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Related Concept Videos

Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

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Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...
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Ion exchange chromatography separates charged molecules from a solution by reversibly exchanging them with mobile, or 'active', ions associated with the oppositely charged stationary phase. This method can be used to separate ions, soften and deionize water, and purify solutions. The polymers comprising the ion-exchange column are high-molecular-weight and chemically stable polymers, crosslinked to be porous and essentially insoluble. They are also functionalized with either acidic or...
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Principles Of Column Chromatography01:13

Principles Of Column Chromatography

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The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used...
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Size-Exclusion Chromatography01:08

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In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
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Types Of Column Chromatography01:29

Types Of Column Chromatography

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The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography
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Chromatography: Introduction01:10

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Chromatography is a technique used to separate compounds based on differences of partitioning between two phases, the stationary phase and the mobile phase.
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Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization
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Ion-Exchange Chromatography: Basic Principles and Application.

Philip M Cummins1, Keith D Rochfort2, Brendan F O'Connor2,3,4

  • 1School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. phil.cummins@dcu.ie.

Methods in Molecular Biology (Clifton, N.J.)
|October 13, 2016
PubMed
Summary
This summary is machine-generated.

Ion-Exchange Chromatography (IEC) separates molecules by charge. This review covers IEC principles and protocols for purifying enzymes from brain tissue using anion-exchange chromatography.

Keywords:
Anion exchangeCaptureCation exchangeContinuous-bedDEAE-Sepharose® Fast FlowLiquid chromatographyPolishProlyl oligopeptidase

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Chromatography

Background:

  • Ion-Exchange Chromatography (IEC) is a versatile liquid chromatography technique for separating ionizable molecules based on charge differences.
  • Its wide applicability, scalability, and automation make it a preferred method for protein and enzyme purification.
  • IEC offers high resolving power, large sample capacity, and moderate cost, contributing to its widespread use.

Purpose of the Study:

  • To review the fundamental principles of Ion-Exchange Chromatography.
  • To discuss criteria for selecting appropriate IEC conditions for effective separation.
  • To provide practical laboratory protocols for the partial purification of a soluble serine peptidase from bovine brain tissue using anion-exchange chromatography.

Main Methods:

  • Review of Ion-Exchange Chromatography principles and selection criteria.
  • Development and description of laboratory protocols for enzyme purification.
  • Preparation of crude tissue extract from bovine whole brain.
  • Partial purification of serine peptidase using anion-exchange chromatography.
  • Assay of total protein and enzyme activity in pre- and post-IEC fractions.

Main Results:

  • Demonstration of Ion-Exchange Chromatography principles through a practical enzyme purification example.
  • Successful partial purification of a soluble serine peptidase from bovine brain tissue.
  • Established protocols for enzyme activity and protein quantification in chromatography fractions.

Conclusions:

  • Ion-Exchange Chromatography is a powerful and adaptable technique for biomolecule separation, particularly for enzymes.
  • The outlined protocols provide a practical guide for researchers purifying enzymes using anion-exchange chromatography.
  • IEC facilitates efficient purification and characterization of target proteins from complex biological samples.