Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.

Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.