Chromatography Elution Order Determination

This question requires understanding ion-exchange chromatography principles, specifically how the charge of molecules at a given pH affects their interaction with a charged stationary phase like carboxymethyl-cellulose (CM-cellulose).

Amino Acid Charges at pH 6.0

CM-cellulose is a cation-exchange resin, meaning it carries a negative charge and binds positively charged molecules. The elution order depends on the net charge of the amino acids at pH 6.0:

• Glutamic Acid: An acidic amino acid. At pH 6.0, which is significantly above its side chain pKa ($\approx 4.25$), it carries a net negative charge ($\approx -1$).
• Valine: A neutral (aliphatic) amino acid. Its isoelectric point ($\text{pI}$) is around 5.96. At pH 6.0, it is very close to its neutral state, with minimal net charge.
• Arginine: A basic amino acid. At pH 6.0, which is below its side chain pKa ($\approx 12.48$), its side chain is protonated, giving it a net positive charge ($\approx +1$).

Elution Principle with CM-Cellulose

The negatively charged CM-cellulose column will interact differently with each amino acid:

• Repulsion: Negatively charged molecules (Glutamic Acid) are repelled by the negative stationary phase and elute first.
• Weak Interaction: Neutral molecules (Valine) have minimal interaction and elute next.
• Strong Binding: Positively charged molecules (Arginine) bind strongly to the negative stationary phase and elute last.

Determining Elution Order

1. Glutamic Acid: Elutes first (negative charge).
2. Valine: Elutes second (neutral charge).
3. Arginine: Elutes last (positive charge).

Therefore, the elution order is Glutamic acid, Valine, Arginine.