Use of correction factors in mobility shift affinity capillary electrophoresis for weak analyte – ligand interactions

Abstract

The practical and theoretical problems associated with interpretation of binding isotherms obtained by the investigation of weak analyte-ligand interactions using mobility shift affinity CE were investigated with special emphasis on various correction methods to compensate for media effects due to high additive concentrations. The interaction between 2-hydroxypropyl-alpha-CD and two bile salts (glycocholate and glycodeoxycholate) was studied applying correction factors based on viscosity, CE based conductance and separately measured conductivities. Accurate measurement of the stability constants proved to be difficult; several points of general nature relating to the investigation of the weak analyte-ligand interactions were identified. These included undesired dilution of the BGE due to the high amounts of additive, avoidance of temperature effects by maintaining a constant power during the separations and limited validity of Walden's rule. The relative sizes of the buffer constituents, the nonelectrolyte ligand additive, and the analytes as well as noninteracting marker substances should be considered in order to minimize errors in the correction factors when significant deviation from Walden's rule is observed. Under such conditions, the mobilities should preferably be corrected by the use of a noninteracting marker; and the most generally applicable corrections may be based on conductance measurements.

Complexation of tauro‐ and glyco‐conjugated bile salts with α‐cyclodextrin and hydroxypropyl‐α‐cyclodextrin studied by affinity capillary electrophoresis and molecular modelling

“…This is the case in the present study, where CE was used to investigate the interaction between the neutral a-CD or 2OHpaCD and a number of conjugated bile salts, which are charged at near neutral pH. However, due to the weak interactions a method to correct experimentally determined electrophoretic mobilities for media changes is needed [31]. 1 The CE experiments were conducted in 0.050 M sodium phosphate buffer at pH 7.00 as this reflects the pH of the small intestine [66] and ensures complete ionisation of both PGA 1 and bile salts.…”

“…(6)) is adequate for correcting the mobilities in the low concentration range (0-15 mM) used to create the binding isotherm for the PGA 1 -CD complex. At higher CD concentrations, the current correction method is inadequate [31], but since the mobility of the PGA 1 -CD complex is known, the correction factor in Eq. (7) may be used to correct the mobilities of the bile salt-CD complexes assuming that they are affected the same way by viscosity changes as the PGA 1 -CD complex.…”

“…Accordingly, the effective electrophoretic mobilities may be corrected for viscosity variations due to the presence of CD in the BGE based on the current measured during electrophoresis [31,42]:…”

“…Additionally, the procedure whereby the BGEs are prepared may lead to differences in the ionic strength of the buffer and thereby different temperatures within the capillary (at constant voltage conditions) [31,36]. The electrophoretic mobilities must be corrected for these medium effects in order to obtain reliable stability constants.…”

“…The use of a marker molecule may enable a correction for these changes assuming equal effects on the investigated species. The validity of this assumption will depend on for instance the size/charge relationship of the mobility marker relative to the analyte (bile salt) [31].…”