Chromatography Forum

Is it possible to run reproducible ion pair gradients ?

I have an isocratic method for a mixture of amno acids but suspect there are later running impurites which I am not seeing.

Any tips ?

Ta

Stanlee

Stanlee
Here are a few references for the retention of underivatized amino acids without the use of ion-pairing reagent. Our columns have ion-pairing reagent attached to the surface. You can use any buffer to elute you compound. Egulibration time is very short (for amino acids):

http://allsep.com/makeCmp.php?cmp=Cmp_007
http://allsep.com/brochures/SielcNewsletter_0604.pdf
http://allsep.com/brochures/SielcNewsletter_0105.pdf

You can use sulfuric or phosphoric acid (or buffer) for UV applications
TFA, ammonium formate, ammonium acetate for LC/MS/prep chromatography
for zwitter ions you can use our new BLIS approach (Buffer Less Ion Separation), wher the mobile phase is ACN/water (no buffer)-this will allow you to go to 190 nm in UV

Thanks for the advice SILEC.
I have heard of your columns but unfortunately we are not in a position to totally re-develop and was hoping to just modify existing methods.

Stanlee

Opinions on ion-pair will vary

Here's mine: depends on the ion-pair reagent. The smaller/weaker ones (e.g., TFA) don't pose any particular problems; gradients with TFA are routinely used for things like peptides. As you get into larger/stronger ones (e.g., the classic alkyl sulfonates), then system equilibration time becomes an issue.

That said, it certainly can't hurt to try tacking a gradient segment on to the end of your run.

Tom,
What is the effect of the ion-pair Reagent (alkyl-sulfonates) on the retention time of acid, basic and neutral compounds? Can identical separations be achieved by using different type of alkyl-sulfonates in different concentrations?
Thanks Tom,

stanlee,

As previously mentioned, ion pair gradients can be problematic but there are several things you can do to minimize equilibration problems (arguably, equilibration issues are the main complaint regarding gradient ion pair separations). As a bare minimum, don't change the concentration of the ion pair reagent during the gradient. Second, if possible stay away from changing the solvent composition of your mobile phase as the solvent composition will significantly affect the amount of adsorbed ion pair reagent, greatly lengthening equilibration time. The best option if you want fast re-equilibration is to change the ionic strength of your mobile phase. For example, if you are using tetrabutylammonium ion as the ion pair reagent, hold constant the amount of tetrabutylammonium ion and solvent content and instead vary the concentration of a co-electrolyte such as sodium chloride or sodium sulfate. Ideally, there should only be one anion present in such a system (i.e. the counter ion on the tetrabutylammonium ion should be identical to the anion used as your electrolyte source) but this might not be viable if you need to buffer your mobile phase. One last point, the higher the concentration of your ion pair reagent, the faster your system will re-equilibrate if you find it necessary to change either the solvent or the ion pair reagent concentration during the gradient.

ltnguy3:

To a first approximation, adding more ion-pair reagent has the following effects on retention:
- opposite-charge analytes: retention increases
- neutral analytes: retention decreases slightly
- same-charge analytes: retention decreases greatly

Also to a first approximation, the effects are controlled largely by the concentration of charge on the stationary phase surface. You can get similar results from a low concentration of a long-chain IP reagent or a high concentration of a short-chain IP reagent.

As with any type of chromatogaphy, secondary effects can complicate matters in specific cases

Chris: your ion-exchange background is showing

Actually, I agree with everything you said, but if the strongly-retained stuff is held more by hydrophobicity than by ion pairing, then simply ramping the ionic strength won't help; in that case, more organic will be required, and equilibration time becomes an issue. If the gradient segment isn't too long, it might not be a problem.