Different area responses on ACQUITY systems

Why do I see much higher area responses on one ACQUITY UPLC System compared to another, even with the same standard, column, and mobile phase?


  • This issue is related to the sample loop. Loops on different ACQUITY UPLC Systems, even with the same nominal volume can be very different in volume. If the a loop is measured outside of tolerance, one of the following reasons may be operative. It should be noted, that volumetric accuracy in full loop mode is ultimately limited by the tolerances of the tubing used to construct the sample loop.

    (1) The loop may be out of specification.

    Not every ACQUITY UPLC loop is measured or individually calibrated after it is manufactured. Waters does perform periodic inspections on the loops, but Waters does not inspect 100% of the product, there are simply too many. Sample loops are fabricated based on length and an assumed internal diameter. If a batch of tubing is either too large or too small in terms of internal diameter, the absolute values of the loop can be inaccurate. The 5 uL loop is nominally made from 0.009 inch ID tubing. The specifications of the stainless steel tubing is ± 0.001 inch Internal Diameter (ID). (Please note this is the best the technology and the industry can offer today). One inch of 0.009 inch ID has a volume of 1.04 µL. For 0.010 inch ID the volume is 1.28 µL and for 0.0105 the volume is 1.42 µL. The volumes have ratios of 100:123:137 percent. A 5 µL loop becomes 6.2 and 6.9 µL for these changes in ID. A small change in the ID across the tube can result in a significant change in the actual volume of the loop. This is why the ACQUITY UPLC System "Characterizes" every loop to determine what the actual volume really is. To compare two systems go to the ACQUITY UPLC Console of each and click on the Sample Manager View. Under "Characterize" check to see what the measured loop volume and nominal loop volumes are and compare between systems. If one of the loop volume is smaller than other or is too low, this loop should be discarded.

    (2) The loop may have been installed incorrectly.

    If the ends of the Sample Loop are not firmly pressed into the valve and "bottomed out" before the ferrules are "fixed", a space will result at the end of the tubing. This space will be measured as additional Sample Loop volume. A quick visual check of the ends of the loop can usually determine if the two ferrules are equal in distance from the tubing ends. If one of the two ferrules is noticeably closer to the end of the tube, the loop was not installed properly and this can explain the extra volume. This type of incorrect installation can lead to poor dispersion (visible for isocratic elution and for peaks which are not strongly focused in a gradient separation) and poor carryover because this volume is poorly swept.

    (3) The detection method differs

    Another Reason for differences in response:

    1. Responses can differ also because the detectors differ with respect to tuning or wavelength accuracy especially when peaks are not detected at lambda max (UV) or in a well tuned MS. Different responses from ELS and FLR detectors are to be expected.

    2. Responses can differ because the effects of system dispersion and column efficiency impact the peak concentrations. This is all about configuration, column selection, separation development and column temperature.

    3. Responses can differ because the volume injected varies. Injection modes and local settings (loop volume, overfill factors, etc.) can all have rather dramatic impacts on the volume injected.

    4. The question of how well the sample loop is installed will impact (a) dispersion and (b) carryover. Increased dispersion and carryover can change the apparent peak area, but as secondary effects since dispersion will decrease plat...

  • Christine's test.

  • Hi Elizabeth,

    Concerning the proper installation of loops, is it possible then that Waters can supply sample loops with the ferrules already "fixed" in the proper place on the loop. Currently sample loops come supplied with loose ferrule fittings that are not "fixed" and thus could lead to user installation errors.


    Rob Burgess

  • This is a great suggestion, but unfortunately there are enough differences in depth of the ports that the ferrule needs to be swaged individually for each injection valve port. If the tubes and valves are not swaged properly for each individual port then there will be issues with carryover and peak shape.

    We do swage the 10 µL loop that is shipped as the default loop and then test the Sample Manager before shipment for carryover and peak shape.

    Waters does recommend that you label the loop top/bottom to indicate original installation position when removing or changing the loop. If the loop is to be reinstalled then the loop/valve orientation must be the same as the original installation. We also do not recommend using loops that have been installed on one inject valve, to be used on a different inject valve. We do have a plan to implement colored tabs on each end of the loops so that you will know which way the loop is to be orientated.

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