Characterization of synthetic carrier ampholytes for isoelectric focusing

The synthesis of carrier ampholytes suitable for isoelectric focusing is described. The mixture of hexamethylenetetramine (HMTA), triethylenetetramine (TETA), tetraethylenepentamine (TEPA) and pentaethylenehexamine (PEHA) ampholytes closely resembles commercial Ampholine, and covers the pH range 3-9.5. We have been able to detect focused ampholytes in a gel slab, taking advantage of their different refractive indices, and to assess their relative amounts along the pH gradient. PEHA ampholytes contain up to 20% of chromophoric structures, with two UV peaks at 368 and 315 nm, in a pH-dependent equilibrium, associated with a very weak nitrogen function having a pK of 1.1. This could be the pK6 of the last amino group in PEHA. However, NMR spectra failed to reveal any nitrogen heterocyclic structure formed during the synthesis. This mixture of ampholytes exhibits good conductivity, produces smooth pH gradients and allows sharp protein separations in the pH range 3-9.5. Their synthesis is very easy and their cost is extremely low. Their availability sould make feasible large-scale preparative isoelectric focusing, and attract more interest to continuous-flow techniques, where large amounts of ampholytes are required.