Chromatography Techniques and Components: Size Exclusion, Ion Exchange, and Protein Separation

A technique used to separate individual components from complex mixtures, consisting of a mobile phase and a stationary phase.

Chromatography

The liquid solvent that contains the molecules to be separated and moves through the stationary phase during chromatography.

Mobile phase

The fixed material (such as paper or resin-packed beads) through which the mobile phase travels during chromatography.

Stationary phase

Microscopic glass beads used as the stationary phase in column chromatography.

Resin

A chromatography type that separates molecules by size using porous beads — smaller molecules enter the pores and slow down, larger ones pass around them.

Size exclusion chromatography (SEC)

The mass of porous beads packed within a chromatography column through which the sample travels.

Column bed

The molecular weight threshold above which molecules are too large to enter the bead pores and elute first from the column.

Exclusion limit

The liquid used to dissolve biomolecules and create the mobile phase in chromatography.

Buffer

The mixture of biomolecules dissolved in buffer that is applied to the chromatography column.

Sample

A collected volume of liquid that elutes from a chromatography column at a specific stage of separation.

Fraction

A technique where a biomolecule (often an antibody) attached to beads binds the protein of interest, retaining it while other molecules pass through.

Affinity chromatography

A technique that separates proteins by charge using beads that bind oppositely charged molecules.

Ion exchange chromatography

Ion exchange chromatography in which positively charged beads bind negatively charged molecules.

Anion exchange

Ion exchange chromatography in which negatively charged beads bind positively charged molecules.

Cation exchange

The process of washing a bound protein off the column using a high-salt or altered-pH buffer.

Elution

A large brown protein (65,000 daltons) that is excluded from SEC bead pores and elutes in early fractions.

Hemoglobin

A small pink molecule (1,350 daltons) that enters SEC bead pores and elutes in later fractions.

Vitamin B12