Veterinary Biochemistry- ICAR JRF/SRF 2024

• Size-Exclusion Chromatography Components:
• Stationary Phase: Porous, cross-linked, hydrated microscopic gel beads (e.g., dextran / Sephadex, agarose / Sepharose, polyacrylamide / Bio-Gel).
• Mobile Phase: A continuous flow of aqueous liquid buffer (liquid phase) carrying the dissolved protein mixture through the packed column bed under gravity or pressure.
• nalysis:
• (Liquid): Correct state of the mobile phase.

Correct option: (2) Liquid

• Mechanism of Gel Filtration:
• Assertion (A): Gel filtration chromatography separates macromolecules according to their hydrodynamic volume / molecular weight. (True).
• Reason (R): The column matrix consists of porous gel beads with defined pore dimensions. Large molecules exceeding the pore exclusion limit cannot enter the beads and travel solely through the interstitial void volume, eluting first. Smaller molecules enter the bead pores, traversing a tortuous path with increased retention volume, and elute at a later stage. (True).
• nalysis:
• Both (A) and (R) are correct, and (R) is the precise physical explanation for (A). Hence, Option 1 is correct.

• Gel Filtration Chromatography (Size-Exclusion Chromatography - SEC):
• Principle: Separates molecules based on differences in their hydrodynamic size and shape as they pass through a column packed with porous gel beads (e.g., Sephadex, Sepharose).
• Applications:
• Purification by shape and size: Larger molecules are excluded from bead pores and elute first in the void volume ($V_o$), while smaller molecules penetrate pores and elute later.
• Desalting: Rapid separation of large macromolecules (proteins) from small salt ions or free dye.
• Analyzing protein-protein interactions: Determination of native molecular mass, quaternary structure, and stable protein complex formation.
• Charge-based separation: Carried out by ion-exchange chromatography, not gel filtration.
• Conclusion: Gel filtration is used for (A), (B) and (C) only.

Correct option: (1) (A), (B) and (C) only.

• Cation Exchangers in Chromatography:
• Cation-exchange resins possess negatively charged functional groups that attract and bind positively charged molecules (cations):
• Carboxy-Cellulose and (D) Carboxymethyl-Cellulose (CM-cellulose): Weak cation exchangers bearing carboxylate groups ($-\text{O}-\text{CH}_2-\text{COO}^-$).
• Sulfopropyl (SP) / Methyl sulfonate (S): Strong cation exchangers ($-\text{SO}_3^-$).
• Anion Exchangers: Possess positively charged groups that bind anions:
• DEAE-Sepharose: Diethylaminoethyl, a weak anion exchanger ($-\text{CH}_2\text{CH}_2\text{N}^+\text{H}(\text{CH}_2\text{CH}_3)_2$).
• Q-Sepharose: Quaternary ammonium, a strong anion exchanger ($-\text{CH}_2\text{N}^+(\text{CH}_3)_3$).
• Conclusion: Resins (C) and (D) only are cation exchangers.

Correct option: (4) (C) and (D) only.