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   2
   Introduction
   High-performance liquid chromatography (HPLC) is a standard technique in 
   analytical chemistry to separate, identify and/or quantify compounds that are 
   dissolved in a solution. 
   HPLC instruments consists of a pump, an injector, a separation column and 
   a detector. An aliquot of the sample is injected onto the column. Each 
   compound in the sample interacts slightly differently with the column 
   material, and therefore, causing different flow rates for the different 
   components and leading to the separation of the components.
   For teaching purpose only 
   9/3/22
   © Agilent Technologies, Inc. 2016
   3
   Table of Content (ToC)
   Introduction                                                Van Deemter Equation
   • What happens inside the column?                           • Eddy Diffusion
   Key Parameters                                              • Axial Diffusion
   • Retention Time & Peak Width                               • Resistance to Mass Transfer
   • Resolution – Baseline Separation                          • More on Van Deemter
   • Resolution – The Fundamental Equation                     Peak Capacity
   • Efficiency or Number of Theoretical Plates                • Gradient Analysis
   • Retention Factor                                          • Definition
   • Selectivity or Separation Factor                          • Calculation of Peak Capacity
   How to Influence Selectivity?                               • Peak Width
   • Selectivity – Example 1                                   • Example
   • Selectivity – Example 2
   • Selectivity – Example 3
   • Plate Number
   For teaching purpose only 
   9/3/22
   © Agilent Technologies, Inc. 2016
   4
    Introduction
    What happens inside the column?
                                                                          Time t
                                                              Separation t -t
                                                                                   r2  r1
                                                              Peak width Wb1,2
    ToC
    For teaching purpose only 
    9/3/22
    © Agilent Technologies, Inc. 2016
    5
      Introduction
      What happens inside the column?
                                                           t -t                                                                                                        t -t
                                                             r2  r1                                                                                                     r2  r1
                                          Superior separation                                                    vs                                Inferior separation
                                     W                                             W                                                        W                                              W
                                         b1                                            b2                                                       b1                                             b2
                                          Superior separation                                                 vs                                   Inferior separation
      ToC
      For teaching purpose only 
      9/3/22
      © Agilent Technologies, Inc. 2016
      6