Capillary Zone Electrophoresis (CZE) is an analytical method used to separate components within a complex liquid mixture. This technique applies an electric field to drive charged molecules through a very narrow capillary, typically made of fused silica. The capillary’s internal diameter is often less than the width of a human hair, creating a microscopic environment for separation. CZE achieves extremely high-resolution separations quickly, making it an advanced tool in analytical chemistry and biochemistry. A mixture is injected into one end, and a high voltage is applied across the tube, initiating the movement and separation of molecules based on their distinct physical properties.
Understanding the Separation Principles
The fundamental physics governing CZE separation involves the simultaneous action of two forces: electrophoretic mobility and electroosmotic flow (EOF). Electrophoretic mobility is the tendency of a charged molecule to move toward the electrode of the opposite charge when an electric field is applied. Cations (positive ions) move toward the negative cathode, while anions (negative ions) move toward the positive anode. A molecule’s travel speed is determined by its charge-to-size ratio; smaller molecules with a higher charge move faster.
The second, and often dominant, force is the EOF, which acts as a bulk movement of the entire buffer solution. In a typical fused silica capillary, the inner wall develops a negative charge when contacting the buffer, attracting a layer of positive ions. When high voltage is applied, these positive ions migrate toward the negative cathode, dragging the entire volume of buffer solution along with them.
The combined effect dictates the resulting separation order. Positively charged ions, pulled toward the cathode by both their electrophoretic mobility and the EOF, exit the capillary first. Neutral molecules, unaffected by the electric field, travel only at the speed of the EOF and emerge next. Negatively charged ions are pulled toward the anode by their mobility, forcing them to fight against the bulk EOF, causing them to emerge last.
Unique Benefits of Capillary Zone Electrophoresis
CZE offers several advantages over older separation techniques, such as High-Performance Liquid Chromatography (HPLC), due to its microscale design and reliance on electrokinetic forces. A primary benefit is ultra-high separation efficiency, often resulting in resolution that exceeds other methods. This efficiency stems from the “plug flow” profile of the EOF, where fluid moves uniformly across the capillary’s diameter, minimizing band broadening caused by pressure-driven systems.
The small capillary dimensions allow for minute sample and reagent consumption. Sample volumes are frequently in the nanoliter or picoliter range, making CZE ideal for sample-limited applications like single-cell analysis. This minimal consumption contributes to cost-effectiveness and reduces solvent waste.
Analysis times are often significantly shorter, with separations frequently completed in minutes. High voltages can be applied across the narrow capillary, allowing fast migration without excessive heat generation that could compromise separation quality. The system is also readily automated, enabling laboratories to process a large number of samples quickly and reproducibly.
Real-World Applications
The precision and speed of CZE make it a valuable analytical tool across a wide range of scientific and industrial fields.
Medical Diagnostics
CZE is routinely used to analyze complex biological fluids like blood, urine, and cerebrospinal fluid. It is employed to screen for and quantify different forms of hemoglobin, such as Hemoglobin A2 and Hemoglobin S. This analysis aids in the diagnosis of blood disorders like thalassemia and sickle cell disease.
Pharmaceutical Industry
The pharmaceutical industry relies on CZE for quality control and drug development. It ensures the purity of drug substances by separating the active compound from trace impurities or degradation products. CZE also assists in characterizing complex therapeutic proteins, helping scientists monitor subtle changes in charge variants that impact effectiveness.
Forensic Science
Forensic laboratories employ CZE for high-resolution analysis of trace evidence due to its sensitivity and small sample requirement. It is used for DNA fragment analysis and sequencing, which is fundamental to human identification and paternity testing. The technique can also analyze the chemical composition of illicit substances, inks, or explosive residues found at a crime scene.
Environmental Monitoring
CZE is effective for detecting and quantifying various organic and inorganic pollutants in water samples. The technique separates a wide array of charged contaminants, such as industrial waste byproducts or pesticide residues, even at low concentrations. This high-resolution separation allows environmental scientists to accurately assess water quality and track regulated substances.