Gel chromatography, also known as size-exclusion chromatography (SEC), is a separation technique used primarily for separating molecules based on their size. It is widely used for purifying proteins, polysaccharides, and other macromolecules. In this technique, molecules are separated as they pass through a column filled with porous gel beads, and larger molecules elute (exit) from the column faster than smaller ones, as the larger molecules cannot penetrate the pores of the beads as easily.
Key Features:
- No chemical interaction between the stationary phase (gel) and the analytes.
- Separation is based solely on the size and shape of the molecules.
- It is widely used in biochemistry, molecular biology, and polymer science.
Theory of Gel Chromatography
Gel chromatography operates based on the principle of molecular size and volume. The separation of analytes is determined by their ability to permeate the pores of the gel beads.
Key Concepts:
- Stationary Phase (Gel beads): These are cross-linked polymers (often made of materials like agarose or dextran) with defined pore sizes.
- Mobile Phase (Solvent): A buffer or solvent system that carries the molecules through the column.
- Elution: Larger molecules are excluded from the pores and elute first, while smaller molecules take longer to travel through the column as they enter and exit the gel pores multiple times.
The molecular weight (size) of the analytes determines their elution time. The process is often visualized by a plot known as the calibration curve, which plots the elution volume versus the logarithm of molecular weight.
Instrumentation in Gel Chromatography
The instrumentation used for gel chromatography is relatively simple compared to other chromatographic techniques. It consists of the following key components:
- Column: This is a glass or plastic tube packed with gel beads. The length and diameter of the column depend on the sample size and desired resolution.
- Gel Beads (Stationary Phase): Made of cross-linked polymers such as agarose, dextran, polyacrylamide, or silica. The beads have a specific range of pore sizes, allowing selective penetration based on the molecular size of analytes.
- Eluent Reservoir: The solvent or buffer is stored in a reservoir and pumped through the system.
- Flow Pump: Ensures the steady flow of the mobile phase through the column.
- Detector: A UV or refractive index detector is commonly used to detect and quantify the eluted compounds. The detector measures changes in absorbance or refractive index as the molecules pass through the flow cell.
- Fraction Collector: This device collects the eluted fractions in separate containers for further analysis or processing.
Applications of Gel Chromatography
Gel chromatography is versatile and has numerous applications across various scientific fields:
- Protein Purification: One of the most common applications is the separation and purification of proteins based on their molecular weight. It is used to purify enzymes, antibodies, and other protein complexes.
- Molecular Weight Determination: By comparing the elution volume of a molecule with standards of known molecular weights, gel chromatography can help determine the molecular size of unknown proteins or polymers.
- Polysaccharide Analysis: It can separate and analyze polysaccharides like cellulose, starch, and glycogen based on size.
- Polymer Analysis: In polymer science, it is used to determine the molecular weight distribution of synthetic polymers such as polyethylene and polystyrene.
- Desalting: Gel chromatography is also employed to remove small molecules like salts, reducing agents, or detergents from protein or nucleic acid preparations.
- Pharmaceutical and Biomedical Applications: It is used in drug development for the characterization of biologics, and in biotechnology for the separation and purification of vaccines, hormones, and other therapeutic proteins.