Method Development Guide

All Primesep columns are reverse phase (RP) columns. They differ
from other brands' reverse phase columns by having an additional
charge on a stationary phase surface. This charge allows to explore
another mechanism of interaction - ion exchange in addition to
hydrophobic interaction.
The column surface charge is greatly influenced by the pH of the
mobile phase. Many analytes also change their charge state as a
result of changes in solvent pH. If a compound has a positive or
negative charge, then in order to obtain an ion-exchange interaction in
addition to reverse phase interaction using Primesep columns, the
column surface charge and the analyte charge should be opposite. This
guide helps you to decide which Primesep column to choose when you
just begin to do method development with Primesep mixed-mode
columns.
There are three steps in a process of selecting a correct Primesep
HPLC column for your method:
Step 1. Choose the appropriate bar for your compounds based on their
pKa value or structure, then find the pH range where your analyte
has a charge.
Step 2. Choose the appropriate buffering system with the pH at a
point where analyte has a charge which satisfies your detection and
scale up requirements that you have found in step 1.
Step 3. Choose an appropriate Primesep column, which has a surface
charge opposite to the analyte charge, found in Step 1.

Step 1.
Choose the appropriate bar for your compounds
based on their pKa value or structure, then find the
pH range where your analyte has a charge.

Note: Some compounds have more than one charge state within a working pH range (examples
include: aspartic acid and glutamic acid). Compounds that have no charge state at any HPLC
condition can be analyzed with any Primesep column via the reverse phase mechanism. Besides
the reverse phase or hydrophobic mechanisms, other mechanisms of interaction can be utilized in
this case (normal phase interaction, HILIC, and pi-pi interaction with Primesep P column).

Step 2.
Choose the appropriate buffering system with the pH
at a point where analyte has a charge, which
satisfies your detection and scale up requirements
that you have found in step 1.

Note: If the analyte and the column surface have opposite charges, elution of analyte from the
column requires the presence of other ions in the mobile phase. Water and acetonitrile alone do
not produce enough ions in the mobile phase to efficiently participate in an ion-exchange
process. As a result, charged analytes in Primesep column systems result in very long retention
time and poor peak shape. Typical HPLC buffers are a good source of ions capable of facilitating
an ion-exchange process.
Two distinct types of buffers are available depending on applicable detection requirements:
Non-volatile buffers for low UV application, and
Volatile buffers for MS, ELSD, and preparatory applications
Only TFA-based buffer can be used for both types of detection. Unfortunately it is not always
applicable for MS detection, and does not permit to use lower UV wavelength.
Cation-exchange Primesep columns allow to use non-buffered solutions. The acidic residue
embedded in the column provides the acidic component of the buffer. Ammonium acetate or
ammonium formate salts can be used for MS, ELSD, and preparative chromatography
applications while potassium sulfate can be used for low UV applications.

Step 3.
Choose an appropriate Primesep column, which has
a surface charge opposite to the analyte charge,
found in Step 1.

Note: Primesep columns in transitional pH gradually lose or change their surface charge. This
process is reversible and column restores ionic properties with another mobile phase pH. When
the column is used without a buffered mobile phase, the surface charge corresponds to the charge
at pH 7 on this chart. In order to work and get consistent results in transitional pH, the ionic
mobile phase modifier should have sufficient buffering capacity.
Unlike the other Primesep columns, the Primesep AB column is a zwitterionic reverse phase
column with a surface charge that is affected by the type of the analyte:
With basic analytes the Primesep AB column behaves as if it has a negatively charged surface
With strong acids the Primesep AB column behaves as if it has positively charged surface.
All column except Primesep B are stable in any buffer system within a pH range of 1.5 - 7.
Primesep B column is recommended at a pH range of 1.5 - 4.5.
Use the chart below to help properly select a column that will interact with the analyte by both
the reverse phase and ion-exchange mechanism