HPLC Column Washing Procedure; Purpose
The tenacity of this SOP is to define the procedure for washing of HPLC column to avoid any intrusion on repeated usage & to escalation column lifespan.
HPLC Column Washing Procedure; Scope
This procedure is applicable for all HPLC columns used within quality control laboratory of XX Pharmaceuticals Limited.
Definition:
- COA: Certificate of analysis
- HPLC: High performance liquid chromatography
- QC: Quality Control
- SOP: Standard Operating Procedure
Responsibilities
The roles and responsibility is as follows
Senior Executive/Executive, Quality Control
- To confirm that the instructions of this procedure are correctly followed.
- To accomplish the HPLC column washing properly in accordance with this SOP.
- To keep record of HPLC column washing time properly.
Manager, Quality Control
- To confirm that this procedure is kept up to date.
- To ensure that the SOP reflects the required working practices.
- To assemble training on the SOP to all concerned personnel and to confirm implementation of the SOP after training.
Head of Quality Assurance
- Take initiative to approval of the SOP.
- To confirm the overall implementation of the SOP.
Procedure
- Use HPLC grade solvents for column washing.
- Freshly prepared, filtered and degassed solvent or solution should be used.
- Purge solvent line to make them free from air bubbles.
- Do not change flow rate at increment greater than 0.2 ml/min.
- Keep column with proper end fitting during storage.
- Do not over make tighter the column end fittings.
Pre-Wash (mobile phase run/ Prior to analysis) procedure
- Set column on column compartment of HPLC.
- Select shipping solvent, from COA of respective manufacturer, If not mentioned in COA, store the column based on column properties like Nitrile /ODS/Amine group etc.
- Run shipping solvent (e.g. Azide solution, 65% Acetonitrile,) mentioned in COA of respective manufacturer for 15-20 minutes.
- Gradually change the organic phase composition depending on the organic phase composition in mobile phase to reach the same in mobile phase. For example, gradually decrease the organic phase from 65% Acetonitrile (in shipping solvent) & finally reach 30% Acetonitrile (in mobile phase). For the Gradient HPLC this can be conducted through time program and for Isocratic HPLC, this can be done using intermediate solvents, as appropriate. Accomplish this run within 15 to 20 minutes.
- Change line with mobile phase & execute HPLC analysis.
Post-Wash (mobile phase run /after completion of analysis) procedure
- For mobile phase containing Buffer and counter ion
- Replace mobile phase with purified water. Purge line. Run same for 15-20 minutes.
- Depending on shipping solvent, gradually reach the same. For example, if 65% Acetonitrile is shipping solvent, then start with 100 % purified water and gradually increase the organic phase and finally reach at 65% Acetonitrile. For the Gradient HPLC this can be conducted through time program and for Isocratic HPLC, this can be done using intermediate solvents like 15%, 30%, 45% Acetonitrile, as appropriate. Accomplish this run within 55-60 minutes conducting each solvent composition period 15-20 minutes.
For mobile phase without buffer and counter ion
Depending on the shipping solvent, gradually reach the condition from the organic phase composition of mobile phase. For example, if 65% Acetonitrile is shipping solvent and organic phase composition in mobile phase is 30% Acetonitrile, then start with the initial organic phase of 30% Acetonitrile (70% Purified water) and gradually increase the organic phase and finally reach at 65% Acetonitrile. For the Gradient HPLC this can be conducted through time program and for Isocratic HPLC, this can be done using intermediate solvents as appropriate. Accomplish this run within 55 to 60 minutes conducting each solvent composition period 15 to 20 minutes.
For mobile phase containing tri-ethyl amine
- Replace the mobile phase with a mixture of methanol and 0.05% phosphoric acid (50:50 v/v) & run for 30 minutes.
- Depending on the shipping solvent, gradually reach same. For example, if 65% Acetonitrile is storing solvent, then start with 20 to 30% Acetonitrile & gradually increase the organic phase and finally reach at 65% Acetonitrile. For the Gradient HPLC this can be conducted through time program and for Isocratic HPLC, this can be done using intermediate solvents as appropriate. Accomplish this run within 55 to 60 minutes conducting each solvent composition period 15 to 20 minutes.
- Stop run. Remove column from HPLC unit, do end capping properly both ends & place in respective column box.
- Store column box in an allocated place.
- Record HPLC column washing time after analysis in logbook for operation of HPLC.