Shallow Gradients on UPLC

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Kaye
Kaye
in Instrumentation and Mass Spectrometry
<p><span>I'm using the Aquity calculator to work out the conditions I need for my method transfer. The traditional HPLC method employs a shallow gradient (60-80% MeCN) over 20 minutes. When I put this into the calculator, it throws up the following warning - 'In order to calculate optimally scaled UPLC Methods Gradient Steps (changes in %B) must be greater than or equal to 30%'</span></p><p><span>It will give me conditions, which I've tried and I'm getting worse resolution on UPLC compared to HPLC. </span></p><p><span>Have others seen this warning, and can someone explain what it means. I'm assuming it to do with the mixing / pumping characteristics of the system. </span></p><p><span>Thanks</span></p><p><span>Kaye </span></p>

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  • dougm
    dougm
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    Hello,

    Regarding the error message that you are seeing, this has nothing to do with the instrument, but rather the calculator's ability to predict and supply Optimally Scaled gradients for you to try.

    If you think about the UPLC method choices that you are given, they are broken down into two categories: Geometrically Scaled and Optimally Scaled. The Geometrically Scaled UPLC gradient calculations attempt to maintain the selectivity of your HPLC method by maintaining the same number of UPLC column volumes per gradient step as are calculated in the HPLC method. These calculations take HPLC column dimensions and particle size into consideration. The Optimally Scaled UPLC gradient calculations are much more complex and attempt to predict and present gradient choices (based on LC theory) that either provide the highest efficiency or the fastest run time. The selectivity of the separation will likely change.

    Basically, the Optimally Scaled UPLC gradient predictions fall apart with shallow gradients. The Geometrically Scaled UPLC gradient choices are unaffected by gradient steepness. So, if you wish to maintain the selectivity of your HPLC method and use Geometrically Scaled gradients (most people use this), simply ignore the error message.

    Regarding your resolution comments, could you please provide a bit of background information? For example, please tell me what the brand, chemistry and dimensions of your HPLC column is. For example, XBridge C8, 4.6 x 150 mm, 5 um. Please be as specific as possible. Also, the same goes for the UPLC column that you are using: chemistry, i.d., particle size and length are required. It would also be helpful to provide your detailed HPLC method (everything that you are entering into the UPLC calculator).

    Questions that need to be answered: What calculated UPLC method choice are you using? What are your UPLC column dimensions vs. your HPLC column dimensions. Are the selectivities of your HPLC and UPLC columns radically different or similar? Are you using the same mobile phases, temperature, etc in both your HPLC and UPLC methods? Is anything different?

    There may be other things at play here, but let's start here.

    I hope this information helps,

    --Doug

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