Chromatography Forum

I've got some questions about ion exchange and pH gradients...

First of all, whether it be SAX or SCX columns, should the "rinse mobile phase" that restores the stationary phase resin to it's native state possess weaker or stronger exchangers than that of the mobile phase used for separation? Should it be more concentrated or less? Should the pH be the same or does it matter? I am asking these questions with respect to electrochemical detectors.

Also, for pH/ionic gradients, what's the best way to determine dwell volume?

For these type of gradients: let's say you have a complex mixture containing 12 analytes with similiar pka's...what's the most efficient manner to develop and optimize the pH/ionic gradient? (trying avoid trial and error approach)

Any assistance and insights to these areas are greatly appreciated.

First of all, pH gradients, IMHO, are a nightmare to transfer from one instrument to another, because the column "titrates" the mobile phase. The result is that the shape of the gradient changes as it moves down the column.

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Simplest method development approach (since you used "pKa" rather than pI, I'm assuming you're dealing with "small" molecules rather than proteins):

1. start with an appropriate buffer for a pH 2 units or so on the ionized side of the pKa.

2. Run a linear gradient from 0 to 1N NaCl. If all your peaks are close enough together (say, less than 20% of the gradient time), then you can probably do the separation isocratically. If the peaks spread out over a wider range, then you'll have to stay with a gradient. You can estimate the appropriate ionic strength from where your peaks come out.

3. Assuming you haven't gotten a workable separation, move the pH closer to the pKa (0.5-unit intervals at first, then 0.2-unit intervals once you get closer to the pKa.

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Dwell volume is dwell volume. You can measure it with any detector (UV, conductivity, whatever):
1. Remove the column and run a connecting tube from injector to detector
2. In the A reservoir, put an appropriate solvent that gives a low response (e.g., water).
3. In the B reservoir, put the same solvent, but add something detectable (acetone for UV, nitric acid for conductivity, etc.) at a concentration that will give you a measurable response within the linear range of the detector.
4. Run a gradient and see how long it takes the change to get to the detector.

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If you simply ran a concentration gradient (as suggested above), then you use the same salt, but return to the original concentration. No equilibration time issues involved; you simply have to wash out the column. 10 column volumes is a good rule of thumb.

If you have run a pH gradient or otherwise changed the ionic form of the column, then you need to convert it back to the starting form (another reason why I dislike pH gradients! . Since the starting form is usually more weakly bound, you should run a higher concentration until conversion is complete. How high the concentration (and how many column volumes) depends on the difference in binding. Check someplace like the Bio-Rad web site for information on converting ion exchangers from one form to another.

chembot,

First off, I'm in total agreement with Tom that in general pH gradients are to be avoided unless this is the sole basis for the separation.

First of all, whether it be SAX or SCX columns, should the "rinse mobile phase" that restores the stationary phase resin to it's native state possess weaker or stronger exchangers than that of the mobile phase used for separation? Should it be more concentrated or less? Should the pH be the same or does it matter?

But, let me try to answer some of the questions posed above as they weren't totally addressed by Tom's answer:

The best option is to use the same eluent ion that is used in the mobile phase as in this case one can avoid re-equilibration conversion from one form to another during the elution process and during the regeneration process. If, for some reason, you need to change the eluent ion (here, I mean the eluent ion opposite in charge to the fixed sites in the ion exchange resin) during the regeneration process, the decision as to whether or not to use a high affinity ion or a low affinity ion depends upon the specifics of your method. A high affinity ion in the regeneration process will more effectively remove any contaminating species not eluted by your eluent but will add to the amount of time required to restore your column back into the form necessary for your next separation. If you use a low affinity ion in the regeneration process, this will slow down the regeneration process but speed up the column re-equilibration process. Depending upon the specific analytes involved, the lower elution power of the low affinity ion can be offset by increasing the ionic strength, so whenever possible this option would probably be the preferred one. But, as I said, the best course of action is to stick with one eluent ion throughout the entire separation.

Another point to keep in mind regarding pH is that the situation is significantly affected by whether or not the buffer ion has the same charge as the mobile phase in ion (in which case it may or may not concentrate in the an exchange material and render the stationary phase pH significantly different than the mobile phase pH) or the buffer ion has an opposite charge to that of the mobile phase ion (in this case the stationary phase will have no pH control). Using a buffer ion which is not retained by the stationary phase will give you significantly faster re-equilibration characteristics but as mentioned above will leave you without control of the stationary phase pH.