Answer to Question #303406 in Biochemistry for Rakesh verma

Question

A solution containing Asp (pI=2.98), G (pI=5.97), Thr (pI=6.53), Leu (pI=5.98) and Lys (pI=9.74) in a pH 3.0 citrate buffer was applied to a Dowex-50 cation exchange column equilibrated with the same buffer. In what order will the five Amino Acids elute from the column

Answer 1

Aspartic acid, with two partially ionized (negatively charged) carboxyl groups (and the lowest $\Delta$p) will elute first. Of the three neutral amino acids, threonine has the greatest [AA⁺]/[AA⁰] ratio (i.e., the highest $\Delta$p), but it is highly polar because of the OH group. Consequently, threonine elutes before glycine and leucine. Glycine and leucine have about the same $\Delta$p, but leucine is decidedly more nonpolar than glycine. Therefore, glycine elutes third, followed by leucine. Lysine has a high effective positive charge because of its additional amino group (p$I$= 9.74, $\Delta$p = 6.74).

Therefore lysine elutes last, or not at all, unless the pH and/or the ionic strength of the eluting buffer are increased.