Question

An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC. The stationary phase is aqueous and the mobile phase is a solvent less polar than water. Which of these amino acids will move the fastest? Which one will move the slowest?

Short answer

Phenylalanine will move the fastest; glutamic acid will move the slowest.

Step-by-step solution

- Understand HPLC Components

HPLC stands for High-Performance Liquid Chromatography. In this setup, the stationary phase is aqueous (water-based), and the mobile phase is a less polar solvent.

- Analyze Amino Acid Polarity

Phenylalanine is nonpolar, glycine is very slightly polar, and glutamic acid is highly polar due to its acidic side chain.

- Determine Interactions with Stationary Phase

Highly polar amino acids (glutamic acid) will interact more with the aqueous stationary phase, making them move slower. Less polar amino acids (phenylalanine) will interact less and move faster. Glycine, being slightly polar, will have intermediate interactions.

- Rank Movement Rates

Based on polarity interactions with the stationary phase, phenylalanine will move the fastest, glycine will move at an intermediate speed, and glutamic acid will move the slowest.

Key concepts

amino acid polarity

Amino acids are the building blocks of proteins, and each of them has a unique side chain that influences its polarity. Polarity refers to the distribution of electric charge around the molecule. Amino acids can be nonpolar, polar, or charged.
Nonpolar amino acids, like phenylalanine, do not have a strong preference for water because they lack polar side chains. Polarity influences how amino acids interact with other molecules, crucial for processes like High-Performance Liquid Chromatography (HPLC).
Polar amino acids, such as glycine, which is slightly polar, and glutamic acid, which is highly polar due to its acidic side chain, are more likely to interact with water and other polar substances. Knowing the polarity of amino acids helps predict their behavior during HPLC separation.

stationary phase

The stationary phase in High-Performance Liquid Chromatography (HPLC) plays a critical role in separating components based on their polarity. The stationary phase is a medium that stays in place inside the chromatograph column. In our example, it is aqueous (water-based), meaning it is highly polar.
Because the stationary phase is polar, it forms strong interactions with polar molecules. The more polar the amino acid, the more it will bind or interact with the stationary phase, moving more slowly through the column.
This concept helps explain why glutamic acid, being highly polar, moves the slowest, while phenylalanine, which is nonpolar, moves the fastest. Glycine, which is slightly polar, shows intermediate movement, indicating moderate interaction with the stationary phase.

mobile phase

The mobile phase in High-Performance Liquid Chromatography (HPLC) refers to the solvent that moves through the stationary phase, carrying the sample with it. In our example, the mobile phase is less polar than water.
The polarity of the mobile phase affects how well it can carry different substances through the column. Nonpolar solvents are better at carrying nonpolar amino acids because like dissolves like.
In this case, phenylalanine, being nonpolar, is less attracted to the aqueous stationary phase and more likely to move quickly through it. Conversely, glutamic acid, which is highly polar, will interact more with the water-based stationary phase and move slower. Glycine, slightly polar, moves at an intermediate speed.