Cleaning of HPLC columns

1.0    Purpose   :   To provide an instruction for washing of HPLC columns.

2.0    Objective :   To provide a documented procedure for washing of HPLC columns.

3.0    Scope      :  This procedure is applicable for washing of HPLC columns in QC Department.

4.0     Responsibility   :

·        Primary  :    QC Chemist / QC-Officer

·        Secondary  :  Overall QC – In charge

5.0      Procedure  :

·        After completion of analysis stop the pump, keep the reservoir in the distilled & filtered water.

·        Open the purging valve of the pump; purge the system for 3 to 5 minutes to remove air bubbles. Close the purging valve by decreasing the flow rate up to zero.

·        Wash the HPLC column initial with HPLC grade water for about 15-20 minutes.

·        Then wash the HPLC column with respective solvent mixtures (e.g. 50:50 Methanol: Water or Acetonitrile: Water) for minimum ½ hr to 1 hr.

·        Finally wash the HPLC column with respective solvent (Acetonitrile / Methanol) for about 15-30 minutes.

·        Keep flow rate 0.5 ml / min or 1.0 ml / min. increase flow rate gradually.

·        Check the pH of eluent on pH paper (it should be neutral) 

·        Store the HPLC column in solvent (acetonitrile or methanol).

·        Disconnect the HPLC column and keep it at respective column storage box.

·        Maintain the cleaning record in HPLC column logbook.