In an earlier blog article, we looked at the effect of trifluoroacetic acid (TFA) concentration on retention when the mobile-phase pH was controlled by adjustment with TFA. Much of the time, especially at low pH, you can get consistent results by adding acid or base to the mobile phase to obtain a specific pH. However, this isn’t an ideal case, and may not work in many HPLC methods. A better way to control mobile-phase pH is to use a buffer. When a buffer is used properly, the mobile-phase pH will stay constant even when assaulted by the sample or injection solvent. For this reason, buffered mobile phases are strongly recommended for most reversed-phase HPLC applications.
Remember back to your first-year chemistry class… buffers are effective ±1 pH unit from their pKa. Let’s use phosphate, the most popular buffer for LC-UV work, as an example. It has three pKa values: 2.1, 7.2, and 12.3. Silica-based columns are limited to pH 2-8 for robust operation, so the 12.3 pKa is not useful. This means that phosphate is effective as a buffer from 2.0 < pH < 3.1 and 6.2 < pH < 8.0 (assuming we limit the mobile phase to 2 < pH < 8).
To form a phosphate buffer, we mix an acidic component (usually phosphoric acid) and a basic component (usually KH2PO4) to reach the target pH. So let’s consider preparing a 25-mM phosphate buffer at pH-3.0. The best way to do this would be to make up a 25-mM solution of the base and a 25-mM solution of the acid and blend them together by adding the acidic solution to the basic solution until the desired pH was obtained. Now we have a 25-mM phosphate buffer at pH-3.0. There’s an easier way to do this, which most of us use. This is to make up the 25-mM solution of the base, then titrate with concentrated phosphoric acid until we get a solution of pH-3.0. Now we have a pH-3.0 phosphate buffer, but the concentration is not 25-mM – it will be a higher concentration because we used concentrated acid rather than 25-mM acid to make the pH adjustment. Most of the time, you will get the same chromatographic results with either technique, but there are sure to be cases when that is not true. If the separation is sensitive to buffer concentration, such as when ion-exchange or ion-pairing take place, there may be a difference in retention between the two buffers described above.
There is a third way to prepare buffer that should be avoided. That is to mix the organic portion of the mobile phase with the basic component of the buffer prior to pH adjustment. pH meters do not give the same readings when organic solvent is present as they do with aqueous solutions, so a buffer adjusted to pH-3.0 by titration of an organic-containing solution will have a different effective pH than when an aqueous solution is adjusted. Don’t use this technique of adjusting the pH after adding organic, or you are likely to obtain varying results with each batch of buffer.
For HPLC, when the pH of the buffer is stated, it always refers to the pH of the aqueous portion before the organic is added. Don’t worry what happens after organic is added – it will vary with the buffer type, but the pH shift should be consistent. With some buffers, the pH will shift up when organic is added, whereas with others, the pH will shift down. It really isn’t important to know the true pH of the mobile phase in the presence of organic. It is more important to be consistent. Besides, if you know the pKa of your analyte(s), it was determined in aqueous solution, too. When organic is added, the pKa of the analyte(s) is likely to shift, and you don’t know which way, either.
So the bottom line is to prepare your buffer either by mixing equimolar portions of acid and base (the right way), or by titrating a basic solution with acid (the easy way), but avoid pH adjustment after organic solvent is present (the wrong way). And because sometimes the method performance may differ between the first two preparation techniques, always state in the method description which technique wasused.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his ongoing research with Lloyd Snyder, resulting in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com
