HPLC experts: help with a problem I'm encountering?

  1. My method is simple--75% ACN and 25% water. If I premix my ACN and water into a single bottle and run it on a single line, my method works perfectly well. We have a quaternary pump set up. The problem happens if I put line A into 100% ACN and line B into 100% water and try to run 75% line A and 25% line B. The pressure of the system does not stabilize even after thorough degassing and through line priming. When I try to run the same method, it no longer works like if I were running a single isocratic gradient from line A with the premixed 75:25 mobile phase. Any ideas on what issues it may be with the pump?

    Also, the flow rate coming out into the waste looks perfectly fine using both methods, which I find perplexing, because if there were some sort of leak when I ran the binary method, I'd expect a change in flow rate.
     
  2. jcsd
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