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