HPLC Column Performance SOP | HPLC Column Washing SOP |
- HPLC Column management SOP covers below points:
- Handling of Column
- HPLC column ID Number allocation
- HPLC Column Storage and Maintenance
- HPLC Column washing procedure
- Verification for Performance of HPLC Column / HPLC column performance test
- Destruction/Disposal of HPLC column
- HPLC column performance test format
- HPLC column usage log book format
- Chromatographic Column Entry Log Book format
- HPLC Column Disposal Record format
1.0 OBJECTIVE :
- To lay down the procedure for handling, storage, maintenance, verification and disposal of chromatographic column.
2.0 SCOPE :
- This SOP is applicable for handling, storage, maintenance, verification and disposal of chromatographic column in Quality Control Department.
3.0 RESPONSIBILITY :
- Quality Control Officer and above
4.0 ACCOUNTABILITY:
- Quality Control Head
5.0 PROCEDURE :
5.1 Handling of Column:
- Receive the chromatographic column with proper documents i.e. Invoice/ challan, certificate etc., by the person authorized by department head.
- Ensure that the documents are proper and as per requirement.
- Ensure that the column is properly packed and in well condition.
- Make counter sign with date of received in documents.
- Take entry in Chromatographic Column Entry Log Book, Annexure No.: 02.
- After receiving the column, ID Number for the column is allocated by numbering system as,
- A/H-C/XX/YY
- Where, A = for Company name initials,
- H = for HPLC,
- C = for Column,
- XX = for last two digit of the year,
- YY = for sequential number starting from 01.
- Label with ID Number of column is affixed on the column.
- Performance of HPLC column is verified as per verification procedure of performance of chromatographic column.
- After completion of column performance, enter performance observation of chromatographic column in record of performance of chromatographic column – HPLC (Annexure No. 01).
5.2 Storage and Maintenance :
- All chromatographic columns are stored in cabinet, dedicated as per column dimension/type.
- Chromatographic column should be handled with care.
- Use the column for analysis as required.
- Usage of column shall be recorded in Column Usage Log Book as per the Annexure No.: 03
- An analyst shall follow manufacturer’s instruction strictly for usage of the column which was provided with the column.
- HPLC Column washing procedure :
- Column shall be washed properly as per follows;
- After completion of analysis:
- In case of reverse phase column
- Use water as the preliminary solvent and switch over to organic solvent based on the mobile phase used.
- Flush the column with the water for at least 45 minutes followed by the respective 80% solvent which is used for analysis, for 45 minutes and again with water for 45 minutes.
- In case of normal phase column (Silica column)
- The column shall be washed properly using organic solvent such as isopropyl alcohol, n-Hexane and methanol.
- Do not use water as solvent for washing the silica column. Water can be used only when any buffer solution is used in preparation of mobile phase.
- Flush the column with the isopropyl alcohol for at least 45 minutes followed by n-Hexane for 45 minutes and then with methanol for 45 minutes.
- Washing period shall be increased to 1 hour instead of 45 minutes whenever required.
- Then the column shall be flushed with the storing solvent for at least 15 minutes.
- In case of reverse phase column
- Before analysis:
- In case of reverse phase column
- Flush the column with the water for at least 30 minutes followed by the respective solvent which will be used for analysis for 30 minutes.
- If the mobile phase contains methanol or acetonitrile as organic solvent, flush with 100 % concentration of the same solvent for washing, for at least 30 minutes.
- If the mobile phase contains both acetonitrile and methanol as solvent, flush with 100 % of the solvent whose concentration is maximum in the mobile phase, for at least 30 minutes.
- In case of normal phase column (Silica column)
- Flush the column with the isopropyl alcohol for at least 30 minutes followed by methanol for 30 minutes.
- Then the column shall be flushed with the storing solvent for at least 15 minutes.
- In case of reverse phase column
- After completion of analysis:
5.3 Verification for Performance of HPLC Column :
- Checking of New Column
- Column efficiency of chromatographic column is evaluated in terms of number of theoretical plates. Column with high theoretical plate numbers are considered to be more efficient than columns with a lower theoretical plates.
- Check the column with respect to certificate of analysis for retention time, theoretical plates, resolution, tailing factor and method specified by vendor.
- Carry out the column efficacy for theoretical plates, resolution and tailing factor.
- Determination of Column Efficacy:
- Selection Criteria:
- Perform the column performance by one of the procedure (either by procedure A, B or C)
- If the column is used for only one product, then follow the procedure A.
- If the column is used for more than one product, prefer the procedure B.
- In case of unavailability of solvent, Reagent or WS/RS, follow the procedure C.
- Column performance by using System Suitability(Procedure A):
- Perform the system suitability of material for which the column is procured.
- Prepare the solutions used for checking of system suitability of specific material as mentioned in pharmacopoeia.
- Acceptance criteria: The system suitability criteria of monograph should complies with the solution.
- Column performance by using Benzene and Toluene (Procedure B):
- Prepare mobile phase freshly on the day of use with combination of 60:40 v/v Acetonitrile and HPLC water. Filter the mobile phase by using 0.45 µm membrane filter and degas the mobile phase by using sonicator.
- Chromatographic Conditions:
- Flow Rate : 1.0 ml/min
- Wavelength : 254 nm
- Injection Volume : 20 µl
- Temperature : Ambient
- Run the mobile phase to saturate the column.
- Prepare the test solution as follow:
- Take 1 ml of Benzene and 1 ml of Toluene in 100 ml volumetric flask and dilute to volume with acetonitrile. Dilute 1 ml of this solution to 100 ml with mobile phase. Mix well and filter through 0.45 µm syringe filter.
- Inject the filtered test solution and check the number of theoretical plates, tailing factor, resolution between the peak of toluene and benzene.
- Acceptance criteria:
- Resolution between the peak of toluene and benzene should not be less than 2 or as given by manufacturer / supplier criteria.
- Theoretical plate for benzene and toluene should not be less than 2000 or as given by manufacturer / supplier criteria.
- Tailing factor of benzene and toluene should not be more than 2.0 or as given by manufacturer / supplier criteria.
- Column performance as per procedure of Manufacturer(Procedure C):
- Column performance is carried out by using the procedure given on column performance record provided by vendor.
- Prepare mobile phase as mentioned on vendor record of column performance.
- Chromatographic condition such as flow rate, wavelength, injection volume, temperature is adjusted as mentioned on vendor record of column performance.
- Test solution is prepared as per vendor record of column performance.
- Inject the filtered test solution and check the test parameter against the specification mentioned in vendor record of column performance.
- Acceptance criteria:
- The results of defined test parameter should be met with manufacturer / supplier criteria.
- Inform to QC Head for replacement of the column if column does not meet with acceptance criteria of column performance.
- QC Head informs to the purchase department for procurement of the column as replacement.
- Report the observation in Annexure No.: 01 ‘Performance of Chromatographic Column – HPLC’.
- Frequency: On receipt of the new column.
- Selection Criteria:
5.4 Destruction of HPLC column :
- The following HPLC column shall be destructed.
- If the column does not meet with verification for HPLC column performance criteria.
- If system suitability criteria as per method of analysis is not matched.
- Affix the label “REJECTED” on the column and store in containers reserved for rejected column.
- All documents regarding last non-compliance of system suitability data (Chromatogram etc.) are recorded and maintained as HPLC column destruction record.
- Destroy the rejected columns as per following procedure.
- Remove all labels or marking from column with the help of water.
- Open the end of fitting of column with the help of Engineering department.
- Columns are to be sent to scrap yard for further disposal.
- After destruction of column, enter the details in HPLC Column Disposal Record Annexure No.: 04.
6.0 ABBREVIATIONS:
| Abbreviation | Expanded form |
| HPLC | High Performance Liquid Chromatography |
| % | Percentage |
| v/v | Volume per volume |
| °C | Degree Celsius |
| ppm | Part per million |
| mm | Millimeter |
| µ | Micron |
7.0 ANNEXURES:
| Annex. No. | Title |
| 01 | Performance of Chromatographic Column – HPLC |
| 02 | Chromatographic Column Entry Log Book |
| 03 | Column Usage Log Book |
| 04 | HPLC Column Disposal Record |
8.0 SOP REFERENCES
- GLP Guideline
END OF THE SOP
ANNEXURES :
Annex. No. 01 Performance of Chromatographic Column – HPLC
Annex. No. 02 Chromatographic Column Entry Log Book
Annex. No. 03 Column Usage Log Book
Annex. No. 04 HPLC Column Disposal Record
