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HPLC Fundamentals

This course introduces the fundamentally important concepts associated with High Performance Liquid Chromatography (HPLC) analysis including hardware basics, modes of analysis, basic troubleshooting, column chemistry, important parameters of the mobile phase such as amount and type of organic solvents, pH, buffer strength and type, and much more. Suitable as a refresher for the more experienced analytical chemist or as an invaluable introduction to the technique for those with limited experience, this course provides a priceless insight into HPLC principles and practice. This course is not instrument-specific.


  • Course delivered face-to-face in one day or online in two half-days.
  • We limit numbers to 12 per course so that each delegate gets the opportunity to ask questions and fully participate in tutorial exercises 
  • When delivered on-site we can design the course material to suit your specific training needs

Course Overview  | Download course details »

"Chromatography is a physical method of separation, in which the components to be separated are distributed between two phases, one of which is stationary whilst the other moves in a definite direction".

This is the IUPAC definition of Chromatography, with which we begin a thorough investigation of modern Liquid Chromatography, from the underlying theory of analyte distribution to a detailed study of the chemistry of the column, via the importance of mobile phase composition to the instrumentation of the HPLC system. The different components of the chromatograph (pumping systems, injectors, detectors, etc) are fully explored, as well as the fundamentals of data analysis.

Who is this course for
Users of LC in any of its modes (NP, RP, HILIC, IEX, SEC…) or anyone interested in gaining a deeper understanding of the chromatographic separation of compounds amenable to analysis by LC.

Previous knowledge
No previous experience of chromatography is necessary, but a basic knowledge of chemistry is beneficial.

What you will learn

  • How the chromatographic process takes place
  • The essential theory behind chromatographic separations
  • The architecture and working principles of a modern HPLC system
  • The information contained in a chromatogram and the basic chromatographic parameters
  • The essential principles of sample preparation, mobile phases, buffers and eluent additives for HPLC

Course Outline

Basics of the Chromatographic Process

  • Main retention mechanisms in HPLC
  • Distribution constant
  • Retention theory
  • Model of the chromatographic process

Sample Preparation Protocols

  • Principles
  • Matrix elimination
  • Liquid and Solid Phase Extraction

Separation Mode/Retention Mechanisms

  • Adsorption (normal phase)
  • Reverse phase
  • Principles of ionisation (Ion Suppression Chromatography)
  • Ion pairing
  • Ion exchange


  • Integration parameters
  • System suitability testing

Injectors and Columns

  • Sample introduction
  • Rheodyne injectors/auto-samplers
  • Silica as a solid support
  • Column & packing geometry
  • Efficiency - the van Deemter & Knox equations


  • Choosing the right detector
  • Operating principles
  • Optimisation
  • Typical operating conditions
  • UV (Diode Array)/RI/Fluorescence

Measuring and Optimising Chromatographic Parameters

  • Efficiency
  • Capacity factor
  • Selectivity
  • Resolution
  • Interdependence via the resolution equation 

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