INTERNAL ONLY: This information may be obsolete.
In the aqueous normal phase mode (ANP) the organic modifier is usually 60% or higher (up to and including 100%) of the mobile phase. Typically to work in ANP, you should also start with a mobile phase at a low pH. We suggest trying Acetonitrile and DI water with up to 0.5% formic or acetic acid, or up to 0.005% TFA.
If the above suggested trials are unsuccessful, the opposite pH condition for that phase should be tried, i.e. switch from an acidic media to a basic media and vice versa.
The recommended starting acidic media is 0.05% or 0.1% formic or acetic acid. In some cases 0.001 to 0.005% TFA should be tried if the desired results are not achieved with acetic or formic acids. Depending on your detector you can use a higher amount of acid.
The recommended basic media is 0.05% or 0.1% ammonium formate, or 0.05% or 0.1% ammonium acetate. For a basic pH (7-9) ammonium hydroxide is added to the solution. For more acidic pH values, either formic acid or acetic acid is added as appropriate.
TIP: When preparing the organic part of the mobile phase, you cannot use ammonium acetate or ammonium formate in 100% Acetonitrile very easily. In those cases the organic component should be prepared as 90:10 Acetonitrile/HPLC Water with the 0.1% ammonium formate; this is for solubility reasons.
If satisfactory results are not achieved with the organic modifiers suggested, THF or Acetone has been shown to be successful in some cases and is an alternative to the solvents suggested above.
|For more robust methods, prepare the sample in at least 50:50 acetonitrile/water with whatever else is in the mobile phase (acetic acid, formic acid, ammonium acetate, ammonium formate etc.). If possible, even a higher amount of acetonitrile is generally better. Add a small amount of TFA (0.005%) to the sample solvent to improve peak shapes in some circumstances.|