Chromatography Forum

I have used a hplc condition ,which is 20mmol KH2PO4(PH=3.0):acetonitrile=95.2:4.8,for several days.then one day i changed the ph to 7.5 and acetonitrile to 5%.After a few hours,i decided to use the old method again,but i found that the retention of my two objects changed about 1 min.i have checked the mobile phase and the pump which are all normal.the column is C18,250*4.6,5,intex.because the condition is used to detect the serum samples by hplc-uv,a little change in retention will bring me troubles.thanks for your reply in advance!

If all retention times shift by the same percentage, and the dead time changes by the same percentage, then you have a flow rate problem; therefore, the first thing to do is to look at t0 and see if that has changed.

If t0 is unchanged, then you have a chemistry problem. Try preparing a fresh batch of mobile phase and see if than helps. Make sure to allow sufficient equilibration time with the fresh mobile phase (pump at least 25-30 mL of the new mobile phase through the column).

If you have the same result with both batches of mobile phase, then the column is the next potential source of error. Did you inject any samples during your pH 7 interlude? If so, it is possible that you have contaminated the column.

The most cost-effective test may be to simply put in a new column (columns are cheap; time is valuable).

thanks a lot for your reply.i will check my chromatograph again to see if i can solve the problem.