Question:
Which one of the following amino acids will move the fastest, and thus be eluted first, on a reverse-phase HPLC column?
Which one of the following amino acids will move the fastest, and thus be eluted first, on a reverse-phase HPLC column?
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RP-HPLC Motto: Polar Elutes First.
Normal Phase Motto: Non-Polar Elutes First.
Think of the stationary phase as "Glue." In Reverse Phase, the glue is "Oily" (Non-polar). Water-loving (Polar) molecules don't stick to oil!
Normal Phase Motto: Non-Polar Elutes First.
Think of the stationary phase as "Glue." In Reverse Phase, the glue is "Oily" (Non-polar). Water-loving (Polar) molecules don't stick to oil!
Updated On: Aug 21, 2026
- Phenylalanine
- Isoleucine
- Glycine
- Glutamate
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The Correct Option is D
Solution and Explanation
Step 1: Understanding the Question:
The question asks to identify which amino acid among the four choices will have the shortest retention time (elute first) in Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC).
The elution order in chromatography is determined by the relative affinity of the solute for the stationary phase versus the mobile phase.
Step 2: Detailed Explanation:
• Principle of Reverse-Phase HPLC: In RP-HPLC, the stationary phase is non-polar (hydrophobic), usually consisting of silica beads coated with long hydrocarbon chains (like C8 or C18). The mobile phase is polar, typically a mixture of water and organic solvents like acetonitrile.
• Interaction Mechanism: In this system, "like dissolves like." Non-polar (hydrophobic) molecules interact strongly with the non-polar stationary phase and are "retained" longer. Polar (hydrophilic) molecules prefer the polar mobile phase and move through the column more quickly.
• Analyzing Amino Acid Polarity:
- Phenylalanine (A): Contains a bulky benzyl side chain; it is highly hydrophobic.
- Isoleucine (B): Contains a branched aliphatic hydrocarbon chain; it is highly hydrophobic.
- Glycine (C): The simplest amino acid with just a hydrogen atom as its R-group; it is moderately polar/hydrophilic.
- Glutamate (D): An acidic amino acid with a carboxylic acid side chain ($-CH_2-CH_2-COO^-$). At physiological pH, it is negatively charged and highly polar.
• Determining Elution Order: Among the options, Glutamate is the most polar and ionic. Because of its high solubility in the polar mobile phase and minimal interaction with the C18 chains, it will travel fastest.
• Summary of Hydrophobicity: Phenylalanine $>$ Isoleucine $>$ Glycine $>$ Glutamate. The elution order will be the reverse of this.
Step 3: Final Answer:
Glutamate, being the most hydrophilic and charged amino acid in the list, will elute first from a reverse-phase HPLC column.
The question asks to identify which amino acid among the four choices will have the shortest retention time (elute first) in Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC).
The elution order in chromatography is determined by the relative affinity of the solute for the stationary phase versus the mobile phase.
Step 2: Detailed Explanation:
• Principle of Reverse-Phase HPLC: In RP-HPLC, the stationary phase is non-polar (hydrophobic), usually consisting of silica beads coated with long hydrocarbon chains (like C8 or C18). The mobile phase is polar, typically a mixture of water and organic solvents like acetonitrile.
• Interaction Mechanism: In this system, "like dissolves like." Non-polar (hydrophobic) molecules interact strongly with the non-polar stationary phase and are "retained" longer. Polar (hydrophilic) molecules prefer the polar mobile phase and move through the column more quickly.
• Analyzing Amino Acid Polarity:
- Phenylalanine (A): Contains a bulky benzyl side chain; it is highly hydrophobic.
- Isoleucine (B): Contains a branched aliphatic hydrocarbon chain; it is highly hydrophobic.
- Glycine (C): The simplest amino acid with just a hydrogen atom as its R-group; it is moderately polar/hydrophilic.
- Glutamate (D): An acidic amino acid with a carboxylic acid side chain ($-CH_2-CH_2-COO^-$). At physiological pH, it is negatively charged and highly polar.
• Determining Elution Order: Among the options, Glutamate is the most polar and ionic. Because of its high solubility in the polar mobile phase and minimal interaction with the C18 chains, it will travel fastest.
• Summary of Hydrophobicity: Phenylalanine $>$ Isoleucine $>$ Glycine $>$ Glutamate. The elution order will be the reverse of this.
Step 3: Final Answer:
Glutamate, being the most hydrophilic and charged amino acid in the list, will elute first from a reverse-phase HPLC column.
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