HPLC Column

How to regenerate the HPLC Column and improve the efficiencies? Or Regeneration of HPLC column

 

Regeneration Procedure:

Following steps shall be performed
First, open all the HPLC column frits from the both ends and wash frequently with mixture of water and diluted nitric acid. If required the sonicate the frits for 20-30 minutes in sonicator. Then tighten the frits as usual and perform the HPLC column washing in below mentioned sequences

Name of Solvent                                      Time required for flushing
Methanol                                                 20 minutes
Water                                                      30 minutes
Methanol                                                 60 minutes
Acetonitrile                                               25 minutes
Chloroform                                              30 minutes
Acetonitrile                                               25 minutes
Methanol                                                 30 minutes
Water                                                      30 minutes
Methanol                                                 20 minutes

How to check the column efficiencies of regenerated HPLC column?
HPLC equipped with pump and UV detector along with suitable integrator system

Chromatographic condition

Mobile Phase                           :               Acetonitrile: Water (40:60)
Flow rate                                 :               1.0 mL/min.
Column Temperature              :               At ambient
Injection volume                       :               5 µl
Wavelength                             :               254 nm
Run time                                  :               25 min.

Test solution for column performance check:

Take 1.0 mL of Benzene and Toluene in 100 mL volumetric flask and make up the volume up to the mark with mobile phase. Further pipette out 10 mL solution and transferred in 100 mL volumetric flask and make up to the mark with mobile phase.

Inject three replicate injection of above test solution and check the system suitability parameters of all three replicate such as %RSD, RT of solvents peak (Benzene and Toluene), tailing factor and theoretical plates.