What is the basic principle of HPLC?
The separation of components in a mixture based on their different distributive behaviors between a liquid mobile phase and a solid or liquid stationary phase.
What are the main components of an HPLC system?
The system consists of: Solvent reservoir, Pump, Injector, Column (stationary phase), Detector, and Data acquisition system (Integrator).
Why is it called “High-Pressure” Liquid Chromatography?
Because high pressure (typically 50 to 400 bar) is required to force the mobile phase through a column packed with very small particles (3–5 µm) to achieve high resolution.
Define Retention Time (tR).
The time interval between the point of injection and the appearance of the peak maximum at the detector.
What is a Chromatogram?
A graphical representation of the detector response (y-axis) versus time (x-axis).
What is the “Heart” of the HPLC?
The Column, because that is where the actual separation takes place.
What is a Void Volume (V0)?
The total volume of mobile phase in the column; the volume required to elute a non-retained compound.
What is the USP General Chapter for Chromatography?
USP General Chapter <621>.
What is the role of a Frit?
A porous filter at the column inlet/outlet that prevents stationary phase particles from escaping and blocks large contaminants from entering the column.
Explain the difference between Isocratic and Gradient elution.
Isocratic: Uses a constant mobile phase composition throughout the run.
Gradient: Changes the composition over time to elute compounds with widely different polarities.