6.4 Ion exchange and size exclusion chromatography

Ion exchange and size exclusion chromatography are powerful separation techniques in analytical chemistry. They work by exploiting differences in charge and size between molecules, allowing for precise separation and purification of complex mixtures.

These methods are crucial for analyzing and purifying biomolecules like proteins and nucleic acids. By understanding the principles behind these techniques, chemists can optimize separations and gain valuable insights into molecular properties and interactions.

Ion Exchange Chromatography Principles

Separation Based on Charge

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• Ion exchange chromatography separates molecules based on their charge using a stationary phase with charged functional groups and a mobile phase containing ions that compete with the analyte for binding sites
• The strength of the interaction between the ion exchanger and the analyte depends on the charge of the analyte and the competing ions in the mobile phase
• Elution in ion exchange chromatography is achieved by increasing the ionic strength or changing the pH of the mobile phase to disrupt the interactions between the analyte and the stationary phase

Applications and Uses

• Ion exchange chromatography is widely used for the purification and analysis of charged biomolecules, such as proteins, nucleic acids, and peptides
• Applications of ion exchange chromatography include water purification, removal of impurities from pharmaceutical products, and separation of amino acids and sugars
• Ion exchange chromatography can be used to separate and quantify post-translational modifications of proteins, such as phosphorylation or glycosylation, which alter the net charge of the protein
• The combination of ion exchange and size exclusion chromatography can be used to achieve high-purity protein preparations by exploiting differences in both charge and size

Ion Exchangers and Selectivity

Types of Ion Exchangers

• Ion exchangers are materials that contain charged functional groups capable of exchanging ions with a solution, forming the basis for ion exchange chromatography
• The stationary phase in ion exchange chromatography is typically a resin with charged functional groups, such as sulfonic acid (cation exchange) or quaternary ammonium (anion exchange) groups
• Cation exchangers have negatively charged functional groups and selectively bind positively charged ions, while anion exchangers have positively charged functional groups and selectively bind negatively charged ions

Factors Affecting Selectivity

• The selectivity of ion exchangers depends on factors such as the charge density of the functional groups, the size and charge of the ions, and the pH and ionic strength of the mobile phase
• The mobile phase in ion exchange chromatography is an aqueous buffer solution containing competing ions, such as sodium or chloride, that can displace the analyte from the stationary phase
• The selectivity of ion exchangers can be manipulated by changing the type and concentration of the competing ions in the mobile phase or by modifying the functional groups on the stationary phase
• The capacity of an ion exchanger refers to the number of functional groups available for ion exchange and determines the maximum amount of analyte that can be bound to the stationary phase

Size Exclusion Chromatography Principles

Separation Based on Size and Shape

• Size exclusion chromatography, also known as gel filtration chromatography, separates molecules based on their size and shape using a porous stationary phase and a mobile phase that does not interact with the analyte
• The stationary phase in size exclusion chromatography consists of porous particles with a well-defined range of pore sizes, such as cross-linked dextran (Sephadex) or polyacrylamide (Bio-Gel)
• Molecules larger than the pore size of the stationary phase are excluded and elute first, while smaller molecules enter the pores and elute later, resulting in a separation based on molecular size

Factors Affecting Resolution

• The mobile phase in size exclusion chromatography is typically an aqueous buffer solution that does not interact with the analyte or the stationary phase
• The resolution of size exclusion chromatography depends on factors such as the pore size distribution of the stationary phase, the column dimensions, and the flow rate of the mobile phase
• The choice of stationary phase pore size depends on the molecular weight range of the analytes being separated, with smaller pore sizes used for lower molecular weight molecules and larger pore sizes used for higher molecular weight molecules

Chromatography Applications for Biomolecules

Protein Purification and Characterization

• Ion exchange chromatography can be used to purify proteins based on their net charge, which varies depending on the pH of the mobile phase relative to the protein's isoelectric point
• Size exclusion chromatography can be used to purify proteins based on their molecular weight and to remove aggregates or impurities with different sizes
• Size exclusion chromatography can be used to determine the molecular weight and oligomeric state of proteins by comparing their elution volume to that of standard proteins with known molecular weights (molecular weight markers)

Other Biomolecules

• Ion exchange and size exclusion chromatography can be used to purify and analyze other biomolecules, such as nucleic acids (DNA and RNA), peptides, and carbohydrates, based on their charge and size properties
• Ion exchange chromatography can be used to separate and purify oligonucleotides based on their charge, which is determined by the number of phosphate groups in their backbone
• Size exclusion chromatography can be used to separate and characterize polysaccharides based on their molecular weight and hydrodynamic radius, which reflects their size and shape in solution