RT shifting


 I'm into a RS method development of Piperaquine phosphate. Chromaographic condition is given below. RT of main peak and impurities is shortening as mobile phase become older.(RT shifted from 21 min to 12 min in 10-12 hrs for main peak). My HPLC is in good working condition, repeated the experiment using different reagent bottles.

 Mobile phase A= 0.1%Trifluoroacetic acid in water

Mobile phase B= 0.05%Trifluoroacetic acid in mixture of Methanol and Acetonitrile 1:1

Column: inertsil ODS-3 150×4.6mm 3micron

Column temp 40°C

Solutions are prepared in mobile phase A and using a gradient run

Conducted an another trial by replacing methanol from mobile phase B with 0.1% TFA. Result was same.

  • For this kind of problem you should do following steps

    1) first equilibrate the column with initial concn of mobile phase A and B (saturation time as per your column volume for your mention column it takes about 30 min in1ml/min flow rate)

    2) if you are doing run method then let it run whole run time don’t stop if peak come earlier 

    3) always remember that if u start mobile phase A and B in ration of 70:30 then u must end it same ration and let it run for 5-10 min even though it was not mention in your method of analysis

    note: in gradient, column saturation determine the whole system so never hurry as like in isocratic 

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