To define the HPLC column performs satisfactorily and gives reproducible results.  


COLUMN OPERATING AND CLEANING: On receipt of the new column put the date of received on the box of the column. While using the column for the first time, flush the column with shipping solvent (the details available. With manufacturer’s COA) for at least 30 minutes with a flow rate of 0.5 ml/min. 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. Carry out the column calibration .After calibration of the column, flush the column with respective solvents . Store the column in appropriate storage solvent . Remove the column, fit the end caps and store in the column box.

CALIBRATION PROCEDURE : For new columns follow the method of calibration as specified in the manufacturer’s COA. Connect the column to HPLC system. Flush the column with mobile phase as mentioned in the procedure for respective columns. Prepare the test solution in the mobile phase as recommended in the procedure. Note down the required details on the record of analysis. After the column is conditioned, inject 20 micro liter or as per the manufacturer’s COA and record the chromatograph. Calculate the tangent (theoretical plates) as per the USP method. The column shall be accepted for use if the values of theoretical plates obtained, exceeds the limit specified .If the column does not meet the requirements, the same shall be rejected & the information regarding rejection shall be given to Purchase Department. Record the following details of the column in the register and HPLC column usage log register. 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. Allocate an in-house identification no. to the column. Record the requisite details of analysis on log card .

REGENERATION (During given HPLC analysis following steps should be followed): Flush the column, first with filtered and de-gassed water for 30 minutes at the rate of 1.0ml per minute. Then with either Methanol or Acetonitrile for further 30 minutes at the flow rate of 1.0ml per minute .Condition the column with a given mobile phase for at least 30 minutes. Ensure that base line signal is stable, carry out the analysis. 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. If any of the above conditions are not satisfactory, regenerate the column as per the procedure given in 6.0. 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. 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.

PROCEDURE FOR REGENERATION (REVERSED PHASE) : Whenever Column fails in  System    Suitability  Test as per pharmacopoeia Regeneration should be done. For columns C18, C8, C6, C4, Phenyl, Amino, Polymeric follow the sequence given below with a 1.0ml flow per minute flow rate. Flush with filtered and de-gassed water for 30 minutes. Inject 20 µl of 1.0% of Acetic acid in Water. Methanol for 30 minutes, inject 20µ liter of 1.0% DMSO (spectroscopic grade) in Methanol. Acetonitrile for 15 minutes Isopropanol for 15 minutes. Chloroform for 10 minutes. Acetonitrile for 10 minutes. Methanol for 30 minutes. Water for 15 minutes. Check pH of water at inlet and outlet, which should be identical. Mobile phase for the compound to be analyzed.


  1. Column should be kept with proper end fitting during storage.

2. Do not over tighten the column end fittings.

3. Test sample should be clean homogeneous and free from particulate matter.

4. Use dedicated columns only for a given product.

5. Protect the column from rapid changes in solvent’s composition.

6. Do not change the flow rate at increment greater than 0.2ml/min.

7. Prevent the column from mechanical shocks.

8. Always use the column in the specified direction.

9. pH of the mobile phase should not be less than 2.0 and not more than 7.0.

10. Backpressure of the column should be monitored for variation.

11. Clean the solvent reservoir filter in the mobile phase after every analysis by sonication in the Methanol for 30 minutes.

12. Test sample should be clean homogeneous and free from particulate matter.

Bhanu Pratap Singh


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