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Study Notes: Changing Conditions

Changes due to mobile and stationary phases and flow rates have been covered earlier. In this Study Note changing the composition of the mobile phase during the run (gradient elution) is discussed. Note that gradient elution may have an effect on the time of the resolution with a good gradient allowing separation of peaks of interest in a shorter time.

It is obvious that the longer the run (up to a point) the better the separation and the shorter the run, the poorer the separation. The way to remember this is that with zero time - no separation of components occurs! But long runs have the twin problems of being wasteful of materials and band broadening of the peaks at the end of run.

Three chromatograms , showing that as time increases so does separation of the peaks.

When the composition of the mobile phase is unchanged for the entire run the conditions are called isocratic but this can lead to problems with long run times and poor resolution when a sample contains a large number of peaks. Gradient elution, on the other hand, can optimise resolution with shorter run times.

Comparative chromatograms, showing 2 parts side by side. Under isocratic conditions (no gradient) the resolution is poor compared with the gradient elution.

Note that with gradient elution the peaks are more evenly spaced across the chromatogram and that the run is completed more quickly. Notice also that the closely resolving peaks 1, 2 and 3 are now well separated. This is the power of gradient elution.

For any given separation it may take time to develop the best gradient and best time for optimal elution. This is based on experience and the work of others. A number of HPLC manufacturers have lists of operating conditions for different separations. They call these applications.

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