HPLC Image
                                                                 System control- Data storage
                                     HPLC column (C18 packing)
                                                  Injector
                                                  Autosampler
                                             Sample
                                                          Detector(s)
                     Solvent(s)                           (Diode Array &
                                                          Fluorescence)       Waste
                                    Pump(s)
    Separation in HPLC
         • Seperation depends on :
          • Polarity of molecule
            • RP verses NP column, 
            • Hilic, Hydrophilic-Interaction Chromatography
            • Hic, Hydrophobic-interaction Chromatography
          • Electrical charge (ion exchange chromatography).
            • Anion exchanger  attracts” –” charged analyte
            • Cation exchanger attracts “+” charged analyte
          • Molecular size 
            • Size exclusion or gel permeation chromatography
       Columns - Heart of System
    • Solid Support (column packing) - Backbone for bonded phases. Usually 10µ, 
    5µ, 3µ, 2.5µ siloxane or polymeric particles.
    • Porous or solid core
    • Packing has different degrees of polarity depending on functional 
    groups attached 
    • Bonded Phases - Functional groups chemically bound to the solid support. 
   HPLC - Modes
       • Normal Phase 
        - Polar stationary phase and non-polar 
        solvent. 
       •  Reverse Phase (used for caffeine and 
        quinine separation)
        - Non-polar stationary phase and a polar    
              solvent. 
         Normal Phase verses Reverse Phase HPLC Packing
        Polar packing
        Non-polar packing
                       C4                     C8                        C18