IP REAGENTS

[Philip]

Hi all,

Wondering if you might help. Two questions:

1. Would decreasing alkyl chain length (Sodium dodecyl sulfate to sodium octyl sulfate) decrease retention time?

2. Why would one want to vary alkyl chain length and concentration at different organic concentration? For example, sodium doecyl sulfate is to be used in ~5-25mM concentrations for 40-60% MeOH range. If I am using a much higher concentration of SDS, e.g. 40 mM++ in ~30% MeOH, and it is fully soluble, is there any problem with this?

Thank you in advance for your help.


Best Regards,

Philip

[Mark Tracy]

1. Yes. A shorter alkyl chain will give shorter retention time.

2. There is no problem with solubility. The main reason to play with ratios of IP and organic is to change the selectivity. Increasing the IP will cause oppositely charged species to elute later, with little effect on neutrals. (It may also increase the loading capacity for the method.) Increasing the organic will cause both to elute earlier.

Ion-pair chromatography can be a jungle.

[DR]

Correct me if I'm wrong, but doesn't chain length also have something to do w/ pH range (buffering at different pH ranges requires different chain lengths)?

[Mark Tracy]

Not really. IP reagents are typically alkane sulfonic acids or perfluoro-carboxylic acid and these have pKa <1. pH does not affect the ionization of the IP reagent. pH does affect the net charge of the weak base analytes, and lower pH increases retention. So if you want to maintain the retention time while lowering the pH, you would use either a shorter IP or higher organic in the mobile phase.

You can also use ionic strength to alter retention. Higher ionic strength shortens retention of charges species, and slightly increases retention of neutrals.

[AA]

As Mr. Tracy nicely pointed out "ion-pair chromatography can be a jungle". I would only add that as you begin to experiment with these various chain lenght IP reagents, you must be VERY patient and make sure your column is equilibrated and all remnants of the previous IP are gone and the new one is fully in place. You will find that you will have to be much more patient than you want to be, or you will never be able to duplicate the result. I have to tell my self this over and over when I am forced to use ion pairing (and I only use it as a last resort).

AA

PS- Jungles are full of snakes and tigers and unknown nasties, be carefull.

[Mark Tracy]

A piece of advice I used to give for people optimizing amino acid analysis, and I think it applies here: Don't change anything until you have seen the same chromatogram twice in a row.

[ym3142]

Mark, I do not have the Snyder HPLC book with me but it does describe a good percentage range of organic to a specific IP length. So why the range of methanol is 10 to 40% (a fake number) if IP=octanesulfonic acid? In other words, why no selectivity beyond this range?

[Bruce Hamilton]

Mark, I do not have the Snyder HPLC book with me but it does describe a good percentage range of organic to a specific IP length. So why the range of methanol is 10 to 40% (a fake number) if IP=octanesulfonic acid? In other words, why no selectivity beyond this range?

My understanding is that the range is limited by the uptake of the IP reagent onto the column stationary phase in the specific solvent system. As you vary the solvent, the ion pair retention changes, and the retantion behaviour goes outside the optimum. Once you reach 40%, the retention is probably so low that higher % don't significantly improve the chromatography.

It's not that you can't use higher methanol content if you want to, just that the retention still decreases, and probably will adversely affect your separation.

Bruce Hamilton

[tom jupille]

ym3142, I think you are misinterpreting those nomograms. They were originally published by Gyula Vigh et al at Texas A&M (referenced in the book; I'm away from the office right now and don't have the reference handy) as part of a systematic strategy for developing separations of mixtures of neutral and charged analytes.

Essentially, it suggests ignoring the charged compounds at first and optimizing the % MeOH to get a reasonable k' range for the neutrals. The nomograms you referred to then gave recommendations as to suitable IP reagents and their maximum concentration; in practice you would start with a lower concentration and increase it until you obtained the necessary retention and selectivity.

My understanding is that the concentrations shown are the upper end of linearity in the adsorption isotherms (i.e., they represent the maximum concentration at which the charge on the stationary phase is proportional to the concentration of IP reagent in the mobile phase. They are also safely below the CMC (critical micelle concentration) values, so you don't have to worry about that.

[ym3142]

Tom and Bruce, thank you very much.
if I understand correctly:
1) stationary phase charge increases with the increasing IP concentration in the mobile phase;
2) saturated stationary phase charge increases with decreasing organic and the increasing IP chain length;
3)saturated stationary phase is achieved at lower concentration of longer chain IP than shorter chain IP;
4) retaining ability is reach its maximum when stationary phase is saturated by IP; after that retention is shortened due to counterion competition;
5) figure 7-13 of Snyder HPLC (p326) shows the conditions, i.e. IP chain length matches organic percentage, where a significant IP uptake can be achieve using reasonable IP concentration under different mobile strength.