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    Chromatography HPLC

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    Filia Yuniza
    This document describes the procedures for analyzing caffeine using High-Performance Liquid Chromatography (HPLC). Students will learn the basic techniques of HPLC and how intermolecular forces play a role. The lab involves using a HPLC system to separate and analyze caffeine standards and samples of diluted soft drinks to create a calibration curve and determine the caffeine concentration in the original soft drinks. Safety considerations for the lab are also outlined.

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    Chromatography HPLC

    Uploaded by

    Filia Yuniza
    50 views16 pages
    This document describes the procedures for analyzing caffeine using High-Performance Liquid Chromatography (HPLC). Students will learn the basic techniques of HPLC and how intermolecular forces play a role. The lab involves using a HPLC system to separate and analyze caffeine standards and samples of diluted soft drinks to create a calibration curve and determine the caffeine concentration in the original soft drinks. Safety considerations for the lab are also outlined.

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    This document describes the procedures for analyzing caffeine using High-Performance Liquid Chromatography (HPLC). Students will learn the basic techniques of HPLC and how intermolecular forces play a role. The lab involves using a HPLC system to separate and analyze caffeine standards and samples of diluted soft drinks to create a calibration curve and determine the caffeine concentration in the original soft drinks. Safety considerations for the lab are also outlined.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    50 views16 pages

    Chromatography HPLC

    Uploaded by

    Filia Yuniza
    This document describes the procedures for analyzing caffeine using High-Performance Liquid Chromatography (HPLC). Students will learn the basic techniques of HPLC and how intermolecular forces play a role. The lab involves using a HPLC system to separate and analyze caffeine standards and samples of diluted soft drinks to create a calibration curve and determine the caffeine concentration in the original soft drinks. Safety considerations for the lab are also outlined.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 16

    CHM 101/102

    Chromatography II: HPLC

    General Chemistry 101/102


    Laboratory Manual
    University of North Carolina at Wilmington

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    • Purpose
     Learn the basic techniques of Hi-Performance
    Liquid Chromatography (HPLC).
     Study the role played by intermolecular forces in
    HPLC.

    • Safety Considerations
     There are no special safety considerations or
    waste disposal concerns associated with this
    laboratory exercise.

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    Hewlett-Packard
    Series 1100 HPLC

    water methanol caffeine


    (very polar) (polar) (fairly nonpolar)

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Column
    solvent
    (mobile phase) to detector
    and
    sample
    wax coated beads

    nonpolar
    stationary phase

    microscopic view of bead


    3 m
    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    Hewlett-Packard
    Series 1100 HPLC

    solvent

    pump

    injector

    column

    detector

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Column

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Pump

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Autosampler and Injector

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC

    HPLC Detector
    UV/Visible Spectrophotometer

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Waste Collection

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    • Procedure
     Use a 10 mL volumetric pipet to add 10.00 mL of soft
    drink to 10.00 mL of deionized water.
     Mix thoroughly and half fill a HPLC vial with your
    sample. Label the vial with your name and the name of
    the soft drink.

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    • Analysis of Results
     Use the four caffeine standards to prepare a
    calibration curve (graph) that plots peak area vs.
    concentration.
     Draw a “best fit” straight line on your graph.
     Use your calibration curve to determine the
    concentration of caffeine in the sample you prepared.
     Use the concentration of caffeine in your sample,
    along with the dilution equation, to determine the
    concentration of caffeine in the soft drink you used.

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Chromatogram of Standard 1

    0.500 x 10-4 M caffeine

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    HPLC Chromatogram of Standard 2

    1.00 x 10-4 M caffeine

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC

    Laboratory Manual
    CHM 101/102

    Chromatography II: HPLC


    • Calculations
    Remember, the original soft drink was diluted to prepare your HPLC
    sample. The concentration (M) of caffeine in the original soft drink can be
    calculated by using the dilution equation:

    M1V1 = M2V2

    M1V1
    M2 =
    V2

    The concentration (mg caffeine / 500 mL soda) of caffeine in a full can of


    your soft drink can be determine using the following equation:

    M2 x 194.2 g caffeine x 1000 mg x 0.500 L = mg caffeine


    mol caffeine g in bottle

    Laboratory Manual

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