IP-RP-HPLC

[Bryan Evans]

Uwe -

Thank for your input. Yes, I understand that using Xbridge allows scientists to use alkali eluent to analyze the 'neutral form' of the basic solute.
Another option is for scientists to use RP + IEX to analyze the ionic species.

Giving the scientific commmunity options is what it's all about.

[Bryan Evans]

Hans,

Thank you for your input.

Scherzo SM-C18 comes with an instruction manual. If one uses
the product within the recommended guidelines, one can expect
reasonable lifetime of this product. That's all I wanted to say.

[Vlad Orlovsky]

The pH stability is important quality/reproducibility issue. pH inside pores of the basic portion of your "mixed" phase can be different from pH of your mobile phase depending on the buffer used, pH of the buffer, concentration of the buffer and buffering capacity. In ACN/water (pH 7) pH inside pores of silica with amino group on the surface is going to be well above 7 (closer to 10) which will cause this portion of ligand to fly off the surface and "travel" to the particle with acidic sites, where it will be trapped, thus changing selectivity of the column even if you use it within recommended pH range (pH 7). You need to stay in acidic buffered pH within recommended pH range and I would suggest to stay below 7 (at least pH 6.5). Unless this is a "hybrid" material like Waters and Phenomenex the hydrolysis of "amino" ligand in certain mobile phase (pH 6.5-7) is inevitable event, no matter how well material is “blendedâ€

[Uwe Neue]

Dopamine...? Interesting beast. Should have overlapping pKa values of the phenol group and the amino group, so it should be a zwitter at pH 9 to 10. I do not recall a study on the loadability of a zwitter, but I bet that it will have a better loadability when it is doubly charged. This would also be the spot where it has the lowest retention. So you will need to play with the conditions to get it right...

[Vlad Orlovsky]

Uwe I was hoping that phenol would not have too much effect at pH 10. We tried pH 10 and there is no retention even with 0% organic, I probably need to move to something simple (benzylamine for example)

[Vlad Orlovsky]

Uwe,

I switched to purely basic molecules benzylamine (pKa 9.3) and phenethylamine (pKa 10.3). Mobile phase is ACN/water/ammonium carbonate pH 10.7. No matter what I try peak shape is very broad with low plate count (less than 300 plates on 150 mm column packed with 3.5 um silica gel). Neutral compound shows 13,000 plates on the same column. I tried Gemini and xBridge and got the same picture. If buffer concentration and amount of ACN is increased peaks gets a little better but compounds will elute at 3 min (K’ around 1). Would you please suggest what to do? I am trying to set up a fair comparison on loadability but with such low efficiency nothing can be done.
Would you please send me your article on loadability from your previous post? I want to see what compounds were used in the study.

[Uwe Neue]

Strange...

You say "ammonium carbonate". You should use ammonium bicarbonate. Check if this is the problem.
Also, you did not say what the buffer concentration was. Should be 10 mM or more.

[Vlad Orlovsky]

it is bicarbonate, sorry. I tried 10,20 and 40 mmol. retention drops with peak eluting close to the voide when 40 mmol is used in combination with 20% ACN

[aldehyde]

This is a really interesting thread, lot of effort posts!