Separating proteins, DNA, and other biological compounds with breathtaking efficiency inside a hair-thin glass tube
In the quest to understand the building blocks of life, scientists have long sought ways to separate and analyze the intricate molecules that power every living organism. Enter capillary electrophoresis (CE), a powerful technique that performs a fascinating molecular race, separating proteins, DNA, and other biological compounds with breathtaking efficiency inside a hair-thin glass tube. This unsung hero of the modern lab provides the high-resolution data that fuels advances in genomics, drug development, and medical diagnostics, all while using sample volumes so small they are nearly invisible to the naked eye 1 6 .
At its heart, capillary electrophoresis is elegantly simple. It separates molecules based on their electrophoretic mobility—a measure of how quickly they move through a solution when pulled by an electric field 1 .
All molecules are carried by electroosmotic flow (EOF), but their own charge affects their net speed
The journey takes place within a fused silica capillary, a tube so narrow that its internal diameter is roughly the thickness of a human hair. This capillary is filled with a conductive buffer solution and suspended between two vials of the same buffer. When a high-voltage current is applied, two key forces spring into action 1 5 :
The inner wall of the silica capillary itself becomes negatively charged, attracting a layer of positive ions from the buffer. When voltage is applied, this layer of positive ions moves toward the negative electrode (cathode), dragging the entire contents of the capillary along with it in a smooth, plug-like flow 1 5 9 .
The net result is that all molecules, regardless of their own charge, are carried toward the detector by the EOF. Positively charged molecules, moving in the same direction as the EOF, arrive first. Neutral molecules, with no charge of their own, are simply carried along at the speed of the EOF. Negatively charged molecules, which are naturally attracted toward the positive electrode, must fight against the current to reach the detector at the other end, and therefore arrive last 5 9 . This creates a precise order of arrival, allowing scientists to identify and quantify each component in a complex mixture.
| Force | Definition | What It Separates |
|---|---|---|
| Electrophoretic Migration | The movement of charged analytes in an electric field. | Charged molecules based on their charge-to-size ratio. |
| Electroosmotic Flow (EOF) | The bulk flow of liquid through the capillary caused by the charged capillary wall. | Carries all molecules toward the detector; its magnitude and direction can be controlled to fine-tune separation. |
Capillary electrophoresis is not a single technique but a family of methods. By modifying the capillary contents and buffer chemistry, scientists can tailor the system to a vast array of biomolecules 5 8 .
Here, the capillary is filled with a viscous polymer solution that acts as a molecular sieve. This is the workhorse for DNA sequencing and fragment analysis, as it separates DNA strands by their size with single-base-pair resolution 5 3 .
Best for: DNA, RNA, proteins by size
Surfactants are added to the buffer to form micelles—tiny spheres that can trap neutral (uncharged) molecules. This allows CE to separate compounds that have no charge, a task impossible with basic CZE 2 5 .
Best for: Neutral molecules and charged molecules in complex mixes
Used primarily for proteins, this method creates a pH gradient inside the capillary. Proteins migrate until they reach the point in the gradient where their net charge is zero (their isoelectric point), allowing for extremely high-resolution separation of protein variants 5 .
Best for: Proteins and peptides
| Technique | Best For | Key Feature |
|---|---|---|
| CZE | Ions, small charged molecules, peptides. | Simple, fast, minimal preparation 5 . |
| CGE | DNA, RNA, proteins by size. | Uses a sieving matrix for size-based separation; essential for genetics 5 . |
| MEKC | Neutral molecules and charged molecules in complex mixes. | Uses micelles as a pseudo-stationary phase 2 5 . |
| CIEF | Proteins and peptides. | Separates based on a molecule's isoelectric point (pI) 5 . |
To truly appreciate the power of CE, let's examine a specific, peer-reviewed experiment: the use of Capillary Polymer Electrophoresis (CPE) to separate double-stranded DNA (dsDNA) and double-stranded RNA (dsRNA) fragments, including small interfering RNA (siRNA) 7 .
This experiment used a "home-built" CE system, demonstrating the technique's fundamental principles 7 .
The researchers prepared a sieving matrix by dissolving hydroxyethylcellulose (HEC), a long-chain polymer, in a TBE (Tris-Borate-EDTA) buffer solution. This creates a viscous solution that acts as a molecular sieve. The fluorescent dye SYBR Green II was added to bind to the nucleic acids and allow for their detection.
A coated fused silica capillary was first flushed with pure water. The prepared 0.5% HEC polymer solution was then pumped into the capillary, completely filling it.
The end of the capillary was placed into a tiny tube containing the sample—a mixture of dsDNA or dsRNA fragments. A brief electric field was applied (electrokinetic injection), which pulled a nanoliter-volume sample plug into the capillary.
The capillary was then transferred to a vial containing only the HEC polymer solution, and a high electric field (100 V/cm) was applied. The different-sized nucleic acid fragments began their race through the polymer mesh.
As the separated fragments passed a small window in the capillary, they were illuminated by the light of a fluorescence microscope. A light-sensitive photomultiplier tube (PMT) detected the resulting flashes of light, which were converted into digital data and displayed as an electropherogram—a graph of signal intensity versus time 7 .
The experiment successfully separated a mixture of dsDNA and dsRNA fragments in under three minutes. The resulting electropherogram showed a series of sharp, distinct peaks, each corresponding to a nucleic acid fragment of a specific size 7 .
The peaks represented fragments ranging from 100 to 500 base pairs, clearly resolved from one another.
A similar high-resolution separation was achieved for DNA fragments.
The scientific importance of this experiment lies in its demonstration of CE as a superior alternative to traditional slab gel electrophoresis. It is faster, more sensitive (requiring only nanograms of sample), and fully automated. Furthermore, by showing that replaceable polymer solutions work effectively, it solved the historical problems of reproducibility and limited capillary lifetime associated with traditional gels 7 . This precise sizing of nucleic acids is foundational to modern molecular biology, with critical applications in gene expression analysis, quality control for synthetic biology, and pharmaceutical development.
| Reagent/Material | Function in the Experiment |
|---|---|
| Fused Silica Capillary | The narrow separation channel where the molecular race occurs; its coating ensures stable conditions. |
| Hydroxyethylcellulose (HEC) Polymer | A replaceable polymer that acts as a sieving matrix to separate nucleic acids by size. |
| TBE Buffer (Tris-Borate-EDTA) | The background electrolyte that conducts current and maintains a stable pH for the separation. |
| SYBR Green II Dye | A fluorescent dye that binds specifically to nucleic acids (DNA/RNA), allowing for their detection. |
| High-Voltage Power Supply | Provides the electric field that drives both the electroosmotic flow and the electrophoretic migration. |
Capillary electrophoresis has cemented its role as an indispensable tool in the life sciences. Its advantages are numerous 6 8 :
CE can achieve hundreds of thousands of theoretical plates—a measure of separation power—far surpassing traditional liquid chromatography 6 .
Analyses that once took hours on a gel can now be completed in minutes. CE consumes only nanoliters of sample and milliliters of buffer, making it a cost-effective and green technology 8 .
These qualities make CE the gold standard in forensic DNA analysis for STR profiling 3 6 , a critical quality control tool for pharmaceuticals and biologics like monoclonal antibodies 6 8 , and a powerful partner to mass spectrometry in the emerging fields of proteomics and metabolomics 6 . Recent advancements, such as the use of Deep Eutectic Solvents (DES) as green and efficient buffer additives, promise to make CE even more sustainable and powerful, particularly for challenging separations like distinguishing chiral drug molecules 4 .
From its theoretical foundations in electrophoretic mobility to its practical application in separating the very molecules of life, capillary electrophoresis represents a perfect marriage of simplicity and power. By orchestrating a race of ions within a microscopic tube, it provides a window into the molecular world that is both precise and accessible. As it continues to evolve, coupling with ever-more-sensitive detectors and integrating into handheld "lab-on-a-chip" devices, this remarkable technique will undoubtedly remain at the forefront of scientific discovery, helping us decode the complexities of biology one nanoliter at a time.