Lecture: Ion Exchange Chromatography

Overview

Ion exchange chromatography is a technique based on the attraction between opposite charges and is widely used for the separation and purification of biomolecules such as proteins, peptides, nucleic acids, and polynucleotides.

Key Concepts

• Stationary Phase (Ion Exchanger)
  • Selected for having a particular charge to attract sample molecules with the opposite charge.
  • Two types:
    • Cation Exchanger: Negatively charged, attracts positively charged cations.
    • Anion Exchanger: Positively charged, attracts negatively charged anions.

Functional Groups

• Functional groups for ion exchangers:
  • Cation Exchanger: Carboxyl, carboxymethyl, sulfur, sulfomethyl
  • Anion Exchanger: Amino, trimethylamino, ethylamino

Buffers

• Cation Exchanger Buffers: Contain negatively charged ions, such as acetate and phosphate.
• Anion Exchanger Buffers: Contain positively charged ions, example: Tris buffer.

Protein Separation Process

• Isoelectric pH: pH at which the net charge of the protein is zero (equal numbers of positive and negative charges).
• Adjusting pH:
  • If pH > isoelectric pH by one unit: Protein has net negative charge.
  • If pH < isoelectric pH by one unit: Protein has net positive charge.

Choosing the Right Chromatography Method

• Determine the pH stability of the protein:
  • Stable one unit above isoelectric pH: Use anion exchange chromatography (protein has a negative charge).
  • Stable one unit below isoelectric pH: Use cation exchange chromatography (protein has a positive charge).

Elution

• After binding the protein to the stationary phase, elution can be achieved by:
  • Changing the pH.
  • Changing the ionic strength.

Summary

Ion exchange chromatography involves careful selection of ion exchangers and buffers based on the protein's charge characteristics and stability at different pH levels to achieve effective separation and purification.