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Capillary electrophoresis separates molecules based on charge-to-mass ratio in a high electric field within a narrow capillary. The technique offers extremely high efficiency (theoretical plates in hundreds of thousands) and selectivity for charged species including amino acids, peptides, proteins, and organic acids without requiring elaborate sample pretreatment.
Develop CE methods for protein analysis and amino acid profiling, comparing to HPLC approaches.
Assuming CE requires lower sample volumes than HPLC (often similar or larger due to detection sensitivity). Thinking EOF is constant across all buffer conditions (actually highly dependent on pH and ionic strength).
In chromatography — your prerequisite foundation — separation happens because different analytes interact differently with a stationary phase as a mobile phase carries them through. Capillary electrophoresis takes a fundamentally different approach: there is no stationary phase. Instead, separation occurs because charged species migrate at different velocities through a narrow-bore capillary (typically 25–75 μm inner diameter) under the influence of a strong electric field, often 100–500 V/cm. The driving force is electrophoretic mobility, which depends on each ion's charge-to-size ratio. Small, highly charged ions move fastest; large, weakly charged ions move slowest. This simple principle produces extraordinarily high separation efficiency — hundreds of thousands of theoretical plates — because the flat flow profile inside the capillary avoids the band-broadening that plagues pressure-driven chromatographic flow.
The key phenomenon that makes capillary zone electrophoresis (CZE) practical is electroosmotic flow (EOF). At the pH values commonly used, silanol groups on the inner wall of the fused-silica capillary are deprotonated, creating a negatively charged surface. Cations from the buffer accumulate near the wall, and when the electric field is applied, these cations drag the bulk solution toward the cathode (negative electrode). This EOF acts as a pump that sweeps everything — cations, neutrals, and even anions — toward the detector. Cations arrive first (their electrophoretic migration adds to EOF), neutrals ride along with EOF unseparated, and anions arrive last (their migration opposes EOF but is overcome by it). Understanding that EOF depends strongly on buffer pH and ionic strength is essential for method development: raising pH increases EOF by deprotonating more silanols, while increasing ionic strength compresses the double layer and slows it.
From your knowledge of buffer solutions, you can appreciate that buffer selection is the primary tool for controlling CE separations. The buffer determines pH (which sets EOF magnitude and analyte ionization state), ionic strength (which affects EOF speed, Joule heating, and peak shape), and in some modes provides additives like surfactants (for micellar electrokinetic chromatography) or chiral selectors. Joule heating — the heat generated by current flowing through the buffer — is the main limitation on how much voltage and how concentrated a buffer you can use, because excessive heating degrades separation efficiency and can damage the capillary or denature analytes. The small capillary diameter helps dissipate heat efficiently, which is precisely why narrow capillaries are used.
Detection in CE is typically by UV absorbance measured directly through the capillary itself (on-capillary detection), though fluorescence, mass spectrometry, and electrochemical detection are also used. Because the capillary path length is tiny (equal to the capillary inner diameter), UV detection sensitivity is inherently lower than in HPLC, which is why CE often requires higher sample concentrations despite injecting nanoliter volumes. CE excels for separating charged biomolecules — proteins, peptides, amino acids, nucleic acids, and small ions — where its unmatched efficiency and speed complement the selectivity of chromatographic methods in a modern analytical laboratory.