Determination of Absolute Injection Volume in Capillary Electrophoresis by the “Capillary-Volume–Plateau Calibration (CVPC)” Method

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 pulses. 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 (Vinj) without measuring pressure, time, or viscosity. The working relation, Vinj = VA/(kCtm), 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 tm. If the detector responds linearly with concentration, the constant k is obtained from a single pressure-driven run: a reference solution of known concentration Cref is introduced just long enough to generate a flat-top plateau, and the average plateau signal ⟨S⟩ gives k = ⟨S⟩/Cref. Error analysis shows that the uncertainly in peak area is the dominant contributor to the overall uncertainly in CVPC-determined Vinj, 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 Vinj 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