Ampholytebased

Ampholyte-based refers to systems that use ampholytes to establish a pH gradient for electrophoretic separation. Ampholytes are small, zwitterionic molecules that contain both acidic and basic functional groups and span a range of isoelectric points (pI). When an electric field is applied to a medium containing ampholytes, they migrate and reconfigure into a continuous pH gradient, with the local pH determined by the dominant ampholytes at each position. This gradient enables isoelectric focusing (IEF) of proteins, peptides, and other charged analytes according to their pI.

Applications include traditional carrier ampholyte–based IEF in gel or solution formats and its use as the first dimension in two-dimensional protein electrophoresis. The ampholyte gradient provides high resolution for separating species with close pI values and can be tailored by selecting ampholyte mixtures to cover specific pH ranges.

Advantages of ampholyte-based systems include flexibility in designing the pH gradient and suitability for separating analytes with a wide range of pI. Limitations include gradient instability under certain conditions, sensitivity to temperature and sample composition, and potential interference in downstream analyses due to residual ampholytes. In modern workflows, immobilized pH gradients (IPG) are often preferred for stability, but ampholyte-based IEF remains a foundational approach and is still used in various research and diagnostic contexts.