Chromatography Forum

Is it true that some RP- columns are not 100 % water compatible..??
Why is this so..?? What happen when 100 % water is passed through RP- column..??

Hi rick1112,

RP phase is composed of an alkane chain ( e.g. C8 or C18) chemically bonded to the stationary phase most often silica based.
As organic material the C18 does not like an aqueous environment, that what they mean by not compatible.
The C18 "shrinks' in a high aqueous mobile phase ( depending on the kind of RP columnmaterial) and can result in less or no retention of you compounds of interest.
In certain cases theC18 chain may not recover from this treatment with 100% water and your retentions/selectivity can be lost.

I am sure more information can be found in different textbooks concerning RP chromatography or on the web

regards

philippe

If you need a column that can handle these conditions, look to any polymer column on the market as well as the Waters Aqua.

Many standard, well endcapped C18 columns don't work well in 100% water. Common problem: you got good retention, then you get a glitch in the pump (loss of pressure, air bubble), or you switch the pump off, and your retention has disappeared. Reason: the mobile phase gets pushed out of the pores. This has been called "hydrophobic collapse", which it really isn't.

This will not happen with unendcapped packings (as many of the "aqua" type packings are), with packings with embedded polar groups, or fully endcapped specially designed packings such as Atlantis T3.

The de-wetting is easily reversible: wash the column with degassed acetone then methanol, then your mobile phase. If this does not cure the column, the problem is something else.

Hi Uwe,

Just for the purpose of clarity. Are you saying that there is no such thing “hydrophobic collapseâ€

I disagree that hydrophobic collapse and dewetting are different things. The phenomenon is that you loose retention, and you can recover retention by using a high composition of organic.

What "pushes" the water out of the pores is its surface tension. If you are familiar with mercury porosimetry: it takes a giant pressure to force mercury into pores due to the combination of surface tension and wetting angle. The dewetting of a C18 is the opposite phenomenon. You release the pressure, and a mobile phase with an unfavorable wetting angle moves out of the pores. There are a range of intermediate states of this black-and-white picture described in the last few sentences. These intermediate states depend on the pore size of the packing, the surface coverage (=hydrophobicity), the mobile phase composition (water compared to water with a little bit of something in it), etc...

If the wetting angle is favorable (unendcapped C18s or C18s with embedded polar goop), the dewetting disappears completely. Reason: the wetting angle drops below 90 degrees.

Reference: J. Chromatogr. A 1075 (2005) 177

The "empty" pores contain gases formerly dissolved in the water and water vapor. What holds the liquid in the pores is the liquid/solid interfacial tension plus the external pressure; what pull the liquid out is liquid/gas surface tension and partial pressures of vapor and gas. The column can exist in a metastable condition for quite a while, but once initiated, the dewetting happens rather quickly. It is quite easy to measure this by weight; we had a summer intern do it.

Hi Rick,
As everybody explained above, the main reason of loss of separation or RT shifts with Reverse phase columns (specially C18) is dewetting of pores or matting of carbon chain.

I believe these two are inherently different phemonenon (debatable subject for chromatographers). But reverse phase columns with some polarity introduced are unlikely to pose problems in your results.

I used PrincetonSPHER Ultima C18 and C8 from Princeton Chromatography Inc. (this has poler amide group embedded in alkyl chain) for typical applications where my MP is 100% aqueous and I got excellent results with no such effects like loss of retention..blah.blah.

Princeton has one more typical phase which has Y shaped C18 carbon chains for 100% aqueous mobile phases. You have 4 carbons and then bifurcated chains of 7 carbons each..something like that.... This is based on the principle that Y shaped chains prevent each other from collapsing under highly polar (aqueous) mobile phase environment. Surprisingly this column also work under 100% water conditions. WHERE DOES DEWETTING THEORY STANDS IN THIS CASE??? BUT THIS EXPLAINS PHASE MATTING EFFECT UNDER AQUEOUS CONDITIONS WELL.

Here are two article from LCGC which expplains these theories in simple and detailed way.
1. The Retention Behaviour of Reversed-Phase HPLC Columns When Used Under 100% Aqueous Mobile-Phase Conditions
http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

2. Phase Collapse in Reversed-Phase LC
http://www.lcgceurope.com/lcgceurope/da ... rticle.pdf

Hi Mark and Uwe,

So, what happens is: In one situation gas replaces the mobile phase in the pores and in another, water does it. But let’s say the mobile phase is water (100 % aqueous solution anyway). What would the problem be then? Will the retention be lost? And if yes, what would the particle surface bonded ligands be doing?
Finally, what about non- porous particle stationary phase support? Are they immune towards de-wetting/hydrophobic collapse (assuming these two are one and the same thing)?

I’m not saying that there is no such thing called “de-wettingâ€

The articls to which LCGUY referred both present a very strong case for dewetting as the cause for loss of retention. The second article merely seems to use the term "collapse" in the sense that retention collapses. As a matter of fact, they allude to a reference which apparently claims that the immobilized alkyl chains are always in a collapsed state, no matter how much organic mod. is present. Now, as mentioned before, I remember seeing (but must have lost refs) some NMR and IR studies which seem to conclude that there can be a change in configuration of substituents when solvents change. It wouldn´t surprise me that there might be some cases where this happens. It would very much surprise me that such conformational changes would cause stark changes in retention. It´s in keeping with this that I don´t remember seeing any study which had unequivocal evidence which links any conformational changes to retention. (The terms configuration and conformation are used "loosely" here)
So, LCGUY might have asked, instead, why does bifurcation and Y´ing prevent dewetting?

The Princeton phase has great steric bulk and can't form an ordered, dense covering. Such sterically hindered phases also don't accept endcapping well. As a result, the substrate is more reachable by water. It does not take much hydrophilicity to make a column resist dewetting.

Once again, Mark responded faster, and I agree: to get dewetting, you need a fully hydrophobic phase and small pores. The branching of a phase will prevent a full and high coverage. But even phases with a reasonably high coverage do not show (complete) dewetting, if they are not thoroughly endcapped. Example: My most favorite phase from 30 years ago (Resolve C18) is not endcapped, and is very happy in 100% water. We did sugar separations on this C18.