Effect of Buffer on Separation

[adam]

We are working on a method to separate Minocycline from its impurities. The mobile phase we are using is isocratic 90/10 Water/THF with buffer.

We started with 150 mM ammonium bicarbonate in the mobile phase, and found that we got better peak shapes as we reduced the amount of buffer.

This didn't make much sense to me. I was wondering if anyone out there had any thoughts on why less buffer would be beneficial in a situation like this.

Thanks
Adam

[mattmullaney]

An educated guess--as minocycline is rather polar with a number of possible sites for hydrogen bonding, having so large an excess of minus-fomal charged carbonate ions may have created a "bulky" yet still polar specie in solution that would be somewhat susceptible to "fluxional behavior" as it moved through the column under flow. The carbonate ion(s) could have been "popping on and off" of the minocycline in solution in a sort of loosely-organized mass in solution, maybe a bit like a thinly viscous "colloid," causing a less-than-sharp band than one would normally observe toward the beginning of an isocratic elution. Maybe lessening the carbonate content allows more order to be achieved within this carbonate-minocycline "adduct" fomed with the column, allowing the band that is created in the chromatography to be sharper in appearance?

Keto-enol tautomerism (if an improper pH at separation is used) or some types of esters (if pH is too acidic or basic, not within say 5-7) may cause band distortion as well, not in the same way as I poorly describe above.

As I say...it's only a guess. Let's see what others will think.