Introduction
Capillary Electrophoresis (CE) is a sophisticated electrophoretic technique that utilizes narrow capillaries to achieve high-resolution separations with rapid analysis times.
CE is highly automated and suitable for a wide range of applications, including the analysis of small ions, biomolecules, and pharmaceuticals.
Principle
In CE, an electric field is applied across a thin capillary filled with an electrolyte, causing charged molecules to migrate based on their electrophoretic mobility.
The small diameter of the capillary reduces band broadening, enhancing separation efficiency.
Methodology
Capillary Preparation: A fused silica capillary is cleaned and conditioned before use.
Sample Injection: Small volumes of the sample are introduced into the capillary via electrokinetic or pressure-based methods.
Buffer Filling: The capillary is filled with an appropriate running buffer.
Application of Electric Field: A high-voltage power supply drives the migration of analytes through the capillary.
Detection: As molecules exit the capillary, they are detected using methods like UV absorbance, fluorescence, or mass spectrometry.
Data Analysis: The resulting electropherogram is analyzed to determine the presence, concentration, and characteristics of the analytes.
Modes of Capillary Electrophoresis (CE)
Capillary Zone Electrophoresis (CZE): Separates analytes based on charge-to-size ratio in a buffer-filled capillary.
Capillary Gel Electrophoresis (CGE): Uses a gel medium for separating DNA fragments.
Micellar Electrokinetic Chromatography (MEKC): Employs micelles to separate neutral and charged analytes.
Capillary Isoelectric Focusing (cIEF): Separates proteins by their isoelectric point (pI).
Capillary Electrochromatography (CEC): Combines CE and HPLC, using chromatographic material for separation.
Detection Methods
UV/Vis Absorption: Common, requires UV-active analytes.
Fluorescence: Detects native or tagged fluorescent compounds.
Mass Spectrometry (MS): Coupled with CE for precise molecular identification.
Advantages
High Resolution: Exceptional separation efficiency due to minimal band broadening.
Speed: Rapid analysis times, often completed within minutes.
Small Sample Volume: Requires only nanoliter to microliter sample volumes.
Automation: Highly automated systems allow for high-throughput analyses.
Versatility: Can analyze a broad range of analytes, including ions, small molecules, and biomolecules.
Disadvantages
Complexity: Requires specialized equipment and expertise.
Cost: High initial investment for instrumentation and maintenance.
Sensitivity: May require sensitive detection methods for low-abundance analytes.
Buffer Requirements: Strict buffer conditions are necessary to maintain capillary integrity and performance.
Sample Preparation: Some samples may require extensive preparation to be compatible with CE.
Applications
Pharmaceutical Analysis: Drug purity testing and pharmacokinetic studies.
Clinical Diagnostics: Analyzing biomarkers and metabolic profiles.
Genomics and Proteomics: High-throughput DNA and protein analysis.
Environmental Monitoring: Detection of pollutants and toxins.
Forensic Science: Identification and quantification of substances in forensic samples.