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.

Hydrolytically stable, low- and high-pI isoelectric hydrogel membranes were prepared from poly(vinyl alcohol) (PVA) as alternatives to polyacrylamide-based isoelectric membranes that hydrolyze in acidic and basic solutions. Low-pI membranes were made by attaching an isoelectric buffer of a welldefined pI value (such as iminodiacetic acid, IDA, aspartic acid, ASP or glutamic acid, GLU) to the PVA backbone and crosslinking the PVA strands, in situ. The pH in these membranes does not change significantly with slight variations in the amount of isoelectric buffer that gets incorporated. The pI values of these membranes were pI is greater then 1.7 but less then 2.0 (IDAPVA), pI is greater then 2.0 but less then 2.6 (ASPPVA) and pI was greater then 2.6 but less then 3.4 (GLUPVA).The membranes were used as anodic membranes in isoelectric trapping (IET) experiments. Sugars, cyclodextrins (CDs), and certain polyhydroxy compounds have pKa values between 11.5 and 14. Thus, high-pI hydrogels were obtained by incorporating (i) quaternary ammonium derivatives of Beta-CD (QCDPVA) (ii) quaternary ammonium groups and Beta-CD (CDQPVA) and (iii) quaternary ammonium groups alone (QPVA) into the crosslinked PVA hydrogels. All three membranes had pI values greater than 11 and served as effective cathodic membranes for the IET of small ampholytic molecules and proteins. In pH-biased IET, proteins are collected into solutions of isoelectric buffers that set the pH to keep the proteins in a charged state affording high solubility and preventing precipitation. Thus, a series of isoelectric buffers (biasers) with high buffering capacity, high conductivity, and pI values covering the useful pH 2-10 range are needed. Two sets of such buffers were designed (i) with pI values between the pKa values of two carboxylic acid groups and (ii) with pI values between the pKa values of the conjugate acid form of two amine groups. Six of these buffers were synthesized and their synthesis was optimized. The products were obtained in their pure, isoelectric form and were extensively characterized.

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.