Tech Compare: Capillary vs. Gel Electrophoresis

Electrophoresis methods are used in a wide range of applications to separate DNA, RNA and protein macromolecules, and many different techniques and technologies are available to achieve electrophoretic separations. Two of the most common electrophoresis formats used today are gel electrophoresis, also known as slab-gel electrophoresis (SGE), and capillary electrophoresis (CE), which includes capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE), among others. 

Gel electrophoresis separates macromolecules by size and charge using an electric field to move the sample through a vertical or horizontal gel slab matrix, usually made from agarose or polyacrylamide. The separated components form distinctive bands in parallel lanes through the gel, which can be viewed after staining and compared or analyzed for applications like DNA fingerprinting or protein detection. 

Capillary electrophoresis can also separate molecules by size and charge, but instead of a slab gel, samples are moved through a narrow-diameter tube filled with a fluid or gel matrix, depending on the specific technique. The separated components are measured by a detector, such as a UV/visible absorbance or fluorescence detector, and appear as peaks on an electropherogram that can be compared and analyzed. 

Capillary electrophoresis is a newer technique than slab-gel electrophoresis, and can offer significant advantages in terms of speed, resolution and automation abilities, but SGE is still widely used in many fields as a well-established method with some of its own benefits and advanced applications. 

Read on for a more in-depth comparison of these two valuable separation techniques:

Resolution

In general, capillary electrophoresis is a higher-resolution method than slab-gel electrophoresis, mainly because the higher surface-to-volume ratio within the thin capillary tubes allows heat to dissipate faster; thus, much higher voltages can be used without causing the matrix to overheat.¹ The use of narrow capillary tubes also increases separation efficiency by reducing lateral diffusion of molecules.² For nucleic acids, CE can provide single-nucleotide resolution, making it an especially powerful technique in sequencing, single-nucleotide polymorphism (SNP) analysis and human identification applications. Capillary techniques for protein analysis, such as capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), also provide higher resolution and quantitative data; for example, CE-SDS can aid in detection of non-glycosylated species that are difficult to resolve using SDS-PAGE (polyacrylamide gel electrophoresis).³ 

Although CE has clear advantages in terms of resolution, traditional gel electrophoresis still provides sufficient separation efficiency for many applications, such as analysis of polymerase chain reaction (PCR) products, restriction mapping and nucleic acid purification.⁴ SGE resolution can also be improved by adjusting parameters such as well size, gel percentage, gel thickness and run time.⁵ Polyacrylamide gels provide higher resolving power for smaller molecules than agarose. Additionally, some variations of gel electrophoresis provide better separations for certain analytes, such as 2D gel electrophoresis, which separates proteins by both isoelectric point and molecular mass, and pulsed-field gel electrophoresis, which improves resolution of larger DNA fragments. 

Speed and Throughput

As already mentioned, capillary electrophoresis allows for much higher voltages to be used, and separations can be completed in just minutes, in contrast to slab gel separations that can take up to an hour or hours to reach completion. This also doesn’t include the time needed to prepare the gel slab, load the samples and stain the gel. SGE typically includes more manual steps than CE, and CE is easily automated with fully-automated systems widely available. For this reason, CE is more widely used in high-throughput applications than SGE. 

SGE throughput can be improved through solutions such as high well-count gels (ex. 96-well gels), robotic loading systems and use of low conductivity buffers that allow higher voltages to be used. Preparing many gels in advance, or using precast gels, can also help cut back on time. 

Cost

One of the benefits of traditional gel electrophoresis is its relatively low cost. Most capillary electrophoresis systems include at least some automation¹ and the upfront cost of these systems will generally be greater than that of less complex slab-gel systems. You will also need to consider the cost of software to collect and process CE data, as well as compare the costs of CE consumables like capillaries/cartridges to SGE consumables like gels and stains to determine overall costs per sample. While CE systems, especially fully automated, high throughput systems, may come with a higher price tag, factoring in the time and labor savings of a CE system can help determine whether it may be more cost-effective in the long term.⁶ 

Multiplexing

Another aspect that many see as an advantage of slab-gel electrophoresis is the ability to visualize the results of multiple samples in a parallel format. A single slab gel can be used to run up to dozens of samples simultaneously, while each capillary can only run one sample at a time and multichannel systems are needed to complete runs in parallel. However, the speed advantages of CE, and the capability of on-capillary detection, can make it more advantageous for running multiple samples, either in succession or in parallel, depending on the number of samples and channels. The ability to visually inspect bands on a slab-gel versus the peaks on an electropherogram may be a matter of personal preference, but any system you choose for running multiple samples should be assessed to ensure both data quality and overall efficiency are optimized to meet the needs of your lab. 

Other Considerations

Here are a few other things you should keep in mind when deciding between gel electrophoresis and capillary electrophoresis methods:

• Slab gels can be more susceptible to contamination from the environment, gel material impurities or human error during casting or handling. They are also more susceptible to overheating, which can cause the gel to distort and melt. On the other hand, capillaries are also susceptible to their own problems, such as leakage and clogging. 

• Capillary electrophoresis systems typically require a smaller amount of sample than slab-gel systems, which can be especially beneficial when only a small amount of sample is available. 

• Gel electrophoresis can be used as a preparative/purification technique as samples can be recovered from slab gels with relative ease. However, this is also possible in a capillary format, using wide-bore capillaries (with diameters greater than 100-200 μm) and low voltage.⁷

• Capillary electrophoresis can be considered a more versatile method than gel electrophoresis, due to the wider range of separation matrices that can be used, as well as the wide range of detection platforms that can be used with CE, including surface enhanced Raman spectroscopy (SERS) and mass spectrometry (MS).

References

1. "Capillary Eletrophoresis Systems for High-Resolution Separations," Caitlin Smith, Labcompare (2014). https://www.labcompare.com/10-Featured-Articles/133264-Capillary-Electrophoresis-Systems-for-High-Resolution-Separations/ 

2. "Advantages and Disadvantages of Capillary Electrophoresis," Chromatography Today (2014). https://www.chromatographytoday.com/news/electrophoretic-separations/35/breaking-news/advantages-and-disadvantages-of-capillary-electrophoresis/32342 

3. Davies, L.; Dwyer, P.; Devanur, D. Benefits of Capillary Electrophoresis (CE) instrumentation in monoclonal antibody analysis. Poster. http://www.deltadot.com/index_htm_files/NHS_poster.pdf 

4. "Nucleic Acid Electrophoresis Applications–Preparative and Analytical Electrophoresis," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-applications.html 

5. "Nucleic Acid Electrophoresis Additional Considerations–7 Aspects," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-considerations.html 

6. "Cost Benefit Analysis of a Multicapillary Electrophoresis System," Varouj Amirkhanian, Ming Lui, Andras Guttman, Eszter Szantai, American Laboratory (2006). https://www.americanlaboratory.com/913-Technical-Articles/35725-Cost-Benefit-Analysis-of-a-Multicapillary-Electrophoresis-System/ 

7. Lauer, H.H.; Rozing, G.P. High Performance Capillary Electrophoresis; Publication Number 5990-3777EN; Agilent Technologies: 2018; pp. 50. https://www.agilent.com/Library/primers/Public/5990-3777EN.pdf