Semester 7
BP701T
Ion Exchange, Gel & Affinity Chromatography
This unit covers three specialized chromatographic techniques for separating ionic, macromolecular, and biospecific compounds. Ion Exchange Chromatography — separation based on ionic interactions with charged resins, widely used for water purification, amino acid analysis, and protein purification. Gel (Size Exclusion) Chromatography — separation based on molecular size, used for molecular weight determination and desalting. Affinity Chromatography — the most selective technique, exploiting specific biological interactions for ultrapure purification of proteins, enzymes, and antibodies.
Syllabus & Topics
- 1Ion Exchange Chromatography – Introduction: Separation based on reversible exchange of ions between the sample and the ion exchange resin (stationary phase). Ions in solution exchange with counter-ions on the resin depending on their relative affinities. Cation exchange: resin has negative fixed charges → attracts/exchanges cations (R⁻–H⁺ + Na⁺ → R⁻–Na⁺ + H⁺). Anion exchange: resin has positive fixed charges → attracts/exchanges anions (R⁺–OH⁻ + Cl⁻ → R⁺–Cl⁻ + OH⁻). Uses: water softening/deionization, amino acid analysis (amino acid analyzer), protein purification, sugar analysis, removal of ionic impurities.
- 2Ion Exchange Resins – Classification: (1) Strong acid cation exchange: functional group = –SO₃⁻H⁺ (sulfonic acid). Active over entire pH range. Examples: Dowex 50, Amberlite IR-120. Fully ionized → pH-independent. (2) Weak acid cation exchange: functional group = –COO⁻H⁺ (carboxylic acid). Active only at pH > pKa (~pH 5-6). Partially ionized at low pH. Example: Amberlite IRC-50. (3) Strong base anion exchange: functional group = –N⁺(CH₃)₃OH⁻ (quaternary amine). Active over entire pH range. Examples: Dowex 1, Amberlite IRA-400. (4) Weak base anion exchange: functional group = –NH₂, –NHR (amine). Active only at pH < pKa (~pH 7-8). Example: Amberlite IR-45. Resin matrix: polystyrene cross-linked with divinylbenzene (DVB). Degree of cross-linking: higher DVB % → harder, tighter beads → higher selectivity but slower exchange.
- 3Ion Exchange – Properties & Mechanism: Properties: (1) Exchange capacity: total number of exchangeable ions per unit weight (meq/g dry resin). Determined by titration. Typically 3-5 meq/g for strong resins. (2) Selectivity: different ions have different affinities for the resin. For cation exchange (with dilute solutions): Li⁺ < Na⁺ < K⁺ < Rb⁺ < Cs⁺ (affinity increases with atomic number). Ca²⁺ > Mg²⁺ > Ba²⁺ (divalent > monovalent generally). For anion exchange: F⁻ < Cl⁻ < Br⁻ < I⁻. (3) Swelling: resin absorbs water and swells — degree depends on cross-linking (higher DVB → less swelling) and ionic form. Mechanism: equilibrium process — Resin-X + Y ⇌ Resin-Y + X. Selectivity coefficient K = [Y_resin][X_soln] / [X_resin][Y_soln]. Higher K → stronger binding of Y to resin.
- 4Ion Exchange – Factors & Methodology: Factors affecting ion exchange: (1) Nature of ion (valency, hydrated radius). (2) Nature of resin (functional group, cross-linking). (3) Concentration of sample solution. (4) pH (affects ionization of weak resins and analyte). (5) Temperature (↑T slightly ↑exchange rate). (6) Particle size of resin (smaller → faster kinetics but higher back pressure). Methodology: (1) Pack column with ion exchange resin. (2) Condition/equilibrate with starting buffer. (3) Load sample. (4) Wash to remove unbound. (5) Elute with increasing salt concentration (NaCl gradient disrupts ionic interactions) or changing pH (alter ionization of protein). (6) Collect fractions, analyze. Regeneration: after use, resin regenerated — cation exchange with HCl or NaCl; anion exchange with NaOH or NaCl.
- 5Ion Exchange – Applications: (1) Water deionization/purification: water passed through cation exchange bed (H⁺ form) → removes metal cations. Then through anion exchange bed (OH⁻ form) → removes anions. H⁺ + OH⁻ → H₂O. Produces deionized (DI) water. Mixed-bed deionizer: both resins mixed in one column → more efficient. (2) Amino acid analysis (amino acid analyzer): amino acids separated on cation exchange column (sulfonated polystyrene) using pH and ionic strength gradient → post-column derivatization with ninhydrin → quantified spectrophotometrically. (3) Protein purification: DEAE-cellulose (anion exchange) and CM-cellulose (cation exchange) widely used for protein chromatography. (4) Drug analysis: isolation of drugs from biological fluids. (5) Removal of ionic impurities from non-ionic pharmaceuticals.
- 6Gel Chromatography (Size Exclusion): Gel chromatography (SEC/GFC/GPC): separation based on MOLECULAR SIZE. Stationary phase: porous gel beads with defined pore sizes. Principle: Large molecules CANNOT enter gel pores → excluded → elute FIRST (in void volume, V₀). Small molecules ENTER pores → longer path → elute LATER (in V₀ + Vi). Intermediate molecules: partially enter pores → elute in between. No chemical interaction with gel — purely a size-based sieving effect. Elution order: LARGEST molecules first → SMALLEST last (opposite of most other chromatography). Terms: V₀ = void volume (inter-bead space); Vi = internal pore volume; Vt = total volume = V₀ + Vi. Molecules larger than exclusion limit: all elute at V₀ (unresolved). Molecules smaller than permeation limit: all elute at Vt (unresolved). Separation range: between these limits.
- 7Gel Chromatography – Gels & Instrumentation: Gel matrices: (1) Sephadex (cross-linked dextran): G-10 to G-200 (number indicates water regain capacity; G-200 separates up to ~200 kDa). For aqueous solutions. (2) Sephacryl: polyacrylamide-agarose composite. Higher exclusion limits. (3) Bio-Gel P (polyacrylamide): P-2 to P-300. (4) Bio-Gel A (agarose): for very large molecules (>1 MDa). (5) Styragel (polystyrene-DVB): for organic solvents (GPC — gel permeation chromatography for polymers). Instrumentation: similar to HPLC → pump, injector, column (30-100 cm), detector (UV, RI). Calibration: plot log(MW) vs Ve (elution volume) using standard proteins of known MW → linear relationship in the separation range → determine MW of unknown.
- 8Gel Chromatography – Applications: (1) Molecular weight determination: calibrate column with MW standards → run unknown → determine MW from calibration curve. (2) Desalting/buffer exchange: protein solution passed through G-25 column → protein (large) elutes at V₀, salt (small) elutes at Vt → rapid, simple buffer exchange. Most common application. (3) Protein purification: separate proteins by size — complement ion exchange and affinity chromatography. (4) Polymer MW distribution (GPC): MW averages (Mn, Mw, PDI) of synthetic polymers. (5) Removal of aggregates: separate monomeric protein from aggregates (larger → elute first). (6) Group separation: separate high-MW from low-MW species (antibiotics from fermentation broth).
- 9Affinity Chromatography – Principle: Most SELECTIVE chromatographic technique — exploits specific, reversible biological interactions between a LIGAND (immobilized on matrix) and its TARGET (in sample). Like lock-and-key: only the target molecule binds. Interactions: antigen-antibody, enzyme-substrate/inhibitor, receptor-ligand, lectin-glycoprotein, protein A-IgG, His-tag-Ni²⁺, biotin-avidin/streptavidin. Methodology: (1) Immobilize ligand on inert matrix (agarose/Sepharose beads) via spacer arm (prevents steric hindrance). (2) Apply sample → target binds specifically (binding). (3) Wash → remove non-specifically bound impurities. (4) Elute → disrupt interaction → release purified target. Elution methods: change pH (low pH for antibodies), increase ionic strength, competitive elution (add free ligand), chaotropic agents (urea, guanidine — harsh).
- 10Affinity Chromatography – Applications: (1) Antibody purification: Protein A or Protein G Sepharose → binds Fc region of IgG → elute at pH 2.5-3. Industry standard for monoclonal antibody purification (capture step in mAb manufacturing). (2) Enzyme purification: immobilize substrate analog or inhibitor → specific binding of enzyme. (3) His-tag protein purification (IMAC — Immobilized Metal Affinity Chromatography): recombinant protein expressed with 6×His tag → binds Ni-NTA column → elute with imidazole. Most commonly used for recombinant protein purification. (4) Lectin affinity: ConA (Concanavalin A) binds glycoproteins → purify glycoproteins from cell lysate. (5) DNA-binding protein purification: DNA immobilized on matrix → bind transcription factors. (6) Drug-receptor interaction studies. Advantages: excellent selectivity (often >90% purity in single step), high capacity. Disadvantages: expensive ligands, limited reusability, gentle handling required.
Learning Objectives
Resin Types: Classify ion exchange resins into 4 types with functional groups, pH range, and examples.
Water Deionization: Explain the process of water deionization using cation and anion exchange resins.
SEC Principle: Explain the principle of gel chromatography with V₀, Vi, and the concept of exclusion/permeation limits.
MW Determination: Describe how molecular weight is determined using gel chromatography calibration.
Affinity Specificity: Explain why affinity chromatography is the most selective technique and describe the His-tag purification method.
Exam Prep Questions
Q1. Why do large molecules elute FIRST in gel chromatography?
In gel chromatography, the gel beads have pores of defined sizes. Large molecules are TOO BIG to enter the pores → they can only travel through the space BETWEEN the beads (void volume, V₀) → they take the shortest path → elute first. Small molecules CAN enter the pores → they diffuse in and out of many pores → much longer path → elute last. Think of it like a maze: big people can’t fit through narrow passages and walk straight through, while small people explore every passage and take longer.
Q2. What is the IMAC/His-tag purification system?
IMAC (Immobilized Metal Affinity Chromatography) with His-tag is the most widely used method for purifying recombinant proteins. A 6×Histidine tag is genetically fused to the protein of interest. Histidine’s imidazole side chains have strong affinity for divalent metal ions (Ni²⁺, Co²⁺) immobilized on NTA-agarose beads. Process: load cell lysate → His-tagged protein binds → wash away everything else → elute with free imidazole (competes for Ni²⁺ binding) → pure protein. Simple, fast, works under native or denaturing conditions.
Q3. How is ion exchange different from ion-pair chromatography?
Ion exchange: the stationary phase itself carries charges (ionic functional groups on resin) → ionic interaction between resin and analyte ions → separation.
Ion-pair chromatography: the stationary phase is a regular RP-HPLC column (C₁₈, non-ionic). An ion-pairing reagent is added to the MOBILE PHASE (e.g., tetrabutylammonium for anions, heptanesulfonate for cations) → forms neutral ion pair with analyte → ion pair partitions into C₁₈ → retention. Ion-pair is done on HPLC; ion exchange uses specialized resins.