Experience to use AcQuity for regular HPLC column

<p>Just want to see if anyone can share their experience to use ACQUITY to run the regular HPLC method, for instance, using 4.6 x 150 mm column? Any advantage and limitation? </p>


  • That depends on your method.

    If you're running flow rates near 2 ml/min don't expect the Acquity to work as well as an ordinary hplc system. The cell volume and the capillares are designed for flow rates below 1 ml/min and for highest (= uplc)performance. With higher flow rates the small ID of the capillares adds significant to back pressure and the small cell will lead to an additional baseline noise. Compared to an average hplc cell the uplc cell shows large scale secondary effects from refractivity, so you will have an higher increase in baseline when using uv active modifieres with gradient runs (eg organic acid).

    Second, the very low system dead volume (or dwell volume or however it is called) makes gradient runs incompatible to typicall hplc gradient runs. On the other hand, the design of the uplc can lead under cetain circumstances to ultra sharp seperations which cannot be realised on a typical hplc system.

    If you are most interested in isocratic runs you may have the "problem" that the uplc gives you lower RTs for the same method as an hplc system. When this effect is of no importance and you are running with flow rates around or below 1 ml/min you have a wonderful working hplc system (but it is like using an formula one racing car for driving to the baker three corners ahead), which has most likely a significant lower downtime than your common hplc system - after all, when using the aquity as a hplc system there has to be some benefits after paying more money for it.

  • KWB

    In my experience HPLC methods run very well on ACQUITY. Of course the flow rate must be met (under 2 mL/min) and your particular column manager must be able to hold the HPLC column if the method requires column heating. If your method is a gradient method, the larger volume of most HPLCs will require you to add some initial hold time to the method when run on the ACQUITY. For example, an HPLC system with a precolumn volume of 1 mL running a method at 1mL/min will create a built -in initial hold of 1 minute. You would in this case add a 0.9 minute initial hold to the method when running this method on ACQUITY (assume about 0.1mL precolumn system volume on ACQUITY). You are changing the gradient table, but preserving the actual gradient. For HPLC columns longer than 150mm or those with very large end fittings, the ACQUITY 30cm column holder is available.

    The only real issue I have seen has been with an occasional method which was run previously on a HPLC system with relatively poor dispersion (ie relatively high band or peak broadening for even an HPLC). In this case, the UPLC optimized system volumes of ACQUITY contribute much less extra-column peak broadening and you may see small peaks on the AQCUITY which you did not see on the HPLC. This may or may not appear to be a good thing to you, depending on the purpose of the separation. The additional peaks now seen in your sample were always there - they were just lost because of the system volume. You could probably add some additional volume back into the system by increasing the tubing ID between the column and the detector. Just don't forget to put the proper tubing back when you are doing UPLC!!!

    High sensitivity HPLC methods do not usually work at the sensitivity scale which ACQUITY detectors are capable of working at. This means that although (as a previous responder has pointed out) baselines may be more affected by the use of UV additives such as TFA, when you are using HPLC columns and methods on ACQUITY, you are generally working at UV scales which are not going to be troubled by this. The ACQUITY light guided flow cells are designed to minimize refractive index effects and work very well at this as long as the cells are kept clean. They are 10mm in pathlength like most HPLC analytical cells and can work at high or low pH.

    K Blakeslee