Column regeneration – what to do if column performance decreases

What is meant by column regeneration?

UHPLC or HPLC column regeneration aims to restore the performance of a column that has been affected by deposits at the frits or samples adsorbed by the packing material. These are removed by the procedures outlined below. Regeneration is therefore different from simple column washing in that it is performed at irregular intervals and removes impurities from the column that have accumulated over time.

When is column regeneration necessary?

The deposits will change the behavior of the columns. The following list contains parameters that point to degraded performance and may require the regeneration of the column.

lower resolution
higher asymmetry
different retention times
lower theoretical plates
differences in selectivity
broader peaks
Backpressure increase

How is a column regenerated?

The general procedure to clean HPLC columns is similar, independent of the column type. However, the cleaning solutions used to remove adsorbed substances differ between column types. This is how to proceed:

Clean your column in the reverse flow direction. To this end, disconnect the column from the system and reconnect in reverse flow
Do not connect the column to the detector.
Run the column at half of the maximum recommended flow rate, taking special care to monitor the pressure as the cleaning solutions may be of different viscosities than your normal mobile phase. The maximum pressure can be found in the column specifications of the manufacturer.
If you are cleaning with a high or low pH solution, make certain that the rest of your chromatographic system (pump, pump seals, injector, etc.) is compatible.
If multiple cleaning solutions must be used, always rinse the column between cleaning solutions with 3-5 column volumes of Milli-Q or deionized water.
Dependent on the column type, clean the UHPLC or HPLC column with the following cleaning solutions:

Silica-based size exclusion columns (SW, SWXL columns)

1. Clean with 5 column volumes of 1 mol/L sodium sulfate (pH 7.0 or down to pH 3.0)
2. Clean with 5 column volumes of Milli-Q or deionized water
3. Clean with 5 column volumes of 20% Acetonitrile.
4. Clean with 5 column volumes of Milli-Q or deionized water

Polymer-based size exclusion columns (PW and PWXL)

1. Clean with 5 column volumes of 1 mol/L sodium sulfate (pH 7.0)
2. Clean with 5 column volumes of Milli-Q or deionized water
3. Clean with 5 column volumes of 20% Acetonitrile.
4. Clean with 5 column volumes of Milli-Q or deionized water

Ion-exchange columns, silica based (SW type)

1. High concentration salt (e.g. 0.5 mol/L - 1.0 mol/L) of your normal run buffer
2. Buffered solutions at low pH (e.g. 2.0 - 3.0)
3. Water soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer
4. Urea (8 mol/l) or non-ionic surfactant in buffer solution

Ion Exchange columns, polymer-based (PW, STAT or NPR type columns)

1. 0.1 - 0.2 mol/L NaOH
2. 20% - 40% aqueous acetic acid
3. Water-soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer

Hydrophobic Interaction columns

1. 0.1 - 0.2 mol/L NaOH
2. 20% - 40% aqueous acetic acid

Reversed-Phase, silica-based (SW-type columns)

1. Acetonitrile or methanol
2. Gradient from 10% - 100% acetonitrile in 0.05% trifluoroacetic acid

Affinity chromatography (FcR-IIIA-type columns)

1. Make 3-5 injections of 0.5 - 2 mL of a buffer containing 0.5 mol/L NaCl or 20 % ethanol

Affinity chromatography (Protein A-5PW)

1. Wash column with 0.5 mL of 0.1 mol/L NaOH solution at 0.5 mL/min.
2. Equilibrate column with 20 mmol/L sodium phosphate buffer, pH 7.4, for 20 CV at 0.5 mL/min

      7. Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes.
      8. If the cleaning solutions listed above do not improve the resolution of the column, then urea or non-ionic surfactant
      may be tried (not compatible with affinity columns).