<p><span>Dear all,</span></p><p><span>I am using UPLC for 3 years, and my lab managers are interested for buying a new UPLC H-Class system. I just read the UPLC H presentation, technical data and some application examples. It is mentioned that transfer from Alliance to UPLC H is very easy. I believe, because I am dealing with method transfers HPLC-UPLC. But I have one question: do you have data regarding UPLC to UPLC H reproducibility? The dwell volumes are so different (80 ul for regular UPLC and 400 ul for UPLC H), and I believe that band spreading due dead volume will affect peak shape and resolution, especially in the gradient methods for impurities determination. </span></p><p></p><p><span>Regards,</span></p><p></p><p><span>Milos </span></p>


  • Milos,

    Thank you for your message.

    While it's true that the ACQUITY UPLC and the ACQUITY UPLC H-Class have different system volumes, what's important to understand is that only the system components through which the sample passes contribute to band broadening resulting in loss of resolution. In both the ACQUITY UPLC and the ACQUITY UPLC H-Class, almost all of the volume is upstream of the injector. As such, the sample does not pass through these components so they do not contribute to band broadening. When we measure band broadening of the system, injector-to-detector (exclusive of the column), both systems are essentially identical with respect to dispersion.

    To address your reproducibility question, the larger system volume of the ACQUITY UPLC H-Class that is upstream of the detector may shift retention times, but, that shift is the same for all peaks eluted in the gradient. For a variety of samples and analytical methods, we have found that the ACQUITY UPLC and ACQUITY UPLC H-Class give comparable reproducibility of resolution, peak areas and retention.

    I hope you find this useful.

    Jeannine Jordan

  • The extra dwell volume will affect the precision of your gradient. If you are performing complex peptide mapping, there may be a high chance of some peptides switching order in a shallow gradient. I am actually testing 50ul, 100ul, and 425ul mixers in term of UV baseline noise and retention time shift out of necessity. In reality, it is not as simple as adding the same delta t to all peaks with larger volume.


  • Thanks for quick replies. Jeannine`s message explains a lot but I still have a doubt concerning peak RT shifts in gradient methods with dozen of peaks. For example, we introduced Waters` UPLC AA application with AccQ Tag Ultra reagents and we do not have big problems with it. My question is: did somebody in Waters try AA application on UPLC H-Class? And was there any difference between chromatograms obtained on UPLC and UPLC H, concerning RT`s and peak shape of amino acids? We also have introduced UPLC methods for very complex impurity profiles such as Simvastatin and Azithromycin and I am concerned whether I will have the same chromatograms on UPLC H or not?



  • Thank you for your comments.

    You are absolutely correct that changes in gradient slope can alter the selectivity, indeed the elution order, in a peptide map. In my original answer, I was using precision and reproducibility in the context of a single instrument, or a single instrument design, running a particular method - not in the sense of different instrument designs making exactly the same gradient, or, of how the composition generated by the system compares to the programmed percentage in an absolute sense. The attached data and application notes specifically show performance of a single system design.

    In comparing two different system designs there are many details that may affect how exactly the two systems create the same separation. The system volume and design of any mixers certainly enter into this. The comparison would also reflect the way column temperature control may differ between the instruments and subtleties in the fluidic desgin of the injector. I also agree with you that different mixer designs will introduce subtle changes in the shape of the gradient and I would add that the mixer effects are different when flow rates are varied.

    In our testing, directly measuring accuracy of proportioning we have observed that the ACQUITY UPLC and ACQUITY UPLC H-Class match very closely over most of the range of compositions. As expected, over this range, the offset is constant. At the extremes of the gradient, the first few and the last few percent, we do observe the effect of differences in common volume. Interestingly, one of the most pronounced differences between the ACQUITY systems, as well as among the several HPLC systems we have measured in the same way, is the volumes required for re-equilibration.

    To briefly summarize, our current understanding is that most separations will be insenstive to the small differences in proportioning accuracy between the ACQUITY systems. I certainly expect to find particular samples that would be effected by some of these differences and peptides would represent that kind of sensitivity. Our experience however, is that retentions would still be reproducible on repeated runs on the same system.

  • Hi

    Thank you for your comments about transferring very complex methods between the different UPLC systems. We are in the midst of ongoing investigations of all the parameters that can affect such transfers. Some have been very straightforward rquiring only adjustment of a gradient offset. Other samples have proven somewhat more challenging in ways that require more careful exploration of the parameters that control retention and selectivity. The most significant such parameter has proven to be the re-qulibration time between injections. Of almost equal importance and significance, the effective column temperature is almost always different bewteen two systems. Some chromatography is altered by 1-2C change. The temperature of the transferred method may require adjustment by that small amount. Beyond that some methods may require more attention.

    Our AAA method, the UPLC Amino Acid Analysis solution is a slightly special case because we provide it as a turnkey solution complete with chemistry and software support. We never suggest any alternative instrument or method alternative until it is not only completely tested but also the documentation software , suport, and training are completely updated.

    Some of those ideas may be appropriate when you consider the complex methods that you have developed. Only your group can judge how closely the transferred methods must match. You have probbaly already done some robustness testing that tells you how much variation can be allowed as a column ages or is replaced. Similarly, some range of retention variability is usually expected between batches of mobile phase. Thos limits are are a good starting point in judging how well a particular method has transferred.