Capillary electrophoresis

Capillary electrophoresis separates analytes based on their electrophoretic mobility, which is the product of charge and inverse of hydrodynamic radius, under an electric field. The narrow capillary minimizes Joule heating and convective mixing, enabling high efficiency. The fundamental equation for migration time is t = L_d / (μ_e E), where L_d is the effective length, μ_e is the electrophoretic mobility, and E is the field strength. Electroosmotic flow (EOF), generated by the ionized silanol groups on the capillary wall, drives the bulk solution toward the cathode, allowing both cations and anions to be detected in a single run. Instrumentation typically includes a high-voltage power supply, a fused-silica capillary, a detector (often UV-absorbance or laser-induced fluorescence), and buffer reservoirs. Sample injection is performed hydrodynamically or electrokinetically, with volumes in the nanoliter range. The choice of buffer pH and composition critically affects separation, as it influences analyte charge and EOF magnitude. Modern CE systems often incorporate temperature control to maintain reproducibility, and capillary coatings (e.g., polyacrylamide or polyethylene glycol) can suppress EOF or reduce analyte-wall interactions.

Capillary zone electrophoresis (CZE) is the simplest mode, where separation occurs solely in free solution, ideal for charged species like peptides and inorganic ions. Micellar electrokinetic chromatography (MEKC) adds surfactants above the critical micelle concentration to separate neutral molecules by partitioning into a pseudo-stationary phase, enabling analysis of drugs and environmental pollutants. Capillary gel electrophoresis (CGE) uses a polymer matrix (e.g., polyacrylamide or sieving polymers) to separate DNA fragments and proteins by size, forming the basis of modern DNA sequencing. Capillary isoelectric focusing (cIEF) separates amphoteric molecules by pH gradient, providing high-resolution protein characterization. Capillary isotachophoresis (CITP) concentrates analytes into sharp zones, often used as a preconcentration step. CE is widely applied in pharmaceutical analysis for chiral separations using cyclodextrins as chiral selectors, in clinical chemistry for monitoring metabolites and therapeutic drugs, and in forensic science for DNA profiling. It also supports single-cell analysis, detecting metabolites in individual cells, and has been coupled to mass spectrometry (CE-MS) for proteomics and metabolomics, offering complementary selectivity to liquid chromatography.

Beyond mainstream uses, CE has niche applications that are often overlooked. For instance, CE can analyze single molecules using laser-induced fluorescence, enabling studies of enzyme kinetics at the single-molecule level. In the 1990s, CE was instrumental in the Human Genome Project, with capillary array instruments accelerating DNA sequencing. A lesser-known mode is capillary electrochromatography (CEC), which combines CE with liquid chromatography by using a packed or monolithic stationary phase, offering high efficiency for neutral compounds. CE has also been used to measure physicochemical constants such as acid dissociation constants (pKa) and binding constants via affinity CE. In the field of astrobiology, CE has been proposed for detecting amino acids on Mars, as part of the Mars Organic Molecule Analyzer (MOMA) instrument. Additionally, CE can separate nanoparticles and viruses, and it has been applied to characterize exosomes in cancer research. A historical note: the concept of electrophoresis dates to the early 20th century with Arne Tiselius's moving-boundary method, but CE as a modern technique emerged in the 1980s with the work of James Jorgenson and Krynn Lukacs, who demonstrated high-efficiency separations in narrow capillaries.

CE offers several advantages over traditional slab-gel electrophoresis and high-performance liquid chromatography (HPLC). It requires extremely small sample volumes (nanoliter to picoliter), reduces solvent consumption, and provides high separation efficiency with theoretical plates exceeding 10⁵ per meter. Analysis times are often short, ranging from seconds to minutes. CE is also highly versatile, with multiple modes available for different analyte types. However, it has limitations: the small injection volumes can lead to poor concentration sensitivity, often requiring preconcentration techniques such as solid-phase extraction or field-amplified sample stacking. Reproducibility can be affected by EOF variations and capillary surface adsorption, which are mitigated by coatings or dynamic modifiers. The technique is less robust for preparative separations due to low loading capacity. Additionally, method development can be complex, requiring careful optimization of buffer composition, pH, and voltage. Despite these challenges, CE remains a powerful tool in research and industry, with ongoing innovations in microchip CE and multidimensional separations expanding its capabilities.

Electroosmotic flow (EOF)

The bulk flow of buffer solution in a capillary induced by an applied electric field, driven by the charged capillary wall.

Electrophoretic mobility

The velocity of an ion per unit electric field, dependent on its charge-to-size ratio.

Micellar electrokinetic chromatography (MEKC)

A CE mode that uses surfactants to form micelles, enabling separation of neutral analytes.

Capillary gel electrophoresis (CGE)

A CE mode using a gel or polymer matrix to separate molecules by size.

Capillary isoelectric focusing (cIEF)

A CE mode that separates amphoteric molecules based on their isoelectric point in a pH gradient.