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

Accurate knowledge of injection volumes is essential for quantitative capillary electrophoresis (CE), yet volumes predicted by the Hagen-Poiseuille equation become unreliable at low pressures or with short injection times. We present a self-referenced approach, termed the Capillary-Volume-Plateau Calibration (CVPC) method, that uses capillary volume as a reference and requires only a one-point calibration to obtain absolute injection volumes (V_inj) without measuring pressure, time, or viscosity. The working relation, V_inj = VA/(kCt_m), depends only on five readily measured quantities: (i) the capillary volume, V = πr²l (r is the inner radius and l is the injection-to-detector length), (ii) the detector constant k (signal per concentration unit), (iii) the analyte concentration C, (iv) the peak area A, and (v) the peak migration time t_m. If the detector responds linearly with concentration, the constant k is obtained from a single pressure-driven run: a reference solution of known concentration C_ref is introduced just long enough to generate a flat-top plateau, and the average plateau signal ⟨S⟩ gives k = ⟨S⟩/C_ref. Error analysis shows that the uncertainty in peak area is the dominant contributor to the overall uncertainty in CVPC-determined V_inj, which remains below 4% under typical CE conditions. A test on a commercial instrument with a built-in Hagen-Poiseuille-based predictor revealed overestimates in V_inj of up to approximately 120%, a bias eliminated by the CVPC method. Because the CVPC procedure relies solely on a detector calibration and fixed geometric dimensions, it is based on first principles and remains valid unless detector settings or buffer composition change, offering a practical and broadly applicable route to accurate volume determination in CE.