Chromatography Forum

What is your theory on why the MeCN washes restored original retention time?

What kind of pump are you using? (metal-free?)

I have no theory about washing and restoring original retention times, it doesn't even restore fully...

The pump is just a regular Alliance 2690 HPLC.

A possible source for your problems could be a buildup of transition metals on your column. This certainly would be expected to interfere with ion chromatography. Also, the improvement in performance following phosphoric acid wash is consistent with the "transition metals" hypothesis.

My suggestion is to passivate your pump with 6 M HNO3. You also might try flushing your column with 20 mM disodium EDTA o/n, or else treat yourself to a polymeric (PS-DVB) SAX column.

Is a polymeric SAX column "resistant" against transition metals?
We have 14 HPLC, running all reversed phase chromatography, it is a bit impractical to passivate them one by one, just for 1 application.
Would adding EDTA to the mobile phase be sufficient?

Ace

I'm using an Hypersil SAX column with 0.33g/l phtalic acid, brought at pH 6.4.
Column temperature is 35°C.

When I injected yesterday morning my first reference, retention time was about 11.5 minutes.
After injecting 9 references (Na2SO4 in water), the retention time was already decreased to 9.7 minutes.
After several hours, the RT was about 6 minutes, and peaks are getting distorted/unseparated from other peaks in my sample.
Any ideas about this decrease, or how to get my retention back?

Could you do a test run that came with the column? If the retention times of those standard anions are significantly lower, it means that the anion exchanger has lost some capacity. Sulfate ion (or divalents) are very sensitive to capacity changes on the stationary phase and any changes in the mobile phase composition (including CO2 contamination).

Secondly, are you using the recommended mobile phase? I am not sure that a totally aqueous phase is recommended on a silica substrate. There is decent work from the Carr's group which shows that the major metallic contamination comes from the metallic frits rather than the column body (or the pumps)-so passivating might not work in your case.

The suggestion to use polymeric substrates comes from the fact the polymers can be subjected to harsh cleaning steps with acids and bases - these reagents would simply chew up a silica column.

Is a polymeric SAX column "resistant" against transition metals?
We have 14 HPLC, running all reversed phase chromatography, it is a bit impractical to passivate them one by one, just for 1 application.
Would adding EDTA to the mobile phase be sufficient?

Ace

Polymeric SAX are not "immune" from collecting transition metals, however, they don't trap them as notoriously as silica-based columns. For inorganic ion analysis, I'd tend to think polymeric SAX are more popular (by far?) compared to silica-based supports. Dionex, Metrohm, Hamilton-- all manufacture PS-DVB SAX.