Tips for the equilibration of HPLC columns

To ensure rapid equilibration of an HPLC column, the change of eluents should be performed in several steps (see also the tips below).

Before using eluents, always rinse the column with 25:75 acetonitrile-water at least ten times the column volume.

After changing to the desired eluent, equilibrate the column with at least ten times the column volume of eluent before the first injection. The following formula is used to calculate the column volume :

Volume V = π r2 L
with    V = column volume [ml]
          r = column radius [cm]
          L = column length [cm]

Switching to the eluent

Some basic rules when switching to solvents/solvent mixtures:

  • Make sure there is no separation of the mobile phase as this can damage the packing material of the column!
  • If switching from eluents with buffer to mobile phases with very organic solvents (> 50 %), always rinse the column in between to avoid buffer deposits on the column!
  • Never rinse with eluents that denature the stationary phase, the fittings or the capillaries!