SOP for Washing of HPLC Column After Use

1.0 OBJECTIVE

To clean the column thoroughly after its each use to avoid any interference on repeated usage.

2.0 SCOPE

This procedure is applicable for different groups of H.P.L.C columns used in Quality Control laboratory.

3.0 RESPONSIBILITY

2.1 Doing: Technical Assistant
2.2 Checking: Executive /Manager

4.0 ACCOUNTABILITY

Head of the Department

5.0 PROCEDURE

5.1 For C18, C8, C6, Phenyl, (CN, Amine, Silica column.:)
5.1.1 Wash the column with the solvent which is use in individual product mobile phase preparation. eg. suppose water is use in mobile phase preparation then wash the column with water.
5.1.2 Allow the minimum 30 ml washing medium to flow through the column.
5.1.3 Disconnect the column from the HPLC unit and store in an allocated place.
5.2 For Protein pack column:
5.2.1 Wash the column with minimum 30 ml of 2.5 M acetic acid.
5.2.4 Disconnect the column from the H.P.L.C unit and store in an allocated place.
5.3 For L17 column :
5.3.1 Change the mobile phase to a mixture of 0.01 N H₂SO₄ : ACN : 85:15 v/v and flush at flow rate of 0.6 ml/min. minimum for 2 hrs.
5.3.2 Take care that flow rate should not exceed 0.6 ml/minute.
5.3.3 Flush the column with water at flow rate of 0.6 ml/min, for 2 hours.
5.3.4 Disconnect the column from the H.P.L.C unit and store in an allocated place.

6.0 ABBREVIATIONS

6.1 H.P.L.C. = High Performance Liquid Chromatography
6.2 PSI = Pound per square inch
6.3 ACN = Acetonitrile
6.4 H₂SO₄ = Sulphuric acid
6.5 nm = Nanometer
6.6 ml = Millilitre
6.7 v/v = Volume by Volume
Related: Relative Response Factor in HPLC

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