Column problem ????

[ltnguy3]

My MP composition: 45% 0.05M KH2PO4 (pH=4.55 with 0.1MKOH) and 55% ACN. FR= 1.5mL/min. @ 225nm. Run time= 5 min. The first day, I ran my sample with this method. Everything came out fine. Peak came out at 3 min. At the end of the run, I flushed the column with ACN : water =50%:50%. Next day, I ran it again, Main peak did not show up. There was one negative peak at 2 min (-7mAU). I ran it again without column. Main peak came out early with normal base line. This column (Inertsil ODS-2) is very new. Void volume = 1 min. If any one has any ideas what went wrong with my column or my mobile Phase, please help me. Thanks

[DR]

I suspect we'll need more information - system pressures for both 'with column' runs would be a good start.

[HW Mueller]

Presumably your compound, when it saw this non-buffering brew, inverted itself and comes out negative, henceforth. ....Just a joke...
What is your compound? Is your column equilibrated?.....

[ltnguy3]

System pressures for both 'with column' runs were around 170 bars. It was Methohexital in ACN. We’ve bought the HPLC (Agilent 1100) and the column (Inertsil ODS-2 4.6 x 150mm, 5um) few months ago. I did equilibrate the column. For first few days, I ran this column under different MP ratios. Everything came out fine. And then I increased MP B (ACN) from 45% to 55%, everything was fine until the next day . The MPA (buffer) was prepared 8 days ago.

[Uwe Neue]

HW joked about it, but the problem could really be there. Phosphate at pH 4.55 has no buffering capability whatsoever. You look at the mobile phase, and the pH will change....

[ltnguy3]

This afternoon, I flushed the column with 50% ACN:50%Water for 30 min. Then 100%ACN for 1 hour. And back to 50%ACN:50%Water for another 30 min before injecting my samples again. The peak came out at 3 min again with a nice base line like before. . I hope that my problem was solved.

[Einar Ponten]

OK, the problem disappeared and that is fine.

It is not explained however. Could it be a compatibility problem with 45% 50 mM phosphate buffer and 55% acetonitrile?

I suppose that you can influence the retention of this cation by the choise of counter ion in the mobile phase. In your case it is [H2PO4- or HPO42-] and as pointed out above while working in a non buffering interval pH is uncertain as is the counter ion.

Try another pH and/or add a counter ion like perchlorate to make the ion pair more hydrophobic (to increase retention) if desired.

[HW Mueller]

Maybe there is an explanatiion?? I mentioned the equilibration, because there was the suspicion that this non-buffer could have trouble (especially if applied for too short a time) with equilibrating the ionic surface (silanols). So, maybe, Itnguy 3 had too many SiO- after his H2O/CH3CN flush which he was able to reduce with possibly a longer flush of his pH = 4.5 solution?
If that is the case his problem is not solved, he should expect, at least sometimes, strange peak shapes, etc., unless he changes to a buffer.