Some of the Learning Outcomes

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


  • 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


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

Sample introduction

  • Types of autosampler
  • Sample filtration

Column oven and column temperature effects


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


  • 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

Benefits of In-house Trainings

Feel free to suggest the topics that you would like to learn about at our HPLC Training Course.

Request an Offer

Request detailed Agenda - HPLC Theory In-house Training

Who should attend?

This course is intended for analytical scientists working in pharmaceutical QC and development roles:

  • Analytical Scientist
  • QC Chemist/Senior QC Chemist
  • Development Chemist/Senior Development Chemist
  • Product Development Scientist
  • QC Manager
  • Analytical Development Manager

Our Pharmaceutical events were attended by these companies