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Swaying Retention Times Due to Insufficient Equilibration in LC

13 Dec 2023

Fluctuating retention times in your LC can have various causes including irregularities in the flow rate or gradient mixture, temperature fluctuations, incorrectly prepared eluents, etc.

However, it may also be due to the method, mainly the equilibration time in the gradient.

  • Early eluting substances are usually more affected than later eluting ones.
  • Working isocratic, the problem occurs only in the first runs.

Analytes are retained by the interaction with the stationary phase, which takes place INSIDE the particles of the column. Consequently, all particles must be completely filled with eluent and the entire INNER surface must be wetted for a stable retention time. This process is very slow, since the movement inside the particles exclusively takes place through relatively slow diffusion. 

There is no general answer on how long equilibration takes. It depends on: 

  • The column dead volume
    • Rule of thumb: Use approximately 10 column dead volumes for equilibration.
    • The correct column dead volume can be calculated from the dead time and the flow rate.
    • You can find approximate values for the column dead volume in the table below: 
Column Dimension approx. Dead Volume 
(= Vleer·ε)
2.0 x 50 mm 0,12 mL
2.0 x 100 mm 0,24 mL
2.0 x 150 mm 0,35 mL
3.0 x 50 mm 0,26 mL
3.0 x 100 mm 0,53 mL
3.0 x 150 mm 0,79 mL
3.0 x 250 mm 1,32 mL
4.6 x 50 mm 0,62 mL
4.6 x 100 mm 1,25 mL
4.6 x 150 mm 1,87 mL
4.6 x 250 mm 3,11 mL


  • The dead volume of your system.
  • The mechanism and its kinetics can result in fewer or – significantly – more column volume for equilibration:
    • RP interactions are quite fast, whereas ionic interactions are slow.
    • HILIC chromatography is special. First, a stable aqueous layer must form on the polar stationary phase, which can take time.

"Equilibrate" means "to bring into equilibrium" (source: Duden Online), and the name says it all: A stable retention time can only be achieved when the stationary phase and eluent are in equilibrium, which can only occur when the stationary phase is completely filled and wetted with the starting eluent.

If you have problems with fluctuating retention times in the gradient:

  • significantly prolong the equilibration time with the starting eluent and make a series of injections to see the effect.
  • Flush the column thoroughly with high levels of the strong eluent (e.g., 95% methanol or acetonitrile for RP mechanism).
    • This can fix problems caused by matrix accumulation on the column, e.g. due to insufficient flushing at the end of the gradient.
    • Permanent contamination on the column is indicated by continuously decreasing retention times. However, it is possible that part of the contamination comes off again which prolongs the retention time again.