Why Buffers?

[bgiles]

I understand the need to adjust pH so that your analyte is protonated or de-protenated to effect retention time. But why buffers. Why not simply adjust the pH of H2O.

the dude

[Mark Tracy]

buffers are the most reliable way to adjust the pH of the aqueous phase

[bgiles]

not sure I understand Mark. I add the buffer then I adjust with and acid or a base accordingly. Why not just adujst with the acid or base in water. I am allways reading how phosphtes are bad for columns, can't be used with MS. ...there must be some additional benefit from using these other than ease of adjusting pH?

the dude

[Mark Tracy]

First of all, just try setting the pH of water to 6.0 with HCl. You can't do it because the dissolved CO2 has the pH already at 5.5, so you have to use NaOH. Now wait 24 hours and see if it is still 6.0. Now ask your buddy to make it and see if you both get the same chromatogram. All these reasons are why people use buffers: they reduce variation in the results.

Phosphates have an unnecessary bad reputation. This has been explained elsewhere on the forum. The short version: when you adjust the pH to some value in water, then add organic solvent, the effective pH rises; all anionic buffers do this. This effect only affects column life under alkaline conditions.

[JA]

Basically the answer to your question lies in the definition of a buffer, and as Mark mentions, the control they give you. I've had some colleagues who like to dissolve up some phosphate/acetate/etc. salt and treat that as a buffer. I simply ask why?

[Uwe Neue]

The ionization of your analyte(s) is determined by the pH of your mobile phase. The retention of a compound can change by a factor of 30(!) from the ionized form to the non-ionized form. If the concetration of H+ or OH- in your mobile phase is not enough to control this ionization, your retention time may change a lot with the weather or with the exact composition of your sample. Plus, chances are high that you may end up with lousy peak shapes.

If you use a buffer, the pH does not wiggle, and the ionization of your analyte is constant (unless you inject so much that your sample concentration exceeds your buffer capacity, in which case you are back to square one).

So if you want reproducible and controllable retention for ionizable analytes, it is a good idea to control the pH and, with other words, use a buffer.

[Consumer Products Guy]

Uwe's an expert, so heed his advice. At my company we mostly don't use actual buffers, usually acidify the aqueous portion with acetic acid or phosphoric acid, which works fine FOR US (that's they key). Then we don't need to flush out any buffers containing dissolved solids, or keep the mobile phase moving.

[Mark Tracy]

That is a good point. Dilute H3PO4 gives pH ~2 and HOAc gives pH ~3. If those pH's work for your separation (and they do for many cases) there is no need to complicate things. But sometimes you need exact pH control, and then a buffer is the only way to go.

[Victor]

Oh no! All this stuff about "true" buffers, "real buffers" and "actual buffers" is coming out again. The reason why things like phosphoric acid work is that they ARE quite good acid pH buffers when added on their own to distilled water in reasonable concentration. Why not get hold of one of those buffer capacity calculation programs and convince yourselves... here is a supporting quotation from Bill Tindall many years ago.....

By bill tindall on Sunday, July 29, 2001 - 08:36 pm:
At high and low pH, one does not need both an acid and its salt, or a base and its salt, to make an effective buffer. These are but limited definitions of a buffer. A buffer is something that changes only a little when a small increment of acid or base is added.
Most rigorously, the "goodness" or effectiveness of a buffer is related to the slope of the titration curve of the respective acid or base, dpH/d(increment of acid or base added). So, what does the titration curve of a strong acid or base look like at the begining of the titration (small increment of acid or base added)? Flat, little change in pH. Or, do the calculation. If you start with 0.01 M HCl and add enough base to neutralize 10% of the acid, the pH stays about the same, ie it is buffered. In fact, at the extremes of pH, strong acids and bases are several times better buffers than a 0.1M mix of a weak acid and its salt at a pH = pKa.

Certainly any solution with a pH of 12 or greater or 2 or lower is well buffered, even if one got to these pH's with strong acids or bases. Now in chromatography it seems to me that there isn't much present but a tiny amount of sample and some silanols to alter the pH of a solution, so the buffering capacity needed in chromatography is small. Hence, in chromatography one could make the case that 11 and greater and 3 and lower is adequately buffered, no matter how one achieved these pH's. One can achieve these pH's with ammonia and phosphoric acid. Therefore, I would argue that ammonia and phosphoric acid are respectable buffers for chromatography in the range of 11-12 and 2-3 respectively.

So, does anonymous have a buffer when they add 10 mL of ammonium hydoroxide to a L of water? Most certainly, yes. Does it have much buffering capacity? No. Will it work? Probably, because one doesn't need much capacity in chromatography. Will it be reproducible? Most likely, and that is the main issue, assuming the pH is high enough to do what ever it is one needs to do with the base.


To quell the howls of protest in this misunderstood facet of buffering, I will provide a reference, Treatise on Analytical Chemistry, Kolthoff and Elving editors, Part 1 Section B page 458 Acid-Base Strength and Protolysis Curves in Water by S. Bruckenstein and I. M. Kolthoff.

[Uwe Neue]

Victor: I don't know what your giant "Oh no!" is all about. Neither Mark nor I said anything that would be different from what you said.

However, there are limits on how dilute a buffer can be, and that depends on the sample concentration as well as the concentration of other stuff in the sample. Think about a sample from a dissolution test with the giant amount of acid in the sample!

[danko]

Victor’s points are OK, but are not valid in protein separations, where the salts are a major part of the analyte and the solvent management with respect to their polarity etc.

Best Regards

[Victor]

Mr Neue-the comment was not directed at your posts, but at the remark from Consumer products guy, who implies that phosphoric acid is not an actual buffer.

Mr Tracy also seems to imply that phosphoric acid cannot give exact pH control.

[Mark Tracy]

My point about dilute acids is not that you can't get a reproducible pH, but that you have relatively little control over it. You could make a pH 3.3 solution of phosphoric acid, but the concentration would be ~ 0.5mM, and hence not very stable and hard to make. At pH 2.2 the concentration is ~10mM which is adequately stable and convenient to make. So if your separation works at pH 2.2, and your column can withstand it, there is no reason not to keep things simple. On the other hand, if you need pH 3.3 to resolve a critical pair, the dilute acid approach will never be reliable. (Even a phosphate buffer is stretched rather thin, and formate would be better.)