Chromatography Forum

hi all

I keep reading that the salt content in buffers for mobile phases should be at least 10mM however i have only read this on various websites . Are there any reputable published reviews or papers or reviews that anyone knows of which explains why this should be ?? What is the reasoning behind this anyway ? as i sometimes wonder if this is a random figure plucked from the air

Friend,

Perhaps there are some reviews or good articles somewhere about buffer concentrations and their effect on HPLC separations. In practical terms, the usual recommendation I give is not to use any more than 0.02M unless there is a good reason.

All the practical reasons are as follows: you always need some buffering capacity if you add a buffer. Very low values, lets say 0.005M tend to be weak in this consideration. More than 0.02M may give you solubility problems in aqueous/organic mixtures. It is rare that anyone employs more than 0.05M, and if the reason for it is not explained, the probably the figure was just pluck it as you say.

Chromatographies other than RP can use larger concentrations to control sample elution or sample solubilities.

Good Luck,

josebenjamin

There is a fairly extensive discussion of buffers for reversed-phase LC in Chapter 7 of the Snyder, Glajch, & Kirkland textbook Practical HPLC Method Development (ISBN 0=471-00703-X), which elaborates on josebenjamin's post. They don't cite specific examples, but recommend buffer concentrations between about 5 and 50 mM as adequate in most cases.

In our Method Development course, we have one figure (see below) illustrating the problem of insufficient buffer. The figure is at least 20 years old (I don't have the original reference, but I think it came from a DuPont Zorbax applications note). Presumably the tailing in this case is caused by secondary interactions with the silanols.

Reversed-phase separations in general are fairly robust with respect to changes in buffer concentration, so there is usually little to be gained by optimizing; most people just pick a "reasonable" value (like 10 mM or 25 mM).

In macromolecule LC one often sees much higher buffer (frequently + "neutral " salt) concentrations, even 0.3M not beeing uncommon. Here one may also need to control ionic strength and other factors. Again, this is not always optimized for each case, but experience from other fields is sometimes transferred.

The buffer concentration needs to be sufficient to accomplish the purpose for which it was included in the procedure. Presumable the buffer is there to either put the sample components or the the column in some desired state of acid/base equilibrium.

Here is a summary of what the buffer may be there for:

Adjust some acid/ base equilibrium in the sample. This need may present the biggest challenge to buffer capacity. For example the sample could be a saponification of some ester, in which case it would be highly basic. It could be necessary to separate the acid components of this sample at low pH (keep them undissociated). Rather than tediously adjusting the sample pH it is common to either underneutralize or over neutralize the sample and rely on a large buffer capacity of the eluant to make the spearation work, as well as protect the column from acid or base attack.

Adjust some acid/ base equilibrium of the column, eg silanols. The column is exposed to large volumes of buffered eluant so it does not take much buffer capacity to hold the column contents at some desirable pH.

Provide ionic strength.

Additional consideration: Buffer capacity depends on BOTH concentration as well as how far from the pKa of the acid constituents the buffer was prepared.

It should be obvious why there is not one one concentration that is BEST for all separations.

bottom line, use as little as you can get by with and still protect the column and get a good separation.