Uses of Carrier Ampholytes

Posted by Biophoretics on March 16th, 2021

Carrier Ampholytes are tiny, absurd molecules with both positive and negative charge groups. They sort based on their isoelectric points in an electric field, which establishes a pH gradient in solution or contributes to the slope when used with IPG strips.

Carrier Ampholyte pH ranges. Each horizontal line acts for the pH range covered by an ampholyte blend.

The mechanism of isoelectric focusing allows the separation of solutes based on their isoelectric points. Most announced applications are for proteins, but any zwitterionic solute can be separated via this approach. Using CIEF, there is no need to develop a shield system to separate solutes based on mobility. If the solutes have adequately different pI values, they will separate.

This device of separation contrasts sharply with CZE and CGE, where the bases for detachment are respectively the charge-to-mass ratio and molecular size. Righetti's elegant textbook (1) is suggested for further background on conventional IEF methodology. Many of the phenomena observed in the slab gel are directly related to events in the capillary.

Carrier Ampholytes and zwitterions are molecules with at least two pKa values, at least one of which is acidic, and at minimum one is basic. Both molecules have two pKa values. In clioquinol, the base pKa is lesser than the acid pKa, so at pH values between the pKa values, the molecule is neutral. But, the acid pKa for ampicillin is lesser than the base pKa, and the molecule is charged over the entire pH scale. Ampholytes are almost lipophilic and least soluble in their neutral form. However, zwitterions are always charged; they are seldom lipophilic, and they are often soluble in water at any pH.

Chosen ampholytes and zwitterions are listed in Table 2. Diprotic molecules with one acidic and one basic pKa that far apart behaves unmistakably as ampholytes or zwitterions. But, those whose pKa values are separated by less than about three units may display both zwitterionic and amphoteric properties. Note that the species XH in the equations above represent a composite of the neutral species XH0 and the charged species XH±, which can both survive at the same time.

To decently describe the ionization of Carrier Ampholytes and zwitterions with close pKa values, a more detailed picture of the ionization equilibria needed. This should include macro constants, which are the PKA values for the ionization of the microspecies.