Question

REFLECT AND APPLY 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 the properties of amino acids

Phenylalanine, glycine, and glutamic acid have different polarities. Phenylalanine is hydrophobic, glycine is neutral and less hydrophobic, and glutamic acid is acidic and polar.

Analyze the stationary phase

The stationary phase is aqueous, meaning it is polar. Polar substances will interact more with the stationary phase and thus move slower.

Analyze the mobile phase

The mobile phase is less polar than water. Nonpolar substances will move faster in this phase.

Determine relative movement

Since phenylalanine is the least polar, it will move the fastest. Glutamic acid, being the most polar, will interact more with the stationary phase and move the slowest. Glycine will be in between.

Key concepts

stationary phase

In High-Performance Liquid Chromatography (HPLC), the stationary phase is the material that stays in place inside the column. It interacts physically or chemically with the components of the mixture being separated. In the context of amino acid separation, the stationary phase is aqueous, meaning it's polar. Polar substances tend to interact more strongly with this type of stationary phase.

As a result, polar amino acids will spend more time bound to the stationary phase and will move more slowly through the column. Simply put, the stationary phase works like a trap for substances similar in polarity.

mobile phase

The mobile phase in HPLC is the solvent that moves through the column and carries the sample with it. In this exercise, the mobile phase is less polar than water. This means it has a lower affinity for polar substances.

Nonpolar or less polar amino acids will not bind as strongly to the mobile phase and will be carried along faster. The polarity difference between the stationary and mobile phases is what drives the separation of the amino acids.

amino acid polarity

Polarity in amino acids is a key characteristic that affects how they interact with both the stationary and mobile phases in HPLC.

• Phenylalanine: Hydrophobic and nonpolar.
• Glycine: Neutral and less hydrophobic.
• Glutamic Acid: Acidic and highly polar.

Polar amino acids will interact more with the polar stationary phase, causing them to move slower. Nonpolar amino acids will prefer the less polar mobile phase, moving more quickly through the column. This differentiation is the basis for their separation.

phenylalanine

Phenylalanine is one of the amino acids in the mixture. It is hydrophobic, meaning it does not like to interact with water. This hydrophobic nature makes phenylalanine nonpolar.

When phenylalanine goes through the HPLC system with an aqueous stationary phase and a less polar mobile phase, it will interact minimally with the stationary phase. This allows it to move quickly through the column, resulting in it being the fastest-moving amino acid in this mixture.

glycine

Glycine stands out among amino acids for being the smallest and simplest. It is neutral and less hydrophobic. This means glycine is neither strongly attracted nor strongly repelled by the polar stationary phase.

In the HPLC system, glycine will move at an intermediate speed compared to phenylalanine and glutamic acid. Its partial interaction with both the stationary and mobile phases gives it a moderate retention time. Glycine will not be the fastest nor the slowest to move through the column.

glutamic acid

Glutamic acid is highly polar because of its acidic nature. In the context of HPLC, this high polarity means that glutamic acid will strongly interact with the aqueous stationary phase.

Due to this strong interaction, glutamic acid will move very slowly through the column as it retains longer on the polar stationary phase. Among phenylalanine, glycine, and glutamic acid, glutamic acid will be the slowest to move. This high polarity significantly increases its retention time in the column.