chromatography_hplc by huanghengdong

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									                                             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 mm
    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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