SOP for HPLC Column Maintenance and Washing Procedure

OBJECTIVE

To lay down a procedure for the maintenance and washing of HPLC columns

SCOPE

This SOP shall apply to different types of HPLC columns used in the Quality Control Laboratory.

RESPONSIBILITY

Execution: Chemist and above – QC department.

Checking: Manager and above – QC department.

PROCEDURE

Receipt of New Column

• On receipt of the new column check the column as per the index.
• Check the column for any physical damages that may occur during its transportation.
• Check the column with respect to the certificate of analysis received from the supplier for its make, type, Serial number, dimension etc.

• Verify that the column and the instrument fittings are compatible.
• In case of any non-compliance with the user requirement, the column should be returned to the supplier.
• In case of compliance with user requirements, make the entry of the column as per Annexure – 1 and assign the column ID as per LCCYYY, where LC stands for Liquid chromatography, C stands for column and YYY is the serial number starting from 001.

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Issuance and Performance of Column

• The column will be issued according to the requirement mentioned in the standard test procedure of that particular product.
• Upon issuance of the column, the column will be tested to meet the system suitability criteria of the product for which it has been issued.
• If the column meets the system suitability criteria, use the column for further analysis.
• In case the column does not meet the system suitability criteria then the column is to be returned back to the concerned person and should be tested for its performance on any other product.
• If the column fails to meet the system suitability parameter repetitively, then it should be returned to the supplier.

Column Regeneration for Reverse Phase Column

• Flush the column using warm water (55℃) at a flow rate of 0.7 ml/min. for 10 minutes. Maintain the column at (55℃) in the column oven during flushing.
• Inject 100 µl of DMSO four times at an interval of 2 minutes.
• Flush it with water for at least 30 min.
• Flush it with methanol at a flow rate of 0.7 ml/min in the reverse direction for 60 minutes.
• After flushing with methanol, flush the column with chloroform for 60 minutes at a flow rate of 0.7 ml /min in a reverse direction.
• At the end, wash the column in methanol for 60 minutes at a flow rate of 0.7 ml/min. in a reverse direction.
• After regeneration of the column, perform the system suitability test as per the standard test procedure of the concerned product.
• Store the column in methanol, after flushing with methanol for 20 minutes at a flow rate of 1.0 ml/min. in the normal direction.
• Make the entry of regeneration in the column history card.

Column Regeneration for Normal Phase Column

• Flush the column with Hexane for 60 minutes at a flow rate of 2.0 ml/min.
• After flushing with Hexane, wash the column with Methylene chloride for 60 min at a flow rate of 2.0 ml/min.
• After flushing with methylene chloride, wash the column with Isopropyl alcohol for 60 minutes at a flow rate of 2.0 ml/min.
• At the end, flush the column with methanol for 60 minutes at a flow rate of 2.0 ml/min.
• After regeneration of the column, perform the system suitability test as per the standard test procedure of the concerned product.
• Store the column in Hexane, after flushing with Hexane for 20 minutes at a flow rate of 2.0 ml/min in the normal direction.
• Make the entry for regeneration in the column history card.

Storage condition for HPLC columns

Withdrawal of Column

Withdrawal of column from the further analysis is done in case:

• Any physical damage.
• Failure to comply with the system suitability requirement as per specification after regeneration.

Washing of Column

1. Washing of C18, C8, C6, Phenyl, CN, Amine-Reverse Phase Columns

• For the mobile phase containing buffer, use the following procedure for washing of column using a 1ml/min flow rate. Water, 15 min
• Water + ACN (50:50), 15 min
• Methanol (100 %) or solvent which is used for the preparation of mobile phase in that particular analysis, 15 min.
• Disconnect the column from the HPLC unit and store in an allocated place.
• For the mobile phase without buffer (Salts), wash the column with the solvent that was used for the preparation of the mobile phase in that particular analysis using a flow rate of 1 ml per min for 30 min e.g. suppose Acetonitrile is used in the mobile phase preparation then the column can be washed with Acetonitrile.
• Disconnect the column from the HPLC unit and store in an allocated place.

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2. 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 in an allocated place.

3. Washing of L17 column

• Change the mobile phase to a mixture of 0.01 N H2SO4 : ACN: 85:15 v/v and flush at a flow rate of 1.0 ml/min for a minimum of 2 hrs.
• 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 in an allocated place.

4. Washing of Silica or Silica bonded-Normal Phase Columns

• Wash the column with mobile phase (Non-polar) used for analysis or use non-polar solvents like Hexane, and Heptane for washing.
• Flush the column for 30 minutes using 1ml/min. flow rate.
• Disconnect the column from the HPLC unit and store it in an allocated place.
• Concerned persons shall maintain the records of washing of HPLC columns (After use) in the column usage log.

Cautions

• If a column is not properly washed it can generate poor peak shapes, non-reproducible retention times and area, high back pressures, and baseline disturbance. That is why “washing of a column” after each use is necessary.
• Harsh reagents can damage the bonded phase of columns, so avoid the use of harsh reagents.
• It is a good practice to prevent column contamination by using filtered and degassed mobile phases, filtered samples, and standards during analysis that will minimize column exposure to undesirable substances.
• Keep the columns in a vibration-free area to prevent mechanical shocks which lead to void formation.
• Increase the flow rate stepwise.
• Filter water with 0.45-micron filter paper.

ABBREVIATION

SOP: Standard Operating Procedure

No.: Number

Dept.: Department

ml: Millilitre

min: Minute

℃: Degree centigrade

IPA: Isopropyl Alcohol

µl: Microlitre

DMSO: Dimethylsulfoxide

ID: Identification number

ACN: Acetonitrile

H2SO4: Sulphuric acid

ANNEXURE

Annexure-1: Specimen format for HPLC Column list

Annexure-2: Specimen format Column usage log

REVISION HISTORY

Nil

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