hplc column performance evaluation storage and destruction

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hplc column performance evaluation storage and destruction

1.0 Purpose
1.1 To describe a procedure for evaluation of HPLC column performance, storage and discard.

2.0 Scope
2.1 This standard operating procedure is applicable for evaluation of HPLC column performance, storage

and discard in Quality Control laboratory at ABC Ltd, India.

3.0 Responsibility
Officer QC/ Executive QC Preparation of SOP and follow the procedure as per SOP
Assistant Manager Reviewing and Training of SOP
Manager QC Implementation of SOP
4.0 Accountability
4.1 Manager Quality Control is accountable for implementation and compliance with SOP.

5.0 Procedure
5.1 All the HPLC column shall be procured from standard manufacturers.
5.2 At the time of receiving new HPLC column, check the pack of column physically as per indent

and certificate of efficiency of column certified by the manufacturer shall be checked and record by the Manager QC.
5.3 HPLC column performance shall be checked for every new C8 and C18 columns before

starting their actual usage for analysis.
5.4 Set following chromatographic conditions:
Mobile phase : Acetonitrile : Water (65 : 35)
Flow rate : 1.0 ml /min
Detection Wavelength : 254 nm
Column temperature : 40o C
5.5 Solution Preparation and Qualification
5.5.1 Benzene and Toluene: transfer 0.5 ml each of benzene and toluene to a 100 ml volumetric

flask, add 25 ml of Acetonitrile and dilute to volume with mobile phase (Solution A).
5.5.2 Napthalene: Transfer about 30 mg of Napthalene to a 100 ml volumetric flask, add 25 ml of

Acetonitrile, allow to dissolve and dilute to volume with mobile phase (Solution B).
5.5.3 Pipette out 5 ml and 10 ml of solution A and B respectively in a 50 ml volumetric flask and

dilute up to volume with mobile phase. (Solution C).
5.5.4 Inject 20 µl solutions C in five replicates and record the chromatogram.
5.5.5 Compare the chromatogram with that supplied along with the column.
5.5.6 Determine the tailing factor for the entire peak. The mean tailing factor for any component shall not be more than 1.5.
5.5.7 Determine the plate count for naphthalene peak. This shall be around 10,000 USP plates.
5.5.8 Determine the resolution between benzene and toluene peaks shall be NLT 2.0.
5.5.9 Calculate the mean and RSD of peak areas for the five replicate injections. The RSD for the

peak areas for each component shall not be more than 2.0%.
5.5.10 Record all the results in HPLC column performance report (Annexure I).
5.5.11 In case the column doesn’t meets as per established criteria, such columns shall not be used for analysis.
5.5.12 If column performance is not satisfactory by above procedure, the same may be tested using

the manufacturers recommended procedures (if different from the one given above). The matter

shall also be discussed with the column supplier accordingly.
5.6 Cleaning and storage
5.6.1 After completion of the analysis the column shall be cleaned as per the following procedure.
5.6.2 if any buffer solution use in mobile phase wash the column initially with water (HPLC grade)

at flow 1.0 ml/min. for one hour, than wash with methanol: water (50:50) or acetonitrile: water (50:50) for one hour.
5.6.3 If any buffer solution was not use in mobile phase wash the column methanol: water (50:50) or

acetonitrile: water (50:50) for one hour at flow 1.0 ml/min. for one hour.
5.6.4 Now remove the column and then close the column end properly store at specifies place.
5.7 Destruction of HPLC column
5.7.1 All the non-performing columns of HPLC (Not meeting the system suitability

requirements e.g. USP plates count, Tailing factor and Resolution), which shall be confirmed

and approved by the Quality Control-Manager, shall be segregated for destruction purpose Annexure-II.
5.7.2 After due permission from Quality Control-Manager, take out packing of the column,

deface it and cut it into three pieces before sending it to scrap yard for destruction.

6.0 Reference
In House Specification

7.0 Attachments
Annexure I : HPLC Column Performance Record
Annexure II : Destruction Record of HPLC column

8.0 Distribution
Distribution of the controlled copy to concerned departments.
Additional copies may be issued for specific purpose.

9.0 Abbreviations
QA : Quality Assurance
QC : Quality Control
SOP : Standard Operating Procedure
HPLC : High Performance Liquid Chromatography

10.0 Revision History