Unit 3: Ion Exchange Chromatography

Introduction and Classification

Introduction

Ion Exchange Chromatography (IEC) is a chromatographic process that separates substances based on their net electrical charge. It is one of the most powerful and widely used methods for purifying charged biomolecules like proteins, amino acids, and nucleic acids.

The principle involves a reversible exchange of ions between the charged components in the sample (mobile phase) and a stationary phase that has an opposite charge.

Classification

IEC is classified based on the charge of the ions it exchanges:

  1. Cation Exchange Chromatography:
    • Stationary Phase (Resin): Is negatively charged (e.g., with -SO3- groups).
    • Function: It binds positively charged ions (cations).
    • Mobile Phase: A solution containing a mixture of cations.
  2. Anion Exchange Chromatography:
    • Stationary Phase (Resin): Is positively charged (e.g., with -N(CH3)3+ groups).
    • Function: It binds negatively charged ions (anions).
    • Mobile Phase: A solution containing a mixture of anions.

Ion Exchange Resins and Properties

Ion Exchange Resins

These are the solid, insoluble stationary phases used in IEC. They consist of a polymer matrix (or "backbone") to which charged functional groups are covalently attached.

  • Matrix: Typically a synthetic polymer like polystyrene cross-linked with divinylbenzene (DVB). The amount of DVB "cross-linking" determines the resin's hardness and pore size.
  • Functional Groups: The active, charged groups attached to the matrix.
Types of Ion Exchange Resins
Resin Type Exchanger Type Functional Group Example Use
Strong Cation (SCX) Acidic Sulfonic Acid (-SO3H) Strongly binds cations (e.g., Na+)
Weak Cation (WCX) Weakly Acidic Carboxylic Acid (-COOH) Binds cations only at higher pH
Strong Anion (SAX) Basic Quaternary Ammonium (e.g., -N(CH3)3+) Strongly binds anions (e.g., Cl-)
Weak Anion (WAX) Weakly Basic Amine (e.g., -NH2, -NR2H+) Binds anions only at lower pH

Properties of Resins

  • Capacity: The total number of exchangeable ions the resin can bind per gram (total ion-exchange capacity).
  • Porosity: The size of the pores, which determines which molecules can enter the resin beads.
  • Chemical Stability: Must be stable to acids, bases, and solvents.
  • Selectivity: The resin's preference for binding one ion over another.

Mechanism and Factors Affecting Ion Exchange

Mechanism of Ion Exchange Process

The process is a reversible, stoichiometric equilibrium. Let's use a Cation Exchanger (Resin-H) as an example to capture a Na+ ion from solution.

Reaction: Resin-SO3-H+ + Na+Cl- leftharpoons Resin-SO3-Na+ + H+Cl-

The Na+ ion from the solution displaces the H+ ion on the resin because the resin has a certain affinity (selectivity) for Na+. The displaced H+ ion goes into the solution.

For a mixture of ions (e.g., Na+ and K+), the ion with the higher affinity for the resin will displace the H+ and be bound more tightly.

Factors Affecting Ion Exchange

  1. Nature of the Ion:
    • Charge: Higher the charge, the stronger the binding. (e.g., Al3+ > Ca2+ > Na+)
    • Size (hydrated): For ions of the same charge, the *smaller* the *hydrated radius*, the stronger the binding. (e.g., Cs+ > Rb+ > K+ > Na+ > Li+).
  2. Concentration: The equilibrium can be shifted by concentration. A high concentration of a weakly-bound ion can displace a low concentration of a strongly-bound ion (Law of Mass Action). This is how elution works.
  3. pH of the Mobile Phase: This is the most critical factor.
    • It determines the charge on weak exchangers (WCX, WAX). A -COOH resin is only charged (-COO-) above pH 4-5.
    • It determines the charge on the sample (e.g., proteins, amino acids). A protein is positive below its isoelectric point (pI) and negative above its pI.
  4. Ionic Strength (Salt Concentration) of Mobile Phase: The "eluting" solvent. See Methodology.

Methodology and Applications

Methodology (How it's done)

IEC is performed in a column, similar to column chromatography.

  1. Step 1: Packing & Equilibration: The column is packed with the chosen resin (e.g., a cation exchanger). A low-salt "binding buffer" (at a specific pH) is passed through to equilibrate the resin.
  2. Step 2: Sample Loading: The sample (e.g., a protein mixture) is dissolved in the same binding buffer and loaded onto the column.
    • Proteins with the "correct" charge (positive, in this case) will bind to the resin.
    • Neutral proteins and proteins with the "wrong" charge (negative) will pass straight through (flow-through).
  3. Step 3: Washing: The column is washed with more binding buffer to remove all unbound proteins.
  4. Step 4: Elution: The bound proteins are "eluted" (washed off) by changing the mobile phase. This is done in two main ways:
    • a) Salt Gradient: Gradually increasing the salt concentration (e.g., 0 to 1M NaCl). The small, highly concentrated Na+ ions will compete with the bound proteins and displace them from the resin. Weakly-bound proteins elute first (at low salt), followed by strongly-bound proteins (at high salt).
    • b) pH Gradient: Gradually changing the pH of the buffer. This changes the charge on the proteins. As the pH moves towards a protein's pI, its net charge approaches zero, it no longer binds, and it elutes.

Applications

  • Water Softening: This is the most common application. "Hard water" (containing Ca2+ and Mg2+) is passed through a cation exchange resin (Resin-Na).
    Reaction: 2(Resin-Na) + Ca2+ leftharpoons (Resin)2-Ca + 2Na+
    The Ca2+/Mg2+ ions are removed from the water and replaced with harmless Na+ ions. The resin can be "regenerated" by washing with a concentrated NaCl (brine) solution.
  • Deionization of Water: To produce ultra-pure water. Water is passed through *two* columns:
    1. A cation exchanger (Resin-H) to remove all cations: Resin-H + M+ → Resin-M + H+
    2. An anion exchanger (Resin-OH) to remove all anions: Resin-OH + A- → Resin-A + OH-
    The H+ and OH- ions produced combine to form H2O.
  • Purification of Biomolecules: The primary tool for purifying proteins, enzymes, and nucleic acids in biotechnology.
  • Separation of Amino Acids: Amino acids can be separated based on their different charges at a given pH.