Capillary zone electrophoresis (CZE) is the most fundamental and widely practiced mode of capillary electrophoresis. In CZE, the capillary is filled with a continuous, uniform background electrolyte (BGE), and the sample is introduced as a narrow plug at the inlet end. Under the influence of the applied electric field, each analyte migrates with its characteristic velocity, determined by the sum of its electrophoretic mobility and the electroosmotic flow (EOF). Analytes that differ in charge or size migrate at different velocities and are resolved into discrete zones as they travel toward the detector. CZE is applied across a broad range of analyte classes, from small inorganic ions and pharmaceuticals to peptides, proteins, and nucleic acid fragments.
The Mechanism of Zone Separation
The net migration velocity of an analyte in CZE is the vector sum of its electrophoretic velocity (toward the oppositely charged electrode) and the electroosmotic velocity (normally toward the cathode). At pH above 3, the EOF is generally stronger than the electrophoretic mobility of most anions, so all analytes — cations, neutrals, and anions — are swept toward the detector at the cathode end. Cations migrate fastest because their electrophoretic migration is in the same direction as the EOF. Neutral analytes migrate at the EOF velocity and co-elute as a single unresolved peak, which is why CZE is not suitable for neutral species without modification. Anions migrate slowest because their electrophoretic migration opposes the EOF, and those with mobilities greater than the EOF in the opposite direction never reach the detector. The separation window is defined by the migration time of a neutral marker (t_EOF) and the time at which the slowest anion emerges.
Buffer Composition and Selectivity
The choice of BGE is the most powerful tool for controlling selectivity in CZE. Buffer pH determines the ionization state of acidic and basic analytes and therefore their effective charge and mobility. For peptide and protein separations, pH is adjusted to a value where the species of interest have significantly different net charges, typically in the range of 2 to 9. Buffer ionic strength affects both the EOF magnitude and the thickness of the electrical double layer; higher ionic strength reduces EOF and compresses the double layer, improving resolution but increasing current and Joule heating. Organic solvents such as acetonitrile or methanol can be added to the BGE to alter analyte solubility, change the dielectric constant, and modify the EOF. Chiral separations are achieved by adding cyclodextrins or other chiral selectors to the BGE, which form transient diastereomeric complexes with enantiomers.
Theoretical Plates and Resolution
CZE separations are characterized by extremely high plate numbers, often exceeding 200,000 plates per meter. Zone broadening in CZE is dominated by longitudinal diffusion, because the flat EOF profile eliminates the flow-induced broadening that limits HPLC efficiency. The number of theoretical plates (N) is given by N = (µ_app V) / (2 D), where µ_app is the apparent mobility, V is the applied voltage, and D is the diffusion coefficient. Resolution between two peaks depends on the difference in their apparent mobilities, the average number of plates, and the electroosmotic velocity. Resolution can be improved by increasing the applied voltage (up to the limit of Joule heating), extending the effective capillary length, reducing the EOF (by lowering pH or using coated capillaries), or optimizing the buffer composition to maximize the mobility difference between analytes.
Sample Stacking for Enhanced Sensitivity
The concentration sensitivity of CZE with UV absorbance detection is limited by the short optical path length and the small injection volume (typically 1 to 20 nL). On-capillary preconcentration techniques, collectively known as stacking, dramatically improve sensitivity by concentrating the sample zone before the separation begins. Field-amplified sample stacking (FASS) exploits the difference in conductivity between the sample matrix (low conductivity) and the BGE (high conductivity). When voltage is applied, the higher electric field in the low-conductivity sample zone causes analyte ions to migrate rapidly until they reach the BGE boundary, where they slow down and accumulate in a narrow band. Sweeping uses pseudostationary phases such as micelles to capture and focus neutral or charged analytes. Transient isotachophoresis (tITP) employs a leading and terminating electrolyte system to focus analytes into sharp zones, similar to capillary isotachophoresis, before transitioning to CZE separation. These stacking methods can provide 10- to 1000-fold sensitivity enhancement, bringing UV detection limits into the low nanomolar range.
Applications in the Clinical Laboratory
CZE is widely used in clinical laboratories for serum protein analysis, where it has largely replaced agarose gel electrophoresis for routine serum protein electrophoresis (SPEP). Serum proteins separate into five major fractions — albumin, alpha-1, alpha-2, beta, and gamma globulins — and the electropherogram is evaluated for monoclonal gammopathies, inflammatory patterns, and protein deficiencies. CZE is also the method of choice for hemoglobinopathy screening, separating hemoglobin variants such as HbA, HbF, HbS, and HbC at alkaline pH. In pharmaceutical analysis, CZE is used for drug purity testing, counter-ion determination, and chiral impurity profiling. In proteomics, CZE coupled with mass spectrometry (CZE-MS/MS) provides high-efficiency peptide separation for bottom-up protein identification, particularly advantageous for low-volume samples and basic peptides that are challenging by reversed-phase LC-MS.
Method Development Considerations
Developing a CZE method begins with selecting the BGE pH based on the pKa values of the analytes. A phosphate or borate buffer at pH 2.5 is a common starting point for small molecules and peptides. The capillary dimensions (length, inner diameter) and applied voltage are chosen to balance analysis speed with resolution. Column temperature is controlled by forced-air or liquid cooling to dissipate Joule heat, and the capillary is rinsed between runs with sodium hydroxide, water, and BGE to maintain reproducibility. For quantitative analysis, an internal standard is added to correct for injection volume variability, and the method is validated for linearity, precision, accuracy, and robustness according to ICH guidelines.
