Abstract
We present a detailed description of a rapid preparative high performance liquid chromatography (HPLC) procedure for isolation of large scale quantities (a minimum of 200 mg) of rat liver microsomal and mitochondrial phospholipid classes in 40 min on a 25 X 2.5 cm preparative HPLC column of 7-mu silica gel particles using a linear gradient of hexane-isopropanol-water mixtures. A minimum of 1.5 g of phospholipids can be quantitatively separated per day into diphosphatidylglycerol (cardiolipin), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, and a phosphatidylcholine-sphingomyelin fraction. The procedure uses no salts, buffers, acids, or bases, and the isolated phospholipids are suitable for preparing model membranes.
Highlights
Up to 200 mg of phospholipids was separated on a 250 x 25 mm Merck Hibar RT preparative column packed with 7-p LiChrosorb-Si-60silica gel
The initial solvent mixture of hexane-2-propanol-water 6:8:0.75 was run at ambient temperature for 7 min at a flow rate of 18ml/min, and a linear gradient was run for 10 min to achieve the final solvent mixture of hexane-2-propanol-water 6:8:1.55, which was run isocratically for 25 min
When a stored column was to be reused, it was subjected to a start-up program prior to any separation by flushing the followingsolvent mixtures through the column at 5 mumin: solvent A for 10 min, a 5-min linear gradient to achieve a solvent composition of solvent B, solvent B for 30 min, a 5-min linear gradient to return to the initial solvent A mixture, and solvent A for 1 hr
Summary
Up to 200 mg of phospholipids was separated on a 250 x 25 mm Merck Hibar RT preparative column packed with 7-p LiChrosorb-Si-60silica gel The initial solvent mixture of hexane-2-propanol-water 6:8:0.75 (by vol) (solvent A) was run at ambient temperature for 7 min at a flow rate of 18ml/min, and a linear gradient was run for 10 min to achieve the final solvent mixture of hexane-2-propanol-water 6:8:1.55 (by vol) (solvent B), which was run isocratically for 25 min. Re-equilibration of the column was achieved by running solvent A for 20 min
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