The ideal range for potassium phosphate monobasic is +/- 1 from its pK of 2.1, but what happens as the pH range pushes to 5.5 besides losing buffer capacity? When we mix a 25mM buffer with about 30% methanol and 10% THF, it appears to precipitate over time in our C8 column causing pressure increase. Washing the column in acetonitrile/ water restores the column.

Is there a practical range outside ideal buffering pK range without impacting solubility with organics?

A quick question or two- why would you have a phosphate based system at pH 5.5? Why would you want the pH of your mobile phase to be "outside ideal buffering pK range"? If you don't want the buffer in it's proper buffering range, then it is simply acting as a salt in your solution (I would think) and you might be better off using plain water - no ppt there!

I can't definitively answer your question regarding the relative solubility of phosphates at various pH's (sorry), but I wouldn't think that pH would have a huge effect on the solubility of phosphate in water - organic mixtures. Could it be simply that your phosphate concentration is near the solubility limit in your particular water-organic mixture, regardless of pH? Have you tried varying the pH to see if the precipitation is pH dependent?

I would humbly suggest using an organic acid buffer (formic, acetic, etc...) if you're running at pH 5.5. You'll most likely lose the precipitation problem, which I'm sure is wreaking havoc on your column, and get better buffering.

Good luck!

Answers to your 2 questions:
1.) We inherited this "control method" in which the salt does affect resolution of a couple components per the validation documents; however, there is no mention of pH control of the separation. So you're right in that we only have the salt for selectvity issues.

2.) I have routinely used 50mM phosphate at pH 2.5-3 with up to 70% acetonitrile and never had problems like we experience with this blend. And supposedly, methanol should afford higher salt solubility than the acetonitrile. But THF may affect the blend, and I wonder if the pH is an aggrevating factor.

Larry J.

I don't have a solubility figure specifically for your conditions, but let me ask a few questions for clarification:
when you say 25 mM concentration, do you mean in the aqueous component of the mobile phase or in the total mobile phase. If it is per total mobile phase, I bet that your concentration may be borderline. If you are talking about the aqueous part only, I do not see the problem either. We have used such concentrations at pH 7 and 70% acetonitrile or methanol without difficulties.
This gets me to the next question: why do you think that the pressure increase is due to the buffer and not something else?

Uwe,

It's a guess that buffer is the culprit since washing with 50:50 acetonitrile/ water alleviates the blockage and pressure is restored to normal when the mobile phase is restarted.

As for your use at pH 7 with high acetonitrile, again the pK2 for phosphate is 7.2 with an ideal working range of 6.2-8.2, which returns me to me original question of solubility with the organics outside the ideal pK ranges.

thanks,
Larry

Larry,

The solubility of inorganic salts in mixtures of water and organic solvents is quite dependent upon both the amount of organic solvent and the type of organic solvent. I don't think the problem has anything to do with where you are in the buffer range per se. If you take phosphate for example, phosphoric acid is relatively soluble in solvent rich mobile phases. However, as you increase the cation concentration, the solubility of phosphate salts can decrease dramatically but this has a lot to do with the choice of the cation used to adjust the pH. Ammonium salts are more soluble them potassium salts which are more soluble than sodium salts for solvent containing mobile phases. Furthermore, organic amine salts are even more soluble in organic solvents.

THF is relatively intolerant of electrolytes and is undoubtedly the cause of your problem. The fact that pressure builds up slowly is probably due to formation of supersaturated conditions caused by addition of the THF. Since the buffer system is borderline stable, you could probably fix the problem by switching to the ammonium salt or by reducing the concentration of the buffer to 20 mM

The question of solubility of common buffers in mixed organic/aqueous solvents came up recently in the course of a project we're working on. I couldn't find the information I needed in the literature conveniently and ended up having to do a set of quick-and-dirty experiments to get the answer, but I would be really surprised if the data aren't out there *somewhere* (a dissertation, maybe?).

Does anyone know if/where the data are available (specifically, solubility of potassium phosphate buffers as a function of organic solvent type/concentration and temperature)?

-- Tom Jupille