PS: There are ways to minimize problems by decreasing injection volumes etc, but the best practice will be to dissolve your sample to something compatible.

Hi Tim -

I wouldn't use phosphate buffers at that high % organic.
They may precipitate out of solution. If you must use them (low UV), then you can try low concentration of potassium phosphate (10mM).

A MUCH better alternative is volatile buffers (e.g. NH4AcOH and NH4HCOOH). You can use these 10-100mM concentration - precipitation
is not an issue.

As for it's purpose - these buffers are useful to improve peak shape
for ionized compounds. Below are some examples on Unison UK-Amino:

Monosaccharides (neutral sugars, no buffer required):
http://www.silvertonesciences.com/files/TI305E.pdf

Sialic acid & uronic acids:
http://www.imtakt.com/TecInfo/TI333E.pdf

Amino sugars:
http://www.silvertonesciences.com/files/TI334E.pdf

Deoxynojirimycin & glucose:
http://www.silvertonesciences.com/files/TI431E.pdf

I did find some additional separations on Unison UK-Amino
using ~ 85% ACN and 0.5% H3PO4:

Quercetin glycosides: http://www.silvertonesciences.com/files/TI369E.pdf
Flavonoids: http://www.silvertonesciences.com/files/TI368E.pdf