It is frequently come across a term 'column collapse' when the water content of the mobile phase approaching 100% during HPLC analysis. Is anybody know the meaning of this term ? And, what is the phenomenon and problem when the column is collapse ?

Your advice is much appreciated.

no collapse, just no wetting

Try this.

This has been extensively discussed before, the wetting argument (ie, Uwe Neue) seems to be the most plausible (a quick search did not recover this discussion, already out of the archives??). Whether this is actually accompanied by a "phase collapse" was discussed in at least one publication on nmr of stat phases, I still have not found this or a similar article, anybody?. However, I donīt recall anything that would justify the word "collapse".

Anonymus;

Your question is well presented, the word "Collapse" is somewhat misleading. Perhaps a better terminology is "Matted" or Matting". Even though the term is unusual, the idea of a rearrangement of the spatial position of the reversed-phase groups ( i.e c18) from "tentacle-like", to a dense layer or "overgrowth" over the silica surface is better expressed that way.

Obviously this phenomena is observed when the % organic modifier (ACN, MeOH) falls below 10% and the groups do not have enough solvation around them to extend beyond the surface.

I hope this explanation helps you.

Benjamin Esquivel

I think this has been examined nicely using Raman Spectroscopy which can directly analyze the conformation of the alkyl chain.

I appears that the alkyl chains are mostly extended regardless of the organic concentration. The term "phase collapse" is inaccurate and is kept alive via marketing material like that linked above. It makes a nice cartoon that is easy to remember and helps sell columns, but it is not accurate.

The C18 forms a hydrophobic layer and as the organic concentration of the mobile phase decreases its surface tension increases and at some point the mobile phase is no longer able to wet the interior volume of the pores and then there is a sudden reversible loss of retention. Low carbon loaded columns have always been able to tollerate lower concentrations of organic.

Here is a link to an article form Pemberton's group: http://www.chem.arizona.edu/faculty/pemb/research/pdf/chromatography_jessica.pdf

No, I have to disagree with Benjamin. There are several phenomena that happen as you increase the water content. The phenomenon commonly called "phase collapse" is a wetting phenomenon, or better an un-wetting phenomenon. The mobile phase is driven out of the pores due to surface tension and loss of pressure or a conbination of both. As a consequence, you loose retention. In the worst case, it is a nearly miraculous on-off phenomenon, but more gradual changes are not unusual.

The term is also kept alive by authors in LCGC. Perhaps Ewe could write a letter to the editor.........

Maybe someone can create a compelling cartoon for the real phemonena and the maketing departments will be more receptive to offering the correct explanation.

Marketing departments and correct explanation! Great joke, Bill.

We have cartoons with the correct explanation. I will send a copy to anybody who is interested.

Russ, there are companies who have employed more marketing people than chromatographers. Uwe can be happy that he is not working for such a company. Still the question is, why using 100% water in mobile phase? If the problem is no retention of very polar compounds, than other separation modes like NP or HILIC can do a better job, without the "phase-collaps-problem". But than sales for so called "AQ" (from latin "Aqua" = water) columns will decrease. "Marketing companies" don't like this. Gerhard

The problem when trying to change "established" terms even when they are inaccurate is that:

1) You end up with several terms describing the same thing which is rather confusing.
2) You will have several responses of the kind "who you thing you are?".

That was the case with David Sparkman when he tried to make a book with the correct and incorrect mass spectrometry terms (the title is: Mass Spectrometry Desk references). He mentions in the preface that when he send his manuscript for reviewing to several people working in the field he had some answers as the one I describe in case 2.

Let me throw in my two cents' worth here:

First of all, to Gerhard: I think you're right about NP or HILIC being a better approach, but only assuming that *all* the compounds of interest are polar. If there is a range (particularly if most of the compounds are well-retained by RP), then the ability to start a gradient at 100% aqueous is quite useful.

Second, with regard to the terminology:
- I think that the experimental evidence supports the contention that there is a "phase transition" that occurs for many RP columns at a particular critical % organic. I.e., the properties of the stationary phase are "different" above and below that critical %.
- The consensus seems to be that the difference is related to the wetting or solvation of the bonded phase.
- Whether the stationary phase actually "collapses" (whatever that means!) is open to debate.
- Speaking personally, I don't think it matters. While I do know a few people who can grasp the equations involved without resort to the "cartoons", they are few and far between.

Finally, with regard to "marketing" people: believe it or not, we do not spend our time thinking up ways to sell overpriced widgets. Most of us spend our time trying to:
- communicate sometimes abstruse concepts from R&D to an audience that ranges tremendously in technical sophistication, skill, and experience, and
- trying to communicate the users' problems and needs back to the R&D people.


-- Tom Jupille / LC Resources

Could we agree then, that a change in RT is due to dewetting (the interaction area for the analyte changed) and that a change of mobile phase necessarily has to change the conformation (etc.) of the C-18 ..... (change in interaction), the latter being of no or lesser consequence in regard to RT. ??

Tom, those in the business for a long time all know that all "marketing" people are angels.

Once again, there are at least two distinctly different phenomena that occur in highly queous mobile phases. We should not confuse them with each other, since the effects are different.

One is dewetting in highly aqueous mobile phases, close to 100% water. The way it work is that you run the column, get good retention, then you stop the flow, start it again, and you have lost retention. The other one is the selfadsorption of the C18 to itself, which reduces retention in mobile phases as high as 50% organic and happens independently if the stuff has a high silanol content or not. The first one is a temporary thing and is a nuisance, the second is a permanent thing, happens all the time and is normal.

LOL!

Actually, if you accept a broad enough definition of "angel", then yes, all marketing people are angels of one sort or another (as it happens, I live near Oakland, California, the home of the famous -- or infamous -- "Hell's Angels" motorcycle gang).

More seriously, I really don't have a problem with the term "phase collapse" to describe the change in stationary phase properties at low % organic. I also have no problems with the term "sunrise" to describe one of the effects of the earth's rotation.

-- Tom Jupille

Uwe, is there any direct evidence that "selfadsorption" or "change of conformation" or even "collapse" has (or can have) a pronounced effect on RT?
Just now the HPLC of ouabain surprised me again: In changing from H2O/MeOH to H2O/ACN of equal elution strength the RT changed from ~25 to ~9 min instead of staying the same. Also, changing the ACN content from 25% to 10% changed the RT from 5 to 50 min (the rule of three would have predicted a factor below 9). Could that be, in part, due to changes in the C-18? Until now I gave thought only to changes in the ouabain (generally: analyte).
Tom, what has bothered me on "collapse" is that it insinuates that it causes the dewetting (or simply the change in RT), when the evidence is quite good for the dewetting concept which must be caused by a desorption of MeOH on or in the C-18 layer. One can imagine that this desorption also gives rise to a change in the C-18s. I just canīt immagine, though, that one conformation of C-18 attracts water, another repulses it.

The cleanest evidence of changes in the conformation of the stationary phase are studies by Engelhardt and Schmidt, where they followed the t0 from high organic to low organic and then back to high organic again. In the path from high organic to low organic, t0 declined in the mobile phases with a high water content. But then it remained lower, when the organic solvent concentration was increased again, until about 50% organic. Then it met the original curve again. I do not recall, what else they did. I am sure that they measured the retention of some retained molecules over some of the range. I would need to look this up.
Anyway, this hysteresis is what one could interpret as a self adsorption of the C18.
There have been studies by Dorsey on the retention as a function of temperature in highly aqueous mobile phases that implied a similar thing. At low temperature, the retention pattern was different than at at higher temperature, with a clear transition point that can also be interpreted as a transition point of the stationary phase.
I am just spitting this out as I remember it, and I do not have the references to read. Sorry!

Thanks, ...guess one has to keep an eye out for that. The article I mentioned recently, Brooks, et al, "Size exclusion chromatography does not require pores" . . . . . would indicate that some moleculesī interaction (especially a flat one like the steroid ouabain) are highly susceptible to changes in surface, others may not be. A quote from this paper: "Our recent alternate picture (of size exclusion) proposes that the partition coefficient can be calculated from a thermodynamic model for the free energy of mixing of the solute with the gel phase."

I just saw that there is an article in the new LC/GC (US version) that covers the subject of column dewetting etc. It also uses the pictures and the equations that I had offered from our files in my message from June 14.
I had explained the phenomenon to Matt Przybyciel at an EAS conference, and he did a few experiments to check it. Read it it, it is neat....

to tom jupille (posting from June 19.)
I agree with your statement, that the behaviour of the stationary phase changes at a ctitical %B. So in my work with DryLab, i experience unusual high variations from predicted results when working at the gradient-mode (0%B to 100%B gradients and conventional C18-phases). Using phases with i.e. polar embedded groups, which are predicted for aqueous mobile phases, i don't get these variations. Is it possible, that the change in behaviour of the C18-phases (in the range of 0%B to 100%B) causes a not so good precision of DryLab-predictions?

Entirely possible. For isocratic modes, you can work around the issue by using additional calibration runs. I essence, this results in a non-linear fitting function which can accomodate changes in stationary phase chemistry.

For gradient modes, we are locked in to the "linear solvent strength" model (which essentially assumes a constant stationary phase) in order to do things like multi-linear gradients or gradient -> isocratic conversions.

If you want to e-mail me privately, I can help you set up "custom" modes in DryLab that may help.

-- Tom Jupille

To "pillin". Sorry for the second reply but, I hadn't read your post sufficiently carefully the first time.

If you are *really* starting your gradients at 0% organic, then the "phase collapse", "dewetting", or whatever that occurs at low %B will definitely affect your predictability. The phase transition will occur at different rates in a steep vs shallow gradients. I believe the DryLab documentation recommends starting at 5%, for reasons discussed very thoroughly in this thread.

With the right LC column you can most certainly start at 0% organic. Obviously with DryLab the developers were trying to make the procedures as generic as possible for as wide a range of LC columns as possible.

However, with the right column, starting with 100% aqueous conditions can and does work. Waters had posters and Pittcon and HPLC (2002) that elegantly showed the dewetting phenomenon. Uwe can point you in the right direction.