Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in narrow fused-silica capillaries with inner diameters of 25 to 100 micrometers. An electric field of 100 to 500 V/cm is applied across the capillary, driving charged analytes toward the opposite electrode at rates determined by their electrophoretic mobility. CE combines high separation efficiency (often exceeding 100,000 theoretical plates) with short analysis times, minimal sample consumption (nanoliters), and compatibility with aqueous and non-aqueous buffers. It has become an essential platform in pharmaceutical analysis, clinical diagnostics, forensic science, proteomics, and metabolomics, offering a separation mechanism orthogonal to both liquid chromatography and gel electrophoresis.
The Principle of Electrophoretic Mobility
When an electric field is applied across a solution, a charged particle experiences an electrostatic force proportional to its net charge (q) and the field strength (E). The particle accelerates until the drag force from the surrounding medium equals the electrostatic force, at which point it migrates at a constant velocity. The electrophoretic mobility (µ_ep) is defined as the velocity per unit field strength and is proportional to q divided by the hydrodynamic radius (r) of the particle. Small, highly charged ions have high mobility, while large or weakly charged species migrate more slowly. This differential migration is the foundation of all electrophoretic separations.
Electroosmotic Flow
Electroosmotic flow (EOF) is a distinctive phenomenon in CE that arises from the electrical double layer at the fused-silica capillary wall. At pH above approximately 3, silanol groups on the inner capillary surface are deprotonated, creating a negatively charged wall. Cations from the buffer accumulate near the wall to form a diffuse electrical double layer. When the electric field is applied, these hydrated cations migrate toward the cathode, dragging the bulk solution with them. The resulting EOF profile is nearly flat (plug-like), in contrast to the parabolic flow profile of pressure-driven systems such as HPLC. This plug flow minimizes zone broadening, contributing to the exceptionally high separation efficiencies observed in CE. The magnitude and direction of the EOF depend on buffer pH, ionic strength, and capillary wall chemistry, and can be suppressed or reversed through wall coatings or dynamic modifiers.
Instrumentation
A CE instrument consists of a high-voltage power supply (typically 0 to 30 kV), two buffer reservoirs with platinum electrodes, a fused-silica capillary (25 to 100 cm in length), and a detector. The capillary passes through the detector, enabling on-column detection without the need for post-column derivatization plumbing. Sample introduction is performed by replacing one buffer reservoir with the sample vial and applying pressure (hydrodynamic injection) or voltage (electrokinetic injection) for a defined time. Hydrodynamic injection is preferred for quantitative work because the volume introduced is independent of the sample matrix, while electrokinetic injection introduces a bias toward more mobile analytes but can achieve higher sensitivity for trace components.
Detection Modes
UV-Vis absorbance detection is the most common CE detection method, typically performed through a window created by removing the polyimide coating from the capillary. Absorbance is measured across the capillary diameter, which limits the optical path length to the capillary inner diameter (25 to 100 µm), resulting in modest sensitivity (low micromolar). Laser-induced fluorescence (LIF) detection provides dramatically higher sensitivity (attomole to zeptomole levels) for fluorescently labeled analytes, making it the method of choice for DNA sequencing and trace analysis. Electrochemical detection (amperometry and conductivity) is used for electroactive species and small inorganic ions. CE coupled with mass spectrometry (CE-MS) via electrospray ionization provides both high separation efficiency and structural identification, and is widely employed in proteomics and metabolomics for analysis of peptides, proteins, and polar metabolites.
The Separation Modes
CE encompasses several operational modes that exploit different separation mechanisms. Capillary zone electrophoresis (CZE) separates analytes based on their charge-to-size ratio in free solution and is the most widely used mode. Capillary gel electrophoresis (CGE) uses a polymer gel or linear polymer matrix to separate molecules by size, and is the standard platform for DNA sequencing and forensic DNA profiling. Capillary isoelectric focusing (CIEF) separates amphoteric molecules such as proteins according to their isoelectric point (pI) in a pH gradient. Capillary isotachophoresis (CITP) uses a discontinuous buffer system to focus analytes into sharp, concentrated zones and is often used for sample preconcentration prior to CZE. Micellar electrokinetic chromatography (MEKC) introduces surfactant micelles as a pseudo-stationary phase, enabling separation of neutral analytes by hydrophobic partitioning. The choice of mode depends on the nature of the analytes and the separation goals.
Comparison with HPLC
CE and HPLC are complementary separation techniques. HPLC separates analytes based on partitioning between a stationary and mobile phase, whereas CE separates based on differences in electrophoretic mobility in free solution. CE typically offers higher plate counts (100,000 to 500,000 plates per meter) than HPLC, but lower concentration sensitivity due to the short optical path length and small injection volumes. CE is particularly advantageous for charged, polar, and ionic species that may be difficult to retain on reversed-phase HPLC columns. Method development in CE is simplified by the wide range of separation modes and the ability to manipulate selectivity through buffer composition, pH, and additives without the need to change columns.
