OBJECTIVE: To lay down a procedure for HPLC Column Cleaning and their Maintenance.
SCOPE: This Standard operating procedure shall be applicable to HPLC Column Cleaning and maintenance of different types of HPLC columns used in the Quality Control Laboratory.
RESPONSIBILITY:
Officer/Execution QC department to follow this procedure.
Manager QC department for overall compliance with this SOP.
PROCEDURE for HPLC Column Cleaning and Maintenance:
Receipt and Inspection of New Column:
- Upon receiving a new column, carefully cross-check it with the provided index.
- Inspect the column for any physical damages that may have occurred during transportation.
- Verify that the column’s make, type, serial number, dimensions, and other specifications align with the certificate of analysis from the supplier.
- Ensure compatibility between the column and the instrument fittings. If any non-compliance with user requirements is found, return the column to the supplier.
- If the column meets user requirements, record its details in Annexure-1 and assign the column ID as per LC, C, YYY, where LC stands for Liquid Chromatography, C stands for the column, and YYY represents the serial number starting from 001.
Issuance and Performance of Column:
- Issue the column as per the requirements stated in the standard test procedure of the specific product.
- Before use, test the column to ensure it meets the system suitability criteria for the intended product.
- If the column satisfies the system suitability criteria, proceed with further analysis.
- If the column fails to meet the criteria, return it to the concerned person for testing on another product.
- In case of repeated failure to meet the system suitability parameters, return the column to the supplier.
Column Regeneration for Reverse Phase Column:
- Flush the column using warm water (55°C) at a flow rate of 0.7 ml/min for 10 minutes, maintaining the column at (55°C) in the column oven during flushing.
- Inject 100 ul of DMSO four times with a 2-minute interval between injections.
- Follow with water flushing for at least 30 minutes and methanol flushing at a flow rate of 0.7 ml/min in the reverse direction for 60 minutes.
- Conclude the regeneration process by flushing the column with chloroform for 60 minutes at a flow rate of 0.7 ml/min in the reverse direction.
- Subsequently, wash the column in methanol for 60 minutes at a flow rate of 0.7 ml/min in the reverse direction.
- Perform the suitability system test after regeneration following the STP for the Related product.
- Store the column in methanol after flushing the column and store in methanol for 20 minutes at a flow rate of 1.0 ml/min in the normal direction.
- Record the column regeneration details in the column history Record card.
Column Regeneration for Normal Phase Column:
- Use the Hexane to wash the column for 60 minutes at a flow rate of 2.0 ml/min.
- Continue with Methylene chloride washing for 60 minutes at a flow rate of 2.0 ml/min.
- Wash it Followed by IPA washing for 60 minutes at a flow rate of 2.0 ml/min.
- Conclude the regeneration process with methanol flushing for 60 minutes at a flow rate of 2.0 ml/min.
- After regeneration, conduct the system suitability test as per the standard test procedure for the relevant product.
- Store the column in Hexane after flushing it for 20 minutes at a flow rate of 2.0 ml/min in the normal direction.
- Record the column regeneration details for future reference.
Related: Calibration of HPLC and their Operation SOP
Storage condition for HPLC Column in solvent:
| Column Type | Storage Solvent |
| Reverse-phase silica gel | Watery Methanol, Chloroform, Methanol |
| Normal-phase silica gel | 2-Propanol, Chloroform (with 2% 2,2-Dimethoxypropane and 1% Acetic anhydride), Chloroform, n-Hexane |
| Chemically modified packing agent | Methanol, Chloroform, n-Hexane (for reverse-phase system) or Watery Methanol, Chloroform, Methanol (for normal-phase system) |
| Ion-exchange silica gel | Water, 0.1 M EDTA•2Na, Water Methanol, Water (with the amount of each washing solvent being about 10 times the column volume) |
Withdrawal of Column:
Columns may be withdrawn from further analysis in the following cases:
- When physical damage is detected.
- If the column fails to comply with the system suitability requirements after regeneration.
HPLC Column Cleaning/ Washing Procedure:
Washing of C18, C8, C6, Phenyl, CN, Amine-Reverse Phase Columns:
- For mobile phases containing a buffer, follow this washing procedure with a flow rate of 1 ml/min:
- Water for 15 minutes
- Water + ACN (50:50) for 15 minutes
- Methanol (100%) or the solvent used in the mobile phase preparation for the analysis, for 15 minutes.
- After washing, disconnect the column from the HPLC unit and store it in the designated area.
- For mobile phases without buffer (salts), use the solvent used for the mobile phase preparation during analysis with a flow rate of 1 ml/min for 30 minutes (e.g., Acetonitrile if used in the mobile phase).
- Again, disconnect the column from the HPLC unit and store it properly.
Washing of Protein Pack Column:
- Wash the column with a minimum of 30 ml of 2.5 M acetic acid.
- Disconnect the column from the HPLC unit and store it appropriately.
Washing of 117 Columns:
- Change the mobile phase to a mixture of 0.01 N H2SO4: ACN in a ratio of 85:15 v/v and flush at a flow rate of 1.0 ml/min for a minimum of 2 hours.
- Flush the column with water at a flow rate of 1.0 ml/min for 2 hours.
- Disconnect the column from the HPLC unit and store it as required.
Washing of Silica or Silica bonded-Normal Phase Columns:
- Wash the column with the mobile phase (Non-polar) used for analysis, or use non-polar solvents like Hexane and Heptane for washing.
- Flush the column for 30 minutes at a flow rate of 1 ml/min.
- Disconnect the column from the HPLC unit and store it appropriately.
- Maintain records of column washing (post-use) in the column usage log.
Important Cautions:
- Properly washing the column after each use is necessary to avoid poor peak shapes, non-reproducible retention times, high back pressures, and baseline disturbances.
- Avoid the use of harsh reagents, as they can damage the bonded phase of columns.
- Prevent column contamination by using filtered and degassed mobile phases, as well as filtered samples and standards during analysis.
- To prevent void formation, place the columns in an area free from vibrations, which could cause mechanical shocks.
- Gradually increase the flow rate when flushing the column.
- Filter water with a 0.45-micron filter paper.
- By adhering to these best practices, you can ensure the optimal performance and longevity of your HPLC columns in various analytical applications.
ABBREVIATION
SOP: Standard Operating Procedure
No.: Number
Dept.: Department
ml: Millilitre
min: Minute
°C: Degree centigrade
IPA: Isopropyl Alcohol
DMSO: Dimethylsulfoxide
ID: Identification number
ACN: Acetonitrile
H2SO4: Sulphuric acid
