Chromatography Forum

hi all,

We've been having problems with a method recently developed for the assay of three cpds. One is a neutral sugar ester, second is a basic, and third is a neutral steroid.

Basically, we need the ion-pair reagent (Sodium dodecyl Sulphate+0.05%TFA) in mobile Phase A so that we can retain the basic compound. Mobile Phase B is Acetonitrile. The 0.05% TFA is for maintaining a low pH. We are using SDS conc of 0.02M to coat the column surface which is a C18 column, Stable bond. We are having no problem retaining the 3 cpds.

The question is what is the implication of using a higher concentration of SDS of say 0.04M or 0.05M SDS because at this higher concentration, we can achieve better selectivity between the sugar ester and an interfering peak which occur randomly at low concentration of our analytes. Will this high conc of SDS bring up future chromatography problems. Has anybody used ion-pair reagent of this high conc before.

olaniyi,

There is no a priori reason why you can't use higher concentrations of an ion pair reagent (other than implications associated with the cost of the reagent and need for higher purity reagent in some cases). The thing is that the effect of using concentrations beyond the critical micelle concentration will usually be the opposite of what is observed below the critical micelle concentration (i.e. increases in concentration beyond a certain value will actually cause decreasing retention for analytes electrostatically attracted to the surfactant). The catch is that the critical micelle concentration is generally only listed for surfactants in purely aqueous media and the critical micelle concentration is a function of: counterion, solvent content, ionic strength and temperature so the value you find in the literature is largely irrelevant. Since you are not likely to find any tabulated reference information regarding critical micelle concentration in common HPLC eluent systems, it's generally easiest to simply characterize the effect of concentration on retention and selectivity in the analytical system of interest (which it sounds like you've already done). If higher concentrations of SDS give you a good separation, I wouldn't worry about it. One thing to keep in mind, though, is that SDS is only modestly acid stable so I wouldn't keep eluents contain TFA around for a long time. So, you might be better off using an alkyl sulfonate instead.

this also intrigues some question i've carried quite long time.

i was several times told (by Waters technical people here) that ion-pairing reagents such as alkyl sulfonates are quite hard to wash clean from the column once it contacts the column and may change the selectivity permanently, so it is better to use one column exclusively for the analysis in which such reagents are used.

that is ok we use one column for only one analysis task. but i always wonder then how about the method development? when ion-pairing reagents are tested with different concentrations, do that mean we are changing the column selectivity again and again? how can we get assured about the method robust enough when moving to other column?

thanks for your comments.

kiknos,

The story that you should dedicate a column to a specific ion pair reagent is only partially true. It's true that when using silica based reversed phase columns there is a significant risk that a column used with TBA is going to take a long time to restore to original selectivity but the same is not true with anionic ion pair reagents. When using polymeric reversed phase columns, you can freely switch back and forth between one type of ion pair reagent and another without issue. But in no case do you need to dedicate a column to a specific type of ion pair reagent and ion pair reagent concentration.

many thanks, Chris ! that question really puzzled me quite long time.

so, i guess, for silica based RP columns, is that because the silanols which make TBA hard to be washed clean? and anionic ion pair reagent won't be troublesome because of no interaction with silanols?

is there any resource on the effect of ion-pairing reagents on silica-based and polymer-based RP columns?

or at least your experiences?

many thanks in advance!

kiknos,

Sorry, but I can't point you in the direction of any kind of published work regarding conversion of columns from one mode of ion pair to the other. This is solely based on actual experience. For example, we have successfully used SDS as a packing solution additive in order to enable packing of silica based reversed phase materials in totally aqueous media. And, of course, we had to prove that no residual effects were observable for SDS from the packing solution before we could proceed with such an approach. In addition, we have verified experimentally on numerous occasions the ability to convert a polymeric reverse phase column (in this case the MPIC NS1 column) from one ion pair form to the other. We've never had any problem with this with polymeric materials.

thanks for the kind reply, Chris!

as for SDS, i've gotten to know it from some publication by Vydac, in which it was useful to wash sample contaminants(proteins, peptides, etc) and it didn't change the chromatography of mix standards after washing it away.

and now your experiences let me know more. many thanks!

hi Chris,

Thanks for your contributions. It was highly appreciated. You mentioned that it is not good leaving SDS mobile phase containing TFA lying around for a long time. How long do you mean, a week, 1 month, 2 months. We normally give it 1 month expiry after date of preparation based on past experiences but really we should determine its stability which we are now going do ASAP. Thanks

olaniyi,

I can't give you any quatitative estimate since that is dependent upon the specifics of your system but I would be surprised if the stability was an issue for eluent storage times of less than one week unless you had a pretty low mobile phase pH.