Tips for the regeneration and cleaning of HPLC columns

For all cleaning steps, the flow rate should be maintained at 25 – 50 % of the normal working flow rate.

Polar foreign substances

Rinse out the buffer with a mixture of 80 % water and 20 % organic solvent (with 20 times the column volume). Then flush the column with a mixture of highly concentrated organic solvent (70:30 to 90:10, methanol-water or acetonitrile-water) with at least 10 times the column volume.

Note: Most polar foreign substances is eluted when the buffer has been flushed out.

Non-polar foreign substances

  1. Flush column with a high proportion of water (max. 90 % water) to remove buffer substances.
  2. Flush column with 100 % methanol to remove polar organic compounds.
  3. Flush column with 100 % acetonitrile to remove weak polar organic foreign substances (if necessary, raise the temperature to 40 °C).
  4. If necessary, flush column with THF in the reverse flow direction (at approx. 1/5 of the original flow rate).

Proteins and peptides

To remove these substances, we recommend an increase in water - 0.05 % TFA (eluent A) and acetonitrile - 0.04 % TFA (eluent B) with an increase from 25 % B to 100 % B in 20 minutes, then 5 minutes with 100 % B and then back to 25 % B within 5 minutes. Repeat this procedure twice.

A well-cleaned column

An important characteristic for a well-cleaned column is the stability of the baseline. At a constant temperature (without thermal drift), a 70:30 acetonitrile-water mixture, applied for 5 minutes should not cause deviations of more than 2-3 mAU.