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Introduction to the Principles and Practices of HPLC

A one-day short course introducing scientists to the basic concepts and operational principles of High Performance Liquid Chromatography with an emphasis on Reversed-Phase HPLC. The class makes extensive use of computer animation to explain basic principles, show instrument operation and to explain chromatography troubleshooting. Handouts printed in four colors aid in understanding the material presented.

Course Content

  • Introduction to chromatography: A brief historical perspective leads into what chromatography is and how separations are performed.
  • Chromatographic process: The chemical interactions behind chromatographic separations are explained in detail. This provides a basis for discussing the four modes of chromatography: reversed-phase, normal phase, ion exchange and size exclusion. The class focuses mainly on reversed-phase HPLC because of its dominant role in HPLC.
  • Instrument operation: Using computer animation, instrument operation is explained and an example chromatogram is shown. Each part of an HPLC instrument is described in detail, including:
    • The solvent delivery system which provides a constant flow of mobile phase.
    • Sample injection.
    • Connecting tubing and fittings.
    • Commonly used detectors.
  • Instrument operation: Using computer animation, instrument operation is explained and an example chromatogram is shown. Each part of an HPLC instrument is described in detail, including:
  • Chromatograms: An example of a reversed-phase chromatogram is shown and significant aspects of the chromatogram such as column void volume and peak efficiency (theoretical plates) are explained.
  • Columns: The heart of chromatography is the column that separates the sample constituents. Modern reversed-phase HPLC columns are explained and their make-up is described. Proper maintenance of columns is explained and cleaning procedures are described.
  • Mobile phase: The mobile phase carries the sample components through the column and affects the retention and separation. The important attributes of the commonly used reversed-phase solvents are discussed and concepts that affect retention and separation such as solvent strength are explained. The class describes how to adjust solvent conditions to get satisfactory retention. Isocratic and gradient elution are explained.
  • Instrument maintenance: Important steps to proper maintenance of an instrument are described
  • Diagnosing and correcting chromatographic problems: Based on animated figures and the understanding of instrument operation previously discussed, problems that can occur during HPLC are explained and ways to avoid or correct these problems are discussed. Chromatographic problems such as tailing peaks are also discussed and ways to avoid problems are outlined. The three main indicators of chromatographic performance are described.

Course Outline


  • An introduction to high performance liquid chromatography.
  • Chromatographic interactions and the four modes of HPLC.
  • Instrument operation including solvent delivery system, sample injection, fittings and tubing and detectors.


  • Understanding and interpreting HPLC chromatograms
  • Reversed-phase HPLC columns.
  • Reversed-phase HPLC mobile phases
  • Diagnosing and correcting instrumental and chromatographic problems.

Who Should Attend:

New chromatographers or current users who wish to review the basics.


Course NameCourse DurationPart NumberPrice
An Introduction to the Principles and Practices of High Performance Liquid Chromatography1 day065192$725 (contact us for Canadian prices)

Schedule and Registration:

View the most current course offerings and self-register at the Unity Lab Services registration page found here


Please send all training inquiries to unity.training.us@thermofisher.com.