Determination of Absolute Injection Volume in Capillary Electrophoresis by the "Capillary-Volume-Plateau Calibration...

Area of Science:

• Analytical Chemistry
• Separation Science
• Instrumental Analysis

Background:

• Accurate determination of injection volumes is critical for quantitative capillary electrophoresis (CE).
• Traditional methods relying on the Hagen-Poiseuille equation are often unreliable, especially at low pressures or short injection times.
• Existing methods lack precision and can introduce significant bias in quantitative CE analysis.

Purpose of the Study:

• To develop a self-referenced method for accurate absolute injection volume determination in CE.
• To establish a calibration technique that is independent of pressure, time, and viscosity measurements.
• To provide a practical and broadly applicable solution for improving quantitative accuracy in CE.

Main Methods:

• Introduction of the Capillary-Volume-Plateau Calibration (CVPC) method, utilizing capillary volume as an intrinsic reference.
• The CVPC method relies on a simple one-point calibration using a known analyte concentration and detector response.
• Calculation of injection volume (V_inj) using the formula: V_inj = VA/(kCtm), based on readily measurable parameters: capillary volume (V), detector constant (k), analyte concentration (C), peak area (A), and migration time (tm).

Main Results:

• The CVPC method accurately determines absolute injection volumes with an uncertainty below 4% under typical CE conditions.
• Comparison with a commercial instrument's Hagen-Poiseuille-based predictor revealed overestimations of up to 120% by the latter.
• The CVPC method successfully eliminated the bias observed with the traditional prediction method.

Conclusions:

• The CVPC method offers a robust, first-principles-based approach for accurate injection volume determination in CE.
• This technique simplifies calibration by requiring only detector constant determination and relies on fixed geometric dimensions.
• CVPC provides a practical and broadly applicable solution for enhancing quantitative accuracy in capillary electrophoresis experiments.