Chromatography Forum

In our lab, we run several analysis in which the aqueous phase has pH around 1.5-1.7. In some the organic phase is 20% and in others is 80%. How can i be sure of the final pH of my mobile phase so to protect my columns?

If you are running existing validated methods, you run them as written, period. Column lifetime is a secondary issue.

If you are developing methods, the issue is complicated. Read these three articles by Bill Tindall that originally appeared in LC-GC:
http://www.lcresources.com/tsbible/20112002.pdf
http://www.lcresources.com/tsbible/20122002.pdf
http://www.lcresources.com/tsbible/21012003.pdf

nice articles(by the way there were 2 of them) but i didn't find a solution. How you chromatographers handle this issue?

Oops! I copied the second link twice. I've edited the post so all three links are correct.

That said, the answer is in the first article:

However, if I knew an acid sample was 50% dissociated at aqueous pH 5, I have no way to predict what the dissociation will be in methanol–water at pH 7. I don’t know the pKa of the acid in the mixed solvent, and, for the reasons described above, I can’t calculate hydrogen ion activity from the pH measurement. Furthermore, if I knew the sample or column was unstable at aqueous pH 7, I could not predict the stability in the pH 7 mixed solvent because I don’t know the hydrogen ion activity or the effect of the standard state change. By itself, neither the pH 5 measurement of the aqueous buffer nor the pH 7 measurement in the mixed solvent is much use in predicting retention and stability behavior in the pH 7 mobile phase.

So, the short answer to your question is "you can't". To reiterate: if you're running an existing, validated method, you run it as written, period. If you're developing a method, you try to stay within the pH limits specified by the manufacturer for that column, recognizing that the lifetime will be shorter at the extremes of those limits than it will in the middle. My mantra on that is "Columns are cheap; time is valuable."

... and of course the column manufacturers are in exactly the same position. So if they say that a column is stable at pH 9.0, they mean that it is safe to include aqueous buffers at pH 9.0 in any appropriate solvent mix you make up for use in the column.

You still can use the good old fashioned pH indicator paper. Prepare your mobile phase and put some drops on an indicatior paper and see the magic. It is not as accurate as a pH-meter, but it will give you an idea. The dyes are in the same situation as your stationary phase is: They have to decide if they want to protonated or not in presence of organic modifier.

Just two throw in my 2 cents: don't concentrate to much on the (apparent) pH of the mobile phase, also keep an eye on other parameters of the method. Type of buffer, concentration, aqueous/organic ratio and especially temperature all play their role concerning column stability. A phosphate buffer pH 9 can be more demanding for a column than a pyrrolidine buffer pH 10.5

My 2 cents: use the pH meter to figure out how much modifier you need to add by either weight or volume to reach that pH needed for the assay, then use one of those two techniques to make mobile phase in the future (document this). Those other ways are much more accurate.