Test Your Knowledge on High-Performance Liquid Chromatography (HPLC)

Key Concepts in High-Performance Liquid Chromatography (HPLC)

• Diffusion in liquids is slower than in gases, making open tubular columns impractical in liquid chromatography.
• Packed column efficiency increases as stationary phase particle size decreases, with typical sizes of 1.7-5 mm in HPLC.
• Smaller particle sizes lead to increased plate numbers, higher resolution, shorter run times, and lower detection limits.
• Small particles provide more uniform flow, reduce multiple path term, and decrease distance for solute diffusion, improving resolution.
• Small particle size leads to higher pressure and increased frictional heating, which can be managed with narrow columns and small analyte amounts.
• Columns are expensive and require protection from particulate matter, such as centrifugation or filtration.
• Main columns are typically steel or plastic, 5-30 cm long, with inner diameters of 1-5 mm and titanium frits for uniform flow.
• Bonded stationary phases on silica surfaces provide alternate selectivities and improved peak shapes for bases, with limitations on pH range.
• Superficially porous particles (SPP) consist of a 0.25-mm-thick porous silica layer on a nonporous silica core, providing faster mass transfer for macromolecules.
• Elution strength is related to the relative abilities of solvents to displace solutes from the stationary phase, with normal-phase and reversed-phase chromatography using polar and nonpolar stationary phases, respectively.
• Gradient elution can be used when one solvent does not provide sufficiently rapid elution of all components.
• Isocratic HPLC separation becomes better as mobile phase B decreases, while gradient elution starts with low B% and gradually increases for good separation and shorter time.
• Hydrophilic interaction chromatography (HILIC) uses polar stationary phases and more polar solvents for separation of polar compounds.