How to Develop HPLC Methods for Challenging Separations

Course Outline

Implementing a 5-step strategy for method development of complex samples.

Review of a 5-step strategy for HPLC method development with particular attention to reasons why some separations are ‘challenging’:

• Setting suitable objectives for method development
• Assessing all available information
• Selecting suitable samples
• Performing scouting experiments to select suitable initial conditions
• Optimising the method to define method parameters which achieve the desired separation.

Separation theory

Method development objectives: Strategies for maximising resolution in HPLC using retention factor, k’, selectivity, α, and efficiency, N.

Case Study Using Computer Modelling

A case study is used to demonstrate how a computer model may be used to find the best conditions for the separation.

Practical Skills Acquired

This course will enable you to find solutions for difficult HPLC separations. In addition you will be able to:

• Understand why some separations can be challenging.
• Identify potential problem separations.

Apply strategies to achieve satisfactory separations for “complex” samples with respect to:

• Sample preparation
• Detection methods
• Retaining very polar analytes
• Optimising gradient methods
• Selecting columns
• Setting up scouting/screening experiments