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Related Concept Videos

Ion Exchange01:17

Ion Exchange

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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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High-Performance Liquid Chromatography: Introduction01:11

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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

Gas Chromatography: Types of Columns and Stationary Phases

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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.
For an analyte to remain on the column for a sufficient amount of time, it must exhibit some level of compatibility (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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Types Of Column Chromatography01:29

Types Of Column Chromatography

11.1K
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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Silica Gel Column Chromatography: Overview01:10

Silica Gel Column Chromatography: Overview

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Silica gel column chromatography is a technique for separating compounds using a column packed with silica gel as the stationary phase. This method relies on differences in the polarity of compounds. Based on their polarities, compounds move between the stationary phase (silica gel) and the mobile phase (the solvent), forming discrete bands in the column.
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Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
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Characterization of reversed-phase liquid chromatographic columns containing positively charged functionality.

Sophie Jolliffe1, Jennifer K Field2, Melvin R Euerby3

  • 1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom.

Journal of Chromatography. A
|June 21, 2024
PubMed
Summary

A new method quantifies low positive charge on RP columns using 4-n-octylbenzene sulfonic acid and toluene. This aids in selecting equivalent or dissimilar columns for method development and biopharmaceutical analysis.

Keywords:
Anionic exchange retentionCationic repulsion of basic analytesModified Tanaka chromatographic characterization protocolPositively charged surface RP-LC columnsSimilarity / dissimilarity of columns based on Euclidian distances

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Existing RP column characterization methods fail to accurately assess low positive charges.
  • Discriminating between neutral and low positive charge RP columns is challenging.

Purpose of the Study:

  • To develop a robust chromatographic method for assessing low positive charges on RP columns.
  • To introduce a new parameter (α4-OBSA/Tol) for column characterization.
  • To aid in selecting equivalent or dissimilar RP columns for method development.

Main Methods:

  • Utilized electrostatic attraction of 4-n-octylbenzene sulfonic acid (4-OBSA) and retention of toluene (Tol) to determine positive charge.
  • Analyzed 15 commercial RP-LC columns.
  • Combined new parameter with existing Tanaka parameters for column selection.

Main Results:

  • Developed and validated the α4-OBSA/Tol parameter for quantifying low positive charge.
  • Demonstrated that even neutral alkyl phases can exhibit positive charge at low buffer concentrations.
  • Identified mobile phase conditions (low % MeCN) promoting mixed-mode retention.
  • Categorized columns into three subsets based on positive charge character.

Conclusions:

  • The new method and parameter (α4-OBSA/Tol) effectively characterize low positive charges on RP columns.
  • The findings assist chromatographers in selecting appropriate columns for method development and biopharmaceutical analysis.
  • The study highlights the importance of considering column charge characteristics in chromatography.