Chromatographic Separations
⭐ Downstream Processing Matrix
4 Parameters
Protein Chromatography: Gel Filtration vs Ion Exchange vs Affinity vs HIC
Comparative matrix of physical separation basis, stationary phase resins, elution methods, and resolution power.
| Method | Physical Separation Principle | Stationary Phase Matrix | Elution Strategy |
|---|---|---|---|
| Gel Filtration / Size Exclusion (SEC) | Hydrodynamic Stokes radius / Molecular Weight (Shape and size) | Porous uncharged dextran/agarose beads (Sephadex, Superdex) | Isocratic buffer elution (Large molecules elute first, small enter pores) |
| Ion Exchange (Cation vs Anion) | Net surface electrostatic charge at specified buffer pH | Charged groups: Cation exchanger (CM, SP-cellulose) vs Anion exchanger (DEAE, Q-Sepharose) | Linear salt gradient (NaCl/KCl) to displace bound proteins, or pH gradient |
| Affinity Chromatography | Specific biological lock-and-key recognition for ligand/tag | Immobilized substrate, antibody, Ni-NTA (for His-tags), or Glutathione (for GST-tags) | Competitive displacement: Free ligand (e.g. Imidazole, Reduced Glutathione) or pH shift |
| Hydrophobic Interaction (HIC) | Surface hydrophobic patches exposed on native protein | Weakly hydrophobic alkyl/aryl chains (Phenyl-Sepharose, Octyl) | Descending salt gradient (high ammonium sulfate promotes binding, pure water elutes) |
Separation Basis & Elution Pattern
Key Point
Method: Gel Filtration
Basis: Size / Molecular Weight
Elution: Large first, small last
Key Point
Method: Ion Exchange
Basis: Net Charge
Elution: Increasing Salt (NaCl)
Key Point
Method: Affinity
Basis: Specific Binding Tag
Elution: Competitive Ligand (Imidazole)
Key Point
Method: HIC
Basis: Hydrophobicity
Elution: Decreasing Salt
Microscopy & Imaging
⭐ Ultrastructural Imaging Core
5 Parameters
Electron Microscopy: TEM vs SEM
Comparative matrix of electron beam path, specimen preparation, 2D internal vs 3D surface imaging, and resolution limits.
| Parameter | Transmission Electron Microscope (TEM) | Scanning Electron Microscope (SEM) |
|---|---|---|
| Electron Beam Pathway | Electrons pass completely THROUGH an ultra-thin sliced specimen | Electrons scan back and forth across the metallic-coated SURFACE of specimen |
| Image Presentation & Dimension | 2D high-resolution cross-sectional view of internal organelles & ultrastructure | 3D realistic stereoscopic image of surface topography and morphology |
| Specimen Thickness & Sectioning | Ultra-thin sections required (<100 nm sliced on ultramicrotome with diamond knife) | Whole thick specimens permitted; no internal sectioning needed |
| Specimen Staining / Coating | Heavy metal salts (Uranyl acetate, Lead citrate) for differential electron density | Gold-palladium or Platinum sputter-coating to emit secondary electrons |
| Practical Resolution Limit | Sub-nanometer: ~0.1 – 0.2 nm (atomic lattice resolution) | ~1.0 – 3.0 nm (lower than TEM) |
TEM vs SEM Comparison
TEM
Electrons: Transmitted Through
View: 2D Internal Cross-Section
Resolution: 0.1 nm (Highest)
SEM
Electrons: Scattered from Surface
View: 3D Surface Topography
Resolution: 1-3 nm
