In Aqueous Normal Phase ANP HPLC, the strongest solvent is water.

The order in decreasing solvent strength is:

DI water > methanol > isopropanol > acetone > acetonitrile

These are general estimates; as an example, in a comparison of acetone and acetonitrile for different amino acids, retention is somewhat comparable for some but significantly different for others.

NOTE: For methanol or isopropanol, these are typically used as part of the A solvent to help remove adsorbed sample components and keep the column clean from run to run. This can be very helpful for samples like biological extracts. You would not generally use just water and methanol in ANP. Acetone is a good acetonitrile substitute in certain situations, such as in LC-MS where the UV absorbance of this solvent is not a problem.

TYPE-C HPLC Column Information