HPLC | High performance liquid chromatography

Learn that HPLC (High Performance Liquid Chromatography) is a modified column chromatography that uses a high-pressure pump generating up to 40 megapascals to push mobile phase through a column. Understand that small particle size stationary phases provide large surface area for better separation efficiency and resolution compared to gravity-driven column chromatography.

Review the key components of an HPLC system: stainless steel column (5-25 cm length, 4.5 mm diameter) filled with small-particle absorbent material, solvent reservoir containing the mobile phase, high-pressure pump, sample injector, and detector (UV, IR, fluorescence, mass spectrometer, or electrochemical). The detector connects to a computer to collect separation data.

Inject standard samples (e.g., glucose and sucrose) separately through the HPLC system to establish retention times at which each compound elutes from the column. Record the retention time (in minutes) for each standard to create a reference for identification.

Inject an unknown sample and compare the retention times of peaks in the resulting chromatogram to the standard retention times. Match peaks at known retention times to identify sample components; peaks without matching standards remain unidentified.

Prepare and run a series of known concentrations of a standard compound (e.g., glucose) through HPLC and measure the area under each peak. Plot concentration versus peak area to generate a standard curve for quantifying unknowns.

Select a separation technique based on sample properties: normal phase (polar stationary, non-polar mobile) for separating by polarity, reverse phase (non-polar stationary, polar mobile) for separating organic compounds, size exclusion (porous particles) for separating by molecular size, or ion exchange (charged stationary phase) for separating by molecular charge.