Microscale Separation by Electric Field
Capillary electrophoresis (CE) separates molecules based on their movement in an electric field through a narrow capillary tube. Developed in the 1980s as an analytical technique, CE offers exceptional resolution, minimal sample consumption, and versatility that rivals or exceeds chromatographic methods for many applications. It has become particularly important in pharmaceutical analysis, forensic science, and genomics — most notably as the technology behind modern DNA sequencing.
Fundamental Principles
When charged molecules are placed in an electric field, they migrate toward the electrode of opposite charge. The electrophoretic velocity of an ion depends on its charge-to-size ratio:
v_ep = mu_ep * E
where v_ep is the electrophoretic velocity, mu_ep is the electrophoretic mobility, and E is the electric field strength. The electrophoretic mobility depends on the charge (q) and the frictional coefficient (f): mu_ep = q / f. Small, highly charged ions migrate fastest; large, weakly charged species migrate slowest.
In free solution, electrophoretic separation alone would require that analytes have different charge-to-size ratios — identical for all analytes of the same composition. The magic of CE comes from performing this separation inside a fused-silica capillary (typically 25-75 micrometers inner diameter, 20-100 cm length), where surface chemistry introduces an additional transport phenomenon.
Electroosmotic Flow (EOF)
The inner wall of a fused-silica capillary carries silanol groups (Si-OH) that are ionized above pH 2, creating a negatively charged surface. Cations from the buffer solution accumulate near the wall, forming an electrical double layer. When voltage is applied, this layer of cations migrates toward the cathode, dragging the bulk solution along. This bulk flow is called electroosmotic flow (EOF).
EOF has a remarkable property: because it originates at the capillary wall, the flow profile is flat (plug-like) rather than parabolic as in pressure-driven flow (HPLC). This flat profile means that all molecules in a given zone experience the same velocity, dramatically reducing band broadening and producing extremely narrow peaks.
The magnitude of EOF typically exceeds the electrophoretic mobility of most analytes, so all species — cations, neutrals, and even anions — are swept toward the detector. Cations arrive first (EOF + electrophoretic migration in the same direction), neutrals arrive together (carried by EOF only), and anions arrive last (EOF minus opposing electrophoretic migration).
CE Modes
The versatility of CE stems from its multiple operational modes:
Capillary Zone Electrophoresis (CZE) is the simplest and most common mode. Analytes separate based on differences in electrophoretic mobility in free solution. A single buffer fills the capillary, and separation depends on charge-to-size differences. CZE excels for small ions, peptides, and proteins.
Micellar Electrokinetic Chromatography (MEKC) extends CE to neutral molecules by adding a surfactant (typically SDS) above its critical micelle concentration. Neutral analytes partition between the aqueous phase and the micellar pseudo-stationary phase, separating based on hydrophobicity. MEKC provides HPLC-like selectivity with CE efficiency.
Capillary Gel Electrophoresis (CGE) fills the capillary with a sieving medium (polyacrylamide or linear polymer solutions). Molecules separate by size as they migrate through the gel network. CGE is the standard method for DNA and SDS-protein separations, where charge-to-size ratios are nearly uniform and only molecular sieving provides resolution.
Capillary Isoelectric Focusing (CIEF) separates amphoteric molecules (proteins) according to their isoelectric points. A pH gradient is established within the capillary, and proteins migrate until they reach the pH where their net charge is zero.
Capillary Electrochromatography (CEC) combines electrophoretic separation with chromatographic stationary phases, offering the selectivity of HPLC with the efficiency of CE.
Comparison with HPLC
| Feature | CE | HPLC |
|---|---|---|
| Sample volume | 1-50 nL | 1-100 microL |
| Solvent consumption | minimal (mL/day) | significant (L/day) |
| Plate counts | 100,000 - 1,000,000 | 10,000 - 50,000 |
| Analysis time | 5-30 min | 5-60 min |
| Sensitivity (conc.) | moderate | better (longer path) |
| Sensitivity (mass) | excellent (attomoles) | moderate |
| Reproducibility | good (with internal standards) | excellent |
| Method development | rapid | well-established |
CE's main analytical limitation is concentration sensitivity, because the detection path length through the narrow capillary is short (50-75 micrometers vs. 10 mm for HPLC). Various strategies address this, including bubble cells, Z-cells, and online preconcentration techniques (stacking, sweeping).
Injection and Detection
Injection in CE uses either hydrodynamic (pressure difference) or electrokinetic (voltage) methods to introduce nanoliter sample volumes into the capillary. Electrokinetic injection is biased toward high-mobility ions, which can be advantageous for trace analysis but complicates quantitation.
Detection methods include:
- UV-Vis absorbance: most common, universal for UV-absorbing analytes
- Fluorescence (laser-induced, LIF): 100-1000x more sensitive than UV, essential for DNA sequencing
- Mass spectrometry (CE-MS): provides structural identification, increasingly important in metabolomics and proteomics
- Electrochemical: selective for redox-active analytes
- Contactless conductivity: universal detection for small ions
Applications
DNA sequencing: The Human Genome Project was completed using capillary array electrophoresis with fluorescently labeled dideoxy terminators (Sanger sequencing). Arrays of 96 or 384 capillaries running simultaneously enabled the throughput required for genome-scale projects.
Pharmaceutical analysis: CE methods are official in multiple pharmacopeias for purity testing, enantiomeric separations (using chiral selectors like cyclodextrins), and content uniformity assays. The low sample consumption is particularly valuable for expensive drug substances.
Clinical diagnostics: CE separates hemoglobin variants for thalassemia screening, serum proteins for myeloma detection, and urinary metabolites for inborn error diagnosis.
Metabolomics: CE-MS platforms profile charged metabolites (amino acids, organic acids, nucleotides) with minimal sample preparation, complementing LC-MS for comprehensive metabolome coverage.
Forensic science: CE with fluorescence detection is the standard method for STR-based DNA profiling, the backbone of forensic identity testing worldwide.