HPLC

1. Selectivity

   Nota:

   • Change in: Elution order Elution rate Ionisation state Activity
   1. pH

      Nota:

      • Change in pH of mobile phase. Usually around 2-3pH. Ionic interactions
   2. Temperature

      Nota:

      • Only if the column is held in an oven (HPLC 3&4) Change of conformation results in less surface-surface interactions
   3. Organic Modifier

      Nota:

      • Liquid Parameter
2. Retention Factor
   1. Sample Solvent

      Nota:

      • Can effect interactions when mixing with mobile phase occurs Liquid parameter
      1. Volume of Injection
      2. Concentration of Sample
   2. %B

      Nota:

      • More polar mobile phase = decreased tR Liquid parameter
3. Efficiency
   1. Band Broadening
      1. Eddy Diffusion
         2. Column Length
      2. Longitudal Diffusion

         Nota:

         • Decreased longitudal diffusion with increased flow rate. Does not occur in gradient method
         1. Pore Size
         3. Particle Size
      3. Mass transfer

         Nota:

         • Decreased mass transfer with decreased flow rate
         1. Column Length
         2. Particle size
         3. Pore Size

1. Retention Time
   1. Flow Rate
2. Efficiency
   1. Linear Velocity

      Nota:

      • Effects back pressure
      1. Column diameter

         Nota:

         • Effects linear velocity
      2. Flow rate

Nota:

• Increasing pressure too much can effect the machine functioning

1. Linear Velocity

1. Injector
2. Pump
3. Sampler
4. Sensitivity
   1. Wavelength/Detector

1. Steady %B

   Nota:

   • Liquid parameter
2. Increasing %B

   Nota:

   • Liquid parameters
   1. Resolution >1.5

Nota:

• Changing the column chemistry ie the stationary phase composition will completely change the interactions of the analyte and the stationary phase.

1. Tailing factor
   1. End Capping

      Nota:

      • Incomplete capping will leave free acid silanols that lead to tailing
   2. Free Silanols

      Nota:

      • Free acids on the stationary phase will be able to ionically bond (depending on pH) to any ionised groups in the analyte
2. Stationary phase arms