Chromatography Forum

I have 2 app notes one from Agilent one from Phenomenex using KH2PO4 water and an acetonitrile gradient which goes very high. The Agilent one goes to 80% acetonitrile and the Phenomenex one goes all the way to 100% Acetonitrile (though since the KH2PO4 is in A there is none being put in at 100% B).

Is it ok to go that high acetonitrile with even relative low amounts of phosphate 20mM or so? I thought going above 60% acetonitrile risks precipitation in the system.

Whoever compiled those Agilent and Phenomenex instructions must not have spent much time at the bench actually doing HPLC. At ~ 75% acetonitrile, a saturated solution of KH2PO4 is about 5 mM. A solution to this problem is to prepare triethylammonium phosphate (TEAP), which has an appreciably higher degree of solubility in primarily organic solvents, or alternatively to use the sodium salt of methylphosphonic acid. In either case, we prepare our salts by adding the base to a solution of the acid (of known concentration) until reaching the pH we want.

Be advised that if your analyte is charged, then different counterions will give different retention times and selectivity in reversed-phase and HILIC. See my paper from 2008 (introducing ERLIC) and my paper from 2018 in which this subject is studied at length with regard to HILIC.

That's what I thought.

here are the two apps for artificial sweeteners
https://www.gimitec.com/file/5989-8961EN.pdf
https://www.phenomenex.com/Application/Detail/13339

Some other related app notes just use 0.1% TFA so I will try that first.

From: MODERN HPLC FOR PRACTICING SCIENTISTS by Michael W. Dong

Beware of the possibility for buffer precipitation when mixing it with organic solvents such as acetonitrile (ACN)—the upper limit should be 70–80% ACN. Caution particularly should be exercised in blending ACN with phosphate buffers. Limit the concentration of the phosphate buffer to <15 mM to prevent precipitation, which can cause check valve malfunctions.