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    HPLC and UHPLC Part 2

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    Alex Nitin
    This presentation discusses the use of higher pH for method development & analysis. There are several advantages to using this method that can produce improved results. This can allow compounds to be separated which can not be done at a lower pH.

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    Attribution Non-Commercial (BY-NC)
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    HPLC and UHPLC Part 2

    Uploaded by

    Alex Nitin
    13 views10 pages
    This presentation discusses the use of higher pH for method development & analysis. There are several advantages to using this method that can produce improved results. This can allow compounds to be separated which can not be done at a lower pH.

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    © Attribution Non-Commercial (BY-NC)

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    This presentation discusses the use of higher pH for method development & analysis. There are several advantages to using this method that can produce improved results. This can allow compounds to be separated which can not be done at a lower pH.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    13 views10 pages

    HPLC and UHPLC Part 2

    Uploaded by

    Alex Nitin
    This presentation discusses the use of higher pH for method development & analysis. There are several advantages to using this method that can produce improved results. This can allow compounds to be separated which can not be done at a lower pH.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 10

    HPLC and UHPLC Separations at Mid to High pH: Part 2

    Introduction
    O LabNote continues the discussion of the

    use of higher pH for method development and analysis


    O There are many advantages to working at

    higher pH, but also some items to be aware of

    Advantages
    O Orthogonal selectivity (different elution

    order) can be obtained for many analytes


    O Basic analytes are retained longer and

    have better loading capacity, linearity, and peak shape

    Advantages
    O Signal and signal-to-noise ratios are

    usually improved for basic analytes


    O Chromatographic separations at higher

    pH allow compounds that are unstable at lower pH to be analyzed without oncolumn degradation

    What to Watch Out For


    O Analytes that are unstable at higher pH

    could degrade on column


    O With LC-UV detection the UV spectra and

    values of ionizable analytes often change dramatically


    max

    O Can make peak tracking more challenging

    What to Watch Out For


    O Column lifetimes for all silica-based

    columns will be shorter using buffers


    O On-column reaction or decomposition can

    occur for metal-sensitive analytes

    Method Development
    O Parameters that affect selectivity (relative

    retention) include:
    1. 2. 3. 4. 5. 6.

    Column bonded phase Organic modifier choice Mobile phase pH % organic modifier or gradient steepness Column temperature Buffer identity and concentration

    O Newest addition to the ACE column

    family: the ACE SuperC18 phase

    ACE SuperC18 Features and Benefits


    O Unique bonding chemistry provides

    increased resistance to degradation


    O Can be used at pHs ranging from 1.511

    with all analytes types

    ACE SuperC18 Features and Benefits


    O Designed for use with LC-MS compatible

    mobile phase buffers


    O Does not show hysteresis effects (memory

    effects) when changing between low and high pH mobile phases

    ACE SuperC18 Features and Benefits


    O The ACE SuperC18 column produces

    highly symmetrical peaks for bases, acids, and neutral analytes


    O Baselines were observed to be better

    when NH4OH or NH4OAc were premixed with the organic modifier in the B solvent

    http://www.mac-mod.com

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