A student develops a paper chromatogram of a mixture containing the four amino acids glycine, alanine, serine, and lysine. The stationary phase is cellulose fiber (relatively polar), and the mobile phase is an organic solvent composed of 1-butanol, acetic acid, and water in a 4:1:5 ratio by volume. After the solvent front has traveled 8.0 cm, the four amino acid spots are observed at the following distances (measured from the origin):
• Spot W – 1.7 cm
• Spot X – 3.2 cm
• Spot Y – 4.1 cm
• Spot Z – 6.3 cm
Assume all amino acids remain in their neutral (zwitterionic) form at the pH of the solvent mixture.
(a) Calculate the Rf value for each spot. Rank the four amino acids from lowest to highest Rf. Justify your identification of which spot corresponds to each amino acid, citing specific structural features that influence polarity.
(b) The student repeats the experiment using a mobile phase with a larger percentage of 1-butanol and a smaller percentage of water (all other conditions unchanged). Predict qualitatively how the Rf values of each amino acid will change. Explain your reasoning in terms of intermolecular forces between each amino acid and the two phases.
(c) Describe ONE change to the stationary phase that would cause lysine to migrate farther than alanine under otherwise identical mobile-phase conditions. Explain how the proposed change impacts the relative strength of interactions for both amino acids.