HPLC Column Management – SOP

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.
    • 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.

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.

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:
AbbreviationExpanded form
HPLCHigh Performance Liquid Chromatography
%Percentage
v/vVolume per volume
°CDegree Celsius
ppmPart per million
mmMillimeter
µMicron

7.0 ANNEXURES:
Annex. No.Title
01Performance of Chromatographic Column – HPLC
02Chromatographic Column Entry Log Book
03Column Usage Log Book
04HPLC 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


Also read : Daily Maintenance and Common problems in HPLC