Chromatography Forum

Dear members of this forum,

Yesterday I have an assay where I do not like the composition of the mobile phase.
It consist of: In 250 ml of distilled water, add 3.86 of perchloric acid and neutralize with about 4 ml of sodium hidroxide 40 % (w/v) and after add sodium hidroxide 1 N until pH 10.90. Finally add distilled water up to 350 ml and add 650 ml of methyl alcohol and 4 ml of triethylamine. The column is a RP-18 (not protected of high pH).
I refuse to do this mobile phase and decided to answer to the forum because I like to preserve the columns as soon as posible and I think the high pH of 10.90 is very dangerous when all the manufacturers advice that the pH range should be 2.50 - 8.00 and others reduce the range to 2.5 - 7.00.
I think the mixture of perchloric acid and sodium hidroxide is making in situ the sodium perchlorate salt but this compound is neutral in water because it is a mixture of strong acid and base and do not understand the high pH.
Thanks in advance for your help,

Diego

High pH dissolves your silica backbone, which ultimately will lead to the collapse of your packing sooner or later. This depends what conditions you use such as eg. kind and conc. of buffer, temperature, %B etc. (if interested look into a series of Kirkland papers dealing with this topic.)

On the other hand there are phases (even of the silica range), which seem to be markedly more stable @ high pH. Not for an advertisement but X-Terra or the later generation BEH phases from waters. Also Kirkland has shown that other silica based phases (e.g. stable bond) when using the right conditions can be used for quite a while @ pH as high as 11. Other phases based on Zirconia (look up papers by Peter Carr) or polymer phases are chemically stable (however, there may be a lack in efficiency.)
So if you want to perform high pH chromatography purchase the appropriate column.

Why do you think you have to use high pH at all?

Hallo again:

• Stability of silica-based, endcapped columns with pH 7 and 11 mobile phases for reversed-phase high-performance liquid chromatography
Journal of Chromatography A, Volume 762, Issues 1-2, 21 February 1997, Pages 97-112
J. J. Kirkland, J. W. Henderson, J. J. DeStefano, M. A. van Straten and H. A. Claessens

I'll answer one of your questions. Recall what a titration curve looks like for a strong acid and strong base. A tiny addition of acid or base changes the pH several units. So, there is little chance that preparing sodium perchlorate by neutralizing perchloric acid is going to result in an exactly neutral solution. Then you add the amine which is a fairly strong base. It is this amine that results in the high pH of the final solution. It all seems like a very labor intensive way to prepare sodium perchlorate when one can simply buy it.

Who knows what it means when a column manufacturer says to use a column below pH XXX? Once methanol is added the pH will change. Furthermore pH 10 in water very likely will behave different from pH 10 in methanol /water. So, unless column stability studies have been made in a particular solvent mixture, you can't extrapolate pH stability predictions from one solvent system to another. While a pH 10 aqueous solution may dissolve a silica cloumn, a pH 10 solution of an amine in methanol may, or may not.

Finally let me point out again that Kirkland's stability studies for various buffer systems was flawed. He failed to measure the pH is the solution he acutally used for the stability study and this pH was much different from the aqueous pH he measured. Hence, his conclusions from these studies is incorrect.