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High-performance liquid chromatography (HPLC) is a versatile analytical technique that involves various separation mechanisms. Depending on the classification criteria, different types of chromatography can be identified. From a mechanistic perspective, HPLC can be categorized into adsorption chromatography, partition chromatography, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography. Additionally, based on the polarity relationship between the mobile phase and the stationary phase, it can also be divided into 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 mobile phase is more polar than the stationary phase.
Adsorption chromatography relies on the differential adsorption of sample components onto an adsorbent stationary phase. This method is particularly effective for separating compounds with different affinities for the adsorbent surface.
Partition chromatography is one of the most commonly used techniques in HPLC. Here, the stationary phase is typically a liquid, and the separation is based on the difference in solubility of the sample components between the mobile and stationary phases, leading to variations in their partition coefficients.
Size-exclusion chromatography uses a gel-based stationary phase, where separation occurs based on the molecular size of the analytes relative to the pore size of the gel. Larger molecules are excluded from the pores and elute first, while smaller molecules penetrate the pores and elute later.
Ion-exchange chromatography employs an ion-exchange resin as the stationary phase. The separation is driven by differences in the ability of the ions in the sample to exchange with the charged groups on the resin surface.
Affinity chromatography is a highly selective method that utilizes biologically active ligands immobilized on the stationary phase. It is especially useful for isolating specific biomolecules, such as proteins, based on their unique interactions with the ligand.
Chemically bonded phase chromatography involves attaching functional groups directly to the support material, forming a stable stationary phase. Reverse-phase chromatography, a type of chemically bonded phase method, is widely used due to its excellent resolution and compatibility with a wide range of solvents.