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Practical HPLC Method Development practical course
2 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, and practical laboratory sessions are included to make an all encompassing two day course.

  • We limit numbers to 6 per course so that each delegate gets the opportunity to ask questions and fully participate in practical exercises
  • When delivered on-site we can design the course material to suit your specific training needs
  • Customisable written assessments are available if required

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

All sections are accompanied by participative tutorial exercises using interactive chromatographic 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
  • Effect of column parameters
  • Developing methods for neutral samples
  • Developing a separation for a mixture of neutral, acidic and basic compounds
  • Using a scouting gradient
  • Optimisation strategies
  • Quantitation and system characterisation

Effect of Column Parameters

  • Particle size, flow rate, and column dimensions
  • Impact of system extra column volume
  • Developing methods using sub-2μm particles

Developing a separation for a mixture of neutral, acidic and basic compounds

  • Consideration of the need for ion-pairing and peak shape issues (suppression of tailing)

Developing methods for neutral samples

  • Column choice
  • Optimising selectivity and resolution: solvent choice/solvent strength
  • Optimising the method using column temperature

Using a scouting gradient

  • Developing gradient and isocratic conditions



Putting it all together!

  • Developing a method for the separation of a complex mixture of ionisable compounds from scratch

Training Calendar

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Peak Shape in Gradient HPLC

In isocratic elution the peaks are relatively broad, the peak width increasing with retention time. In gradient elution, the peaks are narrow with almost equal peak widths.

The main reason for the narrow peak shape is the velocity of the peak as it leaves the column. During gradient elution, all compounds accelerate through the column and thus elute at a high velocity. The retention time difference between compounds is a consequence of the percent organic modifier at which each starts to accelerate. All compounds should have approximately the same speed when they leave the column.

Another minor reason for peak focusing is the fact that the front and tail of a peak are residing in different concentrations of organic modifier. The tail will experience a higher percentage of organic modifier than the heart of a peak. The velocity of the tail will thus be slightly higher then the heart of the peak and vice versa for the front. This results in peak focusing. Asymmetric peaks are less
frequently a problem in gradient elution. In practicality, the narrow peaks obtained in gradient elution provide better detection limits and higher loading capacities.

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