CE determination of the thermodynamic pK_a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes‐based IEF and its numeric simulation

Abstract

CE determination of the thermodynamic pK a values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes ( pK a = pKa monoanion -pKa monocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pK a values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pK a values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pK a and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download). Abbreviations:3MHIS, 3-methylhistidine; DNS-Cl, dansylchloride; DNS-IDA, dansylated iminodiacetic acid; GLYHIS, glycyl-histidine; ICIEF, Imaging capillary isoelectric focusing; IDA, iminodiacetic acid; LAB, labetalol; LBB, Leucoberbelin blue I dye; SERO, serotonin compartment has to be filled with a base (the catholyte, e.g. 20 mM NaOH). Fourth, convective disturbances have to be absent or suppressed in the system (either by using an anti-convective medium or a capillary as the separation compartment). Fifth, a sufficiently large DC voltage has to be applied between the anode and the cathode for a sufficiently long period of time to allow any and all of the initially cationic and anionic carrier ampholytes in the mixture to migrate out of sections of the separation compartment in which the local pH value is different from their isoelectric point value. (Since Rilbe considered the isoelectric point value to be very close to the isoprotic point value-which he calculated as (pKa monocation + pKa monoanion )/2-the symbol pI, and the term pI value, became loosely-and interchangeably-used in the literature to designate either "iso" value.)Once all these conditions are fulfilled, adjacent, individual carrier ampholyte bands form across the separation compartment such that the pH in the successive bands increases Color online: See the article online to view Figs. 1, 3-5 in ...

Instabilities of the pH gradient in carrier ampholyte‐based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation

“…This mobility inequality of the species of ampholytes was mentioned as possible source of cathodic drift (see the comment of Bjellqvist on page 303 of ref. [18]), is found in tables that list mobility values of compounds [82,83] and was recently confirmed for low molecular mass UV absorbing ampholytes that were selected for the characterization of a wide range pH gradient [84]. The data of Fig.…”

“…Predicted drifts are either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species). The cation of an ampholyte has typically a greater mobility than its anionic counterpart [28,80,84]. This inequality contributes to cathodic drift and cannot be abolished in IEF without immobilization of the pH gradient [85,86].…”

“…With a 10% mobility difference, the predicted cathodic drift is larger compared to that of the experiment. On the other hand, mobility differences of selected ampholytes could be as large as 15% [84] and carbonic acid in the catholyte can become larger than 10 mM [87]. This suggests for the data of Fig.…”

Simul 6: A fast dynamic simulator of electromigration

“…This example shows a simulation of a large separation task. It is isoelectric focusing of pI markers analogous to one which we used in a previous paper [33]. Here it will be followed by chemical mobilization to enable the single-point detection of the separated markers.…”

Determination of acidity constants, ionic mobilities, and hydrodynamic radii of carborane‐based inhibitors of carbonic anhydrases by capillary electrophoresis

“…Recently, a new computer program AnglerFish was introduced for direct determination of the thermodynamic p K a s and limiting ionic mobilities from the raw CE experimental data [37]. Contrary to previous approaches, there is no need to perform all the measurements at the constant ionic strength of the set of BGEs [37, 38]. The CE determination of the acidity constant has several advantages such as extremely small amounts and volumes of investigated compounds (usually ca .…”