Several times each month I am asked how to "correctly check and adjust the pH of an HPLC buffer solution which has an organic solvent component"? Well, the answer is to always check and adjust the pH of the purely aqueous solution first. Only pure aqueous solutions can be correctly adjusted for pH in the laboratory. Do not mix any organic solvent into the water based solution until after you have correctly adjusted the pH. The addition of an organic solution will throw off the final reading. Once the aqueous portion of your solution has been correctly adjusted to the desired pH value, then you can mix the solutions (or run an organic solvent gradient against the aqueous portion) as needed.
*This procedure also serves to make sure that all solutions used in chromatography are prepared in the same manner. It is true that the pH of the final mobile phase mixture (aqueous and organic mixture) may not be the same anymore, but the prepared stock solutions from which they were made will be the same each time, insuring reproducible results. Developing and describing chromatography methods and procedures which are highly reproducible equates to good scientific technique.
This makes sense and now everyone in our lab knows how to do it right. TY for providing these great tips. You are the expert.
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