Low Pressure Liquid Chromatography (LPLC)
Low-pressure liquid chromatography (LPLC) is an analytical technique that utilizes low pressure to propel a mobile phase through a column packed with a stationary phase, allowing for the separation of complex mixtures based on differential partitioning. Initially performed using open columns that relied on gravity to move the sample through the packing bed, this method is also known as 'open column liquid chromatography.' The different modes of LPLC enable the precise purification of compounds by separating them according to their chemical properties, such as size, charge, or affinity. LPLC is primarily used for studying biomolecules such as proteins, peptides, and monoclonal antibodies due to its non-destructive preparative nature, which allows the sample to be retained for further analysis. The technique is advantageous due to its simple design, efficient siphon action, and relatively low instrumentation requirements, including detectors, low-pressure pumps, and fraction collectors. Its versatility makes it essential across industries such as pharmaceuticals, biotechnology, food and beverage, environmental monitoring, and scientific research. Additionally, by operating at lower pressures and requiring less solvent, LPLC aligns with green chemistry principles, making it an environmentally sustainable method.
Overview
Adsorption Chromatography
Also known as liquid-solid chromatography, adsorption chromatography retains chemicals by adsorbing and desorbing them at the surface of the support, stationary phase. The adsorbent forms the stationary phase, and the solute binds to it by van der Waal forces and steric interactions. Because adsorption sites are usually on the outer surface of the stationary phase, relatively tiny particles are used as the stationary phase. Silica, alumina, charcoal, Florisil, polyamides, celite, and diatomaceous Earth are the commonly used adsorbents.
Partition Chromatography
Partition chromatography separates by distributing the components between two immiscible liquids. One is the liquid mobile phase while the other is the liquid held stationary on a solid support. Materials used as solid support include silica gel, diatomaceous earth, cellulose, polytetrafluoroethylene (PTFE), and polystyrene. The inert solid support provides a large surface area for the liquid stationary phase.
Affinity Chromatography
Affinity chromatography separates constituents by selective and reversible interactions, such as antibody-antigen, enzyme-substrate, or hormone-receptor pairs, with one of the interacting agents as the stationary phase. The 'affinity ligand' is the interacting species immobilized on solid support such as acrylic beads, agarose, and Toyopearl resins, and it serves as the stationary phase for the affinity column.
Gel Filtration Chromatography
Also known as size exclusion chromatography, it separates molecules of different size such as proteins of varying sizes and oligomeric forms. The stationary phase is a resin made up of cross-linked matrix of beads containing pores of a specific size. Gel filtration columns contain beads of polyacrylamide, agarose, dextran, or a mix of any of these. It isolates smaller analytes by providing partial or full access to the pore volume within the column packing particles.
Hydrophobic Interaction Chromatography (HIC)
Hydrophobic interaction chromatography (HIC) separates biomolecules based on differences in their surface hydrophobicity. It is based on the interaction of non-polar hydrophobic groups linked to the column resin, such as butyl, octyl, or phenyl, with the hydrophobic groups on biomolecules. It is widely used to separate proteins while preserving their biological activity since it uses lesser denaturing conditions and matrices.
Ion Exchange Chromatography (IEX)
Ion exchange chromatography separates molecules based on differences in their net surface charges. The surface of the matrix, such as cellulose, silica, or styrene-divinylbenzene, is covalently linked to charged functional groups. The resin's immobilized ion exchange groups interact with molecules that have opposite charges. In cation exchange chromatography, positively charged species in the mobile phase bind to a negatively charged ion exchange resin. In anion exchange chromatography, negatively charged species in the mobile phase bind to the positive charges on the ion exchange resin.
Low-pressure Liquid Chromatography System
A low-pressure liquid chromatography (LPLC) system typically consists of a column filled with a stationary phase for separation, a pump to deliver the mobile phase through the column at controlled flow rates, and a detector to measure the eluent as it exits the column. LPLC systems also feature an injection system for introducing the sample into the mobile phase stream, ensuring precise and reproducible sample introduction. Some setups include a fraction collector to gather separated components for further analysis.