Topic overview

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HPLC drives a liquid mobile phase through a packed column under pressure. Analytes partition differently between the mobile and stationary phases, so they leave the column at different retention times. Column chemistry and separation mode are selected with the analytes’ properties in mind; reversed-phase HPLC, using a non-polar C18 phase and an aqueous-organic eluent, is a widely used approach for pharmaceutical analysis. Reliable results also depend on preparing and degassing the mobile phase, selecting a suitable detector, and calibrating quantitation with an external or internal standard. Chromatographic performance can be assessed using retention time, resolution, peak tailing, plate number, capacity factor, and selectivity. The full lesson develops these fundamentals alongside sample handling, temperature control, validation, and quality considerations.