Isoelectric focusing as a puzzle

The formation of strong complexes of most acidic dyes with Ampholine in isoelectric focusing has been demonstrated by altering the dye to Ampholine ratio, by re-running single dye bands and by measuring the UV-visible spectra of the complexes. These complexes can only be disaggregated by changing the dielectric constant of the solvent or by high temperatures, or both. On an equimolar basis, the order of the dissociating powers of the most commonly used disaggregating agents is dimethylformamide ≈ tetramethylurea > dimethyl sulphoxide > urea > formamide. A two-step binding model is postulated: first a strong, polydentate and undissociated Ampholine-dye salt, followed by hydrophobic dye-dye interaction.