Fast Protein Liquid Chromatography (FPLC) is a specialized form of liquid chromatography widely used for the analysis and purification of proteins and other biological macromolecules. Developed originally by Pharmacia in 1982, FPLC was designed as a medium-pressure chromatographic technique optimized for biomolecules, particularly proteins, that are sensitive to harsh conditions such as high pressure, extreme pH, and organic solvents.
Principles and Mechanism
FPLC separates components of a mixture based on their differential affinities between a mobile phase and a stationary phase. The mobile phase in FPLC is an aqueous buffer solution, whose flow rate is precisely controlled by a positive-displacement pump, typically maintained at 1–5 mL/min under low pressure (below 5 bar). The stationary phase consists of porous resin beads, often cross-linked agarose, packed into columns. These beads vary in size and surface chemistry depending on the type of separation required.
Applications
- Purification of recombinant proteins, antibodies, and enzymes.
- Isolation of nucleic acids like oligonucleotides and plasmids.
- Preparation of proteins for structural and functional studies.
- Removal of contaminants such as endotoxins from protein preparations.
Advantages
- Mild operating conditions: Low pressure and aqueous buffers prevent protein denaturation.
- High resolution and reproducibility: Small bead sizes and precise flow control enhance separation quality.
- Automation: Facilitates gradient elution, sample handling, and fraction collection.
- Scalability: Suitable for analytical scale to preparative or industrial scale purification.
- Versatility: Compatible with multiple chromatographic modes and biological sample types.
FPLC represents a cornerstone technique in protein biochemistry and molecular biology, offering a robust, gentle, and versatile platform for protein purification and analysis. Its development has greatly facilitated the production of high-purity proteins essential for research, diagnostics, and therapeutic applications.
