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HPLC Method Development class room course
1 Day Course | HPLC Level 3.

For the experienced chromatographer, this course provides a step-by-step approach to method development.

The course includes all the crucial aspects of method development, including information gathering, mode of chromatography, development of mobile phase systems, column choice, principles of ionisation/suppression, optimisation of important chromatographic parameters (R, α, k’ and N), isocratic and gradient operation and detector choice and optimisation.

Each aspect is discussed in detail with relevant examples used to demonstrate theoretical principles. Chromatographic modelling software-based exercises are included to give further understanding.

  • We recommend that course numbers are limited to 20 so that each delegate gets the opportunity to ask questions and fully participate in tutorial exercises
  • As the training is delivered on-site, we can design the course material to suit your specific needs
  • Customisable written assessments are available if required

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download course pdfThis one day course provides a step-by step approach to method development for HPLC.

All sections are accompanied by participative tutorial exercises using interactive chromatgraphic modelling software simulations for increased understanding of critical concepts.

Who is this course for

This course is designed for the more experienced chromatographer with a good working knowledge of HPLC separations. Attendance of the Fundamental HPLC and HPLC Troubleshooting and Maintenance training courses is not compulsory but is advantageous.

Previous knowledge

Delegates should have a good knowledge of chromatography and experience as HPLC users. Some experience in method development is recommended. A good grounding in chemistry is also beneficial.

What you will learn

  • Setting method development objectives
  • Sample preparation
  • System choices
  • Choosing a column and mobile phase
  • Optimisation strategies
  • Quantitation and system characterisation


  • Establishing method objectives
  • Literature searching: What is known? What needs to be known?

Choosing a Column and Mobile Phase

  • Choosing the correct phase
  • Computer-based tools for column choice
  • Effects of column geometry
  • Review of modern stationary phases
  • Isocratic vs. Gradient operation
  • Theory and development of eluent gradients

Sample Preparation

  • Sample clean up
  • Analyte extraction
  • SPE explained
  • Mobile phase selection
  • Optimising for sample type/application

Optimisation Strategies

  • Capacity factor, Efficiency, Resolution, Selectivity
  • Resolution equation
  • Step-by-step guide for logical method development
  • Example method developments

System choices

  • How to choose the appropriate injector/detector
  • Typical operating conditions
  • Developing and optimising injection conditions
  • Mobile phase flow & band broadening (Van Deemter)
  • Modes of chromatography

Quantitation & System Characterisation

  • Single and multi-level calibration
  • Internal standards
  • System suitability testing
  • Introduction to validation

Training Calendar

Click on a title below to download a detailed course description or click a date and book your course.

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Gradient Elution Principles

Gradient elution is most useful for reversed-phase and ion exchange liquid chromatography.

The gradient is formed by increasing the percentage of organic solvent. Consequently – at the beginning of the analysis, when the mobile phase strength is low, the analyte will be partitioned wholly into the stationary phase (or ‘focussed’) at the head of the column
(Region A)

As the mobile phase strength increases, the analyte will begin to partition into the mobile phase and move along the column. As the mobile phase strength is increasing continuously, the rate at which the analyte moves along the column accelerates.
(Region B)

At some point within the column elution, the analyte may be wholly partitioned into the mobile phase, and will be moving with the same linear velocity as the mobile phase.
(Region C)

One cannot assign a fixed k value to a compound when gradient elution is applied. k is the retention coefficient and changes during the elution. Calculating k using the formula
k =(tr-t0)/t0 is only correct during isocratic elution!

The relationship between the gradient retention factor and the mobile phase composition depends upon molecular properties. Band spacing may change as column length is altered!

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