HPLC Elution Order Principle

Reverse-phase High-Performance Liquid Chromatography (HPLC) separates compounds based on their hydrophobicity (water-hating nature). The stationary phase is non-polar, and the mobile phase is polar (here, acetonitrile-water). Compounds that are more hydrophobic interact more strongly with the stationary phase and are retained longer, thus eluting later. Conversely, more polar (hydrophilic) compounds interact more with the mobile phase and elute earlier.

Compound Hydrophobicity Analysis

• A. Morphine: Contains polar hydroxyl (-OH) and amine groups. It is relatively hydrophilic.
• C. Codine: Similar to morphine but has a methoxy (-OCH3) group instead of one -OH group. This makes it slightly less polar (more hydrophobic) than morphine.
• B. Mono acetyl morphine: Acetylation of one hydroxyl group increases hydrophobicity compared to codine.
• D. Heroin (Diacetylmorphine): Acetylation of both hydroxyl groups makes it significantly more hydrophobic than the others.

Determining Elution Order

Based on the increasing hydrophobicity:

1. Morphine (A) - Most hydrophilic
2. Codine (C) - Less hydrophilic than morphine
3. Mono acetyl morphine (B) - More hydrophobic than codine
4. Heroin (D) - Most hydrophobic

Therefore, the order of elution from the reverse-phase HPLC column will be from most polar to most hydrophobic.

Final Elution Sequence

The correct elution order is Morphine (A), followed by Codine (C), then Mono acetyl morphine (B), and finally Heroin (D).

Sequence: A, C, B, D