Method Development Guide
Aqueous Normal Phase (ANP)
Aqueous Normal Phase Method Development Protocol for Silica Hydride (TYPE-C) Stationary Phases
Type C stationary phases (based on silica hydride surfaces as opposed to ordinary silica with silanols that are present on all
other commercial stationary phases) have the ability to retain polar solutes at high concentrations of the organic
component in the mobile phase. The MicroSolv Type-C phases (Bidentate C18, Bidentate C8, UDC-Cholesterol, Diamond
Hydride, and Silica-C) all possess this capability. The exact point where composition of the mobile phase where
aqueous normal phase (ANP) retention begins depends on the solute as well as the stationary phase selected. Because of the
attached organic group that is part of the stationary phase, the Type-C columns can also retain nonpolar compounds based on the
typical reversed phase mechanism. The diagram below illustrates how this dual retention capability works for both
polar (metformin) and nonpolar (glyburide) compounds:
A Generic Starting Point for Cogent TYPE-C Columns
It is easy to use a Cogent HPLC column that is made with TYPE-C Silica.
The power of these columns is found in the wide range of solvents that can be used with them and the unique and
strong retention they can have for polar compounds while at the same time retaining non polars. Selection of the
column (C18, C8, Cholesterol or Silica-C) will be determined by your compounds of interest. Mixtures that will be
highly polar and do not contain non polar compounds might be better suited by the Silica-C where compounds with
both polar and non polar might be better suited to the Bidentate C18.
Step 1. After you have properly installed the column, conditioned it according to our suggestions, it is a
good idea to start with a typical gradient run. We suggest starting with an acidified mobile phase of Water and
Acetonitrile. Acidify the mobile phase with up to 0.5% of an acid such as Formic or Acetic Acid. If you are not
using LCMS, TFA is another good candidate.
Step 2. Run about 6 column volumes of the mobile phase in Step 1 at 95% Water.
Step 3. Set up your instrument to run a shallow gradient from 95% Water to 40% Water over 20 minutes on a
75mm long column. For longer columns increase the gradient time proportionally. This long and shallow gradient will
be very beneficial for determining the optimal gradient or isocratic method to run your mixture with later. For
sharper peaks and less retention, run a shorter (Steeper) gradient from the same starting points to end points.
Step 4. Equilibrate the column by running 100% Acetonitrile for approximately 2 minutes.
Step 5. Set up your instrument to run a shallow gradient using the same mobile phase in Step 3 to run from
90% Acetonitrile to 40% Acetonitrile over 20 minutes for a 75mm long column. For longer columns increase the
gradient time proportionally. This long and shallow gradient will be very beneficial for determining the optimal
gradient or isocratic method to run your mixture with later. For sharper peaks and less retention, run a shorter
(Steeper) gradient from the same starting points to end points.
Step 6. Evaluate both gradient runs for retention time, peak shape and elution order as retention on the
column is compound specific where some compounds will not retain in Step 3 (Reverse Phase) and some do not retain
in Step 5 (Aqueous Normal Phase). One column could produce an isocratic run which retains both polar and non polar
Note: The Cogent Bidentate C8 and C18 columns have a unique quality in that they sometimes can retain
polar compounds not retained on other columns while run at 100% Water. You could insert an isocratic run at 100%
Water after Step 3 and before Step 4.