Chromatography Forum

i mae developing HILIC method. i sometimes see people using 20 mM ammoniumformate in water/acetonitrile 10/90 ( for example https://www.agilent.com/cs/library/applications/5991-8830EN_Paraquat_Diquat_Poroshell120_HILICZ_application.pdf). i made 200 mM ammoniumformate in water and dilute 1:9 with acetonitril. i get phase seperation there is clearly a second layer on the bottem. what ame i doing wrong?

Aldijkhuizen wrote:
i mae developing HILIC method. i sometimes see people using 20 mM ammoniumformate in water/acetonitrile 10/90 ( for example https://www.agilent.com/cs/library/applications/5991-8830EN_Paraquat_Diquat_Poroshell120_HILICZ_application.pdf). i made 200 mM ammoniumformate in water and dilute 1:9 with acetonitril. i get phase seperation there is clearly a second layer on the bottem. what ame i doing wrong?

Have you tried making 20mM ammonium formate in water and 20mM ammonium formate in Acetonitrile then mixing? I know it will dissolve in the Acetonitrile but maybe with such a high concentration in the water it is preventing the two from mixing until the concentration equilibrates at the lower value.

I have not tried this but when i ad acetonitrile to the water at first they mix. At a certain point a small layer of water appairs aar the bottem of the botle. this layer idd still there after standing over night. Amw i doing something wrong or do other people just dont look good enough to the mixture they have prepared?

I just wanted to point out that the app note mentions 20mM ammonium formate at pH = 3. This means that it has been acidified, most likely with formic acid. The 20mM concentration can be either the concentration of formate before acidification, or after (total formate). This is something that no one seems to mention when publishing protocols. I don't know if this will help mix it with acetonitrile.

RnDirk made a good point. We for instance have a method using 20 mM ammonium formate at pH 3.8 and prepare it with formic acid (200 mM) followed by adjustment to pH 3.8 with ammonium hydroxide. Then dilute with ACN to 10:90 and there is no phase or precipitation issue. And indeed the way how buffers were prepared is rarely described even if it is essential for reproducing experiments.