A pharmaceutical quality-control laboratory analyzes a tablet extract containing three active ingredients—A, B, and C—whose structures are shown below.
• A: tertiary amine with two ethyl substituents and one phenyl ring
• B: primary alcohol attached to a para-nitrophenyl ring
• C: zwitterionic amino acid derivative containing one carboxylate and one protonated amine at pH 7
The extract is subjected to high-performance thin-layer chromatography (HPTLC) on silica gel plates developed with a mobile phase composed of 60\% methanol, 30\% water, and 10\% ammonium acetate buffer (0.1 M). Densitometry traces (intensity vs. migration distance) show three peaks in the following order and at the indicated Rf values:
• Peak 1 at Rf = 0.65
• Peak 2 at Rf = 0.48
• Peak 3 at Rf = 0.12
Interestingly, Peak 3 is broader and exhibits significant fronting (leading edge distortion).
(a) Assign each peak to compounds A, B, and C. Provide a comprehensive justification referencing acid–base properties, hydrogen-bonding capacity, and overall polarity.
(b) Explain the molecular origin of the fronting observed for Peak 3. Include in your discussion the role of the buffer and any ionic interactions with the stationary phase.
(c) Design an alternative chromatographic system (stationary phase and mobile phase composition) that is likely to sharpen Peak 3 and improve resolution between all three compounds. Justify every element of your design based on chemical principles.
(d) Predict qualitatively how the Rf of compound B would change if the pH of the mobile phase were increased from 7 to 9, keeping all other conditions constant. Provide a mechanistic explanation.