Electrophoretic separation of molecules such as nucleic acids and proteins remains a staple in any modern molecular biology laboratory. This is a vital technique that allows the investigator to move charged molecules through a gel or free fluid medium under the influence of an electric field, allowing for the characterisation of those molecules (National Human Genome Research Institute, 2025). Traditionally, and still currently, slab gels made of polyacrylamide or agarose are the most common for electrophoretic separations. Although still used widely by many laboratories, slab gel electrophoresis generally suffers from inconvenient manual gel casting, long analysis times, low efficiencies, and difficulties in detection and automation.
In contrast to traditional slab gels, capillary gel electrophoresis (CGE) makes use of narrow-bore capillaries to achieve separation (Santos and Brodbelt, 2021; Hajba et al., 2023). Capillaries have very low conductance, so they generate minimal heat and are naturally resistant to convection, overcoming some of the difficulties associated with slab gel techniques. In this blog post, we will touch upon the basics of the CGE technique.
Nucleic Acids Have a Negative Charge
A nucleic acid’s chemical structure influences its behaviour in an electric field, affecting how efficiently it can be separated and thus informing its size (Figure 1.). DNA is a polymer of monomeric units known as nucleotides. A single nucleotide consists of a 5-carbon sugar, deoxyribose, a nitrogenous base and one or more phosphate groups. The 4 bases of DNA are guanine (G), cytosine (C), adenosine (A) and thymine (T) (Saenger, 1984). RNA on the other hand contains a 5-carbon sugar ribose, and instead of thymine, contains uracil (U) as one of its 4 bases. In either case, DNA and RNA hold an overall negative charge due to the negatively charged phosphate groups found in both, therefore in an electric field the molecules will migrate towards the positive end of the field.
“Old-school” Slab Gels
Slab gel electrophoresis is a widely used method for separating DNA and RNA fragments based on size (Barron and Blanch, 1995). To prepare these gels, agarose is dissolved in a buffer such as TAE with heat, poured into a casting tray, and allowed to solidify around a plastic comb that creates sample wells. Agarose is a common polymer used for nucleic acid separations due to its larger pore size when solidified, which is more suitable for DNA separations. Once nucleic acids are loaded into these wells and an electric current is applied, the negatively charged DNA or RNA migrates toward the positive electrode. Smaller fragments move more easily through the gel matrix and travel farther than larger ones, creating a size-based separation pattern. To interpret results, samples are compared to a “ladder” of fragments with known sizes. This not only helps estimate fragment length but can also give a rough, semi-quantitative estimate of concentration by comparing band intensities, either visually, or with the help of imaging systems. Visualisation is typically achieved by staining the gels with dyes such as ethidium bromide or SYBR Safe, which bind nucleic acids and make the bands visible under UV or blue light. Although this article focuses on nucleic acids, it’s worth noting that a similar principle is applied to proteins. As an additional step to account for the variable charges of different proteins, a detergent such as SDS is added, which coats proteins with a uniform negative charge allowing them to be separated by size only in a gel. Typically, acrylamide-based gels are used for protein separations.
Slab gel electrophoresis is a versatile quality-control method in molecular biology, extending beyond simple DNA sizing. It is commonly used to verify PCR products or restriction digests, assess RNA integrity prior to transcriptomic workflows, and screen for genome editing outcomes such as CRISPR-induced changes (Skrypina, 2003; Garibyan and Avashia, 2013; Bhattacharya and Van Meir, 2019). Plasmid preparations can also be checked to confirm the presence and integrity of vector DNA, while DNA extraction success can be quickly confirmed before moving on to more complex downstream experiments (Miller et al., 1999; Schmeer and Schleef, 2014). In addition, specific DNA fragments can be physically excised and purified from a gel, making electrophoresis valuable not only for analysis but also for preparative steps such as cloning (Griffin and Gasson, 1995). While slab gels are useful, their limitations in scalability and precision have led to more modern alternatives like CGE (Table 1.).
Table 1. Pros and Cons of Traditional Slab Gel Electrophoresis
| Pros | Cons |
|---|---|
| Inexpensive and widely accessible | Manual, labour-intensive |
| Simple to run and interpret | Low resolution and reproducibility |
| Useful for visual QC and teaching purposes | Difficult to scale for high-throughput needs |
| No specialised instrumentation required | Poor quantification, semi-quantitative; subjective band comparison |
From Slab Gel to Capillary Gel Electrophoresis
At its most basic, capillary electrophoresis uses a narrow-bore capillary to separate biomolecules such as DNA, RNA, or proteins with high resolution (Figure 2.). The capillary is normally made from a chemically and electrically inert, flexible and robust material such as fused-silica (Lauer and Rozing, 2018). The capillary ends are placed in buffer reservoirs containing the same solution as inside the capillary, along with electrodes that connect to a high-voltage power supply. A sample is introduced by temporarily replacing one reservoir with a sample reservoir and loading the sample via an electric field (electrokinetic injection) or by applying pressure (hydrodynamic injection). After the sample is loaded, the buffer reservoir is restored, and an electric field is applied along the capillary to perform the separation. Molecules are detected optically, often through the capillary wall using UV absorbance or fluorescence. When the capillary contains a sieving polymer, the technique is specifically referred to as “capillary gel electrophoresis” (CGE), which allows size-based separation like traditional slab-gel electrophoresis but with greater speed, sensitivity, resolution and automation capabilities.
Table 2. Strengths of Capillary Electrophoresis Technology
| Strength | |
|---|---|
| Automation & Miniaturization | Capillaries automate many steps of electrophoresis and work on a much smaller scale, reducing manual handling and variability. |
| Resolution, Speed & Quantitation | The narrow format enables sharper separations, faster run times, and more accurate quantitation compared to traditional slab gels. |
| Reduced Heating | Capillaries dissipate heat efficiently, minimizing Joule heating and allowing high voltages to be used without distorting results. |
| Lower Sample & Reagent Use | Only small amounts of sample and buffer are needed, saving valuable material and reducing reagent costs. |
| High-Throughput Workflows | Capillary systems are designed for parallel processing and standardized methods, making them well-suited for higher-throughput applications. |
Migration Principles Through Capillary and Polymer
Separation using CGE relies on the same principle as slab gels: molecules move through a polymer matrix that acts as a molecular sieve (Lauer and Rozing, 2018). Larger fragments are slowed down by the gel network, while smaller ones move through more easily, creating a clear size-based separation (Figure 3. A)). This sieving effect is essential because DNA molecules, regardless of length, all carry a similar mass-to-charge ratio. Without a gel or another sieving medium, they would migrate at nearly the same speed in an electric field, making true separation impossible.
One of the strengths of capillary systems is their flexibility. Different gel polymers can be quickly prepared and exchanged within the capillary, allowing researchers to adapt the system to a wide range of sample types and analytical needs. The term “gel” is used loosely when referring to the polymers used in the capillaries, and it actually refers to liquid-phase polymer networks. Various types of polymers are used in CGE all with different structures and application uses (Figure 3. B)). Capillaries themselves also come in various lengths and diameters, further refining the resolution and separation characteristics. This adaptability makes capillary electrophoresis not only more efficient but also a powerful tool for diverse applications in nucleic acid analysis.
The concept of Electroosmotic flow (EOF) requires brief mention as it is an important phenomenon often seen in bare capillaries, whereby the liquid phase involved in the separation moves toward the cathode due to surface charge on the capillary wall (Verheggen, Schoots and Everaerts, 1990). In CGE, EOF is largely suppressed by the polymer matrix, allowing for purely size-based separations, therefore this will not be addressed in further detail in this article.
Sample introduction into the capillary can be achieved using either hydrodynamic or electrokinetic approaches, each with their own advantages and considerations (Figure 4.) (Breadmore, 2009; Lauer and Rozing, 2018). In hydrodynamic injections, the sample is physically moved into the capillary by manipulating pressure differences. This can be done by applying positive pressure at the inlet, by applying a vacuum at the outlet, or by using a simple siphoning method whereby the sample reservoir is placed slightly higher than the capillary outlet to allow gravity-driven flow. These approaches are generally straightforward and avoid bias toward certain analytes, making them suitable for a wide range of applications.
By contrast, electrokinetic injections rely on voltage to drive sample into the capillary. This method can provide advantages when working with viscous gels or media where pressure injection is less effective. However, it introduces a level of selectivity: analytes with higher electrophoretic mobility are preferentially injected, while sample loading is also influenced by factors such as buffer conductivity, residual EOF, and solute mobility. As a result, electrokinetic injections are more sensitive to matrix effects, particularly in high-salt buffers, but it remains a valuable option in situations where hydrodynamic injections are impractical.
Component Considerations
In CGE, the power supply and field control are central to ensuring high precision (Figure 5.) (Kubáň, Foret and Erny, 2019). A stable, high-voltage DC supply operating in constant-voltage mode provides reproducible migration times across runs. More advanced setups incorporate field programming, where voltage ramps are applied gradually rather than instantaneously. This reduces thermal shock to the capillary and enhances resolution, particularly in complex separations where subtle differences in fragment mobility must be distinguished. Equally important is temperature regulation, as even small fluctuations in temperature can alter buffer viscosity and, by extension, migration times (Rathore, 2004). Effective cooling is typically achieved either by blowing a high-velocity air stream on to the capillaries or immersing them in a temperature-regulated liquid, both of which help maintain reproducibility and protect sample integrity. In addition, the buffer composition, including pH, ionic strength, and additives, can significantly influence separation efficiency and reproducibility, complementing the effects of voltage and temperature control.
Detection in CGE relies heavily on fluorescent dye chemistry and imaging systems. Intercalating dyes such as SYBR Green, GelRed, or less commonly ethidium bromide insert between nucleic acid base pairs and fluoresce when excited by a laser or LED (Kricka and Fortina, 2009; Li et al., 2024). Once bound, these dyes fluoresce under excitation, enabling sensitive, real-time monitoring of migrating DNA or RNA fragments. Because the dyes bind indiscriminately to any nucleic acid present, sample purity directly affects quantification accuracy. For automation, dyes are often pre-mixed into the separation matrix or buffer, streamlining workflows for high-throughput systems. At the detection window, fragments are illuminated by a laser or LED, and the emitted fluorescence is captured by a CCD camera array. This setup offers high sensitivity and allows parallel detection across multiple capillaries, making modern CGE platforms both efficient and scalable.
Table 3. Characteristics of Automated CGE Platforms
| Feature | Description |
|---|---|
| Capillaries | Narrow-bore fused silica (25-75 µm internal diameter) |
| Voltage | High (10-30 kV; 100-500 V/cm) |
| Heat control | High resistance minimises current and internal heating |
| Performance | High efficiency, sensitivity and shorter run time |
| Detection | On-capillary |
| Sample size | Small sample volumes (~1-50 nl) |
| Flexibility | Multiple modes and wide application range |
| Methods | Simple to develop and execute |
| Automation | Fully automated, supports massive parallel runs |
Comparing Results, Slab vs. Capillary
The comparison between traditional slab gel electrophoresis and CGE highlights key differences in data presentation and workflows (Figure 6.). In slab gels, DNA or protein samples appear as fuzzy bands whose clarity can vary, and quantitation is semi-quantitative, relying on band brightness in static images that require manual interpretation. By contrast, capillary electrophoresis produces sharp, high-resolution peaks in electropherograms, where the x-axis represents fragment size or migration time and the y-axis reflects signal intensity, allowing precise quantitation through peak height or area under the curve.
Example Use Case, mRNA Vaccine Production QC
In vitro transcribed (IVT) mRNA has quickly become a powerful tool in biotherapeutics, driving innovations in vaccines, protein replacement and cancer treatments (Aljabali et al., 2023; Vavilis et al., 2023; Sayour et al., 2024). The process begins with a DNA template, often a linearised plasmid, which is transcribed in vitro to generate mRNA (Davidopoulou, Kouvelas and Ouranidis, 2024; Stamoula et al., 2025). To protect the mRNA from degradation and support efficient protein production, a 5’ cap and poly(A) tail are added during synthesis. This IVT mRNA is then purified and packaged into a delivery system such as lipid nanoparticles (LNPs). These LNPs not only protect the mRNA in the body but also improve its uptake by cells and boost translation efficiency. Each step in this workflow is critical. From DNA template to final drug product, robust quality assessments are essential for ensuring the safety, effectiveness, and scalability of IVT mRNA therapies. The use of automated CGE platforms can play an important role in quality control of the IVT mRNA production process (Warzak, Pike and Luttgeharm, 2023).
The development of an IVT mRNA product follows a carefully controlled process, with quality checks at every stage (Figure 7. and Figure 8.). The gene of interest is first synthesised into a plasmid, which serves as the starting material for the IVT reaction. Before moving forward, PCR is often used as a quality control step to verify the plasmid’s identity and purity, ensuring the correct sequence is present. The plasmid is then linearised by restriction digest, a critical step that prepares it for efficient in vitro transcription. Techniques such as CGE can be used to confirm the sizing and integrity of both the PCR product and the linearised plasmid, adding another layer of confidence before mRNA synthesis. Once transcribed in the IVT reaction, the RNA product is cleaned up and tested for key features such as size, purity, poly(A) tail integrity, and proper capping to ensure stability and function. CGE provides a sensitive and reliable assay to assess the size, quality, and purity of the IVT mRNA, and detects incompletely transcribed and degraded RNA products to enable optimisation of the IVT reaction. The purified RNA is then formulated into a LNP delivery system, which protects the fragile molecule and enables it to reach target cells. In the final stage, the complete drug product is evaluated for LNP size, quality, concentration, and encapsulation efficiency. These rigorous checks ensure that each vaccine batch meets the highest standards of safety and effectiveness, with CGE playing an important role in all these various checks.
In Conclusion
CGE effectively merges the size-based separation principles of traditional slab gels with the precision, speed, and automation of modern capillary systems. By employing sieving polymer networks, CGE enables high-resolution separation of macromolecules such as DNA and RNA. Modern instruments, including the Agilent Fragment Analyser, deliver reproducible results with minimal Joule heating, faster run times, and high-throughput capability through multi-capillary arrays, all within fully automated workflows that produce digital data such as electropherogram traces. CGE has become a vital component for applications such as quality control of IVT mRNA and NGS libraries, RNA integrity assessment, and general nucleic acid sizing and purity analysis. While slab gels remain relevant in certain contexts, such as gel excision for cloning or in smaller academic labs, CGE has largely supplanted them for routine, quantitative, and scalable nucleic acid analysis across research, development, and production environments.
Diagnostech is the official distributor of Agilent’s Genomics portfolio throughout most of Sub-Saharan Africa. This includes their range of automated CGE instruments, the Fragment Analyzer systems. Several different instrument models and a broad range of kits ensure flexibility and quality of analysis for your DNA and RNA samples. Please reach out to us for more information on Agilent’s CGE solutions and for expert guidance on your nucleic acid analysis applications.
References:
