HPLC column-Regeneration procedure

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It describes the (Standard Operating procedure) Sop for Receipt, storage, performance, usage, regeneration& destruction of HPLC column

 

Sop for Receipt, storage, performance, usage, Regeneration& Destruction of HPLC column

I: Purpose and Scope :

  • The purpose of this SOP is to lay down a procedure for receipt, storage, performance, usage, regeneration and destruction of HPLC columns.    
  • This SOP is applicable for receipt, storage, performance, usage, regeneration and destruction of HPLC columns in QC department

II: Responsibilities:

  •  All Microbiologist, Quality personnel shall be responsible to follow and implement this SOP.

III: Introductions and Procedures:

Precautions:

  • Use always HPLC grade solvents and high purity chemicals (AR grade) for preparation of mobile phase and diluents.
  • Filter and degas all mobile phase / solvents/ water prior to use.
  • Avoid usage of column when back pressure is above 4000 psi.
  • Increase/Decrease the flow rate in small increments and decrements of 0.1ml/minute.
  • If there is a shoot up of backpressure of column, reverse flush the column.
  • Filter all the solutions before injection to HPLC system.
  • Ensure the end plugs are fixed to column before storage.
  • Handle HPLC column carefully as packing material is sensitive. Do not drop the column on floor. Column should not be handled with wet hands.

Receipt of new Column: 

  • On receipt of new HPLC column, analyst shall verify the details like Lot No. /Batch No., Column Serial No. with the manufacturer’s certificate.
  • On receipt of new HPLC column check the pack of column physically as per indent and also check the performance report or COA of the manufacturer then record the date of receipt in “HPLC Column Inventory Log” as per Annexure I.
  • Read the leaflet of respective HPLC column to know the shipping solvent.
  • Analyst shall allocate the column number as QC/Y-BBBB
  • Where,
  • QCI – Quality control of CEPHA block
  • Y – HPLC Column
  • BBBB – 01,02…… serial of receipt of column, in case serial number is 1000 and above it shall be written as BBBB where BBBB is 1000,1001……
  • Make entry in HPLC Column Inventory Log as per Annexure I.
  • After receipt, perform column qualification check as per following methods i.e. point no. 5.3 and record the details in Annexure II.
  • Analyst shall make necessary entries in the “HPLC Column Usage Log” as per Annexure III.

Column Performance Check : 

  • Column shall be qualified prior to use for analysis.
  • Before usage of the column first time, system suitability criteria of the intended test in product shall be checked. The column is said to be qualified if it meets the system suitability criteria given for the particular test in the product.
  • The product in which Related substance calculation is by area normalization method in that product the diluted standard shall be injected for related substance calculation and the column shall be qualified against the system suitability criteria for diluted standard.
  • If column performance found satisfactory then column shall be issued for the analysis by Head QC or designee and enter the details in “HPLC Column Inventory Log” as per Annexure I.

Usage of Column : 

  • Make necessary entries on every usage of the column in respective “Column Usage Log” as per Annexure III.
  • Column usage log shall be reviewed by Asst. Manager/ Executive or designee and Verified by Head QC or designee.
  • Before analysis reverse phase HPLC column should be initially wash with 100% water with flow rate 1.0 ml/minute for minimum 30 minute, then with 50% ratio of organic solvent (Methanol/Acetonitrile) in water for 30 minutes. Then finally with 100% HPLC grade Methanol/Acetonitrile with flow rate 1.0 ml/min for minimum 30 minutes. Flow rate shall be varied based on respective MOA.
  • Before starting of Normal phase analysis; HPLC system should be flush with initially with IPA then with organic solvent content in mobile phase. Do not use water at the time of normal phase analysis. Flow rate shall be varied based on respective MOA.
  • Before starting of analysis; wash normal phase column initially with HPLC grade Isopropyl alcohol with flow rate 1.0 ml/minute for minimum 30 minutes. Flow rate shall be varied based on respective MOA.
  • After completion of analysis reverse phase HPLC column should be initially flush with 100 % water with flow rate 1.0 ml/minute for minimum 30 minutes and maximum 2 hour then with 100 % HPLC grade organic solvents (Methanol/Acetonitrile) with flow rate 1.0 ml/minute for minimum 30 minutes and maximum 1 hour. The washing time may be increase if required for particular column.
  • Column temperature may be apply during column washing and allowed maximum up to 50°C.
  • Store reverse phase column in Methanol/Acetonitrile with the end plugs firmly in place.
  • After completion of analysis; wash normal phase column initially with organic solvent content in mobile phase with flow rate 1.0 ml/minute for minimum 30 minutes then with HPLC grade Isopropyl alcohol for minimum 30 minutes.
  • Store normal phase column in Isopropyl alcohol with the end plugs firmly in place. If back pressure is more wash with less flow rate for more times.

Note: Do not wash the normal phase HPLC column with water

  • Wash cyano column with 50/50 mixture of Methanol/Acetonitrile and water with flow rate 1.0 ml/minute for minimum 30 minutes. Store Cyano column in Acetonitrile or Methanol with the end plugs firmly in place. If back pressure is more, wash the column with less flow rate for more time.

HPLC Column Regeneration: 

  • HPLC column regeneration shall be done only when the system suitability criteria not passes after several washings of HPLC Column and if new column is not available in stock or the procurement of HPLC column will take longer time than scheduled time of analysis. Also the approved testing laboratory has no facility to analyze the particular sample.
  • Column regeneration shall be done after approval of Head QC.
  • Literature of HPLC Column shall be referred for column regeneration.

Destruction Of column :

  • Discard the column if not meets to the required specification.
  • Make the entries regarding of “Discarded on” “No of cumulative injections” and “Reason for discard” in “Column Inventory as per (annexure I) and also in the “Remarks” column of the “HPLC Column Usage Log” (annexure III).
  • 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 first regeneration. Take the authorization of Head QC or designee before discard.
  • Discard the column in case a column performance check is found unsatisfactory in column and take the authorization of Head QC or designee.
  • The column shall be destroyed by bending the HPLC column with the help of tools available in engineering for bending and it shall be sent to scrap yard.
  • If new column is not available in stock then give requirement of new column. If new column is not available in market as per analysis schedule then send the sample to approved testing laboratory for analysis.
  • The requirement shall be given to Head office for procurement of New HPLC column with details like Column Name, Dimensions, packing material, manufacturer name etc. but not limited.

 IV: Annexure:

Annexure-I: HPLC Column Inventory log

Annexure -II: HPLC Column Performance Record

Annexure-III: HPLC Column Usage Log

 

Annexure-I

HPLC Column Inventory log 

Sr.

No.

 Column

Name

 Column

Number

 Make Column Serial No. /Lot No. Receipt Date Qualified

(Yes/No)

 Qualification Date No. of Cumulative injections Discarded on Reason for Discard Remark

 

 

Reviewed By:___________________                                                                                    Verified By : _________________

 (Department Head)                                                                                                                        (QA)

 

Annexure -II

HPLC Column Performance Record

 

Column Name: ____________________________________________
Make:___________________________________________________
Dimensions: ______________________________________________
Serial No. / Lot No.: ________________________________________
Column ID No.(In House): _________________________________
Date of Receipt: ___________________________________________
Date of Performance: _______________________________________

 

System Suitability Checks

 

Parameters Result Acceptance Criteria

 (To comply as per specification)
Theoretical Plates  
Tailing Factor  
Area Repeatability (% RSD)  
Resolution  

 

Remark: All the system suitability parameters are within / not within the acceptance criteria

Hence, it is concluded that the column is Qualified /Disqualified and it can be used/rejected.

for the routine analysis.

 

Performed By:                                    Checked By:                                 Approved By

Date:                                                     Date:                                            Date:

 

Annexure-III

HPLC Column Usage Log

 

 

Column Name: __________________________ Column ID Number: _____________________
Sr. No. Date Name of Product / Sample / Material Batch No. /

A.R. No.

 No. of Injections System suitability complies

(Yes/No)

 Cumulative

Injections

 Analyst

Sign

 Remark
                 
                 
                 
                 
                 

 

Reviewed By:___________________                                                                                    Verified By : _________________

(Sign. / Date)                                                                                                                                         (Sign. / Date)

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