SOP for HPLC Column Cleaning and their Maintenance

OBJECTIVE: To lay down a procedure for cleaning and maintaining high-performance liquid chromatography (HPLC) columns. This SOP for HPLC column cleaning and their maintenance is a procedure that defines how Quality Control receives, issues, regenerates, washes, stores and withdraws HPLC columns.

SCOPE: This Standard operating procedure applies to HPLC column cleaning and maintenance of the different types of HPLC columns used in the Quality Control (QC) Laboratory.

RESPONSIBILITY:

The Officer/Executive, QC department, shall follow this procedure.

The Manager, QC department, is responsible for overall compliance with this SOP.

PROCEDURE for HPLC Column Cleaning and Maintenance:

This section covers receipt, issue, regeneration, storage and withdrawal of columns, and then the washing steps for each column type. Schedule M requires written procedures for cleaning and maintaining laboratory equipment, and 21 CFR 211.67 sets the same expectation for equipment cleaning and maintenance. This SOP documents how HPLC columns are kept fit for use.

Receipt and Inspection of New Column:

• On receipt, cross-check the new column against the supplier’s 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. Then assign the column ID as per LC, C, YYY. LC stands for Liquid Chromatography, C stands for the column, and YYY represents the serial number starting from 001.

Issuance and Performance of Column:

USP <621> Chromatography sets the system suitability and allowable adjustment requirements that a column must meet before it is used for release testing. For HPLC instrument use and calibration, see the Calibration of HPLC and their Operation SOP.

• 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.
• If the column keeps failing the system suitability parameters, return it 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. Keep 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 system suitability test after regeneration, following the standard test procedure (STP) for the related product.

• After flushing, store the column in methanol. Flush it with 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 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.
• Follow with 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:

The table below lists the storage solvent for each column type.

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.

WHO GMP guidance for quality control laboratories expects instrument and column use to be recorded. ICH Q2(R2) treats system suitability as part of showing that an analytical procedure stays fit for its purpose.

HPLC Column Cleaning/ Washing Procedure:

Wash each column after use with the solvents listed for its type, then disconnect it from the HPLC unit and store it in the designated area.

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, at a flow rate of 1 ml/min for 30 minutes. For example, use Acetonitrile if the mobile phase contained it.
• Disconnect the column from the HPLC unit and store it in the designated area.

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 in the designated area.

Washing of 117 Columns:

• Change the mobile phase to a mixture of 0.01 N H2SO4 and ACN in a ratio of 85:15 v/v. 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 in the designated area.

Washing of Silica or Silica bonded-Normal Phase Columns:

• Wash the column with the non-polar mobile phase used for analysis, or with a non-polar solvent such as hexane or heptane.
• Flush the column for 30 minutes at a flow rate of 1 ml/min.
• Disconnect the column from the HPLC unit and store it in the designated area.
• Maintain records of column washing (post-use) in the column usage log.

Important Cautions:

• Wash the column after each use to avoid poor peak shapes, non-reproducible retention times, high back pressure and baseline disturbance.
• Do not use harsh reagents; they damage the bonded phase of the column.
• Prevent column contamination by using filtered and degassed mobile phases, as well as filtered samples and standards during analysis.
• Filter water through a 0.45-micron filter before use to prevent contamination.
• 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.
• Following these cautions keeps column performance and life consistent.

ABBREVIATION

This list explains the short forms used in this SOP.

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

Related topic: various types of hplc columns

Frequently Asked Questions