Always ensure that the column is fully equilibrated with your starting mobile phase before making any injections.

When you are equilibrating your columns for the first time or whenever changing your HPLC methods it is wise to consider not putting anything into the columns that can precipitate or is immiscible with the storage or current solvent in the column.

Depending on column type, equilibrating could range from 10 to 15 column bed volumes for typical reversed phase to 50 to 100 bed volumes with polar embedded phases or HILIC columns.

Beware of buffers that might be in your column before you change the mobile phase composition with respect to the organic content. This can cause immediate precipitation and ruin your HPLC columns.

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