Gradient Elution

[JHC1953]

Hello - I’m a biologist by nature, but I use HPLC in my work, and I’ve been trying to learn more about it so I don’t have to bug the chemists here so much with what are probably some really basic questions. The subject of this posting is gradient elution. My understanding has always been that in addition to separation of analytes, gradient elution could also be used to influence and improve peak shape, i.e. sharpening broad peaks, etc. For instance, I thought a steeper gradient would result in narrower, sharper peaks, while a shallower gradient would do the opposite. In practice, this rarely seems to do what I want. Also, when I read literature on improving peak shape, it all seems to focus on things such as column selection (materials/construction), mobile phase makeup (solvents, pH, buffers and modifiers), or optimum wavelengths, with little emphasis on the slope of the gradient. Am I to take from this that the steepness or shallowness of the gradient has little influence on peak shape, and that the chief (if not only) function of a gradient is to provide separation of components? Thanks.

[tom jupille]

To grossly oversimplify, gradient steepness (%B/min) affects a separation in much the same way that mobile phase strength (%B) affects an isocratic separation. As gradients get shallower:
- overall resolution improves
- selectivity *may* change (sometimes!)
- peaks get broader
- peaks get shorter
- run time increases

This slide from our HPLC Basics, Equipment, & Troubleshooting course (http://lcresources.com/training/trbeqts.html) illustrates the effect. All three runs covered the same range (5 - 95% B):

[DJ]

What are you separating?

[carlo.annaratone]

Gradient will make later eluting peaks sharper, but only to a certain point. You can never get them sharper than the plate limit of the column! So suppose you are startig with a very shallow gradient and moving towards a steepr one:
1) for a while resolution won't noticeably change, but your peaks will all be narrower and thus sharper.
2) it comes to a point where the gradient is too sharp and peaks start overlapping, therefore with noticeable loss of resolution.

How steep is steep? It depends a lot on what you are analysing and on the flow rate and diameter of the column. As far as I know on a 150x4.6 mm column with a a flow of 1ml/min typical gradient times for small molecules will be in the range 15-30 mins, while for proteins it can be 30-60 mins