Capillary electrophoresis (CE) stands at the forefront of modern analytical techniques, revolutionizing the way researchers and scientists approach the separation and analysis of diverse compounds. This sophisticated method has found extensive applications across various scientific domains, from pharmaceutics to polymers, providing high-resolution separations and nuanced insights into complex samples.
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Principles of Capillary Electrophoresis
At its core, capillary electrophoresis is based on the principles of electrophoresis, leveraging the movement of charged particles under the influence of an electric field. The key differentiator lies in the capillary-scale dimensions of the separation channel, typically a fused-silica capillary with a small inner diameter. This miniature scale allows for enhanced efficiency and resolution compared to traditional electrophoresis methods.
The separation mechanism in CE involves the migration of charged species through a buffer-filled capillary, driven by an applied electric field. The speed of migration is influenced by the charge and size of the analytes, resulting in distinct bands or peaks along the capillary length. This migration is facilitated by the interaction between the charged analytes and the surrounding electrolyte solution.
Types of Capillary Electrophoresis
Capillary Zone Electrophoresis (CZE): This fundamental form of CE separates analytes based on their electrophoretic mobility in a buffer-filled capillary. It is widely applied in the analysis of small ions, peptides, and proteins.
Micellar Electrokinetic Chromatography (MEKC): Combining electrophoretic and chromatographic principles, MEKC utilizes surfactants to enhance the separation of neutral and charged compounds, making it effective for hydrophobic analytes.
Capillary Isoelectric Focusing (CIEF): CIEF exploits variations in the isoelectric points of analytes, allowing for separation based on differences in net charge at specific pH gradients. This technique is valuable in the study of proteins and peptides.
Capillary Gel Electrophoresis (CGE): Using a sieving matrix, typically a gel, CGE separates analytes based on size. It is commonly used for the analysis of nucleic acids and proteins.
Capillary Electrophoresis-Mass Spectrometry (CE-MS): Integration with Mass Spectrometry enhances the capabilities of both techniques, providing not only separation but also identification and characterization of analytes.
Applications of Capillary Electrophoresis
Applications in Pharmaceutics
The field of pharmaceutics has witnessed a transformative impact with the integration of capillary electrophoresis into its analytical toolkit. One of the notable applications lies in the characterization of biopharmaceuticals, with a particular focus on monoclonal antibodies (mAbs). CE, especially capillary zone electrophoresis (CZE), has emerged as an essential tool for characterizing mAbs using both intact and middle-up approaches. Innovative protocols, such as the two-phase-four-step mode, enable rapid method development for top-down and middle-up analysis of mAbs. The screening and optimization of parameters like pH, ionic strength, and viscosity enhancers contribute to efficient and accurate characterization.
Fig. 1 Applications of capillary electrophoresis (Voeten R. L. C., et al. 2018).
Vaccine development has also benefited from CE methodologies. Comparative studies employing CE and liquid chromatography (LC) methods for dose determination of recombinant subunit envelope protein-based vaccines highlight the advantages of CE, particularly in achieving separation of all vaccine components. In the case of tetravalent dengue subunit-based vaccines, capillary electrophoresis demonstrated superiority as a concentration assay.
The detailed characterization of therapeutic antibodies, such as cetuximab, illustrates the versatility of CE in pharmaceutics. Offline CZE-UV/ESI-MS and top-down characterization after sample enrichment using CE-UV/MALDI-MS provide in-depth insights into the structure and variants of these crucial biopharmaceuticals. The charge heterogeneity of mAbs, a pivotal quality attribute, is meticulously studied using CE, offering improved methods for the characterization of acidic and basic variants. Techniques involving immobilized pH gradient isoelectric focusing fractionation and subsequent characterization by CGE and LC-MS/MS exemplify the comprehensive analytical capabilities of CE in addressing complex challenges in biopharmaceutical analysis.
The analysis of antibody-drug conjugates (ADCs) further demonstrates the wide-ranging applications of CE in pharmaceutics. Innovative sheathless CZE-MS methods for middle-up analysis of ADCs like brentuximab vedotin showcase the precision and depth of characterization achievable. Integration with native MS allows accurate mass determination, providing insights into drug to antibody ratios and drug load distribution. Similar approaches are employed for the analysis of the ADC trastuzumab emtansine, employing various analytical techniques, including MS, imaging capillary isoelectric focusing (cIEF), and capillary gel electrophoresis (CGE) for structural characterization.
Despite the success of CE in pharmaceutics, challenges persist, including method-induced artifacts and the incompatibility of separation medium components with mass spectrometry (MS) detection. Studies highlight the importance of molecule-specific methods to minimize artifacts, emphasizing the ongoing need for tailored approaches in the rapidly evolving landscape of biopharmaceutical analysis.
Applications in Low-Molecular-Weight Drugs
Capillary electrophoresis has made substantial contributions to the analysis of low-molecular-weight drugs, providing rapid and sensitive methods for pharmaceutical quality control and environmental monitoring. Innovations in CE methodologies, such as sample stacking approaches and chirality probing, have enhanced sensitivity in drug analysis.
The development of fast CZE methods for the simultaneous analysis of drugs and impurities exemplifies the practical applications of CE in pharmaceutical dosage forms. The case of glibenclamide showcases the framework of quality by design, where critical quality attributes are represented by peak efficiency, critical resolution, and analysis time. The optimized conditions result in the full separation of analytes in less than 2 minutes, highlighting the efficiency and speed of CE in pharmaceutical analysis.
Trace analysis of weak acids, facilitated by CE with electrospray Ionization mass spectrometry (ESI-MS) detection, has broad implications for environmental monitoring and pharmaceutical analysis. The screening of tyrosinase inhibitors from traditional Chinese medicines, coupled with parallel molecular docking, illustrates the high-throughput capabilities of CE in drug discovery. The implementation of a short-end injection in CE, aiming for high throughput and reduced analysis time, further emphasizes the adaptability of CE methodologies to different analytical needs.
Applications in Polymers
Capillary electrophoresis has found increasing applications in the characterization of complex (bio)polymers, contributing to advancements in materials science. CE under critical conditions has proven to be a suitable approach for separating both natural and synthetic charged polymers. The use of CE as a viscometer, as showcased in the characterization of nitrocelluloses, exemplifies the versatility of CE beyond traditional separation techniques.
The development of CE-MS methods for characterizing glycosaminoglycans (GAGs) demonstrates the wide applicability of CE in polymer analysis. Reverse polarity CE separation and negative-mode electrospray ionization, optimized using a volatile methanolic ammonium acetate buffer gas electrode (BGE), provide a detailed analysis of low molecular weight heparin. The integration of orbitrap mass spectrometry facilitates disaccharide compositional analysis and bottom-up and top-down analysis of complex polysaccharides.
CE has also been applied to the qualitative and quantitative determination of oligosaccharides. Reference compounds, including xylo-, manno-, and cello-oligosaccharides, are concurrently measured in a highly alkaline solution without derivatization. This method's application to the determination of oligosaccharides from hot-water extracts of bleached birch and pine kraft pulp showcases its potential in studying the degradation of hemicelluloses into oligosaccharides as a function of time and temperature.
Conclusion
Capillary electrophoresis stands as a transformative force in analytical chemistry, permeating diverse scientific disciplines with its high-resolution separation techniques and nuanced insights. From the meticulous characterization of biopharmaceuticals to the rapid analysis of low-molecular-weight drugs and the intricate study of complex polymers, CE continues to shape the landscape of modern analytical methods.
The applications of CE in pharmaceutics underscore its critical role in drug development and quality control, offering precise and comprehensive characterization of therapeutic compounds. In low-molecular-weight drug analysis, CE's speed and sensitivity make it an invaluable tool for pharmaceutical and environmental applications. The realm of polymers sees CE contributing to the understanding of complex (bio)polymers, offering insights into structure and composition.
As CE continues to evolve, innovations in methodology and integration with other technologies are poised to unlock new possibilities. The journey of capillary electrophoresis from a laboratory technique to an integral component of analytical workflows exemplifies its adaptability and significance in advancing scientific research and industrial processes. The ongoing exploration and refinement of CE methodologies promise a future where its applications will continue to expand, meeting the ever-growing demands of analytical chemistry in the 21st century.
References
- Voeten R. L. C., et al. Capillary electrophoresis: trends and recent advances. Analytical Chemistry. 2018, 90(3): 1464-1481.
- Geiger M., et al. Capillary electrophoresis. Analytical Chemistry. 2012, 84(2): 577-596.
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