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

Study Notes

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.