Small particle liquid chromatography separation

G Jones, HF DeLuca. High-pressure liquid chromatography separation of the metabolites of vitamins D2 and D3 on small-particle silica columns. J Lipid Res 16 448-453, 1975. 

High-pressure liquid chromatography (HPLC) is a variant of the simple column technique, based on the discovery that chromatographic separations are vastly improved if the stationary phase is made up of very small, uniformly sized spherical particles. Small particle size ensures a large surface area for better adsorption, and a uniform spherical shape allows a tight, uniform packing of particles. In practice, coated Si02 microspheres of 3.5 to 5 fxm diameter are often used. 

Many nonvolatile and thermally labile allelochemicals can be well separated by liquid chromatography (LC). Identification of the separated components on-line by mass spectrometry (MS) is of great value. Fused-silica LC columns of 0.22 mm ID packed with small-particle material are used in the described LC/MS system. The shape of the column end allows direct connection to a electron impact ion source of a magnetic sector mass spectrometer. Separations by LC are reported and LC/MS mass spectra are shown for monoterpenes, diterpene acids, phenolic acids and cardiac glycosides. The LC/MS system provides identification capability and high-efficiency chromatography with a universal detector. 

For enhanced throughput, fast RPLC methods using monolithic silica columns [238], small size particles (3 pm) packed columns [173, 195, 226, 235-237, 239, 240], ultra high pressure liquid chromatography (UHPLC) columns packed with sub-2 pm particles [145,196, 227, 245, 247] and 2.6 pm core-shell particles HPLC columns [246] have been proposed for the high-throughput separation and quantification of tamoxifen/metabolites. 

There is much interest in high-efficiency- and high-speed separation media for liquid chromatography. The plate numbers available in practice have been in the range of 10,000-30,000 in HPLC for 20 years or so, but these are low compared to well over 100,000 theoretical plates in capillary gas chromatography or in capillary electrophoresis. This is caused by the limitation in the use of small-sized particles for HPLC, where a particle-packed column is commonly used under a pressure-drop of up to 40 MPa. An increase in column efficiency by using small particles, which is the approach taken in the past, is accompanied by an increase in the pressure-drop, as expected from Eqns. 5.2 and 5.3, below. Eqns. 5.1-3 describe the efficiency (plate height) and flow resistance of a column packed with particles [1-3], where N stands for the... 

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