Use the following process to regenerate/clean a Reverse Phase HPLC Column

Macherey-Nagel HPLCRun each of these at the appropriate column volumes based on how the column has been previously used, number of injections or problematic analytes, in this sequence:

  • 20 column volumes Water/ACN (95/5 v-v)
  • 20 column volumes Acetonitrile
  • 10 column volumes Isopropanol
  • 10 column volumes n-Heptane
  • 5 column volumes Isopropanol
  • 20 column volume Acetonitrile
  • Follow with mobile phase to be used for your separation, until you have a stable baseline

It’s important to never leave isopropanol on the column so ensure you follow with the ACN and mobile phase steps above. Store columns in 80% ACN and 20% water. Make sure you plug both ends of the column before storing in the drawer.

For sticky proteins use the following to clean/regenerate a column

  • Flush with 30 volumes of your mobile phase but unbuffered
  • Then 20 column volumes of 0.1% TFA in water
  • Follow with 20 column volumes of 0.1% TFA in ACN/IPA (1:2)
  • Then flush with 30 column volumes of 100% ACN
  • Follow with mobile phase to be used for separation