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    Chromatography

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    Hanzala Labeeb
    This document discusses internal and external standard calibration methods in chromatography. Internal standard calibration involves comparing the response of target compounds to a reference standard added to samples. The response ratio is used to account for variation. External standard calibration compares sample responses to calibration standards. Key equations relate response or area to concentration for each method. Advantages are discussed such as accounting for variation, while disadvantages include finding appropriate reference compounds.

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    Chromatography

    Uploaded by

    Hanzala Labeeb
    42 views15 pages
    This document discusses internal and external standard calibration methods in chromatography. Internal standard calibration involves comparing the response of target compounds to a reference standard added to samples. The response ratio is used to account for variation. External standard calibration compares sample responses to calibration standards. Key equations relate response or area to concentration for each method. Advantages are discussed such as accounting for variation, while disadvantages include finding appropriate reference compounds.

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    This document discusses internal and external standard calibration methods in chromatography. Internal standard calibration involves comparing the response of target compounds to a reference standard added to samples. The response ratio is used to account for variation. External standard calibration compares sample responses to calibration standards. Key equations relate response or area to concentration for each method. Advantages are discussed such as accounting for variation, while disadvantages include finding appropriate reference compounds.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    42 views15 pages

    Chromatography

    Uploaded by

    Hanzala Labeeb
    This document discusses internal and external standard calibration methods in chromatography. Internal standard calibration involves comparing the response of target compounds to a reference standard added to samples. The response ratio is used to account for variation. External standard calibration compares sample responses to calibration standards. Key equations relate response or area to concentration for each method. Advantages are discussed such as accounting for variation, while disadvantages include finding appropriate reference compounds.

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    You are on page 1of 15

    Internal and external standard method of

    calibration in Chromatography
    Contents

    Internal Pros and Equation Pros and


    Response External
    Standard Cons for Cons
    Factor Standard
    Calibration Calibratio
    Equation Calibration
    n Factor

    2
    Internal Standard Calibration
    Introduction

    Internal standard calibration involves the comparison of


    the responses of reference standards added to the sample
    or sample extract before injection to the instrument
    responses from the target compounds in the sample.

    3
    Internal Standard Calibration
    Introduction

    The response of the target compound is normalized to


    the response of the reference standard. This standard is
    called
    an internal standard as it is contained
    within the aliquot of the sample or sample
    extract that is added into the
    instrumentation.

    4
    Response Factor Equation
    The ratio of the peak area of the target compound in the sample extract
    to the peak area of the internal standard in the sample extract is
    compared to a similar ratio obtained for each calibration standard.

    This ratio is referred as the relative response factor or for short RRF, therefore
    showing that the target compound response is found relative to that of the
    internal standard.
    Response Factor equation

    RF = ((Ax)(Cis))/((Ais)(Cx))

    Where: Ax = Area of compound


    Cx = Concentration of compound
    Ais = Area of internal standard
    Cis = Concentration of internal
    standard

    6
    Advantages
    ✘ It is the reason for routine variation in the response of
    the chromatographic system.
    ✘ It also accounts for the variations in the exact volume
    of sample or sample extract introduced in the
    chromatographic system.
    ✘ The retention times of the target compound and the
    internal standard can be used to find out the relative
    retention time (RRT) of the target compound and
    after that it may be used to compensate for small
    retention time shifts.

    7
    Disadvantages
    ✘ The main disadvantage of internal standard
    calibration is that the internal standards must
    be compounds that aren’t found in the
    samples to be analyzed and also they must
    produce an unambiguous response on the
    chromatographic detector system.

    8
    External Standard Calibration
    Introduction

    For an external standard quantitation, unknown data


    from the sample and known data from a calibration
    standard are combined to generate a quantitative report.
    This is known as external standard because the standard
    or known material is external or separate to the
    unknown material.

    9
    External Standard Calibration
    Introduction

    External standard calibration is one of the most


    commonly used approaches to calibrations. It requires a
    simple comparison of instrument responses from the
    sample to the responses from the target compounds in
    the calibration standards.

    10
    Calibration Factor Equation

    What is Calibration
    Factor?
    The ratio of the detector response to the
    mass of analyte in the calibration standard is
    termed as the calibration factor or CF.

    11
    Equation for Calibration Factor
    (External Standard Curve)

    CF = (Ax)/(Cx)

    Where: Ax = Area of compound


    Cx = Concentration of compound
    Advantages
    ✘ One advantage of external standard
    calibration is that it is very simple to
    perform this type of calibration.
    ✘ Also, this calibration can be applied to a
    wide variety of methods.

    13
    Disadvantages
    ✘ The most prominent disadvantage is that it is
    considerably affected by the stability of the
    chromatographic detector system and the
    presence of chromatographic interferences
    in a sample or sample extract.

    14
    References

    www.Wikipedia.com
    www.researchgate.net
    www.chromatographyonline.com
    www.analyticalsciencejournalsonline.com

    This presentation is made by Hanzala Labeeb CHE-13B

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