Question

The order of elution (first to last) of amino acids Histidine (H), Glutamic acid (E), and Lysine (K) from a cation exchange column with a buffer having pH 6.0 is (Given: The pK values of H, E, and K is 6.0, 4.25, and 10.5 respectively):

• A. Glutamic acid, Histidine and Lysine
• B. Lysine, Histidine and Glutamic acid
• C. Histidine, Lysine and Glutamic acid
• D. Glutamic acid, Lysine and Histidine

Hint:

In cation exchange chromatography, amino acids elute in order of increasing isoelectric point ($\text{pI}$):
\[ \text{Acidic Amino Acids} \rightarrow \text{Neutral Amino Acids} \rightarrow \text{Basic Amino Acids} \]

Answer 1

The Correct Option is A

Step 1: Understanding the Concept:
Cation exchange chromatography uses a negatively charged stationary phase. At a given pH, the net charge of each amino acid determines its binding strength and elution order.
Molecules with a net negative charge are repelled and elute first, while those with a strong positive charge bind tightly and elute last.

Step 2: Detailed Explanation:
Let us analyze the ionization state and net charge of each amino acid at $\text{pH } 6.0$:
1. Glutamic acid (E) ($pK_a = 4.25$ for side-chain carboxyl): At $\text{pH } 6.0$, the side-chain is fully deprotonated and negatively charged ($\text{COO}^-$). Since the net charge is negative, it does not bind to the negatively charged cation exchanger and elutes first.
2. Histidine (H) ($pK_a = 6.0$ for the imidazole side-chain): At $\text{pH } 6.0$ (where $\text{pH} = pK_a$), the imidazole ring is 50% protonated (neutral and positive states are in equilibrium). Since its net charge is weakly positive, it binds weakly and elutes second.
3. Lysine (K) ($pK_a = 10.5$ for side-chain amino): At $\text{pH } 6.0$, the side-chain is fully protonated and positively charged ($\text{NH}_3^+$). Its strong net positive charge causes it to bind tightly to the column, meaning it elutes last (requiring high salt or pH to displace).
Therefore, the correct elution sequence is Glutamic acid, Histidine, Lysine.

Step 3: Final Answer:
The elution order is Glutamic acid, Histidine, and Lysine.