Chromatography Forum

With our Primesep technology the use of ion-pairing reagent should become a history. Check the following link for separation of zwitter ions

http://allsep.com/makeChr.php?chr=Chr_009 (amino acids)
http://allsep.com/makeChr.php?chr=Chr_042 (DOPA)
http://allsep.com/makeChr.php?chr=Chr_044 (catheholamine pathway)

The retention of zwitterionic molecules on a C18 column can be manipulated using the pH of the mobile phase. Usually, it will have less retention in the singly charged state than in the doubly charged state. This means that the retention is best at low and high pH. A good option for such a compound is the XTerra MS C18 column. It also can be run in 100% water, in case that you need to go to a fully aqueous mobile phase to get retention. A very good alternative for very polar molecules is the Atlantis dC18 column, which has been optimized to give maximum retention in 100% water. While XTerra can be run at alkaline pH (e.g. pH with an ammonium bicarbonate buffer), Atlantis can not be used at alkaline pH so you would need to use acidic pH to maximize the retention of the zwitter.

Ion-pair reagents are fairly rarely needed to get retention.

We are aware of customers retaining hydrophilic zwitterions by using our zwitterionic stationary phase ZIC®-HILIC. The pH value may still be an issue since it will influence the selectivity and reflect the properties of the zwitterionic analyte. The use of a high pH is enabled by using our polymeric column.

Check these links - in these cases the peak shape on leading brand C18 column was an issue. Due to the different surface chemistry the peak shape on Primesep columns is much better:. The silanol interactions are masked by stationary phase ligand.

http://allsep.com/makeCmp.php?cmp=Cmp_151
http://allsep.com/makeCmp.php?cmp=Cmp_080

Dear kvgpal,

as Uwe mentioned, there are some critical factors with charged or even zwitterionic molecules. Change of pH and organic modifier conc. are classically the most common parameters to get retention and separation. Besides the XTerra and Atlantis, almost every major supplier offers alkaline stable (Phenomenex Luna, Agilent Zorbax Extend, Macherey-Nagel Gravity.....) and water wettable (aq OR aqua, MN Pyramid...) columns.
I would never recommend using a non-endcapped column, as this kind of technology is quite archaic. Typically You will find huge tailing for most analytes using non-endcapped RP-silica.

The staement about tailing on non endcapped column might be true for RP columns. Mixed mode columns offer different chemistry and we do not observe any tailing on basic (or acidic) analytes. We compared the best columns on the market with our approach, the argument was "that it is not hard to get bad chromatogram on competitors column" but when we asked to show how to use the particular column with particular compound (quaternary amines, strong amines) we did not get any recommendations. We are willing to compare side by side these columns (end-capped or capped).

There were several messages at this point about "commercial" posts and replies. I've split those out into a separate topic and moved it to the "Around the Water Cooler" board. We can fight over it there.

-- Tom Jupille (Chrom Forum admin)

Still, even for an analyte that is a permanent zwitterion the pH may be important when a non-endcapped support is used since ion exchange and silanolic effect may contribute to the selectivity.

A perfectly well endcapped RP material should not be able to retain a permanent zwitterion, or should it?

Einar,
maybe I misunderstood you, but one should expect a highly diffuse (soft) zwitterion to be retained on your theoretical column. Proteins are zwitterions, some can attach doggedly to teflon. Even a "simple" mono-ion like TcO4- (counterion: Na+) is retained in RP at pH 2.5 as well as 7 (where one presumably has SiOH or SiO- , respectively), also, TcO4- is commercially extracted from aqueous solutions with organic solvents.

Hi kvgpal,

I see that up to now we barely replied your questions (except from the first one...).

In reply to your 2nd question: Yes ion-pairing reagents can help you. As it is easier to operate in acidic conditons you could use peflurinated carboxyylic acids or alkylsulfonates in order to create retention for your analyte. Just be sure that you equilibrate appropriately your colum in order to achieve reproducible retention times.

In reply to your 3rd and 4th question: When using ion-pairing reagents, I always use end-capped columns as I have noticed that they provide much higher column lifes than the non-endcapped. End-capped columns used under acidic conditions and ion-pairing reagents can last for really long time (at least all the columns I have tested up to now).

Well, Kostas, for my taste the questions had been answered very well, the gist being: Usually one can solve the problem without perfluorinated acids, and if one can, it is to be preferred.
Now if we were told what the zwitterion is I might have been able to get more specific.

HWM,

I do not agree... Nobody replied the three last questions, which were if ion-pairing reagents can help him, which is the life time of a column when using ion-pairing reagents and which type of reverse phase to use (and from questions 2 and 3 it could be applied to the use of ion-pairing reagents).

Giving alternatives is good, but it is good to answer the original question as well.

As an exagerated example: If someone asks how he can analyze his drug by LC-UV in a matrix, and I answer ohhh... this is very easy by LC-MS and you should avoid using UV due to the complexity of this matrix, would have I answered his question?

I have a sample with triprotic zwitterionic molecule. It has 3 pKas: 2.12, 2.90 and 7.98.
For pH of mobile phase I choose midpoint between pKa 2 and pKa 3, > 2 points above and below them: 5.5 (25 mM ammonium acetate buffer). I add 20 mM TEA in mobile phase to reduce the tail, beside using endcapped column packing.

Regards,
SYX