Dear all,

I have been using ion pair reagent (sodium hexanesulfonate) in HPLC analysis. Is this reagent agressive and would cause damage to the column's stationary phase? I read thru the catalogue that some suppliers may introduce the products which is so called resistant to ion-pair reagent e.g.Alltech Adsorbosphere HS column? Please advise.

Sodium hexane sulfonate is not chemically aggressive, but some care needs to be taken when developing methods with it. First, I would dedicate whatever column you're using SHS with to be run with mobile phases containing ion pairing agents only. Depending on the column you use, how you treat it, and the method you use, you run a good risk of permanently changing the column once you expose it to SHS. With that said, there are things you can do to minimize any problems.

First, allow a significant equilibration time when you set up your run. 30-100 column volumes or even more could be necessary depending upon the chain length and concentration of the ion pairing agent.

Try to avoid gradient methods if at all possible. Gradients can cause spurious peaks and re-equilibration can be lengthy if the gradient is too steep and the SHS conc is too low. Your chances of having reproducibility problems (not to mention spurious peaks) goes way up if you go wrong here. It CAN be done, it just takes careful planning and patience.

Make sure your MP is otherwise well buffered and at the pH where the IP agent will do the most good. Typically with SHS you'll want to run in an acidic MP - check the pKa of your analyte. If you get the pH wrong, there will be no benefit to the IP agent.

Rinse your column well after use with an IP mobile phase. Typically, you can rinse with an water/organic mix similar in composition to that of your MP in order to remove any buffer salts, then rinse with a strong organic to elute the IP agent. For example, if your MP is 50:50 3mM SHS, 100mM phosphate, pH = 3.5:MeCN, rinse with 50:50 water:MeCN, then with 90-100% MeCN.

IP's are really handy if you know how to manipulate them. They can be used to separate basic compounds that are really hydrophilic on a standard RP column very well. I've also had success using IP agents to move basic peaks that were interfering with other analytes.

The most recent edition of Snyder's Practical HPLC Method Development (1997?) has an excellent chapter on the application of IP in LC. Read it, live it, love it.

If I can help any further, just holler.

Chris

Chris,

Thanks for your suggestion. I know from some guys that the RP column should be kept in the MP with IP reagent when storage in order to shorten the equilibrium time. It is however contrary to what you have just said. Are they correct? For method development work, do you have any suggestion on how to start with? Also I'll try to get some time reading Snyder's book. It's an excellent reference. Thanks again.

Uberto

Uberto,

Hmmm...I just saw the same thing. I think Uwe's right for the short-term storage of a column - overnight or a few days at most. Leaving the IP agent will certainly reduce re-equilibration time. However if you're going to store the column for a week or more, I would recommend flushing the IP agent off the column and re-equilibrating "from ground zero" on your next run. I think that's a reasonable compromise between efficiency and safety.

If you need anything further, please holler!

Chris

Chris is right. There is nothing wrong with taking a column out of the ion-pairing system. The only problem is the reequilibration. This is why I would not want to do this every day. In addition, I would think that ion-pair chromatography is much more forgiving than standard RPLC, meaning that retention and resolution do not depend as much on a perfect bonding level as in standard RPLC. This means that I would be less concerned about changes happening to the column with storage in an aqueous system. For longer term storage, say a week or longer, I would store it in an organic solvent.
To go back to the comment up front: I do not think that ion-pairing reagents are more aggressive than the other stuff that is in the mobile phase. Column life is effected by buffers and the buffer pH, together with temperature. But there is no reason to believe that ion-pairing reagents themselves are agressive.