Question

how do buffer ph/pKa/pI values for amino acids affect their elution order during ion exchange chromatography?

what molecular properties affect the migration distance (on the chromatography paper) of amino acids during IEC?

Answer 1

Amino acids exist as zwitter ions i.e they have both acidic and basic part so their elution is affected by ph and pka values as during ion exchange chromatography in acidic medium basic amino acids are eluted first as in acidic medium acidic amino acidic part dominates which is dissolved in acidic medium and basic amino acids are eluted first. Same happens if increasing ph forms basic medium i.e ph is greater than 7 so in that case basic amino acids will be eluted at last and here if the pka value is small then the acid is stronger for amino acid . Elution is affected as the same principle which says that like dissolves like so pka values and ph both affect the elution of amino acids through ion exchange chromatography. So amino acids with lower pka values will elute at last in case of acidic medium in ion exchagers and vice versa.

Molecular properties such as solubility , ionisation, size of side chains, shape and structure all affect the migration distance of amino acids during iec or ion exchange chromatography. Larger molecules elute fast and smaller molecules penetrate into the ion exchangers and thus moves slowly and also if the amino acid is soluble in ion exchange resin then also it will elute at last because its migration distance will increase with the other fast moving amino acids through the ion exchagers.