HPLC Presentation by 5ZAmNTZ

VIEWS: 6 PAGES: 10

									     Organic Chemistry I - Lab

  High Performance Liquid Chromatography

 HPLC is characterized by the use of high pressure to push a
 mobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high resolution.
                              TLC              vs.   HPLC
   Type of Analysis         qualitative only         qualitative &
                                                      quantitative
   Stationary Phase         2-dimensional          3-dimensional
                            thin layer plate           column
   Instrumentation             minimal!           much! with many
                                               adjustable parameters
 Sample Application             spotting              injection
                               (capillary)      (Rheodyne injector)
Mobile Phase Movement       capillary action        high pressure
                         (during development) (solvent delivery)
Visualization of Results      UV lightbox        “on-line” detection
                                                 (variable UV/Vis)
   Form of Results             spots, Rf’s           peaks, Rt’s
                           (retention factors)    (retention times)
               Varian HPLC System
                                                      9060 Polychrom              Computer
                                                   (Diode Array) Detector        Workstation




                                  9050 Variable
                9010 Solvent     UV/Vis Detector
HPLC Solvent   Delivery System
 Reservoirs




                                                          Rheodyne
                                                           Injector

                                                                            Keep an eye on
                                                           HPLC             these 4 screens!
                                                           Column
Varian Solvent Delivery System
              %A %B   %C   Flow Rate    Pressure                  to column
             {H2O}  {MeOH} (mL/min)     (atmos.)
                                                                               load

  Ready
                                                                              inject

                                                                Rheodyne
                                                                 Injector
Varian 9010 Solvent Delivery System             to injector



                                      through pump




                                                                                 Column
   through                                           C
    pulse
  dampener
                               A              B

                                                              from solvent           to
                               Ternary Pump                     reservoir         detector
Variable UV/Vis Detector
         ABS AUFS l RunTime EndTime
         0.001 2.000 238 0.00 min 10.0 min


 Ready
                  Ready
                                    UV Spectrum
         UV Spectrum                {shows full UV abs.}
                                     UVmax
                                             UVmax

         Chromatogram




                             ABS.
Reset
                                       Wavelength


                                        Rt           Rt




                             ABS.
                                         Time

                                    Chromatogram
                                     {shows peaks, Rt}

           Varian 9060
        Polychrom Detector
                                                                     Approximation
                   HPLC Chromatograms                                of peak area by
                                                                     triangulation
Absorbance 




                        Peak A               Peak B


                                                                                      height


               0    1   2        3       4        5       6     7
                            Time (minutes)                             base

                             Rt = 3.0 min.      Rt = 5.2 min.                base x height
                            faster moving      slower moving        Area =
                                                                                  2
                             less retained      more retained
  Chromatography Stationary Phases

     Silica Gel             Derivatized Silica Gel

   O    O     O                  O    O     O
    |    |    |                   |    |    |
OSiOSiOSiOH           OSiOSiOSiOR           Where R = C18H37
    |    |    |                   |    |    |
                                                            hydrocarbon chain
   O    O     O                  O    O     O
                                                           (octadecylsilyl deriv.
    |    |    |                   |    |    |
                                                             silica or “C18”)
OSiOSiOSiOH           OSiOSiOSiOR
    |    |    |                   |    |    |
   O    O     O                  O    O     O

 bulk (SiO2)x   surface        bulk (SiO2)x    surface


 relatively polar surface    relatively nonpolar surface
    “normal phase”               “reversed phase”
       Normal vs. Reversed Phase Chromatography
                        Normal Phase           Reversed Phase
 Stationary phase      Polar (silica gel)     Non-polar (C18)
                          Non-polar                 Polar
   Mobile phase
                      (organic solvents)      (aqueous/organic)
Sample movement       Non-polar fastest         Polar fastest
                      Different polarities        Different
Separation based on
                        (functionality)      hydrocarbon content

								
To top