Drifting Retention Time on Bare Silica

[M_Farooq]

Hello, I was testing a synthesized column material for retention time stability of benzyltrimethylammonium chloride (BTMA) with 75%ACN -25 % ammonium acetate (5 mM total). I was seeing a drift in BTMA retention (slow increase in retention time of BTMA over a period of > 30 hours) on the synthesized column. The material is not end-capped. Using bare silica (fully porous), exactly the same trend is seen, that the retention of BTMA increases slowly but steadily on bare silica column as well over a period of 15 hours. Bare silica is famous for slow equilibration but 15 hours should be enough to stabilize retention.

My earlier assumption was that more and more silanols are forming on the degraded column, however bare silica, which has no functional group, shows the same trend, albeit at a higher retention time. Note that acid washed silica does the same thing. What could be reason behind this drift for BTMA (which I have chosen intentionally) to detect material degradation.

Is this effect due to the finite solubility of silica in any aqueous system, i.e. every time a new surface is regenerated and more silanols are exposed?

Thanks.

[Andy Alpert]

It takes at least 20 mM salt in the mobile phase to form a complete electrical double layer of counterions on the surface of an ionizable stationary phase. Your 5 mM is inadequate. Try 30 or 40 mM, then back off until you find the minimum concentration that results in a reproducible retention time here.

[M_Farooq]

It takes at least 20 mM salt in the mobile phase to form a complete electrical double layer of counterions on the surface of an ionizable stationary phase. Your 5 mM is inadequate. Try 30 or 40 mM, then back off until you find the minimum concentration that results in a reproducible retention time here.

Thanks Dr. Alpert. I had tried your suggestion on the bonded material with a 20 mM (total) and the same drift was observed with BTMA. However, the linker was leaching, which explained the drift. I haven't tried this with bare silica. Your emphasis on 20 mM salts raises a question in my mind. In IC, all phases are fully charged, and eluent concentrations of 2.5 mM carbonate or 5-10 mM NaOH are typical. Why does one get reproducible retention times there because that is also a double layer phenomenon? Is it because of the organic content (low dielectric) issues?

[Andy Alpert]

IC isn't a double layer phenomenon if you don't have enough electrolyte present to form a complete double layer. In IC, you want untitrated charged resides on the stationary phase to be available for interaction with ions in the sample. By contrast, you're running BTMA on silica under conditions that are well in the HILIC range. With too little salt in the mobile phase, the concentration of untitrated charged residues on the surface is going to vary with time. Consequently, the contribution of electrostatic interaction (attractive or repulsive) to the overall retention will vary with time, hence the drifting.

[M_Farooq]

IC isn't a double layer phenomenon if you don't have enough electrolyte present to form a complete double layer. In IC, you want untitrated charged resides on the stationary phase to be available for interaction with ions in the sample. By contrast, you're running BTMA on silica under conditions that are well in the HILIC range. With too little salt in the mobile phase, the concentration of untitrated charged residues on the surface is going to vary with time. Consequently, the contribution of electrostatic interaction (attractive or repulsive) to the overall retention will vary with time, hence the drifting.

That is an interesting idea. Thanks for your insight. I would certainly like to see if higher buffer strength can stabilize retention of bare silica and nobody can beat your experience in HILIC mode. My (limited) understanding was that electroneutrality is maintained at all times on any ionized or ionizable stationary phase i.e. for every charged moiety on the surface, there exists another counter-ion either bound or in the double layer whether it is IC or HILIC mode. It is the thickness of the double layer that varies with concentration of the surrounding electrolyte. Still, I do not understand as to why with little salt (say < 20 mM) in the mobile phase will cause the number of charged sites to vary on the stationary phase? I mean what is the fundamental cause of this time variation that too in one direction only?

[Andy Alpert]

I'm afraid that I do not agree with your concept of electroneutrality. All electrolyte pairs have a dissociation constant; there is some tendency to dissociate as well as to associate, at least in a solvent that's capable of solvating the resulting ions. This is true even if one of the ions is immobilized on the surface of a stationary phase. If the mobile phase contains > 20 mM salt, then any counterion that's lost to the mobile phase and swept downstream will be replaced. If the mobile phase contains < 20 mm salt, then as dissociation occurs and counterions get swept out of the column, there's no guarantee that there will be a replacement counterion for the immobilized charged residue. Result: Leaching away of the counterions, with resulting drifting in retention time of charged analytes as they encounter a surface whose charge characteristics start to vary.

Some surface residues are titrated more easily than others. Example: ZIC-HILIC, which is ostensibly neutral because the functional ligand contains a zwitterion: a sulfonate group at the end and an amine residue that's spaced 3 (CH2)'s away in the interior. These charged centers may associate with each other at salt concentrations < 5 mM, but by 5 mM the amine group's been titrated by an external anion while the sulfonate group remains exposed with no counterion. At that point, ZIC-HILIC acts like a high-capacity cation-exchange material. Not until you get to 20 mm salt in the mobile phase does the sulfonate group reliably get titrated by an external cation, and only then does ZIC-HILIC behave like a neutral material. If you want references, contact me off-list.