Use of correction factors in mobility shift affinity capillary electrophoresis for weak analyte-ligand interactions.
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.
Østergaard
Jesper
J
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. joe@farma.ku.dk
Jensen
Henrik
H
Holm
René
R
D019075:Electrophoresis, Capillary / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D006000:Glycocholic Acid / Q000378:metabolism; D006002:Glycodeoxycholic Acid / Q000378:metabolism; D008024:Ligands; D009844:Oligosaccharides / Q000378:metabolism; D012996:Solutions; D014783:Viscosity; D047391:alpha-Cyclodextrins / Q000378:metabolism
C402302:(2-hydroxypropyl)-alpha-cyclodextrin; D008024:Ligands; D009844:Oligosaccharides; D012996:Solutions; D047391:alpha-Cyclodextrins; D006002:Glycodeoxycholic Acid; D006000:Glycocholic Acid; D005990:Glycerol