Chromatography Forum

Hi everyone,

Thanks for you time!
I'm heaving a hard time understanding this one.

Many sites report reversed phase chromatography to be 75% of world wide chromatography methods, or more.

Why? Shouldn't HILIC be just as effective? Why is so little done in normal chromatography?

Is it because reversed-phase it's much cheaper?
(the most solvent used ends up being water, instead of organic solvents)

All help is welcome! Thanks.

"Normal phase" was first, used mostly non-polar organic solvents and silica columns, and could be tricky.

In the last 40 years, reverse phase column technology improved dramatically and became easy to use. These used typically with aqueous-organic buffers and were hand in hand timewise with improved hardware technology. Reverse phase could typically be used for many analytes of interest by changing organic level, temperature, buffer, etc.

HILIC is still relatively new, and used for very polar analytes.

Folks tend to start with stuff with which they are familiar.

If you get the chance to run some normal-phase, reverse-phase and hilic methods, it gets easier to envisage why people like reverse phase so much.
Proper old-fashioned normal-phase with silica columns goes wrong at the merest sniff of water, so not only must you use nastier solvents anyway, but you also have to keep them specially dry. No one (except Andy Alpert; watch his posts, and read his papers!) really understands how hilic works, and it certainly seems very sensitive to changes in pH of the buffer, changes in sample solvent composition and many other factors too. Reverse phase, on the other hand, is embarrassingly robust; very often even quite inexpert method development gives enough separation to do the job. The solvents are simple to make up; buffering is rarely critical (you can very often get away with a simple water/acid mix). There is a huge range of almost indestructible columns available.
Having said all the above, you are right; hilic is a good method, and we should ideally choose the method that suits the analyte. If you have very polar compounds, it would be better to use Hilic than reverse phase. Of course polar compounds may also be suitable for various ion-exchange approaches, but these aren't always compatible with mass spec, whereas hilic is.

Thank you for your answers. They were a good help.

I also found this board that helps understanding where to use what is case anyone else is battling with this too.



If anyone else would like to add something it would be much welcome.

Thank you all!

I think that graph is not very accurate in the "polar" region. There are many very polar compounds which are not organic-solvent soluble and therefore not amenable to normal phase, but very well suited for HILIC. The most outstanding example are sugars and polysaccharides. And the polarity range should extend more to the left, where Ion Chromatography should be. There also would be some overlap between ion chromatography and HILIC

Thanks biotechno,

As a newbie of this forum i am studying on chromatography. your infographic help me to understand more.
Best of Luck!