desalting a zwitterion compound by HPLC?

[smiley]

i have a small zwitterion compound that has tertially amine and carboxylic acid on it, there is some citric acid and sodium ion in the crude. so i need to get rid of the citric acid and sodium salt part to obtain pure compound in its natural form. is HPLC the best way to do it or other better way?
i test the solubility today it dissolves well in water with methanol, but not well with dichloromethane, do this mean the phase extraction will not work? usually the salt part stays with aqueous phase, now the compound does not seem to go into non polar phase.

[Chris Pohl]

I'm not sure if you'd be interested in doing this but one way to deal with the sample type you describe is as follows: pack a column with high capacity anion exchange resin in the hydroxide form (you don't need particularly high chromatographic efficiency for this, so self-packed columns will work fine). Under these conditions, your target compound will be anionic. You'll need to calculate the size of column required such that total number of equivalents for all anions in your sample is no more than 80% of the column capacity. When you pass this solution over the resin, the sodium should pass through unretained. If you then elute with a weak acid (say, for example, acetic acid) you will elute the zwitterion first, leaving the citrate behind since it is a stronger acid. You can then strip off the acetic acid to leave behind purified zwitterion.

[smiley]

Chris,
what concentration of Acetic acid shall I use when wash it off?
i have about 4 grams crude. how much resins do i need or the resin reusable?
i am new to this.

[Einar Ponten]

I think Chris procedure will work although some development work is required.

Another option is to use a zwitterionic stationary phase in HILIC mode (ZIC®-HILIC). The sodium ions will be unretained and your zwitterion is likely to be separated from citric acid at any pH and I think you can do this on a SPE, although some development work is required

[Uwe Neue]

A lot depends on how hydrophobic your zwitterion is. If it is very hydrophilic, than HILIC (as suggested by Einar) might be a good approach. If not and it is reasonably well retained in reversed-phase, a standard method might work. Zwitterions tend to be roughly 5-times better retained at either acidic or basic pH, where one of the ionic functions is turned off.

[Uwe Neue]

I should have added that either low or high pH can be achieved with additives that can be evaporated easily. Basic: ammonia, acidic: formic acid.

[Chris Pohl]

Smiley,

Sorry I didn't get back to reading your question earlier. Presumably, by now you've already dealt with your purification problem. But just for the sake of completeness:

1). The concentration of acetic acid can be adjusted over a wide range but should take into consideration the capacity of the resin. 2-3 mLs of a 1 molar solution will convert 1 gram of resin during the elution step.

2). The capacity of such resin is typically in the range of 1.8-2.3 mEq per gram. Since I don't know the molecular weight of your compound I can't tell you how much you can load but I doubt you'll want to load more than half the capacity of the resin bed with sample.