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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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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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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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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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Contributions to reversed-phase column selectivity: III. Column hydrogen-bond basicity.

P W Carr1, J W Dolan2, J G Dorsey3

  • 1University of Minnesota, Minneapolis, MN, USA.

Journal of Chromatography. A
|April 20, 2015
PubMed
Summary

This study clarifies hydrogen bonding in reversed-phase chromatography (RPC), identifying three distinct column basicity sites that influence carboxylic acid retention. Understanding these interactions enhances RPC method development.

Keywords:
Column hydrogen-bond basicityColumn selectivityHPLCHydrophobic–subtraction modelRetention mechanismReversed-phase

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Reversed-phase chromatography (RPC) selectivity is governed by five interactions: hydrophobic, steric, and three types of hydrogen bonding.
  • Hydrogen bonding between donor solutes (acids) and stationary-phase acceptor groups is poorly understood.

Purpose of the Study:

  • To elucidate the mechanisms of hydrogen bonding between carboxylic acids and stationary-phase acceptor groups in RPC.
  • To resolve uncertainties regarding column basicity in RPC.

Main Methods:

  • Investigated three distinct stationary-phase sites (column basicity I, II, and III) influencing carboxylic acid retention.
  • Analyzed interactions involving vicinal silanols, metal contamination, and embedded polar groups (EPG).

Main Results:

  • Column basicity I, involving vicinal silanols, universally affects carboxylic acid retention in RPC columns.
  • Column basicity II, linked to metal contamination, also impacts carboxylic acid retention in some type-A columns.
  • Column basicity III, specific to EPG columns, involves the polar group acting as a proton acceptor for acidic solutes.

Conclusions:

  • The study identifies and characterizes three distinct column basicity sites in RPC, crucial for understanding and predicting carboxylic acid retention.
  • This work provides a clearer mechanistic understanding of hydrogen bonding in RPC, aiding in method optimization and column selection.