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  • Warda Wasim,,, Roll No 438889 Presentation

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    The document discusses normal phase and reverse phase separation in HPLC. In normal phase separation, components have stronger affinity for the polar stationary phase and elute slower, while components with greater affinity for the non-polar mobile phase elute faster. Reverse phase is the opposite, with components having stronger affinity for the non-polar stationary phase eluting slower. The key differences are that normal phase uses a polar mobile phase and non-polar stationary phase, while reverse phase uses the opposite - a non-polar mobile phase and polar stationary phase.

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    The document discusses normal phase and reverse phase separation in HPLC. In normal phase separation, components have stronger affinity for the polar stationary phase and elute slower, while components with greater affinity for the non-polar mobile phase elute faster. Reverse phase is the opposite, with components having stronger affinity for the non-polar stationary phase eluting slower. The key differences are that normal phase uses a polar mobile phase and non-polar stationary phase, while reverse phase uses the opposite - a non-polar mobile phase and polar stationary phase.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    3 views12 pages

    Warda Wasim,,, Roll No 438889 Presentation

    Uploaded by

    Its Kazmi
    The document discusses normal phase and reverse phase separation in HPLC. In normal phase separation, components have stronger affinity for the polar stationary phase and elute slower, while components with greater affinity for the non-polar mobile phase elute faster. Reverse phase is the opposite, with components having stronger affinity for the non-polar stationary phase eluting slower. The key differences are that normal phase uses a polar mobile phase and non-polar stationary phase, while reverse phase uses the opposite - a non-polar mobile phase and polar stationary phase.

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    © All Rights Reserved
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    of 12

    SEPARATION PROCESS IN HPLC

    NORMAL PHASE & REVERSE PHASE SEPARATION


    SEPARATION

    SAMPLE COMPONENTS IN THE MOBILE PHASE ARE SEPARATED BY PASSING THROUGH


    COLUMN BY ADSORPTION.

    COMPONENTS HAVING STRONG AFFINITY WITH STATIONARY PHASE = GET ABSORBED


    =TRAVEL SLOWER
    COMPONENTS HAVING GREATER AFFINITY WITH MOBILE PHASE =TRAVEL FASTER
    = ELUTE OUT FIRST
    • SEPARATION PROCESS IN HPLC
    The component of mixture are separated from each other due to their different degrees of interaction.

    The separated components are detected at the exit of this tube (column) by the detector that measures
    their amount.
    • TYPES OF SEPARATION PROCESS

    NORMAL PHASE SEPARATION

    REVERSE PHASE SEPARATION


    Phases

    Stationary Phase:
    Polar ( Silica)
    Non Polar( Long Chain N-c18 Hydrocarbon Attached To Silica)

    Mobile Phase:
    Polar (Water, methanol)
    Non Polar (n-hexane, dichloromethane)
    NORMAL PHASE SEPARATION
    REVERSE PHASE SEPARATION
    SIMILARITIES

    Both Are Separation Techniques

    Same Instrumentation

    Both Operate Under High Pressure

    They Take 2-60min Per Sample

    Both Types Give Quantitative Analysis Of Sample

    The Retention Time Can Be Increased By Increasing The Interactions Of Analytes


    DIFFERENCES
    Evolution Normal phase was evolved in Reverse phase is recently
    the 1970s in form of liquid evolved form the hplc
    chromatography

    Analytes in Mobile phase In normal phase, the mobile In the reverse phase, the
    phase carries non polar mobile phase carries polar
    analytes at the beginning of analytes during separation
    separation
    Reproducibility N.Phase has a poor R.Phase has a higher
    reproducibility of the retention reproducibility of the retention
    time time

    Damage of column The column of N.Phase is easy The column of R.Phase is


    to damage difficult to damage
    DIFFERENCES

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