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Gradient or isocratic elution in preparative reversed phase HPLC

A common question that often arises in the discussion between prep chromatographers is when and why one should choose either isocratic or gradient elution? Since we in the Kromasil group have a long experience of developing large scale separations we would like to share some of these experiences with the readers of KromaNews.

Pros and cons

In general an isocratic elution is preferred, in order to maximize the loading capacity. A gradient will undoubtedly compress the peaks in time, and give less room for peak broadening as a result of the concentration overload. The result is lower loading capacity, and lower productivity. However, sometimes it is necessary to use a shallow gradient, in order to control the elution. The gradient will ensure a more stable separation, with improved peak shapes, and shorter cycle time.

Adsorption process

For larger molecules such as proteins the adsorption process is very much on/off, i.e. up until a certain point in solvent strength, or concentration of organic solvent, the molecule sticks to the surface of the packing material, and once a certain concentration has been reached the protein is desorbed, and travels with the mobile phase. This can be shown in a diagram with log k vs. % organic, where the protein has a very steep dependence, while peptides are in between, and small molecules have a more shallow slope, and much lower dependence from the % organic.

diagram of relation between adsoprtion process parameters
Effect of organic modifier ratio in mobile phase on substances depending on their sizes.
HPLC elution profile
Typical step gradient concentration profile for an isolation of a peptide or a small protein, using either isocratic or gradient elution.

How to choose method

Because of the above it is necessary to use at least a shallow gradient when running RP separations of insulin or larger polypeptides or proteins, in order to control the elution (even though one insulin manufacturer claims to use isocratic elution for the RP HPLC step!). However, the gradient should be kept as shallow as possible to maximize loadability or loading capacity; for insulin typically 0.1-0.2% organic/min.

Small molecules and peptides are usually run using isocratic elution, but very often a step gradient is used. The loading is performed at low concentration of organic, so that the substance of interest is adsorbed at the inlet, or the top of the column. The retention time is then infinite for the substance. After that an elution step is applied, when the substance of interest is eluted out of the column, and finally a washing step is run, to wash out very retentive impurities.


Heads up! To sum it up, use isocratic elution as long as it is possible, go to gradient elution only when you are forced to it, and then use as shallow gradient slopes as possible!