I have a problem in hplc

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AboSaleh
AboSaleh's picture
I have a problem in hplc

 I was working on an HPLC column by gradient elution. I achieved good separation of a number of components. Peaks were sharp, smooth and symmetrical. After I had carried out some runs, I observed peak worsening; very bad peak shape. Peaks became very broad; there was like valleys and splitting in each peak !!. Very broad and well observed peak fronting was also observed. I enumerated a number of possible causes like: -passing buffer individually by mistake without organic modifier
-injecting very high concentration of components under study.
 I washed the column several times. The most recent wash procedure was carried out using 95% water and 5% methanol as the mobile phase is composed of buffer and methanol. However, I repeated my run with the same gradient elution method and no improvement was observed. 
I can't understand what has gone wrong !!