HPLC Column Care Protocol

Shirley Clark 6/4/01

Protocols: Most HPLC protocols use Water + 0.05% TFA for Solvent A

and Acetonitrile (ACN) + 0.06% TFA for Solvent B.

Common protocols start with 95% A and 5% B.

Gradients should normally go from 5% to 80% B.

If your protein is not eluded in this range, then you should consider another solvent system.

Cleaning: Anytime you go to 90% B or higher, you shorten the life of the column.

A gradient may go to 90 or 95% for a few minutes, but should not stay there longer than that.

Never use 95% for cleaning a column! It destroys it!

If a column needs cleaning, use 100% isopropyl alcohol in one direction, then reverse the column and clean it in the other direction.

Equilibrate the column back in 5% acetonitrile.

Storage: Long-term storage can be done in 100% acetonitrile with no TFA or more normally in 50% B with TFA.

Mass Spec: Buffers for the HPLC that also go to the Mass Spec work more efficiently with formic acid than with TFA which represses the signal.

The first part of a gradient that may contain salts should never be directed into the Mass Spec. It must be diverted into another vessel. If you are not sure about the time for this, ASK!