Friday, December 10, 2010

Operation, Cleaning, Storage, Calibration And Regeneration of HPLC Column.

1.0          OBJECTIVE    
            
         To ensure that the HPLC column performs satisfactorily and gives reproducible results.  
          
2.0          RESPONSIBILITY    

Q.C. Chemist / Q.C Executive.

3.0          ACCOUNTABILITY  

  Manager  Q.C.

4.0      PROCEDURE                :

4.1     COLUMN OPERATING AND CLEANING:

Ø      On receipt of the new column put the date of received on the box of the column.
Ø      while using the column for the first time, flush the column with shipping solvent (the details available       
Ø      With manufacturer’s COA) for at least 30 minutes with a flow rate of 0.5 ml/min.
Ø      Change the column to mobile phase, which is to be used for the column calibration. Allow the system to stabilize and condition the column for 30 minutes.
Ø      Carry out the column calibration as mentioned in point no.4.2
Ø      After calibration of the column, flush the column with respective solvents as described in Annexure-I
Ø      Store the column in appropriate storage solvent as mentioned in Annexure-I.
Ø      Remove the column, fit the end caps and store in the column box.

4.2  CALIBRATION PROCEDURE:

Ø      Calibrate the column using the procedure tabulated in Annexure-II. For new columns not mentioned in Annexure-II, follow the method of calibration as specified in the manufacturer’s COA.
Ø      Connect the column to HPLC system.
Ø      Flush the column with mobile phase as mentioned in the procedure for respective columns.
Ø      Prepare the test solution in the mobile phase as recommended in the procedure.
Ø      Note down the required details on the record of analysis.
Ø      After the column is conditioned, inject 20 m liter or as per the manufacturer’s COA and record the chromatograph.
Ø      Calculate the tangent (theoretical plates) as per the USP method.
Ø      The column shall be accepted for use if the values of theoretical plates obtained, exceeds the limit specified in the Annexure-II.
Ø      If the column does not meet the requirements, the same shall be rejected & the information regarding rejection shall be given to Purchase Department.
Ø      Record the following details of the column in the register and HPLC column usage log register:
Ø      Column name, Dimension and serial / batch number of the column at the top of the page followed by Sr. Number, Date of analysis, Number of injection, Product, Signature of chemist and remarks.
Ø      Allocate an in-house identification no. to the column.
Ø      Record the requisite details of analysis on log card as per Annexure-III.


 
4.3  REGENERATION

                  4.3.1.    During given HPLC analysis following steps should be followed:

Ø      Flush the column, first with filtered and de-gassed water for 30 minutes at the rate of 1.0ml per Minute.
Ø      Then with either Methanol or Acetonitrile for further 30 minutes at the flow rate of 1.0ml per minute.
Ø      Condition the column with a given mobile phase for at least 30 minutes.
Ø      Ensure that base line signal is stable, carry out the analysis.
Ø      Check the responses of the Chromatogram of standard component with previous analytical report. If both satisfactory with respect to peak response, resolution, capacity factor, theoretical plates continue the analysis.
Ø      If any of the above conditions are not satisfactory, regenerate the column as per the procedure given in 6.0 .
Ø      After analysis is complete flush with the mobile phase for 15 minutes (flow rate 1.0ml/min) ,followed by water for at least 45 minutes and finally with organic solvents Methanol or Acetonitrile.
Ø      Extra precaution should be taken when organic buffers are being used in a mobile phase; flush the column with filtered de-gassed water for at least 1 Hour.
Note: When inorganic buffers are used in mobile phase do not flush directly with organic solvents like Methanol, Acetonitrile as these reduces the performance & life of the column.

4.4      PROCEDURE FOR REGENERATION (REVERSED PHASE) : Whenever Column fails in              System       
            Suitability  Test as per pharmacopoeia Regeneration should be done.

Ø      For columns C18, C8, C6, C4, Phenyl, Amino, Polymeric follow the sequence given below with a 1.0ml flow per minute flow rate.
Ø      Flush with filtered and de-gassed water for 30 minutes. Inject 20 m liter of 1.0% of Acetic acid in Water.
Ø      Methanol for 30 minutes, inject 20m liter of 1.0% DMSO (spectroscopic grade) in Methanol.
Ø      Acetonitrile for 15 minutes.
Ø      Isopropanol for 15 minutes.
Ø      Chloroform for 10 minutes.
Ø      Acetonitrile for 10 minutes.
Ø      Methanol for 30 minutes.
Ø      Water for 15 minutes. Check pH of water at inlet and outlet, which should be identical.
Ø      Mobile phase for the compound to be analysed.

4.5.     PRECAUTIONS:

Ø      Column should be kept with proper end fitting during storage.
Ø      Do not over tighten the column end fittings.
Ø      Frits of the column should be checked for cleanliness/disposition of the excipients in consultation with the Department Head.
Ø      Use dedicated columns only for a given product.
Ø      Protect the column from rapid changes in solvent’s composition.
Ø      Do not change the flow rate at increment greater than 0.2ml/min.
Ø      Prevent the column from mechanical shocks.
Ø      Always use the column in the specified direction.
Ø      pH of the mobile phase should not be less than 2.0 and not more than 7.0.
Ø      Backpressure of the column should be monitored for variation.
Ø      Clean the solvent reservoir filter in the mobile phase after every analysis by sonication in the Methanol for 30 minutes.
Ø      Test sample should be clean homogeneous and free from particulate matter.

4.6  ABBREVIATIONS

HPLC – High performance liquid chromatography.

USP – United state pharmacoepia

COA – Certificate of analysis.

DMSO- Dimethyl Sulphonic Oxide


ANNEXURE-I

DETAILS OF SOLVENT USED FOR STORAGE OF COLUMN
                                              
REF. SOP NO.:  K/QC/022
Revision No.:  00
Page No. : 1 of 1
Effective from: 




COLUMN TYPE
SOLVENT
C2, C3, C4, C6, C8, C18, Phenyl, Cyano, Amino, Co-polymer columns
Methanol
ION exchange column
10% Methanol



  


Prepared By
Checked By
Approved By
Authorised by



REF. SOP NO.:  K/QC/022
Revision No.:  00
Page No. : 1 of 2
Effective from:


I.D. No.
Name of column & Dimension
(Mm)
Mobile
Phase
Flow
(ml/min)
Test sample & Concentration
Wave
length
Min. no. of theoretical plates

1.

Kromasil 100–5C1– 5 µ
 250 ´ 4.6 mm
75: 25
Methanol: Water
1.0
Dimethyl phthalate, Toluene & Biphenyl
254 nm
NLT 30% of the suppliers specification

2.

Kromasil 100 – C18, 5 µ
 250 ´ 4.6 mm                 
Acetonitrile: Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification

3.

Kromasil 100 – C18, 5 µ
 250 ´ 4.6 mm                 
Acetonitrile: Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification

4.
Waters Spherisorb S5C6 12.5 X 4.6 mm
Methanol: Water
70: 30
1.0
Dimethyl phthalate, Toluene & Biphenyl
254 nm
NLT 30% of the suppliers specification

5.
Zorbax TMS, 5 µ, 4.6 x 250 mm Sr.No.
Methanol : Water
48 : 52
1.0
Uracil, Acetone, Bezyl Alcohol
254 nm
NLT 30% of the suppliers specification

6.
Waters Spherisorb S5C6 150X 4.6 mm                   
Methanol : Water 
70 : 30
1.0
Dimethyl phthalate, Toluene & Biphenyl
254 nm
NLT 30% of the suppliers specification

7.
Waters Symmetry C18, 5µ (3.9 x 150 mm)

Acetonitrile : Water
60: 40
1.0
Acenaphthene, Acetone
254
NLT 30% of the suppliers specification

8.
Kromasil C18, 250 ´ 4.6 mm                                     
Acetonitrile : Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification
9.
Kromasil C18, 250 ´ 4.6 mm
Acetonitrile : Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification


ANNEXURE-II

                                              
REF. SOP NO.:  K/QC/022
Revision No.:  00
Page No. : 2 of 2
Effective from:





10.
Kromasil C18, 250 ´ 4.6 mm
Acetonitrile : Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
Min. no. of theoretical plates

11.
Kromasil C18, 250 ´ 4.6 mm
Acetonitrile : Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification

12.
Kromasil KP – 100 C18 - 25406 mm
Acetonitrile: Water
(80 : 20)
1.0
Toluene, Naphthalene
254 nm
NLT 30% of the suppliers specification

13.
Waters Novapck C18 4 µ (3.9 x 300 mm)

Acetonitrile : Water
60: 40
1.0
Acenaphthene, Acetone
254 nm
NLT 30% of the suppliers specification

14.
Waters Novapck C18 4 µ (3.9 x 300 mm)

Acetonitrile: Water
60: 40
1.0
Acenaphthene, Acetone
254 nm
NLT 30% of the suppliers specification


   
Prepared By
Checked By
Approved By
Authorised by


ANNEXURE-III


HPLC  COLUMN  LOG REGISTER

                                              
REF. SOP NO.:  K/QC/022
Revision No.:  00
Page No. : 1 of 1
Effective from: 


Column Name: -                                               Dimension: -                             Sr./ Batch No: -

Sr. No.
Date of Analysis
No. of Injection
Product
Sign of Chemist
Remarks























Prepared By
Checked By
Approved By
Authorised by



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