5

• Components in HPLC

  • Solvent (MP) reservoir
  • Pump & solvent mixer
  • Injection system
  • Column
  • Detector
• MP reservoir

  • Solvents used as MP must be of high purity
  • Degassed (bubbles & microbubbles are removed)
  • Filtered through 0.45 µm or 0.2 µm filter membrane
• Isocratic elution

  Fixed MP composition throughout the analysis
• Gradient elution

  MP composition changes at certain points during the analysis
• Gradient elution

  • Allows adequate separation of early eluting peaks without the later eluting peaks becoming too dispersed
  • Starts with a weaker eluting strength MP to allow least retained analytes to have more retention in the SP
  • The eluting strength of the MP increases with time to elute strongly retained analytes earlier
• Pump

  • Drives mobile phase through the entire LC system
  • Has an in-built mixing chamber for homogenizing aqueous and organic solvents
• Sample Injection system


  • Can either be manual injection loop or autosampler
• Column


  • Located within a heating element controlled by a thermostat to ensure a constant column temperature
  • Typical column is 5-30 cm in length & 3-4.6 mm in inner diameter
• Detectors used in HPLC

  • UV-VIS
  • Evaporative Light Scattering Detector (ELSD)
• UV-VIS detector


  • Most widely used
  • Quantification based on absorption of UV-VIS radiation
  • Variable wavelength UV-VIS detector can measure across the entire wavelength range
  • Can be used to measure analytes that absorb UV-VIS radiation
  • Wide linear dynamic range
  • Non-destructive
  • Inexpensive
  • Robust
• Evaporative Light Scattering Detector

  • Based on the light scattering properties of the analyte
  • Concentration of the analyte is linearly related to the amount of light scattered
  • Universal detector
  • Can be used for analytes that DO NOT absorb UV-VIS radiation
  • Limited linear dynamic range
  • Lower sensitivity than UV-VIS
  • MP must be volatile
• Common problems in HPLC

  • Pressure (abnormally high or low)
  • Baseline (noisy or drifting)
  • Peak shape (peak broadening or split peaks)
  • Retention (shifting retention time)
• Troubleshooting abnormally high pressure

  1. Plugged inlet frit

  2. Column blockage

  3. Precipitation along the solvent lines
• Troubleshooting abnormally low pressure

  1. Presence of air bubbles between the pump and MP reservoir

  2. Leaking pump valve / seals
• Troubleshooting noisy baseline


  1. Detector lamp failing

  2. Contaminated detector cell

  3. Air bubble in the system / detector
• Troubleshooting drifting baseline


  1. Contaminated MP

  2. Temperature fluctuations

  3. Column bleeding
• Troubleshooting peak broadening


  1. Sample solvent strength is higher than MP

  2. Injection volume too large (column overload)

  3. Low MP flow rate
• Troubleshooting split peaks


  1. Partially blocked inlet frit

  2. Small void at the column inlet

  3. Poor separation of two peaks
• Troubleshooting shifting retention time

  1. Column aging / contamination

  2. Change in MP composition

  3. Presence of air in pump
• Separation principles in TLC

  • Stationary phase (silica plate) is spread in a thin layer on the surface of a plate
  • Analyte prepared in a volatile solvent, are placed as spots
  • Analyte carried by the mobile phase (solvent) which travels up the stationary phase by capillary action
  • Separation is based on the relative affinity of the analyte for the stationary phase vs the mobile phase
  • More polar analytes adsorbed with higher affinity to the silica gel travel slower, decreasing the retardation factor (Rf)
• Stationary phase in TLC

  • Silica gel or silica-gel based materials
  • Can be modified to make it non-polar
• Mobile phase in TLC

  • Volatile liquids, e.g. mixture of chloroform/methanol/water (180:15:1 v/v)