Chromatography Forum

I would appreciate some advice where to start method development on a Zorbax 4.6x150 mm, SAX column.

- The molecules I would like to detect are negatively charged down to a very low pH (<1). I would therefore like to work at lowest pH possible (2.0) to avoid retention of everything else (it is related to the post about flavor peaks)

- I do not want any reversed-phase retention at all.

I assume that SAX columns are usually run with a salt gradient, but I have no feeling of where to start. I was thinking of phosphate buffer pH 2.0 and then a salt gradient with ammonium sulfate. Do I need any acetonitrile? Are the better salts to use? How much salt is needed to really clean the column from all anions? Tricks for good peak shape, plate count and baseline?

Thanks,
Mattias (working 99.9% with reversed-phase )

Assuming UV detection and a silica-based column (which usually has a fairly low capacity) I typically start with 25 mM buffer (in both A and B) and run a gradient from 0 to 1 N NaCl. At pH 2 sulfate might be easier on the hardware than chloride, but being divalent, it is a much stronger eluant. Probably a coin-toss on that. I don't see any advantage to ammonium over sodium as the counterion, and I would expect fewer problems with microbial growth with sodium.

Thanks Tom!

Yes, the Zorbax column is silica based and I will be using UV detection. Are there other types of SAX columns around that have higher capacity and that can be used at even lower pH (than pH 2.0)?

If sulfate is stronger than chloride, then I might just as well use sulfate at lower concentration (I guess). Good point about ammonium being a nutrient for microbes. I'll switch to the sodium salt.

Higher capacity is not necessarily an advantage for analytical purposes, as it requires higher ionic strength for elution. There are a wide range of polymeric columns intended for protein & peptide work. Check witb PolyLC, Tosoh, GE Healthcare (there are more, but those come immediately to mind).

Now that Tom's taken the stopper out of our lamp...

Two points:
1) Below pH 5, both a SAX and a WAX (weak anion-exchange) material will have its full potential (+) charge density. There is then no advantage of a SAX material over the WAX material. There are some disadvantages to SAX materials. Typically they're more hydrophobic, which can introduce some mixed-mode effects that would have to be overcome by including more organic solvent in the mobile phase.
2) At pH 2 the rate of attack of acid on the Si-C bond of silane-based coatings becomes significant (which is why C-18 columns steadily lose capacity over weeks or months of use with 0.1% TFA). PolyLC's PolyWAX LP material doesn't have a silane-based coating. It's the only type of coated silica that has no lower limit to its operation (aside from potential acid attack on the metal of the column).

Thanks!

I have done some tests using higher pH and salt gradient. As expected, I get good retention of my compounds. The problem was that I got quite good retention of citric acid as well (major part of formulation), and of other not wanted peaks.

I have now gone down to pH 2.5 (phosphate) and I still get good retention of my peaks. Even the peak that is zwitter-ionic. That I do not understand... At pH 2.5 this compound bears both a positive and negative charge and I would expect the retention on SAX to be zero. I use 25% ACN in both channels, and I do not expect any reversed-phase activity to be present.

Anyhow, it looks quite good at the lower pH, might have a method soon