This two-day course covers HPLC theory, troubleshooting and method development. Content can be tailored to the client’s requirements and a hands-on, practical element may be included if desired.

Topics include:

HPLC Theory

Important chemical concepts

• Bond polarity
• Hydrogen bonding
• Acid-base theory

Chromatography

• Definitions of basic terms
• Plate number, separation factor, retention factor and resolution
• The Van Deemter Equation

Mobile and stationary phases

• Retention mechanisms
• Normal phase, HILIC, aqueous normal phase, reversed phase, chiral separations, ion
• chromatography and size exclusion chromatography
• Effect of particle size on efficiency/UHPLC
• Core-shell packings
• Reversed-phase retention mechanisms
• Strategies for the retention of ionized compounds

Pumps

• Isocratic, low-pressure and high-pressure mixing
• Gradient delay volume

Sample introduction

• Types of autosampler
• Sample filtration

Column oven and column temperature effects

Detectors

• Detector properties
• UV/visible (including diode array), fluorescence, refractive index, conductivity, electrochemical, light scattering, charged aerosol and mass spectrometric detectors

Troubleshooting

• System suitability as a troubleshooting tool
• History of method and previous problems
• Connecting tubing
• Pump priming and leak testing
• Causes of leaks
• Diagnosing pump, autosampler, column and detector faults
• Column care

Method development

• Analyte properties (acid/base properties, solubility and detectability)
• Matrix properties
• Sample matrix effects
• Achieving adequate specificity
• Injection solvent effects
• Method performance requirements
• Method performance and drug substance/drug product specifications
• Sources of analytical variability
• Typical specification limits
• Sampling error
• Sample preparation
• Analyte extraction
• Sample stability
• Developing the separation
• Regular separations (neutral and ionisable organic molecules)
• Special cases
• Choice of initial conditions
• Stationary phase chemistries and retention mechanisms
• Column temperature effects