Q.27 A mixture of Arginine, Phenylalanine and Histidine was fractionated using cation exchange
chromatography at neutral pH. The amino acids were eluted with an increasing salt gradient. Identify
the correct order of elution.
(A) Arginine, Histidine, Phenylalanine
(B) Phenylalanine, Histidine, Arginine
(C) Histidine, Phenylalanine, Arginine
(D) Arginine, Phenylalanine, Histidine
Answer: (B) Phenylalanine, Histidine, Arginine
In cation exchange chromatography at neutral pH (~7), amino acids separate based on net positive charge: least positive elutes first with increasing salt gradient, as weaker binding occurs with lower charge. Phenylalanine (neutral, near-zero charge) elutes first, followed by histidine (+0.1, weakly positive), then arginine (+1.0, strongly positive, binds tightest).
Option Analysis
(A) Arginine, Histidine, Phenylalanine: Incorrect—arginine’s high +1 charge binds strongest, eluting last, not first.
(B) Phenylalanine, Histidine, Arginine: Correct—Phe (pI 5.5, near neutral) elutes first; His (pI 7.6, slight +) next; Arg (pI 10.8, strong +) last.
(C) Histidine, Phenylalanine, Arginine: Incorrect—Phe’s lower charge elutes before His, not after.
(D) Arginine, Phenylalanine, Histidine: Incorrect—arginine binds too strongly to elute first; violates charge order.
Introduction: Cation Exchange Chromatography Amino Acids Elution Order
Cation exchange chromatography separates amino acids like arginine, phenylalanine, and histidine based on charge at neutral pH using salt gradients. This guide explains the precise elution order—phenylalanine first, then histidine, arginine last—for biotech exam preparation.
Charge at Neutral pH
At pH 7, net charge determines binding to negatively charged resin:
| Amino Acid | pKa (COOH) | pKa (NH3+) | pKa (Side Chain) | pI | Net Charge at pH 7 | Elution Position |
|---|---|---|---|---|---|---|
| Phenylalanine | ~2.2 | ~9.3 | None | 5.5 | ~0 | First |
| Histidine | ~2.0 | ~9.0 | 6.0 (imidazole) | 7.6 | +0.1 | Second |
| Arginine | ~2.0 | ~9.0 | 12.5 (guanidino) | 10.8 | +1.0 | Last |
Least charged (Phe) elutes first; salt competes off stronger binders later.
Separation Mechanism
-
Resin: Sulfonate groups (-SO3-) attract cations.
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Gradient: Rising NaCl displaces bound amino acids by charge competition.
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Neutral pH Effect: Basic residues (Arg > His > Phe) protonate increasingly.
This order holds for protein purification in biochemical engineering labs.
