HPLC

HPLC

1. Resolution

   • Selectivity
     • Change in: Elution order Elution rate Ionisation state Activity
     • pH
       • Change in pH of mobile phase. Usually around 2-3pH. Ionic interactions
     • Temperature
       • Only if the column is held in an oven (HPLC 3&4) Change of conformation results in less surface-surface interactions
     • Organic Modifier
       • Liquid Parameter
   • Retention Factor
     • Sample Solvent
       • Can effect interactions when mixing with mobile phase occurs Liquid parameter
       • Volume of Injection
       • Concentration of Sample
     • %B
       • More polar mobile phase = decreased tR Liquid parameter
   • Efficiency
     • Band Broadening
       • Eddy Diffusion
         • Column Length
       • Longitudal Diffusion
         • Decreased longitudal diffusion with increased flow rate. Does not occur in gradient method
         • Pore Size
         • Particle Size
       • Mass transfer
         • Decreased mass transfer with decreased flow rate
         • Column Length
         • Particle size
         • Pore Size

2. Run Time

   • Retention Time
     • Flow Rate
   • Efficiency
     • Linear Velocity
       • Effects back pressure
       • Column diameter
         • Effects linear velocity
       • Flow rate

3. Back Pressure

   • Increasing pressure too much can effect the machine functioning
   • Linear Velocity

4. Equipment Parameters

   • Injector
   • Pump
   • Sampler
   • Sensitivity
     • Wavelength/Detector

5. Gradient or isocratic method

   • Steady %B
     • Liquid parameter
   • Increasing %B
     • Liquid parameters
     • Resolution >1.5

6. Column Chemistry

   • Changing the column chemistry ie the stationary phase composition will completely change the interactions of the analyte and the stationary phase.
   • Tailing factor
     • End Capping
       • Incomplete capping will leave free acid silanols that lead to tailing
     • Free Silanols
       • Free acids on the stationary phase will be able to ionically bond (depending on pH) to any ionised groups in the analyte
   • Stationary phase arms