HPLC Column Management – SOP & Guideline

Standard Operating Procedure (SOP) and Guideline for Receipt, Qualification, Usage, Regeneration, and Discardation of HPLC/UPLC column.

Procedure for Handling of HPLC/UPLC Column

1.0   Purpose :

    • To define the procedure for the Receipt, Qualification, Usage, Regeneration, and Discardation of HPLC/UPLC column.

2.0   Scope :

    • This guideline is applicable to all HPLC/UPLC columns received at the quality control department.

3.0   Reference, and Attachments:

    • Reference:

    • Certificates received along with HPLC/UPLC Column from the manufacturer like Waters, Whatman, Hypersil, Merck, G.L.Sciences, Supelco, etc.
    • Attachments :

    • Format “Column Inventory/Index  (Attachment – 1)
    • Format “Column Label (Attachment – 2)
    • HPLC/UPLC Column Usage log (Attachment – 3)
    • Format “HPLC/UPLC Column qualification check Protocol (Attachment – 4)
    • HPLC Column Regeneration Protocol (For C18/ C8 /CPS (Cyano) / NH2 / Phenyl Columns)- Attachment – 5.
    • HPLC Column Regeneration Protocol (For Silica Columns) – Attachment – 6

4.0   Responsibility :

    • Analyst :

    • Take column inventory, check the performance, and maintain the column as per the guideline.
    • Maintain the Column Usage log.
    • Regenerate the column and document it as per SOP.
    • Intimate the head QC or designee in case of malfunctioning of the column.
    • Head Quality control or Designee :

    • Training to be given to the analyst before the implementation of the SOP
    • Ensure proper documentation is done as per SOP.
    • Evaluate the performance of Column efficiency and regeneration.
    • Proceed for procurement of new column and discarding of the column.
    • Supervise the Column qualification and Regeneration procedure.

5.0   Procedure – HPLC Column Management

    • General Notes & Precautions during handling of HPLC Column :

    • Increase the flow rate gradually over a period of 2-5 minutes.
    • Never expose the HPLC column to the pH below or above the pH tolerance limit specified by the manufacturer.
    • Use HPLC grade solvents to prepare the mobile phase.
    • Filter all prepared buffer through 0.45μm filter before using them in the HPLC system.
    • Avoid the HPLC/UPLC column from fall down or any other mechanical shock.
    • To prevent HPLC/UPLC columns from drying out, they should be tightly capped for storage under the recommended solvent.
    • Flushing column with pure water for long periods should be avoided because of stationary phase De-wetting.
    • Receipt of the New HPLC/UPLC Column :

    • On receipt of the HPLC/UPLC column, the Analyst shall verify the details like Batch No. / Column serial No. with the manufacturer’s certificate.
    • The analyst shall allocate the Column No. in serial of receipt of the column and make necessary entries in “Column Inventory/Index”. (Attachment – 1)
    • The analyst shall mention the Column No. on the Certificate and put initials and date.
    • File the Certificate in the respective files.
    • The analyst shall prepare the “Column label” (Attachment – 2) and affix it to the column.
    • The analyst shall make necessary entries in the “HPLC/UPLC Column Usage Log” (Attachment – 3).
    • HPLC/UPLC Column Efficiency Check (Column Qualification):

    • Before usage of the column first time, the system suitability criteria of the intended test in the product shall be checked.
    • The HPLC/UPLC Column is said to be qualified if it meets the System suitability criteria given for the particular test in the product.
    • HPLC/UPLC Column qualification shall be documented in the Column qualification protocol. (Attachment – 4)
    • The tests in which system suitability criteria are not given in method then the efficiency check shall be confirmed by verification of the peak shape of the interested peak.
    • Once column performance found satisfactory then the column shall be issued for the analysis by Head QC or designee.
    • Usage of the HPLC/UPC Columns :

    • Before starting the analysis, wash the column (other than the Silica column) with water for at least 30 min. and then, saturate for at least 30 min. with the mobile phase at a suitable flow rate.
    • Make necessary entries on every usage of the column in the respective “Column Usage log” (Attachment – 3).
    • Put notes in the “Remark” column of the log for the failure of system suitability criteria.
    • After completion of analysis wash the column with Water for about 1hr. or more depends on the concentration of buffer used in the mobile phase and finally with water and methanol or acetonitrile (80:20) for at least 30 min.
    • For the Silica column, before starting analysis wash the column with Isopropyl alcohol for at least 30 min. and saturate with the mobile phase for at least 30 min.
    • After completion of the analysis, wash the Silica column with Isopropyl alcohol for at least 1hr. and finally with methanol for at least 30 min.
    • Note: The above-suggested column washing procedures are generalized. The column washing procedure if required can be varied as per column manufacturers’ instructions.
    • After completion of analysis and washing disconnect the column, fix column ends tightly, and keeps the column at a designated place.
    • HPLC/UPLC Column Regeneration :

    • In case of failure of the system suitability, the column shall be regenerated and checked for the qualification.
    • The regeneration shall be done as per “HPLC COLUMN REGENERATION PROTOCOL” (Attachment –5 & 6 )
    • Note: If column regeneration procedures are defined by column manufacturer and are different as per attachment–5 & 6, in such cases separate column regeneration protocol needs to be prepared.
    • After regeneration, HPLC/UPLC column shall be checked for its efficiency and recorded in the Column qualification protocol ( Attachment No.4).
    • If column performance is found to be satisfactory continue the usage for analysis of the same product.
    • If the column is not qualified for the same product then the column shall be used for other products or different tests of the same product.
    • In case of the HPLC/UPLC column meets System suitability of the other product (i.e. If qualifies for the other product or qualifies for the different test of the same product) then it can be used in the product for which it is qualified and qualification shall be recorded in Column qualification protocol ( Attachment No.4).
    • Discarding of the HPLC/UPLC Column

    • After regeneration, if the column is not qualified then the column shall be discarded.
    • Bend the HPLC/UPLC column and remove all packing material from the column & destruction of the material to be done as per solid waste destruction procedure.
    • Discardation of the column and transfer of the column from one product to another or from one test to another of the same product shall be documented in the column usage log and authorization shall be taken from the quality head.
    • Head QC or designee shall make necessary entries regarding of “Discarded on”, “No. of cumulative injections” and “Reason for discard” in the “Column Inventory/Index” (Attachment-1). And also in the “Remarks” column of the “HPLC/UPLC Column Usage Log” (Attachment-3).
    • Discard the column in case a loss of packing material is found in the column and take the authorization of Head QC or designee.
    • Discard the column if it gives high backpressure even after cleaning the frits and regeneration.
    • Take the authorization of the Head QC or designee before discardation.

6.0   Attachments – HPLC/UPLC Column:

Attachment – 1 : Format “Column Inventory/Index 

SR. NO. DATE OF RECEIPT MAKE/ COLUMN TYPE COLUMN SERIAL NO. COLUMN DIMENSION REFERENCE PAGE  NO. OF  COLUMN USAGE  LOG DEDICATED FOR
QUALIFICATION DATE DATE OF ISSUE DISCARDED ON NO. OF CUMULATIVE INJECTIONS REASON  FOR DISCARD REMARK

Attachment – 2 :  Format “Column Label

HPLC/UPLC Column Label
  • Type : ____________________________
  • Particle size :______________μm
  • Dimension :_________________________
  • Column No. :  _____________
  • For : _______________________________

Attachment – 3 : HPLC/UPLC Column Usage log 

COLUMN NO.:_______________          MAKE:_______________

COLUMN TYPE:                                               DEDICATED FOR:__________

Date

Product/Sample B.No. /A. R. No. No. of  Injections Cumulative Injections
Washing Time(Before Analysis)
Start End
Saturation with the mobile phase Usage Time
Start End Start End
Washing Time (After Analysis)
Start End
Washed

with

Sign Remarks

Attachment – 4: Format “HPLC/UPLC Column qualification check Protocol 

Column Type :  
Column No. :   No. of cumulative  injections :  
Product name :
Test for :
Reason for Efficiency check :
Reference ATP No. :
Reference document No. ( Handbook/ Template) :
Procedure:

  • Carryout the injections as per the requirement of the respective tests of product/material in ATP/pharmacopeia.
  • Record and review the chromatograms and report the System suitability results in the following table.
  • Compare the results against the system suitability criteria and confirm whether the column performance is satisfactory/unsatisfactory for the analysis of a particular test of the product.
  • Attach the system suitability chromatogram along with the “HPLC/UPLC COLUMN QUALIFICATION CHECK PROTOCOL” and submit for checking.
System suitability criteria :
Parameters Observations Limits
Theoretical plate    
Tailing factor    
Resolution    
Capacity factor    
Others    
Conclusion: The Column qualifies / Not qualifies for the intended use.
Remarks  :

Attachment – 5: HPLC Column Regeneration Protocol

(For C18/ C8 /CPS (Cyano) / NH2 / Phenyl Columns)

Column Type   : C18 / C8 /CPS (Cyano) / NH2 / PHENYL
Column No.
Reason for Regeneration:
Remark:

  • Remove the frits of the column. Wash the frits with water and sonicate for 15 min. in 6M Nitric acid.
  • Discard the Nitric acid, wash the frits with water and sonicate the frits for about 15 mins. in water.  Assemble the frits to the column.
Procedure:

  • Attach the column to be regenerated to the HPLC pump.
  • The set flow rate of about 1.0 ml/min. Wash the column with hot water at about 55°C or if a column oven facility is available, set column oven temperature 55°C and wash the column with water for a specified time.
  • While washing with water inject 4 x 100 ml of DMSO.
  • On completion of water, washing change the mobile phase solvents as specified in the following table and in the same sequence as specified in the following table.
  • Note the time of each washing.
  • Check the column efficiency after regeneration.
Solvent Flow Rate Time
Test Condition Applied Condition Test Condition Applied Condition Test Condition Applied Condition
DMSO Injection
Water at 55°C 1.0 ml 50 min. 4  of 100 ml
Methanol 1.0 ml 50 min.
Chloroform 1.0 ml 50 min.
Methanol 1.0 ml 50 min.
Conclusion  :

Attachment – 6: HPLC Column Regeneration Protocol (For Silica Columns)

Column Type   :  SILICA
Column No.  
Reason for Regeneration: 
Remark:

  • Remove the frits of the column.
  • Wash the frits with water and sonicate for 15 min. in 6M Nitric acid.
  • Discard the Nitric acid, wash the frits with methanol and sonicate the frits for about 15 mins. in methanol. 
  • Assemble the frits to the column.
Procedure  :

  • Attach the column to be regenerated to the HPLC pump.
  • Set flow rate 1.0 ml/min.
  • Wash the column with Hexane for a specified time.
  • On completion of Hexane, washing change the mobile phase solvents as specified in the following table and in the same sequence as specified in the following table.
  • Note the time of each washing. Check the column efficiency after regeneration.
Solvent Flow Rate Time

Test Condition

Applied Condition Test Condition Applied Condition Test Condition

Applied Condition

Hexane 1.0 ml 50 min.
Methylene chloride 1.0 ml 40 min.
Isopropyl alcohol 1.0 ml 40 min.
Methylene chloride 1.0 ml 30 min.
Hexane 1.0 ml 30 min.
Conclusion  :

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pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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