Poor linearity on my ACQUITY UPLC system

Why do I see poor linearity on my ACQUITY UPLC System?


  • If the system is working correctly, then there are two possibilities:

    Figure 1


    Figure 2


    (1) A common mistake for analysts that are less familiar with the ACQUITY UPLC system and its loop based Sample Manager, is accidental mixing injection modes. Above is an example where the calibration curve is made up on Partial Loop Uses Needle Overfill Mode (PLNO) injection volumes and a full loop (FL) injection volume. Quite correctly the analyst has used 0.5 µL through 3.5 µL injections, keeping within the 10% to 75% allowed range for a 5 µL loop. However, the inclusion of the 5 µL full loop volume has skewed the calibration curve for this analyte.

    Full loop injections virtually eliminate the dispersion (dilution) contribution of an injector. During the injection process he original sample concentration is maintained by flushing the sample loop several times with the sample. This is termed "overfill factor", which is the number of times a loop is overfilled. There are defaults, which provide larger overfills for the smallest loops and smaller, usually two for the 20µL or 50 µL loops. The result is a sample loop volume full, from end to end, with undiluted sample. Peak shape, column efficiency, precision and injection accuracy will all be best, and closest to theoretical, when employing full loop injections. Thus the increased accuracy/recovery of the full loop is greater than for a partial loop injection, where dilution occurs. Thus a full loop injection always delivers a greater amount on column than a partial loop injection.

    Partial loop injections, even under the most carefully controlled conditions, never attain the same accuracy of delivery as full loop injections. There will always be some level of dispersion in partial loop injections. This is why partial loop injections cannot be compared to a full loop injection in terms of absolute area or response. The partial loop injections will be diluted and so the slope of their response curve will be less.

    (2) The second possibility is adsorption.

    This is more likely to occur when multi-sample components have differing polarities. In this case, the sample diluent chosen to solubilize the components cannot solubilize all the constituents equally. If the sample diluent is more aqueous, polar sample components will solubilize most readily, but non-polar hydrophobic components will be less soluble. The same is true if the sample diluent is more organic, hydrophobic compounds will be readily solubilized and not the hydrophilic compounds. When this occurs the sample component that is "mis-matched to the sample diluent" may adhere more strongly to the needle surface than to the sample diluent. This can result in a reduction in accuracy. This is one reason why additional types of sample needles have been added to the ACQUITY UPLC Sample Manager and there are several choices available, including one that is optimized for use with very hydrophobic compounds.

    This behavior can also be compensated for in Partial Loop Uses Needle Overfill (PLNO Mode) by adjusting the "Overfill Flush Volume" method parameter. With version 1.30 ACQUITY UPLC System Driver, the Sample Manager Instrument Method Editor provides an Advanced Settings tool which contains this parameter. Users can adjust "Overfill Flush Volume" if necessary to reduce either sample consumption or minimize analyte retention on needle surfaces.

    Figure 3


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