- Capillary electrophoresis (CE) is a powerful analytical technique used to separate ionic species based on their size-to-charge ratio. It is a modern evolution of traditional gel electrophoresis and operates by applying an electric field across a narrow capillary tube filled with an electrolyte solution.
- Because of its high resolution, speed, and small sample requirements, CE has become a valuable tool in diverse fields such as molecular biology, forensic science, pharmaceutical analysis, and clinical diagnostics.
- At the core of capillary electrophoresis is the principle of electrophoresis—the movement of charged particles through a fluid under the influence of an electric field. In CE, the sample is introduced into a thin fused silica capillary (usually 25–100 micrometers in internal diameter and 20–100 centimeters in length), which is filled with a background electrolyte. When a high voltage (often 10–30 kV) is applied, ions in the sample migrate toward the electrode of opposite charge. Smaller and more highly charged particles move more quickly, while larger or less charged particles move more slowly, enabling their separation within the capillary.
- One key feature that enhances separation efficiency in CE is electroosmotic flow (EOF). In silica capillaries, the inner wall carries negative charges, which attract a layer of positively charged ions from the buffer. Under an electric field, these ions drag the surrounding liquid toward the cathode, creating a bulk flow of the buffer solution. EOF allows both positively and negatively charged species to be carried through the capillary in the same direction, improving the speed and uniformity of the separation.
- Detection in CE is typically performed using ultraviolet (UV) absorbance or laser-induced fluorescence (LIF), depending on the nature of the analytes. In DNA sequencing applications, for example, DNA fragments are labeled with fluorescent dyes, and a laser excites the fluorescent tags as the fragments pass a detection window. The emitted light is captured by a detector, and the resulting data is used to determine the composition or sequence of the sample.
- Capillary electrophoresis offers several advantages over traditional gel-based techniques. It provides high resolution, allowing the separation of molecules that differ by as little as a single nucleotide. It also consumes very small volumes of sample and reagents, making it cost-effective and suitable for precious or limited samples. CE systems are often automated and computer-controlled, which minimizes human error and allows for high-throughput analysis.
- In addition to DNA analysis, CE is used for the separation and quantification of proteins, peptides, amino acids, pharmaceutical compounds, and inorganic ions. It also plays a crucial role in clinical diagnostics, such as detecting hemoglobin variants or analyzing cerebrospinal fluid proteins. Due to its versatility and efficiency, capillary electrophoresis remains a fundamental technique in both research and applied sciences.
