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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 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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Types Of Column Chromatography01:29

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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.
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High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term "high pressure" refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
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Gas Chromatography: Types of Columns and Stationary Phases01:17

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Gas chromatography (GC) relies on stationary phases to separate and analyze components in a sample. There are two main types of stationary phases: liquid and solid. Liquid stationary phases are non-volatile, thermally stable, and chemically inert liquids coated onto the column. Solid stationary phases are particles of adsorbent material, such as silica gel or molecular sieves.
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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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Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach
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Pepsin-modified chiral monolithic column for affinity capillary electrochromatography.

Tingting Hong1, Cuijie Chi, Yibing Ji

  • 1Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing, China.

Journal of Separation Science
|August 23, 2014
PubMed
Summary

A novel pepsin-modified affinity monolithic capillary electrochromatography system was developed for chiral separation. This stable, reusable system shows promise for enantiomeric analysis and enzyme-based applications.

Keywords:
Affinity electrochromatographyCapillary monolithsEnantiomeric separationPepsinSilica

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Separation Science

Background:

  • Enzyme-modified materials offer unique separation capabilities.
  • Monolithic capillary electrochromatography provides a high-surface-area stationary phase.
  • Chiral separations are critical in pharmaceutical analysis.

Purpose of the Study:

  • To develop a pepsin-modified affinity monolithic capillary electrochromatography system.
  • To evaluate its performance for chiral separation of (±)-nefopam.
  • To assess its stability and reusability for enantiomeric analysis and protein digestion.

Main Methods:

  • Covalent bonding of pepsin onto a silica monolith.
  • Chiral separation of (±)-nefopam using the modified column.
  • Optimization of electrochromatographic parameters (pH, voltage, temperature, etc.).
  • Characterization of proteolytic activity for online protein digestion.

Main Results:

  • Successful chiral separation of (±)-nefopam achieved.
  • High column stability demonstrated with low %RSD values for retention time.
  • No significant loss in performance over 50 days and 100 injections.
  • Demonstrated operational stability and reusability for at least 20 protein digestions.

Conclusions:

  • Pepsin-modified affinity monoliths are effective for enantioseparation.
  • The system exhibits excellent stability and reusability.
  • This approach opens new avenues for enzyme-modified affinity capillary monoliths in chiral recognition.