HPLC (High-Performance Liquid Chromatography)

HPLC (High-Performance Liquid Chromatography)

Principle:

• Separation of molecules based on their interactions with a stationary phase and a mobile phase
• High pressure is used to force the mobile phase through the column, resulting in faster separation and higher efficiency

Instrumentation:

• Pump: delivers the mobile phase at high pressure (up to 400 bar)
• Injector: introduces the sample into the mobile phase
• Column: contains the stationary phase, where separation occurs
• Detector: measures the absorbance or fluorescence of the separated compounds
• Data system: collects and analyzes the chromatographic data

Types of HPLC:

• Reversed-phase HPLC: uses a non-polar stationary phase and a polar mobile phase
  • Most commonly used type of HPLC
  • Suitable for separating non-polar and moderately polar compounds
• Normal-phase HPLC: uses a polar stationary phase and a non-polar mobile phase
  • Suitable for separating polar compounds
• Ion-exchange HPLC: uses a stationary phase with ionic functional groups
  • Suitable for separating ions and molecules with ionic functional groups
• Size-exclusion HPLC: uses a stationary phase with pores of specific sizes
  • Suitable for separating molecules based on their size

Advantages:

• High resolution and sensitivity
• Fast separation times (typically 10-30 minutes)
• Can be used for both analytical and preparative purposes
• Can be coupled with various detection methods (e.g. MS, UV, FL)

Applications:

• Pharmaceutical analysis (e.g. drug development, quality control)
• Biotechnology (e.g. protein analysis, bioprocess monitoring)
• Environmental analysis (e.g. water and soil analysis)
• Food analysis (e.g. food safety, quality control)
• Clinical analysis (e.g. disease diagnosis, biomarker discovery)