FPLC (Fast Protein Liquid Chromatography) can use a variety of chromatography methods including gel filtration (size exclusion) chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography and reversed phase chromatography. These chromatography modes allow for proteins to be separated based on their size, charge, hydrophobicity, specific ligand binding as well as other properties. Gel filtration (size exclusion) chromatography and ion exchange chromatography are the most typically used modes in FPLC systems.
Gel Filtration Chromatography is also known as Size-Exclusion Chromatography. This technique separates molecules based on their size. It is used to separate both proteins and other molecules. Gel Filtration chromatography is a form of partition chromatography used to separate molecules based on their molecular size. Molecules are partitioned between the mobile and stationary phase. The gel medium is comprised of specific size distribution of spherical beads. The separation takes place when the sample is passed through the beads (the gel medium). Smaller molecules pass through the beads and flows through the column faster than larger molecules. The proteins are eluted from the column in order of decreasing size. Therefore, molecules are separated based on their size as they pass through the column and may be eluted in order of decreasing molecular weight. For this reason this mode of FPLC is sometimes refered to as Molecular-seive chromatography. Gel filtration chromatography is used for protein purification, determining molecular weight and for analyzing protein complexes.
Ion Exchange Chromatography (IEC) is a technique that separates charged molecules (proteins) based on their net charged by binding them to a charge resin with opposite polarity. Charged compounds are absorbed and retained by the ion exchanger with the opposite charge. The neutral or similarly charged compounds pass through the void volume and are eluted from the column. Absorbed compounds are eluted with a salt or pH gradient. It is used to analyze ionic compounds, purify proteins, and separate ions and ionizable molecules.
Affinity Chromatography is a technique used to purify a specific molecule from a complex mixture based on its affinity for a complementary molecule, this complementary molecule is called a ligand. The ligand is bound to a solid support, the resin (column material). Next, the sample is run in a column and the target molecule (usually protein) binds to the ligand. None of the bound components are washed away leaving only the protein of interest bound to the ligand. In the elution stage, the target protein is released from the ligand.
Affinity Chromatography is used for protein purification, antibody purification, enzyme purification, drug discovery and biomarker detection. Reversed-Flow Chromatography is for Affinity Purifications..
Hydrophobic interaction chromatography (HIC) is a technique used to separate and purify molecules (primarily proteins) based on their hydrophobicity. Molecules with more hydrophobic regions bind more strongly to a hydrophobic stationary phase on a chromatography column, allowing for separation based on their relative hydrophobicity. The salt in the buffer reduces the solvation of sample solutes. This separation is typically achieved by gradually decreasing the salt concentration in the mobile phase. High salt concentrations in the mobile phase promote hydrophobic reactions. Therefore, proteins are usually loaded onto the column in a high salt buffer. This causes less hydrophobic molecules to elute first. To elute bound proteins, the salt concentration is gradually decreased. This weakens the hydrophobic interactions. Proteins can then separate based on their relative hydrophobicity. Hydrophobic Interaction Chromatography is widely used for antibody purification, vaccine development, plasma protein fractionation and drug discovery. Different than other FPLC techniques, HIC operates under relatively mild conditions, this helps to preserve the native structure and function of the proteins. Using HIC, researchers can isolate proteins based on how much of their surface area is exposed as hydrophobic patches, allowing the removal of impurities and aggregates with different hydrophobic properties. HIC is widely used to purify proteins from complex mixtures like cell lysates.
Reversed Phase Chromatography (RPC) is not commonly used. It is a technique used to separate, analyze, and purify organic compounds. RPC separates compounds based on their hydrophobicity. The stationary phase is nonpolar, while the mobile phase is polar. Reversed phase chromatography is used to separate and purify molecules like proteins, peptides and antibodies. RPC is easy to handle and has high resolution. Reversed phase is used to analyze peptides and proteins, separate drugs and metabolities, study environmental contaminants and to purify biological molecules.
There are one step purifications and multi step purifications depending on what matrix your protein is in and how complex the separation is. In the simplest of cases, there is a huge difference in the molecular weight in the protein of interest vs other proteins. In that case, gel filtration or sizing might be the purification method if choice. In most cases, a combination of two or more techniques (Gel filtration/size exclusion chromatography, ion exchange chromatography, affinity chromatography and reversed phase chromatography) are needed to separate and highly purify your protein of interest.
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