I have recently run into a couple of HPLC Ion-Pair methods which involve gradient elution. I have always run IPC under isocratic conditions, so this is new to me. I was under the impression that the concentration of the IP reagent should remain constant, but in these gradients, the %B is ACN with no IP reagent added. Specifically, the IPC does NOT involve TFA or HFBA, but does involve sodium perchlorate in one example and 1-decanesulfonic acid, sodium salt in the other. Shouldn't the IP concentration be the same in MP A as in B? Do these gradient IPC methods make chromatographic sense? Thank you.

Gradient IPC is not normally recommended because column equilibration times can be significant. This is more of a problem with more hydrophobic reagents like decanesulfonic acid. I don't think perchloric acid/perchlorate are IP reagents

It is not mandatory to have IP reagent in the orgainic, but depending on the wavelength sometimes it helps with the baseline. It will also change the amount of IP reagent that is on the column and will therefore affect selectivity. So you can't just add it to the organic if the method was not developed that way.

At the initial conditions, the column will load to some equilibrium concentration of ion pair reagent on the column then when you execute the gradient the equilibrium will shift and the column will start releasing ion pair reagent. This will occur whether you have IP reagent in the organic or not.

Make sure you are adequately equilibrating the column and that your retention times are stable. To see what is going on you can also run a couple of blank gradients without a column at a short wavelength where the IP reagent will have some background absorption. Then turn autozero off and run a couple of gradients with the column. Good luck.

Thanks, Tom. These are very useful insights. I am not looking to change these methods, but just looking to get inside the head of the original developer so that I could understand the process a little better. Thanks, again.

Kevin

I was under the impression that we use IPC in case that our analyte doesn't "come out" of column due it's high polarity. Please correct me if I'm wrong but if you use alkaline enviroment, pH~8, and start the gradient from Buffer to AcCN shouldn't do the same as by using the IPR and start from AcCN to Water?

Chempara,

In the first place, ion pair is used to enhance retention of ionic solutes which otherwise exhibit little, if any, retention under standard conditions. Since the original questioner was using a sulfonate ion pair reagent, it is possible that working at high pH might be an alternative but the analyte would have to be a fairly weak base for this to work. Typically ion pair is applied in cases where I am suppression provides inadequate retention. For analytes with a pKb greater than 8, ion pair would be a better choice.

Anonymous (original),

Generally, as mentioned above, operating in the gradient mode is not the greatest idea with ion pair as the reequilibration time can be fairly long in this mode. However, I agree it does sound a bit unusual to make no attempt to maintain a constant feed concentration of ion pair reagent. Perhaps, the thinking was that increasing solvent concentration would reduce the amount of adsorbed ion pair reagent and so keeping the feed concentration constant is unnecessary. However, using this approach will only increase the reequilibration time.

In the case of perchlorate (which is not a true ion pair reagent in that it doesn't adsorb to any substantial extent on the surface of the reversed phase column except perhaps in the case of purely aqueous eluents), there shouldn't be significant reequilibration issues since there is very little adsorption of perchlorate in the first place. Gradients in this mode are fairly common. Even so, I would favor keeping the concentration of this reagent constant during the gradient.

Chris has a good idea. Instead of ion pairing the basic compound, you can get excellent retention by making it non-ionic. Use a column that is compatible with this, such as the XTerra column. Then set up an alkaline mobile phase such as an ammonium carbonate buffer at pH 10. Under thes conditions most bases are not ionized, and you get about a factor of 30 higher retention than in the acidic pH.