HPLC Peak retention time shift


I am facing a peak retention time shift while using HPLC /DAD with a C18 column , system containing methanol and water .

while using a system ACN and buffer for other samples molecules didn't face this problem .

The peak shifting in an irregular manner (backward , forward) between different injections even at same day or at different days at week .

some time (rarely) it may not shift  between 2 injections .

  • Hi, are you using pre-mixed mobile phase or online-mixing i.e. A = water B= ACN?

    Just to confirm, you have run other methods on the same system but do not have the same issue?

    My first thought is this is an issue with the valves on the pump. I'd suggest running a leak test if you have LabAdvisor software available, or monitor the system pressure and see if that is fluctuating.

  • Hi 

    I am using online mixing (A =water , B =MeOH)  , I try also a pre-mixing but still facing same problem of shifting .

    when trying another method(C=ACN , D = Phosphate Buffer ) for another samples there was no shifting ( This most indicate that no problem in the machine?)

    Concerning pressure , I compare between to injections there was no difference ( 1 to 2 bar only)

    No leaks are detected .

  • Hi

    From what you have said it sounds like you are using a pump with a quaternary valve (low pressure mixing). The issue might be a problem with either line A or B. Most likely the valve for line B on the proportioning valve. I'd suggest trying the method you are having a problem with using lines C and D and see if the problem reoccurs. 

  • Hi ,

    I have try the method (Isocratic) on the different lines (A,B,C,D) and compare the results . It appear that there was a difference (shift in retention time about 2 mins)  in the result were line (A) is used in the method . Thus the problem might be in Line (A) . While there is little difference between other lines (B,C,D) concerning the retention time (+-0.2min.) this while using isocratic method.

    While using gradient method there was no great difference between lines (A,B,C,D) only a little shift (+-0.2 min.) and problem didn't appear at line A .

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