100% aqueous and C18 columns

[Bruce Hamilton]

Just read an earlier thread about C18 columns and water.

I'm aware that there are now C18 columns from most major suppliers that are comfortable with 100% water, but recently I've encountered new methods from different clients that use 100% water with and without TFA on columns such as Luna C18(2) and Zorbax XDB-C18, which I've usually used with gradients that stopped at 95% water.

The other aspect is that several methods I've been given often have dramatic gradients at the end of runs from 90% acetonitrile back to 20%, along with short or non-existant post-run times to give fast turnaround.

When I've run the methods, rather than test out my columns, I've usually changed the gradient slope to a 20% maximum change for acetonitrile and 10% for methanol. With the clients consent, I've usually also changed the gradient to start at 95-97%, but I've no idea whether that is sufficent organic, but the assays are OK.

My columns usually last from several years up to a decade, but recently I purchased some new C18 columns. What's the conventional stance on gradient slope and water/C18 with modern columns?. Are they far more robust than I believe.

If there's already a thread covering this, pointing me to it would be appreciated.

Bruce Hamilton

[Uwe Neue]

You may want to look at this fairly recent publication by Pete Carr:
Isocratic and gradient elution chromatography: A comparison in terms of speed, retention reproducibility and quantitation. Journal of Chromatography A, Volume 1109, Issue 2, 24 March 2006, Pages 253-266, Adam P. Schellinger and Peter W. Carr. They reference also their primary study, which was published not too long ago.

In principle, you are never in equilibrium in a gradient. However, you can get to the same state of non-equilibrium at the beginning of a gradient if you faithfully carry out the exact same procedure. In practice, this means what all of us have done since a long time, i.e. run one or two blank gradients before we begin our analysis runs.

The other article that is relevant is the article by Walter on the dewetting issue, commonly called "hydrophobic collapse". I'll post the reference in a second post. Dewetting is primarily an issue in isocratic chromatography. I would think that if one is following the Carr scenario, one rarely will have to worry about dewetting, since the column never manages to get in full equilibrium with the highly aqueous phase.

Of course, if you do not want to worry at all about these things, you have the option to use a column designed not to exhibit this problem to start with.

[Uwe Neue]

Here is the Walter reference:
Mechanism of retention loss when C8 and C18 HPLC columns are used with highly aqueous mobile phases, Journal of Chromatography A, Volume 1075, Issues 1-2, 20 May 2005, Pages 177-183
Thomas H. Walter, Pamela Iraneta and Mark Capparella

[Bruce Hamilton]

Thank you!.

I'll request copies of those articles.

Bruce Hamilton