ECHMET

ECHMET analyzes and characterizes pH gradients and carrier ampholytes for isoelectric focusing (IEF) to derive thermodynamic pKa values, limiting ionic mobilities, and to enable reality-based IEF simulations.

Key Features:

• Selection of carrier ampholytes: Identifies low molecular mass UV-absorbing ampholytes that are commercially available and meet Rilbe's criteria (ΔpKa < 2) based on a computational study using ChemSketch and SPARC.
• Electrophoretic mobility analysis: Measures electrophoretic mobilities of selected ampholytes in background electrolyte solutions (BGEs) at 10 mM ionic strength across a pH range of 2 to 12.
• Effective mobility recalculation: Recalculates effective mobilities to zero ionic strength using AnglerFish based on Debye-Hückel and Onsager-Fuoss laws to enable thermodynamic interpretation.
• Nonlinear regression for pKa and mobilities: Applies a Henderson-Hasselbalch equation-type nonlinear regression to directly calculate thermodynamic pKa values and limiting ionic mobilities from recalculated data.
• pH gradient characterization: Characterizes overall and narrow-segment pH gradients in IEF to address errors in analyte ampholyte pI assignments caused by assuming linear pH gradients.
• Simulation capabilities: Performs reality-based numeric simulations of the IEF process using Simul to model and predict IEF outcomes.

Scientific Applications:

• IEF accuracy improvement: Improves the reliability and accuracy of isoelectric focusing by providing thermodynamic pKa and limiting mobility data for carrier ampholytes.
• Protein pI assignment correction: Mitigates errors in protein pI assignments arising from incorrect pH gradient assumptions.
• Experimental planning and optimization: Supports experimental design and optimization of IEF separations through numeric simulation and ampholyte characterization.
• Proteomics and related fields: Provides ampholyte and pH-gradient data applicable to proteomics and other protein-separation research areas.

Methodology:

Computational selection with ChemSketch and SPARC; measurement of electrophoretic mobilities in BGEs at 10 mM ionic strength over pH 2–12; recalculation to zero ionic strength using AnglerFish with Debye-Hückel and Onsager-Fuoss laws; Henderson-Hasselbalch-type nonlinear regression to obtain thermodynamic pKa and limiting ionic mobilities; numeric IEF simulation using Simul.