Buffer isoelectric focusing revisited

• Published in: Journal of Chromatography A 1988-05, Vol.440, p.367-377
• Main Authors: Righetti, Pier Giorgio, Fazio, Marco, Tonani, Carlo
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Others; Various methods and equipments
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(00)94540-0

It has been experimentally demonstrated that buffer isoelectric focusing (IEF), as epitomized by the most elaborate mixture commercially available (Poly-Sep 47), cannot perform satisfactorily for the following reasons: (a) the mixture of buffers is too impoverished, amounting to less than eight species per pH unit (assuming a uniform p I distribution along the pH axis), when at least 20 amphoteres are needed per pH unit to generate a stepless pH course; (b) most of the amphoteric buffers commercially available (and present in the above mixture) have much too wide Δp K values (in general well above 4 pH units) and therefore, at pH = p I, they cannot buffer and conduct the current, i.e., cannot be utilized as “carriers”. Hence there seems to be no alternative, for a well behaved IEF system, to the random synthesis of carrier ampholytes (CA), as epitomized by the classical Vesterberg approach. In fact, the only valid alternative to conventional CA-IEF, and much superior, is the revolutionary methodology of immobilized pH gradients, as introduced in 1982 in the biochemical field.