METHODS AND TECHNOLOGIES

Chromatography is a technique used to separate a mixture into its individual components by exploiting their differing interactions with a mobile phase and a stationary phase. In High-Performance Liquid Chromatography (HPLC), the analyte mixture is dissolved in a solvent and separated within a column packed with solid particles. The separation process requires elevated pressure (100–300 bar) to move the sample through the column efficiently.

Device design

The HPLC system consists of the following components:

1. Mobile phase: Storage containers hold various liquids (organic solvents, aqueous solutions, or buffers) that serve as the mobile phase.
2. High-pressure pump: Moves the mobile phase through the column at a constant flow rate.
3. Mixer: Solvents are combined in a mixer to achieve a desired composition, which can be adjusted during the separation process (gradient separation).
4. Autosampler: Introduces a precise volume of the prepared sample into the system.
5. Column: This is where the separation of analytes occurs based on their interactions.
6. Detector: Converts the analytes chemical signals into electrical signals. Optical detectors such as UV/VIS or fluorescence are commonly used, depending on whether the analytes absorb UV light or emit fluorescence.
7. Evaluation unit: Software generates a chromatogram for analysis.
8. Waste container: Collects the processed samples after detection.

Separation principle

The sample is injected at one end of the chromatography column, which is packed with small, solid particles. The column is flushed with the mobile (moving phase), causing the mixture to move continuously through the column. The chemical properties of the analytes, primarily polarity, affects interactions between the analyte and the mobile or stationary phase.

●Substances with the strongest interaction with the mobile phase move faster through the column in the running medium, thus reaching the detector sooner.

●Substances with greater interaction with the stationary phase move more slowly, eluting later.

By altering the mobile phase composition, the separation of complex mixtures can be optimized.

Chromatogram and data analysis

The separation process is visualized in a chromatogram, which plots retention time (time analytes spend on the column) against signal intensity measured by the detector. The detected measurement signal changes in proportion to the analyte concentration. The resulting peaks have Gaussian distribution. To evaluate the data, the peak areas are integrated and compared with calibration data from known concentrations for quantification.

Advanced HPLC technology

Ultra-performance liquid chromatography (UPLC) improves upon HPLC by utilizing smaller column particles and higher total pressures (up to 1000 bar) to improve separation performance with shorter analysis times.