Ion Exchange Chromatography, B.Pharm

• Instrumental analysis, B.Pharm, 7th semester

Ion Exchange Chromatography

• Defined as a process allowing the separation of ions and polar molecules.
• Separation based on their affinity for the exchanger.
• The technique is usable for any kind of charged molecules.
• Examples include large proteins, small nucleotides, amino acids, and peptides, etc.
• It is based on the exchange of ions in solution with those present in the ion exchange resin.
• Two classifications exist: cation exchange and anion exchange.

Cation Exchange

• It is carried out with buffer having a pH between 4-7.
• Cations present in solution separate and exchange for similar ions in cation exchange resins.
• Cations are retained by the solid matrix.

Anion Exchange

• Separation uses anion exchange resin.
• The chromatography is carried out with buffer having a pH between 7-10.
• Anions in solution separate for similar ions in anion exchange resin.
• Anions are retained by the solid matrix.

Mechanism of Ion Exchange Process

• Cation and anion exchange resin columns are used for the separation of cations and anions.
• Separation also relies on the binding of analytes to positively or negatively charged groups fixed on a stationary phase.
• Ions compete with similarly charged ions to bind to oppositely charged ionic functional groups.

Cation Exchange Chromatography

• Exchangeable ions are cations, represented by a given equation.
• In this process, the eluent cation (M⁺) is replaced with the analyte cation (C⁺) that is bound to the anion (X⁻) fixed on the chromatographic support (S).

Anion Exchange Chromatography

• Exchangeable ions are anions, and are represented by a given equation.
• In this process, the eluent anion (B⁻) is replaced with the analyte anion (A⁻) that is bound to the cation (X⁺) fixed to the chromatographic support (S).

Factors Affecting Ion Exchange

Nature and Properties of Ion Exchanged Resins

• More cross-linking agent: resin becomes more rigid with smaller pore size, and swelling decreases.
• When swelling decreases, separation of different sizes is difficult.

Concentration and Charge of Ions

• If resin has a higher charge, a solution has a lower charge.
• Exchange is favored at higher concentration.
• If resin has a lower charge, a solution has a high charge.
• Exchange is favored at low concentrations.

Solvated Size of the Solute Ions

• Smaller solvated sized ions exhibit a greater binding affinity, when compared to larger ions.
• A smaller ion gets easily retained in the resin pores.

Porosity

• High porosity offers a large surface area covered by charged groups.
• High porosity provides a high binding capacity.

Properties of Ion Exchange Resins

Ion Exchange Capacity

• The property is determined by the number of functional groups per unit weight of the resin.
• Ion exchange capacity is measured with a particular ion.
• Ion exchange capacity of a resin is useful.

Concentration Estimation

• It measures the concentration of the competing ion.
• Resins with higher capacity perform well, when using more concentrated eluents.

Swelling Characteristics

• Organic resin exchangers have cross-linked polymer chains.
• When coming in contact with water, outer functional groups get solvated and the polymer chains unfold.
• The degree of swelling depends on the solution's composition.

Ion Exchange Selectivity

• Selectivity coefficient can determine the relative affinities of ion exchangers for different ions.
• A well-defined affinity series for anions and cations are obtainable by simple experiments.

Applications

• Total content of cation in a solution
• Concentration of traces of an electrolyte
• Softening of hard water
• Analysis of natural and industrial water
• Separation of complex mixtures of biochemical compounds
• Separation of amino acids and sugars.

Methodology, Column Method

• The technique involves separation of mixture components on the basis of differences in the selectivity coefficients for the resin.
• The apparatus consists of a glass column fitted with a glass wool plug at the low end, and a sintered glass disc.
• A slurry of resin is made in distilled water, then gradually poured into the column.
• Proper packing is key: ensure no air bubbles remain.
• The column is back-washed with distilled water.
• Water flow stops and the resin settles.
• Excess water will then drain off.

Gel Chromatography

• The technique's component separation is based on the difference in molecular size.
• Techniques: gel filtration and gel permeation.
• Molecules are separated based on size.
• It is also called molecular sieve chromatography.

Theory of Separation

• A column is made up of swollen gel particles.
• The solvent is used to swell the gel into a suitable tubular container.
• The equation below displays this relationship where Vt is the total volume of the column.
• Vt = Vo + Vi + Vm
• Vo = volume of the mobile phase outside the gel particles
• Vi = volume of the internal pores of the gel particles
• Vm = volume of the stationary phase, is the gel matrix itself

• A column is also filled with swollen gel beads, and acts as a molecular sieve.
• A mixture with molecules is poured over the column.
• Large molecules quickly elute, while smaller are retained for longer within the pours.
• Large molecules are collected first.

Instrumentation

Stationary Phase

• Porous and permeable composition.
• Well defined and ranging polymer gel beads.
• Examples: Dextran, Agarose gel, Acrylamide gel.

Mobile Phase

• Uses a liquid which will dissolve the biomolecules and form the mobile phase.
• Examples: Toluene, water, and tetrahydrofuran.

Pump

• Pumps: syringe or reciprocating.
• Pumps must have a highly consistent flow rate.

Detectors

• Refractive index detectors
• Ultraviolet absorbent detectors
• Light scattering detectors

Application

• Determination of molecular weight
• Determination of Purity
• Protein fractionation
• Separation of sugar, proteins, and peptides.

Affinity Chromatography

• The used technique separates biochemical mixtures based on biological interactions, such as those between an antigen and antibody, or an enzyme and substrate.
• It involves a single purification step, but is time consuming.
• Separation occurs from a substance's affinity, to a molecule where it can specifically bind.
• There are following components: Matrix, Spacer Arm, and Ligand

Componants of Affinity Chromatography - Matrix

• Support used for ligand coupling.
• Materials are available in a range of particle and pore sizes.
• Examples include agarose, polyacrylamide, and dextran.

Componants of Affinity Chromatography - Spacer Arm

• Used to improve binding between a ligand and target molecule
• Efficient binding is achieved by introducing a spacer arm between a matrix and ligand

Componants of Affinity Chromatography - Ligand

• Molecules that bind reversibly to a group of molecules
• Ligands exhibit specific and reversible binding for that target substance.

Procedure

• A sample is injected into the affinity chromatography column.
• Only substances with an affinity for the resin, will be retained by the column.
• Substances with no affinity for the ligand, elute off the column.
• Retained substances can be eluted by altering pH levels.
• Other ways to change substance: salt concentrations and organic solvents.

Application

• Genetic engineering
• Nucleic acid purification
• Vaccine production
• Antibody purification from blood serum
• Determination of biological compounds