My LC Blog: Isocratic vs. Gradient LC

Since we're still very much at the beginning of the weird and wonderful world of HPLC, this introduction to gradient chromatography can by no means be seen as an in-depth explanation of all there is to know about HPLC gradients. I'm merely trying to cover the basics and include this common technique at an early stage to provide an overview of the different aspects of HPLC. These concepts will all be explained in more detail later on, when talking about method development, where the decision whether to run an isocratic or gradient analysis is a crucial one.

When discussing RPLC, I've explained that the different combinations of water and organic solvent in the mobile phase will have different effects on the separation and elution behavior of the compounds to be separated – they have different solvent strength. A weak solvent takes longer to carry a substance through a column, while a strong solvent elutes them off quicker. Deciding the appropriate solvent conditions is an important step in method development. There are different ways to achieve an LC separation, that differ in the way the mobile phase is delivered.?In an isocratic run, this composition remains the same throughout the runtime of the analysis, while in a gradient run the composition changes over time, so that solvent strength is increased. It is a way to elute a range of compounds with varying polarity in a reasonable timeframe.

I will explain it in an example - in figure 1 you can see two chromatograms from an isocratic separation of the same sample. In this case methanol is a strong and water a weak solvent. In the combination of 6 parts methanol and 4 parts water, the first 4 compounds are well separated with nice sharp peaks, while the two later eluting substances produce broad lumps. Also, waiting for them to elute means the runtime of this analysis is quite long.

In the second separation, the portion of the stronger solvent – methanol – was increased to 8 parts and only 2 parts of water remain in the mobile phase. This causes the two strongly retained compounds to elute earlier, and in higher, sharper peaks, but the early eluting compounds are no longer separated properly – they all run together now. The solvent combination was too strong to maintain good retention and separation for them. So, in this case – a combination of the first and second scenario would have been ideal – which is what a gradient run can offer.

In a gradient run the mobile phase composition changes during the run. In this example shown in figure 2 the run starts off at 30 % Methanol – we call that solvent B - and the %B is gradually increased over time up to 95 %?- while the water content - or solvent A - decreases from 70 to only 5 %. So, the mobile phase running through the column, that carries the sample, changes its composition from fairly weak, to quite strong within the course of the run time. This changing mobile phase gives the early eluting compounds the time to interact with the column at low % B and pushes the strongly retained analytes out of the column with a high % of strong solvent.

This is realized by using a gradient time program in the software and can significantly shorten analysis time and improve peak shape for otherwise late eluting peaks. However, one thing to realize is that in gradient analysis the mobile phase is very different at the end of the run from the method's starting conditions. Therefore, before injection of the next sample, the solvent composition must be reversed and left to run at the starting conditions for some time – that is called re-equilibration of the column – so that each sample is analysed with the same mobile phase composition. Bearing this re-equilibration time in mind, a gradient analysis may not always be the faster separation method.

In any case, to be able to set-up a gradient, the HPLC system must be capable of delivering and mixing two solvents at the same time, which requires either two separate pumps or a low-pressure gradient valve. So next time, I will talk about HPLC system configurations.