Use of Sep-Pak cartridges for on-line preparative high-performance liquid chromatography.
Sep-Pak C18 cartridges have found wide application to the extraction, concentration, and fractionation of peptides. Their use has hitherto been limited to stepwise loading, washing, and eluting procedures. By the use of commercially available plastic column end-fittings it was possible to connect steel tubing to both ends of the cartridges and allow low-pressure operation in a high-performance liquid chromatography (HPLC) instrument. The cartridge was then able to function as a disposable, reversed-phase (RP) preparative column. Operation in a gradient mode gave several advantages over step-wise elution, including the direct UV absorption monitoring of the eluant. Gradient conditions could be optimised for sample recovery. The cartridge was compared with standard analytical RP-HPLC columns for the separation of both proteins and peptides. A test protein gave a single peak with good recovery. Experiments with peptide mixtures showed that the cartridge could resolve some components sufficiently to allow fractions of pure peptides to be collected.
Southan
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Department of Haematology, Charing Cross and Westminster Hospital Medical School, Hammersmith, London, U.K.
D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D005340:Fibrinogen / Q000032:analysis; D006801:Humans; D010316:Particle Size; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
D002021:Buffers; D012997:Solvents; D005340:Fibrinogen