At the core of this process are the basic principles of chromatography: differential partitioning between the mobile and stationary phases causes components in a mixture to move at different speeds. These principles rely on the chemical and physical properties of each analyte — such as polarity, molecular weight, and solubility — to drive separation. The greater the interaction with the stationary phase, the slower a compound will travel; less interaction leads to faster elution. These interactions form the foundation of HPLC’s ability to isolate and quantify compounds with high precision.

The mobile phase is usually a mixture of organic solvents such as methanol, acetonitrile, and water, or a combination of these. The choice of solvent depends on the type of sample being analyzed and the type of separation needed. As the mobile phase passes through the column, the different components will interact differently with the stationary phase, causing them to move at different rates and eventually separate. The components can then be detected and quantified using a detector, such as a UV-Vis detector or a mass spectrometer.

Common HPLC Techniques

Several HPLC techniques are employed to separate compounds based on their chemical properties and the specific goals of the analysis. The two main modes are reverse-phase and normal-phase chromatography, with additional specialized techniques used for targeted separations:

• Reverse-phase HPLC (RP-HPLC): The most widely used technique, featuring a nonpolar stationary phase (commonly C18-bonded silica) and a polar mobile phase such as water mixed with organic solvents. It is ideal for separating small to moderately polar compounds across many industries.
• Normal-phase HPLC: This technique uses a polar stationary phase (like bare silica) combined with a nonpolar mobile phase such as hexane. It is particularly useful for separating polar analytes that are less retained in reverse-phase systems.
• Ion-exchange HPLC: Separates molecules based on charge through stationary phases with fixed ionic groups. It is commonly applied in analyzing charged species like amino acids, peptides, proteins, and nucleotides.
• Size-exclusion HPLC (SEC): Also known as gel filtration chromatography, this technique separates molecules based on size. Larger molecules elute first, making it suitable for proteins, polymers, and other large biomolecules.
• Chiral HPLC: Uses chiral stationary phases to separate enantiomers — molecules that are mirror images but cannot be superimposed — critical for pharmaceutical and biochemical analysis.
• Affinity HPLC: Employs stationary phases designed for specific biological interactions, such as antibody-antigen or enzyme-substrate binding, allowing selective retention and purification of target molecules.

Each technique is selected based on the nature of the analyte and the required separation performance.

HPLC Uses

HPLC is used for a wide range of applications, including the analysis of pharmaceuticals, food and beverages, petrochemicals, and environmental samples. It is also a popular technique for protein and peptide analysis, due to its high resolution and sensitivity.

This type of liquid chromatography offers high resolution, sensitivity, and accuracy and can be used to analyze a wide range of samples. It is also relatively easy to use and can be used to analyze complex mixtures with ease.