In a high-tech lab, a sample of DNA fragments, proteins, or even a sip of soda is injected into a hair-thin glass tube. Minutes later, a sophisticated instrument reveals its precise chemical composition.
Imagine trying to separate a mixture of charged particles by having them race through a tiny tube under the influence of a powerful electric field. Capillary Electrophoresis (CE), a powerful analytical technique, does exactly that. It separates ions based on their electrophoretic mobility, which is determined by their size, shape, and charge 3 6 .
Since the introduction of modern CE by Jorgenson and Lukacs in 1981, the technique has evolved into a highly mature and versatile separation method 2 .
It has become indispensable in fields ranging from pharmaceutical development and biotechnology to food analysis and environmental monitoring 5 .
At its core, CE is elegantly simple. The primary components include a narrow glass capillary, two buffer reservoirs, and two electrodes connected to a high-voltage power supply (often 10,000–20,000 V) 5 8 . The capillary, typically no wider than a human hair, is filled with a conductive background electrolyte (buffer) 5 .
This is the ability of a charged molecule to migrate through the buffer under an applied electric field. Its velocity is determined by the molecule's charge and its frictional coefficient, which is related to its size and shape, as well as the viscosity of the solvent 3 .
This is the movement of the entire buffer solution through the capillary. In uncoated silica capillaries at a pH above 3, the silanol groups on the inner wall ionize, creating a negative charge 3 .
This attracts a layer of positive cations from the buffer, which, when an electric field is applied, are pulled toward the cathode, dragging the entire solution with them 3 5 .
EOF acts as a "conveyor belt" moving all molecules toward the detector
The combination of these two forces—the analyte's own electrophoretic mobility and the "conveyor belt" of the electroosmotic flow—results in the high-resolution separation that makes CE so powerful 5 .
CE is not a single method but a family of related techniques, each optimized for different types of analyses .
| Technique | Abbreviation | Separation Mechanism | Primary Applications |
|---|---|---|---|
| Capillary Zone Electrophoresis | CZE | Separation based on charge-to-size ratio in a free solution 3 . | Small ions, drugs, metabolites . |
| Micellar Electrokinetic Chromatography | MEKC | Uses surfactant micelles to separate neutral molecules based on their partitioning between micelles and solution . | Neutral molecules, complex mixtures 2 . |
| Capillary Gel Electrophoresis | CGE | Uses a polymer or gel matrix to sieve molecules by size . | DNA fragments, proteins, SDS-protein complexes . |
| Capillary Isoelectric Focusing | cIEF/icIEF | Separates amphoteric molecules (like proteins) by their isoelectric point (pI) within a pH gradient 4 . | Protein characterization, biopharmaceuticals 4 . |
| Capillary Electrophoresis-Mass Spectrometry | CE-MS | Couples CE's separation power with the identification capabilities of a mass spectrometer . | Proteomics, metabolomics, identifying unknown compounds 2 5 . |
Most common CE mode, separates based on charge-to-size ratio.
Extends CE to neutral molecules using micelles.
Size-based separation ideal for DNA and proteins.
To illustrate how CE works in practice, let's examine a real-world application: the analysis of caffeine, aspartame, and benzoic acid in soft drinks 8 . This example showcases the speed, efficiency, and practical utility of CE.
A CE instrument is powered on. The capillary, typically made of fused silica, is prepared by flushing it with sodium hydroxide to remove contaminants, followed by rinses with water, acid, and finally the background electrolyte (e.g., sodium borate buffer) 6 8 .
A small volume of the soft drink (e.g., Diet Pepsi) is mixed with a sample buffer. It may be heated and centrifuged to remove any insoluble material that could clog the capillary 6 .
The sample is introduced into the capillary—either by applying pressure or a brief voltage—creating a tiny plug. A high voltage (e.g., 10-20 kV) is then applied across the capillary 8 . The molecules begin to separate based on their unique electrophoretic mobilities.
In the analysis of diet Pepsi, three distinct peaks are observed corresponding to caffeine, aspartame, and benzoic acid, each migrating at a consistent time 8 . In regular Pepsi, only the caffeine peak is present, as expected 8 .
The entire separation is remarkably fast, being completed in just 3–4 minutes 8 .
This experiment demonstrates CE's ability to rapidly and efficiently quantify specific ingredients in a complex mixture, a task highly relevant for quality control in the food and beverage industry 8 .
High Efficiency & Speed
A successful CE experiment relies on a suite of specialized materials and reagents. The following table details the key components used in a typical CE protocol 6 .
| Item | Function/Description |
|---|---|
| CE Instrument | The core system, comprising a high-voltage power supply, capillary cartridge, temperature controller, and an on-capillary detector (e.g., UV-Vis) 5 . |
| Fused Silica Capillary | The separation channel. Typical inner diameters are 50-75 μm. The silica walls can be bare or coated with polymers to minimize unwanted analyte interactions 3 6 . |
| Background Electrolyte (BGE) | The running buffer that fills the capillary (e.g., sodium borate or phosphate buffers). Its composition, pH, and ionic strength are critical for controlling separation and electroosmotic flow 6 . |
| Sample Buffer | A solution (e.g., Tris-HCl) used to dilute and prepare the sample, ensuring compatibility with the separation conditions 6 . |
| Standard Marker Solution | A mixture of known compounds used to calibrate the instrument, correlating migration times with analyte identity 6 . |
Critical for controlling separation and EOF
Hair-thin separation channel
10,000-20,000 V power supply
CE remains a vibrant field of research. Scientists are continuously developing new methods to overcome its main limitation—relatively low concentration sensitivity due to the tiny sample volumes injected 2 7 .
To prevent biomolecules like proteins from sticking to the capillary wall, new stable and biocompatible coatings are being developed, such as covalently bound polyethylene glycol (PEG) and polydopamine/polyethylenimine layers, which enhance reproducibility 2 .
Coupling CE with mass spectrometry (CE-MS) has become a cornerstone of modern proteomics and metabolomics, allowing for the high-resolution separation and definitive identification of complex biological molecules 2 .
From ensuring the quality of our medicines and food to unlocking the secrets of the human genome, capillary electrophoresis has proven to be an indispensable tool in the scientist's arsenal. Its unique combination of high efficiency, minimal sample requirements, and versatility has secured its place as a powerful alternative and complement to other separation techniques like liquid chromatography.
As it continues to evolve, this "invisible race" within a tiny capillary will undoubtedly keep driving discoveries and innovations across the scientific landscape for years to come.
Capillary electrophoresis continues to revolutionize scientific analysis through its unparalleled efficiency and versatility.