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5C · The chemistry of life
Separation and purification
Lab methods that separate mixtures by exploiting a physical difference — extraction (polarity/solubility), distillation (boiling point), chromatography (affinity for a stationary vs. mobile phase), and gel electrophoresis (size and charge). Mostly recognition points: know what each separates by.
Extraction
Liquid–liquid extraction separates solutes by partitioning them between two immiscible solvents (typically water and an organic layer) based on relative solubility — "like dissolves like."
A solute distributes between the two layers according to its partition coefficient; polar/ionic species favor the aqueous layer, nonpolar species the organic. Acid–base extraction adds a switch: protonating or deprotonating a compound flips its solubility (a carboxylic acid becomes water-soluble carboxylate in base, then returns to the organic layer when reacidified), letting you pull one component out cleanly. The denser layer (usually the aqueous, unless a halogenated solvent is used) sits on the bottom.
Acid–base extraction
Adjust pH to toggle a compound's charge: deprotonate an acid (or protonate a base) to send it into water, then reverse to recover it in the organic layer. Separates acids, bases, and neutrals from one another.
Because charged species are water-soluble and neutral species organic-soluble, sequential washes at chosen pH peel a mixture apart: wash with base to remove carboxylic acids as carboxylates, wash with acid to remove amines as ammonium salts, leaving neutrals behind. This is a direct application of the acid–base strength logic from 5A.
Distillation
Distillation separates liquids by boiling point: heat the mixture, the more volatile component vaporizes first, then recondenses in the condenser. Simple distillation for far-apart boiling points; fractional for close ones.
The lower-boiling (more volatile, weaker-IMF) component enriches the vapor and is collected first. Simple distillation works when boiling points differ by more than ~25 °C; fractional distillation adds a column that provides many vaporize–condense cycles to resolve components with close boiling points. Vacuum distillation lowers the pressure to boil heat-sensitive compounds at lower temperature. The driver is volatility, which traces back to intermolecular forces.
Chromatography
Chromatography separates a mixture carried in a mobile phase over a stationary phase: components that bind the stationary phase more strongly move slower. Variants differ in what they separate by — polarity, size, charge, or specific affinity.
In normal-phase TLC/column chromatography the stationary phase is polar (silica) and nonpolar compounds elute first; reverse-phase flips this. Gas chromatography (GC) separates volatile compounds by boiling point and stationary-phase affinity. For biomolecules: size-exclusion (gel-filtration) separates by size (large molecules elute first, bypassing the pores — a common trap), ion-exchange by charge, and affinity chromatography by specific binding (e.g., antibody–antigen, His-tag to a nickel column). The key is matching the technique to the property it exploits.
Don't confuse
In size-exclusion chromatography, large molecules elute first (they can't enter the beads' pores, so they take the short path), while small molecules are retained longer. The intuitive "small things slip through faster" is backwards here.
Gel electrophoresis
An electric field drives charged macromolecules (DNA, RNA, proteins) through a gel; smaller molecules travel farther. SDS-PAGE coats proteins with uniform negative charge so they separate by size alone; isoelectric focusing separates by pI.
DNA (uniformly negative from its phosphate backbone) migrates toward the anode, with small fragments moving fastest through the gel mesh — separation by size. For proteins, SDS-PAGE denatures and coats them with a constant charge-to-mass ratio so migration reflects size only; native gels and isoelectric focusing (which stops each protein at the pH equal to its pI, where net charge is zero) separate by charge instead. This overlaps directly with the protein and nucleic-acid lab techniques in the Bio/Biochem textbook — learn it once.
Worked question
A mixture of three globular proteins — 12 kDa, 65 kDa, and 240 kDa — is run through a size-exclusion (gel-filtration) column. In what order do they elute (first → last)?