Change in retention time between Acquity Binary system and H-Class

<p> I have been working to show some robustness of a method as it transfers from our original Acquity binary based system with a PDA to a H-Class quaternary based system with a TUV.  The baseline and responses are good, but I am getting a strong shift in retention time as I move to the H-Class (approx 1 minute for everything).  The void volume seems to be the same as the non-retained compounds all elute at 0.4 min for both systems, and I'm having some trouble explaining this shift.  Any help would be appreciated, thanks.</p>

Best Answer

  • atus
    Answer ✓

    Hi Brian,

    The system dead volume in H-Class is much bigger than the regular ACQUITY. Do not forget the proportional valves/mixer in the Quad low pressure pumps are higher as well. I had late peaks shifted by 4min at 0.2ml/min in a 240min peptide mapping gradient when tested between these 2 systems. Unfortunately, I do not think you can change any of that.

    We have H-class, regular ACQUITY, and nanoACQUITY. I choose a particular system that is most suited for my application, and stick with it.

    Best of luck,



  • RD


    As Albert correctly noted, the system delay volume of an ACQUITY HClass system is much greater than that of a Classic ACQUITY.  While there is slightly less volume for an HClass from the injector through the solvent pre-heater and onto the column, this volume is insignificant.  The retention of unretained components only depends on this volume   which explains why you see no difference in the elution time for the unretained components.

    The bulk of the delay volume for any system is found between the pump and the injector.  For a Classic ACQUITY which employs binary blending, the solvents meet after the pump heads at the gradient mixer and thus need only to pass through the mixer and the tubing connecting it to the injector to reach the sample.

    The HClass employs quaternary blending where the solvents meet before the pump heads. Thus the blend must pass through both the primary and accumulator heads, the tubing connecting the accumulator to the purge valve, the tubing connecting the purge valve to the gradient mixer and then the tubing connecting the mixer to the injector. Reasonable estimates for the volumes of the 2 systems are 125 uL for the Classic ACQUITY and 400 uL for the HClass.

    To compensate for the greater delay, when transferring a method that was developed on a Classic ACQUITY to an HClass, the HClass method must make the injection after the gradient starts.  Using the above estimates, the gradient for the HClass method would need to start and allow 275 uL of solvent to flow before injecting the sample.

    The good news is that this is very easily accomplished with logic built into the pump's operating software and is facilitated by using the ACQUITY Calculator.  See the attached document for a description of this.

    If you are still struggling, consider enrolling in one of our Web-based Method Transfer Seminars or contact your local Waters Support Representative.

    Good Luck,