Column Selection

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rporter
rporter
in Instrumentation and Mass Spectrometry
<p><span>Hello, </span></p><p></p><p><span>I'm new to UPLC and trying to convert a method from HPLC to UPLC. What UPLC column is the most similar to the Waters Symmetry C18? I only have BEH columns available so I tried the BEH C18 and the resolution is worse than HPLC. I am considering ordering a HSS column. I'm thinking the HSS C18 would be a better match? </span></p><p></p><p><span>Thanks, </span></p><p><span>Becky</span></p>

Answers

  • ACQUITYEPM
    ACQUITYEPM
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    Greetings Becky,

    The HSS column might be a good option, but if you could provide a little more information, I'm sure we can offer several suggestions for a successful conversion. Is your method isocratic or gradient? What are the solvents and flow rates? What would you like to optimize; resolution, speed, sensitivity, solvent usage? Are you free to alter any, and all, parameters or are you under certain constraints?

    Due to the reduced system volume of the ACQUITY UPLC, gradients will tend to develop more quickly. That is, for any given unit of time (or fraction of a column volume), the ACQUITY will more closely match the requested gradient composition. This can cause the relative peak retention to decrease and the peaks will trip over each other trying to get out of the column. Sometimes, a slight reduction in the overall slope of the gradient will put them back in their "proper" order. Even with this reduction in slope, it is almost always possible to define a flow rate and gradient time that will decrease the separation time when compared to HPLC.

    Regards,

    pcb

  • rporter
    rporter
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    Thanks pcb. I went ahead and ordered the HSS C18 but here is more info. My HPLC method is isocratic, waters symmetry C18 5um, 4.6x150mm, 1.2ml/min, MP is 67 MeOH:33 phos buffer pH 7, 15 min. I'm trying to optimize speed while keeping the MP the same. On UPLC I tried the equal efficiency parameters from the columns calculator using the BEH C18 2.1x50mm column and obtained less resolution than HPLC. I'm assuming this is b/c the column chemistries are different? I also tried the max efficiency parameters with the 100mm column and resolution was great but run time was the same as HPLC. Today I'm going to decrease the flow rate and see how that effects it.

    Do you know where I could get a copy of the RP column selectivity chart? I know there is a waters program you can download but I would prefer a paper copy.

  • ACQUITYEPM
    ACQUITYEPM
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    Greetings Becky,

    Since your separation is isocratic, there are fewer parameters that can be adjusted for a conversion to UPLC. With the constraint of maintaining the current mobile phase composition, column chemistry must be called upon to control selectivity and retention. I believe you will find that the HSS column is more similar to the Symmetry material and is you best option under the circumstances. I will investigate the selectivity chart and reply.

    Regards,

    pcb

  • dougm
    dougm
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    I agree with pcb's recommendation that the closest UPLC chemistry is the ACQUITY UPLC HSS C18 column. My apologies for stating the obvious, and it goes without saying, but chromatographically please keep everything the same: same mobile phase, temperature, samples, etc.

    The eSelectivity chart is here.

    Regarding a 'hard copy' I have attached a pdf of a slide that I have used in the past.

    Best of luck and please keep us posted as to your progress.

    --Doug

  • rporter
    rporter
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    Doug,

    Thank you! The pdf chart is exactly what I was looking for because I am unable to download the program due to computer restrictions. I received the HSS CI8 column yesterday and will try it today. I'll keep you posted.

    Becky

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