Isoelectric focusing uses a pH gradient to separate molecules by their isoelectric point.

Isoelectric focusing uses a pH gradient to separate molecules by their isoelectric point.

• A. It separates by isoelectric point in a pH gradient
• B. It separates by size using a gel
• C. It separates by charge in a capillary
• D. It separates by hydrophobicity

Answer: (A)

Isoelectric focusing relies on a fixed pH gradient to separate molecules by their isoelectric point. Each molecule has a pI where its net charge is zero. As proteins migrate in the electric field, their charge changes with the local pH: they move toward regions where their charge would neutralize, and when they reach the region where pH equals their pI, they have no net charge and stop migrating. This focusing along the gradient yields separation based on small differences in pI with high resolution. The other options describe different separation principles. Size-based separation uses a gel to distinguish molecules by how easily they migrate through a sieve-like matrix, not by pI. Separation by charge in a capillary refers to general electrophoresis driven by electrophoretic mobility rather than focusing to a pI. Separation by hydrophobicity relies on interactions with a hydrophobic stationary phase, not on pI.

Isoelectric focusing relies on a fixed pH gradient to separate molecules by their isoelectric point. Each molecule has a pI where its net charge is zero. As proteins migrate in the electric field, their charge changes with the local pH: they move toward regions where their charge would neutralize, and when they reach the region where pH equals their pI, they have no net charge and stop migrating. This focusing along the gradient yields separation based on small differences in pI with high resolution.

The other options describe different separation principles. Size-based separation uses a gel to distinguish molecules by how easily they migrate through a sieve-like matrix, not by pI. Separation by charge in a capillary refers to general electrophoresis driven by electrophoretic mobility rather than focusing to a pI. Separation by hydrophobicity relies on interactions with a hydrophobic stationary phase, not on pI.