Chromatography Forum

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

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

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?.....

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.

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....

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.

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.

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.