“How do you calculate the gradient program in HPLC method development?”
“The aim of an HPLC method is to enable the separation of a mixture of components. This may be achieved by selecting a suitable mobile phase composition for a particular column which results in a peak for each component that is separated from other peaks, and is retained at a suitable retention time. The composition of the mobile phase may be isocratic, where it is held constant throughout each injection, or gradient, where the amount of the stronger solvent is increased throughout each injection. Whether you use isocratic or gradient conditions depends on the nature of your mixture of analytes. For example in reversed phase HPLC a mixture of analytes which have widely differing hydrophobicity is likely to require an unfeasibly long run time under isocratic conditions and will need to be analysed under gradient conditions.
For RP-HPLC, I suggest that you run your test sample using a full range gradient, e.g. 5 to 95 %B. An inspection of the resulting chromatogram will indicate whether the method is suitable for isocratic analysis (if the difference between the first and last peak is less than 25% of the gradient time) and if not, provides a starting point for gradient method development. Changing the gradient time, tG will make the gradient more or less steep, corresponding to stronger and weaker mobile phase composition in isocratic analysis.
For a detailed discussion on gradient method development you may wish to attend one of my training courses. ‘How to Develop HPLC Methods’ will be held next week in London and Milton Keynes. Future events will be publicised on this blog.”