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This lecture covers the factors influencing the separation in HPLC, such as the properties of the eluent and optimization criteria. It discusses the influence of stationary phases, solutes, and physical aspects of the column on resolution and peak capacity. The lecture also explores the impact of temperature on column performance, particle size distribution, and the use of different solvents in the eluent. It delves into the chromatographic peaks obtained under various conditions and the importance of pressure, temperature, and chemical properties of the eluent in achieving optimal separation.