Chromatography Forum

I`ve a little problem with my baseline drift. My mobile Phase contends 20mM NH4H2PO4 buffer and MeOH, the used column is a based endcapped C18. My baseline is reproduceable going up and down in a range of 10 minutes. It`s not a detector failure, I´ve checked, and I also switched between two same columns, the problem is the same. Has everyone an idea?

What is the pH of your mobile phase and how much MeOH present in your mobile phase?

Ananda

What is the wavelength for detection. If short you may detecting the methanol gradient.

What is the pH of your mobile phase and how much MeOH present in your mobile phase?

Ananda

My pH is 3,3 and the mobile phase contents 50% of MeOH. Have you any idea?

Dear St. Torres,

Without having more information, such as that requested in other replies, it is difficult to tell what the problem is.

However, I can advance some information. If you are using wavelengths lower than 220 nm, it is very likely that you will always see baseline problems. One other consideration is the solubility of ammonium phosphate in MeoOH, in my experience, when it is higher than 65% you may start having solubility problems.

When using gradient elution in systems like these, the best thing to do is to keep the salt concentration constant throughout the gradient. Another recommendation I can pase to you is to adjust the pH of this buffer with H3PO4 or NH4OH as needed.

Good Luck,

Benjamin

If I understand you correctly, you are running an isocratic method with 50% methanol and a 20 mM phosphate buffer at pH 3.3. We do not yet know what the detection wavelength is or how you control the temperature. It would also be good if you would tell us your column brand name, since "base deactivated" can mean a lot of things. Some manufacturers endcap with an amine, others base deactivate their packings by holding a bottle of base next to their reactor (I am joking, of course...)

Many years ago had a similar problem with amm phosph buffer at around 225nm. Really strange because there was a difference between making the buffer from solid ammonium phosphate or by adding ammonia to phosphoric acid to appropriate pH [much worse]. Never foudn the explanation but the problem occured with several different detectors---it was nothing to do with the colum from what I remeber.

Previous threads on the topic of buffers have established that, in most cases, use of 'straight acid' (like H3P04) is just as effective in lowering and stabilizing the pH as a 'conventional buffer' (equal molar amounts of a weak acid and its conjugate base).

It is generally acceptable to use this approach at pH's that are somewhat removed from 7, which covers the majority of cases in reversed phase HPLC (where the pH is usually 2.5 - 3.5).

By force of habit, most people use the 'conventional buffer' approach but it is far (!!) preferable to use the 'straight acid' approach (in situations where it is appropriate) as it is very easy, doesn't require wieghing or filtering, and virtually eliminates the problem of precipitation: which is what seems to be the problem in this discussion.

I think the previous thread was "necessity of buffers in HPLC" or something like that.

adam.
it seems appropriate to qualify some of your statements somewhat. A "straight acid" (if far enough from its pKa) does not stabilize pH at all in the sense of buffering (simple def. of buffering: If you add x equivalents of acid to a solution and get less than x change in H+ concentration you have buffering, if your H+ changes by x you do not have buffering, etc. for bases).
Such a non-buffering acid can, nevertheless, create robust chromatographic condition if the resulting pH is far from the pK of analyte. (Careful here, you may trip yourself if your pH is near the point where your silica is dissociating, or when you inject large amts of acid or base, as was apparently the case in a recent example).
Also, some of your other advatages listed ....????

HWM- if your "simple" definition of a buffer was universally aceepted, then I think that you are right. A strong acid does not resist pH change through the same mechanism as a weak conjugate acid-base pair.

However, a simple and more useful definition of a buffer for a chromatographer, and one that is also used by physical chemists, is that a buffer is a solution that resists pH change on the addition of acids or bases. If you look at the titration curve of an acid against a base, you can see that e.g. a solution of phosphoric acid is a good buffer. Adam- I'd forget about these definitions and carry on. Just be careful that you are aware of the titration curve of your acid. YOu can find these in any elementary text book.

Victor,
your buffer definition is exactly the same as "mine", resisting pH change means changing the solution´s [H+] less than the added [H+], etc.