SOP for Washing of HPLC Columns

1.0 OBJECTIVE

To describe the procedure for washing of HPLC column.

2.0 SCOPE

This SOP is applicable for washing of HPLC column.

3.0 RESPONSIBILITY

Officer/ Executive - Quality Control

4.0 ACCOUNTABILITY

Manager - Quality Control.

5.0 PROCEDURE

5.1 For C18, C8, C6, Phenyl, CN, Amine column:
5.1.1 Change the mobile phase to filtered distilled water.
5.1.2 Allow the water to flow through the column at the rate of 1ml / min. For 30 minutes. Check that the pressure does not exceed 400 BAR.
5.1.3 Change the mobile phase from water to HPLC grade 80% Acetonitrile.
5.1.4 Set the instrument to flow rate 1 ml/ minute and wash the column for 30 minutes.
5.1.5 Ensure that the column contains 80% Acetonitrile.
5.1.6 Disconnect the column from the HPLC unit and store in an appropriate place.
5.2 For protein pack column:
5.2.1 Change the mobile phase to 1:10 solution of Isopropyl Alcohol and HPLC water.
5.2.2 Allow mobile phase to flow through the column at the rate of 1 ml/ min. for 30 minutes. Check that pressure does not exceed 400 bars.
5.2.3 Ensure that the column contains mobile phase.
5.2.4 Disconnect the column from the HPLC unit and store in an appropriate place.
5.3 For L17 column:
5.3.1 Change the mobile phase to a mixture of 0.01 N H₂SO₄: ACN (85: 15).
5.3.2 Allow the mixture to flow through the column at the rate of 0.6 ml/min for 2 hours. Flow rate should not exceed 0.6 ml/min.
5.3.3 Change the mobile phase from (H₂SO₄, ACN) to water.
5.3.4 Set the instrument to flow rate 0.6 ml/ min. and wash the column for 2 hours.
5.3.5 Ensure that the column contains water.
5.3.6 Disconnect the column from the HPLC unit and store in an appropriate place.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 ACN - Acetonitrile
6.3 HPLC - High Performance Liquid Chromatography

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