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 sonicate. 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
minutesWater 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 systemChromatographic 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. 
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