Gel filtration (GF) chromatography separates proteins solely on the basis of molecular size. Separation is achieved using a porous matrix to which the molecules, for steric reasons, have different degrees of access--i.e., smaller molecules have greater access and larger molecules are excluded from the matrix.
How does size exclusion chromatography work?
Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while large molecules do not enter the pores and are eluted in the column's void volume.
What is the meaning of void volume?
The void volume is the volume of mobile phase (Vm or V0) in a column. For example, if the stationary phase occupies 40% of the total column volume, the void volume would be 60% of the total column volume.
What is absorption chromatography used for?
a form of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column. thin-layer chromatography. that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate.
What is the purpose of ion exchange chromatography?
Ion chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.
What is molecular sieve chromatography?
A molecular sieve is a material with pores (very small holes) of uniform size. Some molecular sieves are used in chromatography, a separation technique that sorts molecules based on their size. Other molecular sieves are used as desiccants (some examples include activated charcoal and silica gel).
What does SDS do to proteins?
For nucleic acids, urea is the most commonly used denaturant. For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to coat proteins in order to impart two negative charges (from every SDS molecule) to every two amino acids of the denatured protein.
What is the size exclusion limit?
exclusion limit - in SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit.
What is molecular exclusion chromatography?
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.
What is the stationary phase in an affinity chromatography column?
Affinity chromatography. The principle of affinity chromatography is that the stationary phase consists of a support medium (e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.
What is gel permeation chromatography?
Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates analytes on the basis of size. The technique is often used for the analysis of polymers.
What is the affinity chromatography?
Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.
What is meant by partition chromatography?
medical Definition of partition chromatography. : a process for the separation of mixtures in columns or on filter paper based on partition of a solute between two solvents one of which is immobilized by the substance in the column or by the paper.
What is the mobile phase in gel filtration chromatography?
In a gel filtration chromatography column, the stationary phase is composed of a porous matrix, and the mobile phase is the buffer that flows in between the matrix beads. The beads have a defined pore size range, known as the fractionation range.
How does protein affinity chromatography work?
Affinity chromatography is a separation process used to purify molecules or a group of molecules that are in a biochemical mixture. Specific molecules from the moving phase will bond to the stationary phase based on their properties whilst the rest of the solution passing through unaffected.
What is the anion exchanger?
Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE). In solution, the resin is coated with positively charged counter-ions (cations).
What is the isoelectric point of a protein?
Isoelectric point (pI): The pH at which the net charge on the protein is zero. For a protein with many basic amino acids, the pI will be high, while for an acidic protein the pI will be lower.
What is elution volume?
The "elution time" of a solute is the time between the start of the separation (the time at which the solute enters the column) and the time at which the solute elutes. In the same way, the elution volume is the volume of eluent required to cause elution.
What is the hydrodynamic volume?
Quick Reference. (of a molecule in solution) the sum of the time-average of the molecular volume and the volume of the solvent molecules associated with it. From: hydrodynamic volume in Oxford Dictionary of Biochemistry and Molecular Biology »
What is exclusion limit in gel filtration?
The table shows the useful range for the most commonly used gel filtration media - the lower and upper molecular sizes (in kDa) over which they can be used to separate macromolecules. The upper limit is known as the exclusion limit of the gel - the size above which proteins will elute in the void volume of the column.
What is Sephadex used for?
Sephadex is a trademark for cross-linked dextran gel used for gel filtration. It was launched by Pharmacia in 1959, after development work by Jerker Porath and Per Flodin. The name is derived from separation Pharmacia dextran. It is normally manufactured in a bead form and most commonly used for gel filtration columns.