A biochemist separates a mixture of nucleotides by reverse-phase HPLC using a C18 column (nonpolar stationary phase). The mobile phase starts as 95% water/5% acetonitrile (v/v) and is ramped to 60% water/40% acetonitrile over 10 minutes. Two analytes are injected: compound M (more hydrophobic) and compound N (more hydrophilic). Detection is by UV absorbance.
Which outcome is most consistent with this method?
Assume both compounds are stable and have similar UV response factors.
- Compound N elutes after compound M because hydrophilic compounds bind more strongly to C18
- Compound M elutes after compound N, and increasing acetonitrile promotes elution of M (correct answer)
- Both compounds elute earlier as the mobile phase becomes more aqueous
- Elution order depends only on molecular weight, so M and N cannot be ranked by hydrophobicity
Explanation: This question tests understanding of reverse-phase HPLC separation principles. In reverse-phase chromatography with a C18 column (nonpolar stationary phase), compounds are separated based on their hydrophobicity, with more hydrophobic compounds having stronger interactions with the nonpolar stationary phase. The mobile phase starts highly aqueous (95% water) and becomes more organic (40% acetonitrile), which increases its elution strength for hydrophobic compounds. Compound N, being more hydrophilic, has weaker interactions with the C18 stationary phase and will elute first when the mobile phase is still highly aqueous. Compound M, being more hydrophobic, binds more strongly to the C18 phase and requires a higher proportion of organic solvent (acetonitrile) to disrupt these hydrophobic interactions and promote elution. A common misconception is that hydrophilic compounds bind more strongly to C18 (choice A), which reverses the fundamental principle of reverse-phase chromatography. To predict reverse-phase elution order, remember: hydrophilic compounds elute first, hydrophobic compounds elute later, and increasing organic solvent promotes elution of hydrophobic compounds.