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Related Concept Videos

Affinity Chromatography01:03

Affinity Chromatography

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Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile...
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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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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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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

Chromatography: Introduction

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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.
The phase in which the compounds linger or on which the compounds adsorb is called the stationary phase, whereas the mobile phase is the solvent that carries the solutes to be analyzed. In traditional column chromatography, the mixture flows through the stationary phase, and the compounds partition between the stationary and mobile phases...
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Chromatographic Methods: Classification01:12

Chromatographic Methods: Classification

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Chromatographic techniques are classified in three ways: the classification is based on the physical state of the stationary and mobile phases, how the mobile phase and the stationary phase contact each other, or through the chemical or physical processes that isolate the components of the sample. Typically, the mobile phase is either a liquid or gas, while the stationary phase is either a solid or a liquid layer applied to a solid surface.
Chromatographic techniques are typically named by...
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Related Experiment Video

Updated: Apr 16, 2026

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study
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Mixed-bed affinity chromatography: principles and methods.

Egisto Boschetti1, Pier Giorgio Righetti

  • 1JAM-Conseil, 9-11 rue Boutard, 92200, Neuilly sur Seine, France, egisto.boschetti@gmail.com.

Methods in Molecular Biology (Clifton, N.J.)
|March 10, 2015
PubMed
Summary

Mixed-bed chromatography, a bioseparation technique using multiple sorbents in one bed, aids in discovering low-copy proteins and purifying biopharmaceuticals. This chapter details its applications and sorbent selection for effective protein purification.

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Area of Science:

  • Biochemistry
  • Chemical Engineering
  • Biotechnology

Background:

  • Mixed-bed chromatography is an emerging bioseparation technology utilizing a single column packed with multiple sorbent types.
  • Its primary development over the last decade has focused on reducing the dynamic concentration range in complex proteomes.
  • This technique has enabled the discovery of numerous low-copy proteins.

Purpose of the Study:

  • To introduce the fundamental concepts of mixed-bed chromatography.
  • To provide detailed protocols for preparative applications of this technique.
  • To guide the selection and utilization of sorbents for mixed-bed protein purification.

Main Methods:

  • Description of basic principles governing mixed-bed chromatography.
  • Development of specific application protocols for preparative chromatography.
  • Guidance on sorbent selection and integration into mixed beds.

Main Results:

  • Mixed-bed chromatography effectively reduces protein dynamic concentration range, facilitating the discovery of low-abundance proteins.
  • The technology is applicable for trace impurity removal in late-stage bioprocessing.
  • Sorbent selection criteria and mixed-bed configurations are detailed for optimized protein purification.

Conclusions:

  • Mixed-bed chromatography offers significant advantages for protein purification and discovery in complex biological samples.
  • The chapter provides practical guidance for implementing mixed-bed chromatography in bioseparation workflows.
  • Further development and application of mixed-bed chromatography are expected to enhance bioprocessing efficiency.