Which electrophoretic technique employs a pH gradient to separate molecules with similar isoelectric points?

Which electrophoretic technique employs a pH gradient to separate molecules with similar isoelectric points?

• A. Zone electrophoresis
• B. High-resolution electrophoresis
• C. Isoelectric focusing
• D. Immunoelectrophoresis

Answer: (C)

Isoelectrical focusing uses a pH gradient in a gel so molecules migrate until their net charge becomes zero at their isoelectric point. Because each molecule reaches its own pI, it stops moving and forms a focused, narrow band. This creates very high resolution for separating proteins that have similar isoelectric points, since even small differences in pI cause the bands to settle at different positions along the gradient. The gradient is typically generated with carrier ampholytes or an immobilized pH gradient strip, providing a continuous range of pH values for precise focusing. In contrast, zone electrophoresis uses a uniform buffer pH and separates mainly by charge-to-mass ratio, not by pI, so closely related proteins may not be resolved as well. Immunoelectrophoresis combines electrophoresis with antibody reactions to identify or quantify components, rather than relying on a pH gradient for separation. High-resolution electrophoresis is a general designation for sharper separation under appropriate conditions but does not inherently imply separation by a pH gradient focused on isoelectric points.

Isoelectrical focusing uses a pH gradient in a gel so molecules migrate until their net charge becomes zero at their isoelectric point. Because each molecule reaches its own pI, it stops moving and forms a focused, narrow band. This creates very high resolution for separating proteins that have similar isoelectric points, since even small differences in pI cause the bands to settle at different positions along the gradient. The gradient is typically generated with carrier ampholytes or an immobilized pH gradient strip, providing a continuous range of pH values for precise focusing.

In contrast, zone electrophoresis uses a uniform buffer pH and separates mainly by charge-to-mass ratio, not by pI, so closely related proteins may not be resolved as well. Immunoelectrophoresis combines electrophoresis with antibody reactions to identify or quantify components, rather than relying on a pH gradient for separation. High-resolution electrophoresis is a general designation for sharper separation under appropriate conditions but does not inherently imply separation by a pH gradient focused on isoelectric points.