Introduction

Ion‑exchange chromatography questions often ask which peptide elutes first from CM cellulose at pH 7.2, testing understanding of peptide charge and resin chemistry. CM‑cellulose is a negatively charged cation exchanger, so separation depends on how strongly different positively charged peptides bind at the working pH.

Step 1: Understand CM‑cellulose and the pH

CM‑cellulose carries a deprotonated carboxymethyl group, so the matrix is negatively charged at neutral pH and behaves as a cation exchanger. Cation exchangers bind species with net positive charge; peptides with greater positive charge bind more strongly and elute later as salt concentration or pH is changed.

At pH 7.2:

• Side chains of Lys and Arg are largely protonated and positively charged because their pKa values (about 10.5 and 12.5) are well above 7.
• The side chain of Glu (pKa ≈ 4.1) is deprotonated and carries a negative charge at this pH.
• Side chains of Val, Pro, Gly, and Tyr are essentially neutral around pH 7 (Tyr’s phenolic OH has pKa ≈ 10.9, so it is mostly uncharged at 7.2).

Therefore, peptides with more Lys/Arg residues have higher net positive charge; peptides with Glu have negative charge; neutral residues contribute mainly to hydrophobicity, not ionic binding.

Step 2: Determine net charge of each peptide

For this exam‑style question, the N‑terminus (NH₃⁺) and C‑terminus (COO⁻) of all peptides are the same and largely cancel each other, so relative binding is dominated by side‑chain charges.

(A) PKKRK

Sequence: Pro‑Lys‑Lys‑Arg‑Lys (PKKRK)

• Charged side chains: 3 Lys (+1 each), 1 Arg (+1).
• Net side‑chain charge ≈ +4 at pH 7.2.

This peptide is strongly cationic, giving the strongest interaction with the negatively charged CM‑cellulose and thus very late elution.

(B) RGERV

Sequence: Arg‑Gly‑Glu‑Arg‑Val (RGERV)

• Charged side chains: 2 Arg (+1 each), 1 Glu (−1).
• Net side‑chain charge ≈ +1 at pH 7.2.

RGERV is positively charged but less so than PKKRK; it will bind, but weaker than PKKRK, so it elutes earlier than PKKRK but later than a neutral peptide.

(C) RYRGV

Sequence: Arg‑Tyr‑Arg‑Gly‑Val (RYRGV)

• Charged side chains: 2 Arg (+1 each).
• Tyrosine side chain is neutral at pH 7.2.
• Net side‑chain charge ≈ +2.

RYRGV is more cationic than RGERV (because it lacks the negative Glu); therefore it binds more tightly and elutes later than RGERV but still earlier than PKKRK.

(D) LVVYP

Sequence: Leu‑Val‑Val‑Tyr‑Pro (LVVYP)

• All side chains (Leu, Val, Val, Tyr, Pro) are non‑ionizable at pH 7.2.
• Side‑chain net charge ≈ 0; overall peptide is essentially neutral (only the termini contribute ±1 and approximately cancel).

A nearly neutral peptide has the weakest electrostatic interaction with the cation exchanger and therefore flows through the column first or elutes at very low salt. Thus, LVVYP (option D) elutes first.

Why neutral peptides elute first in cation exchange

In cation‑exchange chromatography, elution is usually achieved by gradually increasing salt concentration or changing pH while monitoring UV absorbance. Peptides with low or zero net positive charge are displaced by counter‑ions at lower ionic strength and elute before strongly cationic species, which require higher salt to be displaced.

In this question, because LVVYP carries no extra positive charge, it has the lowest affinity for CM‑cellulose and therefore emerges first from the column.