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Mobile Phase Optimization Strategies For Reversed Phase HPLC PDF
Mobile Phase Optimization Strategies For Reversed Phase HPLC PDF
Moderator
Dave Walsh
Editor In Chief
LCGC Magazine
Mobile Phase Optimization Strategies
for Reversed Phase HPLC
Aims & Objectives
1. Review of Reversed Phase
Retention Mechanisms
2. Methanol or Acetonitrile - which
one is best?
3. Eluotropic Strength - quick ways
to reach the ideal!
4. Changing Solvent - useful tools
and approaches
5. Mobile phase pH - understand
the effects
6. Optimising pH vs. retention
7. Which buffer - what strength?
8. Strategies for when all else fails
Reversed Phase Retention Mechanisms
1. Mobile Phase - mixture
of organic and
aqueous solvents
2. Stationary phase -
hydrophobic moiety
chemically bonded to
silica
3. Mobile phase MORE POLAR than Stationary Phase
4. Analyte partitions between the two phases depending upon its
chemistry (hydrophobicity)
5. Increasing the % organic in the mobile phase increases the
elution power of the mobile phase
6. Retention and Selectivity are altered by changing the chemistry
of the stationary phase mobile phase and the temperature
Controlling Retention and Selectivity
We will discuss the pH of the mobile phase and choice of buffer shortly
Simulation to Optimise %B
1. Use modelling for predicting separation
2. This DryLab model was achieved using 2 injections at 30% B
and 50% B