ChromFAQ:PHAdjust

From ChromFAQ

What's the best way to adjust the pH of a mobile phase?

Arguably, the best way is not to attempt to adjust pH at all, but rather to prepare the mobile phase by weight. Weighing is much more precise (a 20% error in weighing translates to only a 0.1 unit error in pH). You can find an on-line calculator for buffers here (http://www.bi.umist.ac.uk/users/mjfrbn/buffers/makebuf.asp)





If you do use a pH meter, never, ever put the pH electrode in the bulk mobile phase. To measure pH, transfer an aliquot of the solution to a test tube or small beaker, measure the pH, and then discard the aliquot. Contamination from the pH electrode can contribute significantly to "garbage peaks", especially in gradient separations, as shown in the example at the right.


The second-best way to adjust pH is to blend equimolar solutions of the acid and base as necessary to obtain the desired pH. This allows fine adjustment of the pH while maintaining a constant buffer ionic strength.

The common technique of adding concentrated acid or base to a starting solution introduces variability in the procedure because the ionic strength of the final solution is uncontrolled. While ionic strength differences have only a small effect in most reversed-phased separations, they can be a significant factor in ion-pair chromatography.

In most cases, pH and buffer ionic strength are specified for the aqueous component of the mobile phase before the addition of organic modifier. It is possible to measure pH in a mixed (aqueous/organic) system, but the values obtained will be different from 100% aqueous measurements. A post by Mark Tracy (http://www.sepsci.com/chromforum/viewtopic.php?t=3780) on Chromatography Forum (http://www.chromforum.com) sums up the issues:

  • You can measure the pH after addition of the mobile phase, and it will be valid if you understand a few things. First, the pH scale is no longer 0-14 with 7 being neutral. Any given organic/water mixture will have its own pH scale, with a fixed relation to the familiar aqueous one. This means that you can calibrate with conventional buffers, and still have a repeatable measurement of your mobile phase pH. Second, the reference electrode will have a different liquid junction potential depending on the filling solution and the organic/water mixture; fortunately, this effect is usually minor and you can ignore it. Third, the pKa (and thermal coefficient) of your buffer salt will shift in the organic/water mix. This effect can be as much as one pH unit either way.
  • There are a couple of practical issues also. The liquid junction of the reference electrode may suffer from crystallization if the mobile phase is mostly organic. There are reference electrodes with more generous junction area that will work better.
  • If you attempt to transfer to another lab a method that measures pH after organic addition, be sure to specify the type of electrode you use. This will minimize the hassles for the other lab. The exact brand is not (usually) important, just the type.

For more detailed discussion of mobile phase pH issues, look at a series of artcles by Bill Tindall appearing in LC/GC Magazine (http://www.lcgcmag.com/lcgc/):
November, 2002 (http://www.lcgcmag.com/lcgc/article/articleDetail.jsp?id=38298)
December, 2002 (http://www.lcgcmag.com/lcgc/article/articleDetail.jsp?id=40823)
January, 2003 (http://www.lcgcmag.com/lcgc/article/articleDetail.jsp?id=45059)

Also check the following threads on Chromatography Forum (http://www.chromforum.com):

September, 2004 (http://www.sepsci.com/chromforum/viewtopic.php?t=779)
January, 2005 (http://www.sepsci.com/chromforum/viewtopic.php?t=1268)
March, 2005 (http://www.sepsci.com/chromforum/viewtopic.php?t=1603)
March, 2005 (http://www.sepsci.com/chromforum/viewtopic.php?t=1719)


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