Question

A mixture of four peptides, PKKRK, RGERV, RYRGV and LVVYP, is loaded onto an ion-exchange column at pH = 7.2. If carboxymethyl (CM) cellulose is used as the stationary phase of this column, then which peptide elutes first? 

• A. PKKRK
• B. RGERV
• C. RYRGV
• D. LVVYP

Hint:

In ion-exchange chromatography, always check whether the resin is cation- or anion-exchange. Neutral molecules elute first, followed by progressively stronger charged ones.

Answer 1

The Correct Option is D

Step 1: Nature of the stationary phase
Carboxymethyl (CM) cellulose is a cation-exchange resin.
- It contains negatively charged carboxyl groups at pH 7.2.
- Therefore, it binds to positively charged peptides (cationic residues).
- Neutral or negatively charged peptides will bind weakly or not at all, and thus elute first.
Step 2: Analyze the peptides at pH 7.2
We must consider the approximate charges of side chains at pH 7.2: - Lysine (K), Arginine (R): positively charged (+1 each).
- Glutamic acid (E): negatively charged (–1 each).
- Tyrosine (Y), Proline (P), Valine (V), Glycine (G), Leucine (L): neutral.
Step 3: Calculate net charges
PKKRK: Contains 3 Lys (+3), 1 Arg (+1), 1 Pro (0). Net charge $\approx +4$. Strongly cationic $⇒$ binds strongly to CM-cellulose. 
RGERV: Contains Arg (+1), Glu (–1), Arg (+1), Val (0). Net charge $\approx +1$. Moderately positive $⇒$ binds moderately. 
RYRGV: Contains Arg (+1), Tyr (0), Arg (+1), Gly (0), Val (0). Net charge $\approx +2$. Positive $⇒$ binds. 
LVVYP: Contains Leu (0), Val (0), Val (0), Tyr (0), Pro (0). Net charge $\approx 0$. Neutral $⇒$ weak/no binding. 
Step 4: Elution order
- Peptides with higher positive charge bind more strongly to the negatively charged CM-cellulose.
- Therefore, the order of elution will be from least positive to most positive.
- Neutral peptide LVVYP (charge 0) will elute first.
Therefore, the peptide that elutes first is (D) LVVYP.