Summary
Highlights
Absorption chromatography involves the solute being absorbed by the stationary phase and later desorbed into the mobile phase; strongly bound solutes elute slower. In partition chromatography, the stationary phase is a liquid retained on a solid support, and separation occurs due to the analyte's distribution between the mobile and stationary phases. This can be normal phase (polar stationary, non-polar mobile) or reverse phase (non-polar stationary, polar mobile).
Chromatography is a set of techniques used to separate mixtures based on the differential interaction of each component with another substance. It's a physical separation method where components are distributed between two phases: a stationary phase (where the sample is placed on a fixed support like paper) and a mobile phase (a substance that moves over the mixture on the static support, like a liquid moving through paper).
Chromatographic techniques can be classified into three main categories: column chromatography, planar chromatography, and high-resolution chromatography. Column chromatography includes ionic exchange, exclusion, affinity, absorption, partition, and reverse phase.
This type of chromatography is based on the charges of molecules. Charged molecules reversibly attach to ion exchangers, allowing them to be bound or unbound by changing the ionic environment. Ion exchangers are typically polymers with attached charged groups; anionic exchangers have positive charges and bind anions, while cationic exchangers have negative charges and bind cations.
Affinity chromatography relies on the reversible binding between the analyte of interest and a specific ligand immobilized on an inert solid support. It boasts high specificity and is primarily used for purifying biological substances like enzymes, antibodies, and recombinant proteins, and also for removing harmful substances like pathogens.
Liquid exclusion chromatography separates molecules based on differences in size, form, or charge. It uses a porous stationary phase, allowing differential elution of products according to their molecular sizes. Larger molecules pass through faster than smaller ones, as there is no chemical or physical interaction between the analytes and the stationary phase.
This technique is widely used in biotechnology for separating and purifying proteins. The mobile phase is a high-concentration saline solution that bathes a layer of inert particles (stationary phase). It's effective for separating monoclonal antibodies and proteins based on their reversible interaction with the hydrophobic part of the stationary phase. Elution requires decreasing the ionic strength in the mobile phase.
Paper chromatography is a qualitative analysis process that doesn't require specialized equipment. It relies on the differential displacement speed of solutes as they are carried by a mobile phase (usually water) over a stationary phase (usually filter paper). Components with higher affinity for water travel faster.
Gas chromatography volatilizes the sample and injects it into a column, with an inert gas as the mobile phase that doesn't interact with the analytes. SFC is useful for purifying low molecular weight molecules, using a supercritical fluid (often CO2 due to accessible critical temperature and pressure) as the mobile phase to dissolve large, non-volatile molecules.
HPLC (High-Performance Liquid Chromatography) is a column chromatography type needing mobile phases with opposite polarity, where all solutes are soluble and ideally well-separated. Metal chelate affinity chromatography uses a matrix with covalently bound metal chelating agents to purify recombinant proteins. Chromatofocusing separates solutes based on their isoelectric point, often employing an ion exchanger.
Covalent chromatography involves a reversible covalent bond between the stationary phase and the biomolecule to be separated. Immunoadsorption is a technique used to remove unwanted immunoglobulins from blood complexes, treating them with columns containing beads that bind to specific antibodies.