Biochemical Separation Techniques

Chromatography and electrophoresis methods

Chromatography separates the components of a mixture by distributing them between a mobile phase, which carries the sample, and a stationary phase, which retains each solute to a different extent. Each solute moves at a speed that depends on how strongly it interacts with the stationary phase, so the chosen matrix determines which property is exploited. The main principles are molecular size, net charge, hydrophobicity or polarity, and specific biological binding. Electrophoresis, in contrast, separates charged molecules by their migration through a medium under an applied electric field, and its resolution depends on charge, size, and the gel matrix. A useful study strategy is to learn each method by the property it separates.

Gel filtration uses an inert porous matrix, such as cross-linked agarose (Sepharose), as the stationary phase, and it separates molecules by size rather than by chemical interaction. Blue dextran is not a stationary phase; it is a high-molecular-weight dye that is fully excluded from the pores and is used to measure the void volume of the column. Carboxymethyl (CM) cellulose is a cation exchanger and diethylaminoethyl (DEAE) cellulose is an anion exchanger, so both separate by charge.

Affinity chromatography depends on a specific, reversible biological interaction between a ligand fixed to the matrix and the target molecule. A 6×His tag binds strongly to immobilized Ni^{2+} on a nitrilotriacetic acid (NTA) resin. This format is called immobilized metal affinity chromatography (IMAC), which is a form of affinity chromatography, and the bound protein is released by competition with imidazole.

In paper chromatography, separation is expressed by the retention factor, the ratio of the distance moved by the solute to the distance moved by the solvent front, both measured from the point of origin.

$R_f = \frac{\text{distance moved by solute}}{\text{distance moved by solvent front}}$

Because both distances are measured in the same units, R_f is dimensionless and lies between 0 and 1. For example, a spot that moves 3 cm while the solvent front moves 12 cm has R_f = 3/12 = 0.25.

SDS-PAGE separates polypeptides mainly by mass. Sodium dodecyl sulphate denatures proteins and coats them with negative charge in proportion to their mass, so they migrate toward the anode, the positive electrode, and smaller polypeptides move through the gel pores more easily and travel farther. β-mercaptoethanol is a reducing agent that cleaves disulphide bonds so that the chains are fully unfolded. Bromophenol blue is the tracking dye that marks the migration front, whereas ethidium bromide is a nucleic acid stain and plays no role in protein tracking.

Several methods separate proteins by molecular weight, including size-exclusion chromatography, SDS-PAGE, and sucrose density gradient centrifugation. Ion-exchange chromatography, by contrast, separates by net charge and does not sort proteins by mass.

Each physical property is matched to a particular separation method. Solubility is exploited by salting out, net ionic charge by isoelectric focusing, polarity by reverse phase chromatography, and molecular size by ultracentrifugation.