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High-performance liquid chromatography (HPLC) is a powerful analytical technique that encompasses several separation methods. Depending on the criteria used for classification, different categories can be identified. One common way to classify HPLC is based on the separation mechanism. Under this approach, HPLC can be divided into adsorption chromatography, partition chromatography, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography. Another classification method considers the polarity of the mobile and stationary phases, leading to normal-phase and reverse-phase chromatography. In normal-phase chromatography, the stationary phase is more polar than the mobile phase, while in reverse-phase chromatography, the opposite is true.
Adsorption chromatography involves an adsorbent as the stationary phase, where separation occurs due to differences in the adsorption capacities of the sample components. This method is often used for separating non-polar compounds.
Partition chromatography is the most widely used mode in HPLC. Here, both the stationary and mobile phases are liquid, and separation is based on the differential solubility of the sample components between the two phases. The partition coefficient determines how each component moves through the column.
Size-exclusion chromatography, also known as gel filtration, uses a gel as the stationary phase. Separation is based on the molecular size of the sample components relative to the pore size of the gel. Larger molecules elute first, while smaller ones penetrate deeper into the pores.
Ion-exchange chromatography employs ion-exchange resins as the stationary phase. It separates ions based on their ability to exchange with the functional groups on the resin surface. This technique is commonly used for analyzing ionic substances like proteins and nucleic acids.
Affinity chromatography relies on specific molecular interactions. A biologically active ligand is immobilized on the support, and the target molecule binds selectively to it. This method is particularly useful for isolating specific biomolecules such as enzymes or antibodies.
Chemically bonded phase chromatography involves attaching functional groups to the support material to create a stable stationary phase. Reverse-phase chromatography, which uses a non-polar stationary phase and a polar mobile phase, is one of the most popular techniques in modern HPLC due to its versatility and efficiency.