Capillary Electrophoresis (CE) is a powerful analytical technique used to separate components within a complex chemical mixture. This method evolves traditional electrophoresis by moving the separation process from a large gel slab into a microscopic environment. The separation takes place inside a narrow, liquid-filled tube, known as a capillary, under the influence of a high-voltage electric field. Different molecules move through the capillary at different speeds, allowing scientists to isolate and identify them with high precision. CE is widely employed across science and industry to analyze compounds ranging from small ions to large biomolecules like DNA and proteins.
The Fundamental Principle: How Capillary Electrophoresis Works
The mechanism of separation in capillary electrophoresis relies on the precise movement of charged molecules through a buffer solution contained within a narrow fused-silica capillary. A typical system consists of two buffer reservoirs, a high-voltage power supply that applies up to 30,000 volts across the capillary, and a detector positioned near the outlet. The components of a sample are separated based on their unique velocities, which are determined by the interplay of two primary forces: electrophoretic mobility and electroosmotic flow.
Electrophoretic mobility describes the movement of a charged molecule when an electric field is applied. Positively charged molecules (cations) are attracted to the negatively charged electrode (cathode), while negatively charged molecules (anions) move toward the positively charged electrode (anode). The speed of this movement is directly proportional to the molecule’s charge and inversely proportional to its size and the buffer solution’s viscosity. Molecules with a higher charge-to-size ratio move the fastest, causing the initial differentiation of the sample components.
A second, powerful force driving the separation is electroosmotic flow (EOF), which is the bulk movement of the entire buffer solution inside the capillary. EOF originates because the inner wall of a fused-silica capillary contains silanol groups that become negatively charged at neutral to high pH levels. These negative charges attract positive ions from the buffer, creating a layer of positive charge near the wall. When the high voltage is applied, these positive ions migrate toward the cathode, dragging the entire volume of solvent with them.
The resulting flow profile of EOF is nearly flat across the capillary’s diameter, which is a significant advantage over the parabolic flow seen in pressure-driven methods like High-Performance Liquid Chromatography (HPLC). The overall velocity of a molecule is the sum of its electrophoretic mobility and the velocity of the EOF. Since the EOF is typically strong enough to pull all components toward the cathode, regardless of their charge, all molecules—cations, neutrals, and most anions—are detected in a single run. This combined movement provides the high-resolution separation characteristic of the technique.
Key Advantages Over Traditional Separation Methods
Capillary electrophoresis provides technical superiority over older separation techniques, such as slab gel electrophoresis and column chromatography, primarily through enhanced efficiency and speed. The method achieves exceptionally high separation efficiency and resolution. The narrow diameter of the capillary, typically between 20 and 100 micrometers, facilitates the rapid dissipation of heat, known as Joule heating, which is generated when high voltage is applied.
Efficiently removing this heat prevents the temperature gradients that cause band spreading, or diffusion, in traditional methods. This allows for the use of much higher electric fields, which leads to fast analysis times. Many separations are completed in just a few minutes, representing a substantial improvement over methods that can take hours.
CE requires only minute amounts of sample, often in the nanoliter range, which is especially valuable when working with precious or limited biological samples. The low consumption of both sample and buffer reagents translates to lower operating costs and less waste compared to large-scale techniques. Modern instrumentation is automated, meaning the entire process, from sample injection to data analysis, is performed with minimal manual intervention, increasing throughput and reproducibility.
Major Applications Across Science and Industry
Capillary electrophoresis is a primary tool across scientific and industrial applications, particularly in biotechnology and genomics for high-throughput DNA analysis. It is foundational to Sanger sequencing, where it separates fluorescently labeled DNA fragments that differ in length by only a single base pair. This provides the high resolution necessary to accurately determine the genetic code.
CE is used for fragment analysis, such as genotyping and DNA fingerprinting, precisely sizing DNA segments to identify genetic markers or establish relationships. The pharmaceutical industry relies on the technique for quality control and drug development. CE checks the purity of drug compounds and separates chiral pharmaceuticals, which are mirror-image molecules. Separating these isomers is often necessary to ensure drug safety and efficacy.
Forensic science utilizes CE for analyzing trace evidence, benefiting from its ability to handle extremely small sample volumes. This includes identifying and separating complex mixtures, such as various ink components, to authenticate documents. CE is an integral part of modern forensic DNA analysis, separating the short tandem repeats (STRs) used in human identification profiles after amplification by the polymerase chain reaction (PCR).