An Introduction to Capillary Electrophoresis: Theory, Practice and Applications

Capillary electrophoresis (CE) is the name of a family of analytical techniques that involve the separation of charged molecules within a narrow capillary tube under the influence of an electric field.

The origins of these techniques (Figure 1) can be traced to the early development of electrophoresis in the 1930s by the Swedish biochemist Arne Tiselius.¹ In the 1950s, scientists, including the Swedish biochemist Stellan Hjertén, introduced the use of chromatographic capillary tubes to the electrophoretic techniques in response to a need to use smaller amounts of sample and improve separation efficiency.² In the 1980s, James W. Jorgenson and Krynn D. Lukacs introduced the use of fused silica capillaries of less of 100 μm, significantly improving the performance of the technique, which lead to its popularization.³  Today, CE is a powerful separation and analysis technique that offers quick and efficient results, performing better in many cases than other techniques such as liquid chromatography (LC). For this reason, CE is widely employed in pharmaceutical and biotechnological industries.⁴

Figure 1: Historical milestones of CE development. Credit: The Author.

In general, electrophoresis techniques are based on the premise that different charged chemical species in a sample move at different velocities when an electric field is applied. These distinct mobilities, which depend on the charge and size of the molecules, enables the separation of the analytes present in a sample.^{5, 6, 7} Although all electrophoresis techniques share these fundamentals, there are many methodologies that can be classified into four groups (Figure 2):⁸

Figure 2: Classification of electrophoretic techniques. Credit: The Author.

• Gel electrophoresis: separation takes place through a gel matrix.
• Zone electrophoresis: molecules are separated into zones as a function of their size.
• Free flow electrophoresis: separation occurs through a buffer flow between two glass plates.
• Capillary electrophoresis: analytes separate within a fused silica capillary.

What sets CE apart from other electrophoretic techniques is its utilization of a fused silica capillary as the separation medium. This microscale system offers numerous advantages, including high separation efficiency, rapid analysis, minimal sample consumption and compatibility with various detection methods such as UV-absorbance, fluorescence and mass spectrometry. Furthermore, CE can be performed in various modes (which will be discussed later), each tailored to specific separation requirements. Unlike the widespread gel electrophoresis, CE does not require extensive gel preparation or offer limited separation lengths. Instead, it relies on the electroosmotic flow phenomenon, wherein a controlled flow of buffer solution moves the analytes through the capillary, enhancing separation efficiency. In contrast to other electrophoretic techniques, CE offers superior resolution due to its ability to mitigate diffusion effects and enhance reproducibility. The small capillary dimensions and precise control of experimental parameters grant CE a distinctive edge over conventional methods. While gel electrophoresis depends on the migration of molecules through a porous gel matrix, and other electrophoretic methods may involve larger separation platforms, CE stands out for its high-resolution separation on a microscale. ^{5, 6, 7}

As explained before, electrophoretic separation is possible thanks to the different velocity at which chemical species move when an electric field is applied. This velocity is proportional to a physical magnitude known as ionic or electrophoretic mobility, μ_ep, which depends on the charge (q) and size (r_i) of the molecule in the following manner:

With η being the viscosity of the medium, this expression shows that the electrophoretic mobility is proportional to the charge/size ratio (q/r_i) and that the displacement direction is determined by the sign of the charge (Eq. 1). ^{5, 7}

μ_ep =        q            Eq. 1
6πηr_i 

In CE, the mobility of ions is affected by an additional phenomenon called “electroosmotic flow” (EOF). Fused silica capillaries are the type most commonly employed in CE and their inner surface displays ionizable moieties that present negative charge at pH values above 4. As a consequence, positive ions from the passing buffer solution become attached to the inner surface of the capillary, creating a very static layer (called a Stern layer). Next to the Stern layer, a more diffuse layer of ions is created (known as the outer Helmholtz layer) (Figure 3A). When an electrical field is applied, positive ions from the outer Helmholtz layer move to the cathode, dragging associated solvent molecules, which results in the generation of an EOF inside the capillary. This flow displays a uniform front, in contrast to the parabolic front of the more common hydrodynamic flow (Figure 3B), which results in a highly enhanced efficiency of the separation compared to other techniques. ^{5, 7}

Figure 3: A: Scheme of the ion distribution at the inner surface of a fused silica capillary. B: Comparison of the front profile of the flow for both electroosmotic and hydrodynamic flows. Credit: Technology Networks.

Because of the presence of the EOF, the mobility of the ions is redefined as an apparent mobility, μ_app:

                                                μ_app = μ_ep + μ_EOF                        Eq. 2

Subsequently, the separation of the chemical species in CE is determined by the joint action of the electrophoretic mobility and the electroosmotic mobility (Figure 4).^{5, 7}

Figure 4: Scheme of the migration of chemical species occurring in CE and its dependence on charge and size. Credit: Technology Networks.

The equipment for CE is relatively simple. In the case of the most commonly used CE technique, capillary zone electrophoresis (CZE), the capillary is filled with an electrolyte solution, and an electric field is applied across it. The capillary itself consists of three key regions: the injection end, separation region and detection point (Figure 5). At the injection end, a small sample volume is introduced. The separation region, where the actual separation takes place, is typically coated with a polymer to minimize interactions with the capillary wall. As the electric field is applied, positively charged ions (cations) migrate towards the negatively charged cathode, while negatively charged ions (anions) move towards the positively charged anode. Bearing in mind Eq. 1, smaller, highly charged molecules will move faster than larger or less charged ones. Neutral molecules will migrate unseparated to the cathode by the action of the EOF. When the analytes reach the detection point, they are registered by the detector (UV-visible, fluorescence, mass spectrometry…) and then displayed as peaks in a spectrum called an “electropherogram”. In this electropherogram, different peaks corresponding to different analytes are displayed at the time they reach the detector, with the peak area being directly proportional to the amount of analyte in the sample. ^{5, 6, 7, 9}

Figure 5: Schematic representation of a typical CE instrument. Credit: Technology Networks.

As mentioned before, CE encompasses a set of techniques with different characteristics that allow adaptation to diverse separation or sample requirements. The most relevant CE techniques are summarized in the next paragraphs and compared in Table 1.

Capillary zone electrophoresis (CZE)

CZE is the most commonly used CE technique and its basic working principles have been explained in the previous section. CZE is characterized by its minimal sample preparation requirements and speed. This technique is particularly suitable for ions and small molecules, but its resolution might be limited when dealing with complex mixtures, as closely related species can elute in broad peaks.¹⁰

Capillary gel electrophoresis (CGE)

CGE enhances separation by incorporating a sieving matrix, typically a cross-linked polymer, in the capillary. This matrix retards the migration of larger molecules more than smaller ones, resulting in high-resolution separations. CGE is particularly well-suited for the separation of nucleic acids and proteins, enabling accurate analysis of molecular weight differences. However, CGE's reliance on the gel matrix makes it slower compared to other techniques.^{11, 12, 13}

Capillary isoelectric focusing (CIEF)

This technique exploits differences in the isoelectric points (pI) of analytes to achieve separation. The capillary is filled with an ampholyte buffer, creating a pH gradient. Analytes migrate to their respective pI, where they become electrically neutral and halt. This technique is excellent for proteins and peptides with distinct pIs. However, CIEF is time-consuming due to the need for pI calibration, and complex samples can challenge resolution.^{14, 15, 16}

Capillary isotachophoresis (CITP)

In CITP, the separation of analytes is based on their ionic mobilities in a stepwise manner. A leading electrolyte with lower mobility and a trailing electrolyte with higher mobility sandwich the analytes, driving them toward the detector. CITP excels in separating complex mixtures, ensuring that each analyte occupies a distinct migration zone. However, concentration effects can lead to distortion of peak shapes.^{17, 18}

Micellar electrokinetic capillary chromatography (MEKC)

MEKC is a hybrid technique that merges electrophoresis and chromatography. It involves adding surfactant micelles to the electrolyte. Neutral analytes partition between the aqueous phase and the micelle core, while charged analytes migrate according to their charge-to-mass ratio. MEKC's versatility allows for the separation of both neutral and charged compounds. However, micelle stability can affect reproducibility.^{19, 20}

Capillary electrochromatography (CEC)

CEC combines chromatography and electrophoresis principles. A stationary phase coats the inner capillary wall, interacting with analytes similarly to traditional chromatography. This interaction, combined with electrophoretic mobility, offers high-resolution separations. CEC is compatible with various detection methods and is capable of separating complex samples. However, the optimization of stationary phases and mobile phases can be intricate.^{21, 22, 23, 24}

Table 1: Summary of electrocapillary separation techniques with their strengths and limitations.

CE and LC are powerful separation techniques widely employed in analytical chemistry, each offering distinct principles and applications. Both techniques share some common features: Firstly, they involve the separation of complex mixtures based on the interaction of analytes with a mobile phase and a stationary phase. Then, both techniques can achieve high-resolution separations of a wide range of compounds, including small molecules, ions, proteins and biomolecules. And finally, they are compatible with various detection methods, such as UV-absorbance, fluorescence and mass spectrometry, enabling robust and versatile analyses.^{5, 7, 25, 26}

The fundamental difference between CE and LC lies in the driving force behind separation. Whereas CE relies on the differential migration of charged species under an electric field, LC capitalizes on differential interaction between analytes and a stationary phase within a column. In addition, CE typically offers rapid separations, while LC allows for greater control over separation parameters, making it suitable for complex mixtures.^{5, 7, 25, 26} 

Since both CE and LC present advantages and limitations (summarized in Table 2), some criteria must be taken in consideration in order to choose the technique that fits better to the studied sample and the available resources (Table 3).

Table 2: Summary of the advantages and disadvantages of CE and LC.

Table 3: Criteria for the selection of CE or LC as separation and analysis technique.

CE has emerged as a versatile analytical technique standing out because of its ability to separate charged molecules with remarkable precision and speed. For this reason, CE is utilized in diverse fields and applications, such as:

• DNA/RNA analysis: CE plays a pivotal role in genomics, facilitating DNA sequencing, fragment analysis and mutation detection.^{27, 28} For instance, Sanger sequencing utilizes CE to unravel genetic codes.²⁹ In RNA analysis, CE aids in transcriptomics, revealing gene expression patterns.^{30, 31} Additionally, CE's rapid separations and high-resolution capabilities enhance genotyping, something crucial for personalized medicine.^{32, 33}
  
  
• Food analysis: CE contributes to food safety and quality control. Detection of allergens, additives and contaminants is streamlined by CE's sensitivity.^{34, 35} It enables the quantification of vitamins, amino acids and fatty acids, ensuring nutritional label accuracy.³⁶ CE also helps to detect food product adulterations.³⁷
  
  
• Environmental monitoring: CE stands as an indispensable tool in environmental testing of pollutants, heavy metals and organic compounds in water and soil samples.^{38, 39, 40} Detection of pesticide residues and pharmaceutical pollutants aids in safeguarding ecosystems.^{41, 42} CE also assists in monitoring water quality, allowing the quantification of relevant anions like nitrates and sulfates.^{43, 44}
  
  
• Pharmaceutical analysis: CE expedites drug development, allowing quantitative determination of drugs to both assure maximum purity and the absence of dangerous impurities, ensuring product safety and efficacy.^{45, 46} Furthermore, the production of many drugs involves the selection and isolation of enantiomers. In this regard, CE-based chiral separations enable enantiomeric purity assessment, which is crucial since the presence of undesirable enantiomers may result in adverse effects.^{47, 48}
  
  
• Diagnostics: CE can be used as a tool for diagnostics through biomarker detection or protein profiling. It is possible to detect and quantify electrolytes, enzymes and hormones in body fluids, aiding disease diagnosis.^{49, 50, 51} In this context, CE-based immunoassays have been developed offering rapid and precise protein quantification.^{52, 53}
  
  
• Proteomics: CE applied to the study of proteins and peptides allows post-translational modifications to be uncovered and protein interactions to be identified.^{54, 55, 56, 57} This information contributes to the discovery of potential drug targets and disease biomarkers.
  
  
• Forensic science: CE facilitates forensic DNA analysis, vital in criminal investigations and paternity testing. The technique's sensitivity enables the analysis of minute samples, often crucial in solving cold cases.^{58, 59, 60}
  
  
• Biotechnology: CE is employed by biotechnological companies to ensure the quality of products such as recombinant proteins or monoclonal antibodies. CE-based assays assess protein folding and glycosylation, pivotal for biotherapeutic efficacy and safety.^{61, 62, 63}

CE has become one of the most versatile techniques to perform analysis of samples of a wide variety of molecules, from metal ions to organic compounds or biomolecules. Due to its resolution, speed and efficiency, it surpasses other conventional techniques such as LC in many aspects, and has become a routine technique in many diverse fields.