By Anonymous on Thursday, June 3, 2004 - 10:19 pm:

please sombody tell me what should be the differnce in pH of mobile phase and Pka of a compound in reverse phase hplc for better separtion and analysis.

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By A.Mouse on Thursday, June 3, 2004 - 11:54 pm:

For good separation, anything is fine. If the mobile phase pH and the pK of the compound are too close (less than 2 pH units), you need to be careful with the pH adjustments to get reproducible retention times. That's all.

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By bill tindall on Sunday, June 6, 2004 - 05:22 am:

If you want to keep an acid protonated during injection and separation (to get good peak shape and retention) then eluant pH should be about 2 units lower than acid pKa. So long as the compound and column are stable more than 2 units is acceptable.

For acidic compounds that have inadequate resolution one can diddle with the eluant pH and maybe get them separated by taking advantage of their pKa difference. Usually this must be done empirically because the relevant Pka data is unavailable (need pKa in actural mobile phase composition not their water pKa)