I am currently studying Individual and joint effect of PAHs and surfactants on CYP 1A enzymes in earthworms, looking at the EROD activity using the UPLC. My current mobile phase is K-phosphate/methanol (55:45) pH 2.5, though the method works, K-phosphate is not compatible with the column because of its crystallisation. I have not seen any other literature that has used a different mobile phase yet, so my first question would be: why is K-phosphate used as a constant mobile phase, and what other mobile phase can I use, bearing in mind that tris and hepes are no good either because they interfere with the mass spec.
K-Phosphate has a pKa that is close to the required pH of your mobile phase. You can use Na-Phosphate instead but you will have to more Phosphoric Acid to get to pH 2.5. However, both K and Na ions are very soluble. You have not described what is your precipitate.
Be sure both your mobile phases and samples are ultrafiltered (~0.1 um).
What concentrations of potassium phosphate are you using? Have a look at this article to see what is maximum solubility. Potassium salts are more soluble in organics than sodium, and ammonium salts are far more soluble than potassium. A pH of 2.5 is near the borderline of damaging a silica based column.
http://quimica.udea.edu.co/~carlopez/cromatohplc/buffer_sol subility_2004.pdf
Please check any precipitation in your mobile phase after sitting 1-2 days. If you see some crystallization on the bottom, then its concentration is too high. I did see it before when the organic solvent was ACN rather than MeOH. Usually, 20 mM K-phosphate should be good enough.
Thank you all for your answers, we are using 10mM of K-phosphate/methanol (55:45) (0.1um filtered) and so far we have no precipitation.
I surely agree that K-phosphate or Na-phosphate is difficult to use with methanol. Therefore, I have previously used acidic-Na-phosphate (0.1 M, pH 2.0)-2-propanol-ethyleneglycol (three phase system; please see file, Lysozyme by RP-HPLC). I have used Na-phosphate instead of K-phosphate only due to lower cost.
The three phase system is very widely applicabe (please see files; PTH Roma RP-HPLC and Netherlands biotin).
Its not clear to me how your phosphate eluent is compatible with mass spec. Can you clarify what you mean by K-phosphate is not compatible with the column because of its crystallization when you later say it does not precipitate.
In most cases analytes are separated from sample matrix and major buffering is not needed. 50 mM formic acid will get you the same pH in water and will evaporate totally before a mass spec although it is hard to say what the pH will be in the hydrorganic medium. If u want ESI-MS Compatibility, thats what I'd try. At that pH and concentration it will have sufficient self-buffering and will work wee with positive ion ESI-MS.
Your experimental condition supposed to be described in detail for adequate suggestions. For the better choice on mobile phase, the name of anionic surfactant with pKa, 1st. CMC, 2nd CMC need to be shown in advance. Let me agree with Prof. P. Dasgupta's suggestion for your next experimental pathway.
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