Chromatography Forum

1 Gram?? That's a lot!!

Are you sure that you want to use prep LC?

Even with an ideal system, I have only used prep LC for a few mg of material (impurities). For larger amounts (a few hundred mg) I generally use open column/flash chromatograhy (but this will probably not work for you since ioninc compounds tail in silica systems).

I wonder if there is such thing as prep electrophoresis.

I need more than a few mgs, it is for synthetic work. Again I am new to reverse-phase LC, but I used separate enantiomers on prep scale Chiralpak columns. I would often isolate about a gram, with loadings of up to 80mg per run. Maybe typical loadings are smaller in RP LC?
As for flash chromatography, I wish I could use it, but there is no way my compound would move on a silica column, even with high concentrations of methanol.

As for why zwitterions are least soluble at pI: maximum dipole moment and minimum net charge so inter-solute dipolar interaction is also maximum and repulsion minimum.

As to the question of C8 versus C18: If you survey across the whole industry, there is no statistically significant difference between the "average" C8 and "average" C18 because the standard deviations (in multiple dimensions) are so large that the distributions overlap. On the other hand, if you compare C8 versus C18 on the same silica substrate, using the same bonding techniques, you will find that the capacity factor for C8 is consistently lower, owing to lower hydrophobicity.

Makes sense Mark, had to digest this a little, apparently still made the wrong prediction in that I thought that the zwitter of an amino acid would come out later on a HILIC (zwitterionic, zic-HILIC) column than the cation version. Anybody out there with some info on this comparison? (Zwitter ionic peptides generally come out ahead of net charged counterparts on RP?, according to p.130 in "Practical HPLC Method Development", Snyder, Glajch, Kirkland, 1988, as I just learned).

It would have been good if you would have told everybody up front that you will need to do preparative chromatography at the 100 mg to 1 g scale. This changes things drastically, since one needs to consider the cost of the medium. You do not want to do 1000+ injections on an analytical reversed-phase column...

Based on this constraint, I recommend to develop a method on an analytical silica column either using very polar solvents or better using HILIC, i.e. acetonitrile or methanol with some water. Your problem might be the solubility of your compound in such a medium; zwitterions commonly prefer water, but this problem can be solved using at-column dilution. Contact me and I send you some literature.

To follow up on the previous post: your compound will move on a silica column, if you think beyond methanol...