Column Tips Series # 1 – RP HPLC Column Washing
To prolong column lifetimes, it is recommended to wash the HPLC column after every single analysis or whenever possible. Presence of buffer salts, acids or ion-pairing reagents in the column can cause hydrolysis of the stationary phase, leading to shorter column lifetimes. Also, if the column is stored in highly aqueous mobile phases, sample matrices such as lipids can accumulate in the column, leading to peak splitting or reduced column efficiency.
When the mobile phase contains no buffer
A. Flush the column with mobile phase until all samples elute out completely
B. Flush with 20 column volumes of 90% organic solvent like Acetonitrile, Methanol etc.
C. If highly lipophilic contaminants are present, the column can be flushed with 100% organic solvent for 5 column volumes.
D. If high backpressure is observed when increasing the organic solvent concentration, reduce and adjust the flow rate.
When the mobile phase contains a buffering agent
A. Flush the column with the mobile phase until all samples elute out completely
B. Flush the column with 20 column volumes of intermediate mobile phase (original mobile phase without the buffer)
C. Flush with 20 column volumes of 90% organic solvent like Acetonitrile, Methanol etc.
D. If highly lipophilic contaminants are present, the column can be flushed with 100% organic solvent for 5 column volumes.
E. If high backpressure is observed when increasing the organic solvent concentration, reduce and adjust the flow rate.
F. When using the column again for the analysis, follow the procedure to avoid buffer precipitation in the column:
– Equilibrate the column with 20 % acetonitrile in water at 1 mL/min for at least 30 minutes.
– Equilibrate the column with the buffered mobile phase to be used at 1 mL/min for at least 30 minutes.
– The column may be considered fully equilibrated once a constant back pressure and stable baseline are observed.
When the mobile phase contains an ion-pairing agent
A. Flush the column with the mobile phase until all samples elute out completely
B. Flush the column with 20 column volumes of intermediate mobile phase (original mobile phase without the buffer)
C. Flush the column with 20 column volumes of 50% Methanol
D. Flush with 20 column volumes of 90% organic solvent like Acetonitrile, Methanol etc.
E. The content of organic solvent can be increased further when using ion-pairing reagents containing long alkyl chains to effectively remove out from the column.
Note: To maximise column lifetimes, always follow the operating guidelines described in the Glantreo Certificate of Analysis, supplied with the column.