Open Question
What is the basis for the separation of proteins by the following techniques?
5. Protein Techniques
HPLC
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What is the order of elution (first → last) of the following amino acids in reverse-phase HPLC: Ala, Arg, Leu.
A
Leu → Ala → Arg.
B
Leu → Arg → Ala.
C
Arg → Leu → Ala.
D
Arg → Ala → Leu.
E
Ala → Leu → Arg.
F
Ala → Arg → Leu.
Verified step by step guidance1
Understand the principle of reverse-phase HPLC: In reverse-phase high-performance liquid chromatography (HPLC), the stationary phase is non-polar (hydrophobic), and the mobile phase is polar. Molecules are separated based on their hydrophobicity; more hydrophobic molecules elute later.
Identify the properties of the amino acids: Alanine (Ala) is small and relatively non-polar, Leucine (Leu) is larger and more hydrophobic, and Arginine (Arg) is polar due to its charged side chain.
Predict the elution order based on hydrophobicity: In reverse-phase HPLC, the least hydrophobic (most polar) amino acid will elute first, and the most hydrophobic will elute last.
Apply the hydrophobicity concept: Arginine, being polar, will elute first. Alanine, being less hydrophobic than Leucine, will elute next. Leucine, being the most hydrophobic, will elute last.
Conclude the order of elution: The order of elution from first to last is Arg → Ala → Leu.
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