As pharmaceutical and biochemical research advances, the need for highly selective, accurate, and reproducible separation techniques has become increasingly critical. Chromatography continues to serve as a cornerstone of analytical and preparative sciences, especially in drug development, biotechnology, and quality control. Unit 5 focuses on three powerful and specialized chromatographic techniques—Ion Exchange Chromatography, Gel Chromatography, and Affinity Chromatography—each offering unique mechanisms and applications in separating complex biological and chemical mixtures.
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Ion Exchange Chromatography: Separation Based on Charge
Introduction and Classification
Ion exchange chromatography is a technique that separates molecules based on their electrical charge. It is particularly useful for analyzing inorganic ions, amino acids, proteins, peptides, and nucleotides.
Based on the nature of ions exchanged, it is classified into:
Cation exchange chromatography, where positively charged ions are exchanged
Anion exchange chromatography, where negatively charged ions are exchanged
This classification allows flexibility in selecting the method according to the analyte’s charge properties.
Ion Exchange Resins and Their Properties
Ion exchange resins are insoluble, cross-linked polymeric materials containing charged functional groups. Cation exchangers possess acidic groups such as sulfonic or carboxylic acids, while anion exchangers contain basic groups like quaternary ammonium ions.
Key properties of resins include exchange capacity, degree of cross-linking, particle size, and chemical stability, all of which influence separation efficiency.
Mechanism of Ion Exchange
The ion exchange process involves reversible replacement of ions bound to the resin with ions of similar charge present in the mobile phase. When a sample passes through the column, ions with greater affinity displace weaker ions from the resin. Elution is achieved by altering pH, ionic strength, or salt concentration of the mobile phase.
Factors Affecting Ion Exchange
Several factors influence separation efficiency, including pH of the mobile phase, concentration of counter ions, temperature, and resin characteristics. Careful optimization of these parameters ensures reproducible and selective separations.
Methodology and Applications
The method involves column equilibration, sample loading, washing, and controlled elution. Ion exchange chromatography is extensively applied in protein purification, water analysis, amino acid separation, and quality control of pharmaceuticals, making it indispensable in analytical laboratories.
Gel Chromatography: Separation by Molecular Size
Introduction and Theory
Gel chromatography, also known as gel filtration chromatography or size exclusion chromatography (SEC), separates molecules based on molecular size rather than chemical interaction. The stationary phase consists of porous gel beads that allow smaller molecules to enter the pores, while larger molecules are excluded.
Mechanism of Separation
Large molecules are unable to penetrate the pores of the gel matrix and therefore elute first. Smaller molecules diffuse into the pores and take longer to pass through the column. This purely physical separation minimizes chemical interactions, preserving molecular integrity.
Instrumentation
The system includes a column packed with gel particles such as Sephadex, Sepharose, or polyacrylamide gels, a suitable mobile phase, and a detector. Since no binding occurs, the mobile phase is often a simple buffer compatible with the sample.
Applications of Gel Chromatography
Gel chromatography is widely used for:
Molecular weight determination
Protein desalting and buffer exchange
Separation of biomolecules like enzymes, antibodies, and polysaccharides
Purification of vaccines and biological products
Its gentle nature makes it ideal for fragile biological samples.
Affinity Chromatography: Highly Selective Molecular Recognition
Introduction and Principle
Affinity chromatography is one of the most selective chromatographic techniques available. It relies on specific biological interactions between a target molecule and a ligand immobilized on the stationary phase. These interactions may involve antigen–antibody, enzyme–substrate, receptor–ligand, or metal–protein binding.
Theory of Affinity Interaction
When a mixture passes through the column, only molecules with specific affinity for the ligand bind to the stationary phase, while others are washed away. The bound molecules are then eluted by changing pH, ionic strength, or by adding a competitive ligand.
Instrumentation
The chromatographic setup includes an affinity column packed with ligand-bound matrix, buffer delivery systems, and sensitive detectors. The choice of ligand and matrix determines selectivity and efficiency.
Applications of Affinity Chromatography
Affinity chromatography is extensively used in:
Purification of monoclonal antibodies
Isolation of enzymes and hormones
Recombinant protein purification
Vaccine and biopharmaceutical production
Clinical diagnostics and proteomics
Its high specificity reduces purification steps and enhances product purity.
Comparative Advantages and Limitations
Advantages
Ion exchange chromatography offers high resolution and scalability
Gel chromatography preserves molecular structure and requires mild conditions
Affinity chromatography provides exceptional selectivity and purity
Limitations
Ion exchange chromatography requires precise pH control, gel chromatography has limited resolution for similarly sized molecules, and affinity chromatography can be expensive due to ligand costs and stability issues.