is this sepperation accepted ?

[WESAM]

For pH control, use a phosphate buffer at pH 3. This may do wonders already.
.

Thanks Uwe , just to confirm in this particular as i am not in this area at all .

The Pka of aniline = 3.46 ( as i know it a weak base so i need to go for PH =5.5 at least to make it nutral am i correct ? )

[Uwe Neue]

First of all, do not worry about the pKa.

Second, you will get higher retention and a drastic change in selectivity with a phosphate buffer at pH 6 compared to a phosphate buffer around pH 3.

[juddc]

Echoing what others said, if I had no idea what the peak between 1 &2 is, I would definitely start playing with selectivity with the aim of improving peak shape & resolution. If your lab is cash poor, the cheapest way to do that is with pH and ionic strength. As long as you remain within the pH constraints of your column and your analytes of interest, you have no worries about degradation. If working with formates or acetates, you can do some subtle (or not so subtle) manipulation of selectivity with pH, concentration of modifier, and the nature of the cation (ammonium, potassium, etc..), which can have a real effect. Depending upon how that peak behaves when you change pH or use ammonium acetate at a given pH versus potassium acetate, you can get a few clues as to its nature and these will help guide you. If your analyte is basic, perhaps TFA or TEA will help, though they come with attendant issues. After that, solvent, gradient, and temperature changes can be made and optimized.

My advice: Do a few simple experiments, changing only one variable at a time. Start with your 72% MeOH separation, make incremental changes, and follow any line that brings about improvement in your separation to it's logical conclusion.