Abstract
A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.
Highlights
A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described
As a first step towards the goal of molecular species separation of phospholipids, we have attempted the separation of sphingomyelin species by reversedphase high-performance liquid chromatographic (HPLC) technique
We described the HPLC analysis of phosphatidylcholine and sphingomyelin with detection in the region of 200 nm without derivatization [9].Here we describe the HPLC separation of individual molecular species of sphingomyelin from various sources on a reversed-phase HPLC column with detection in the region of 200 nm
Summary
Sphingomyelin from beef and pig brain as well as pig and sheep erythrocytes was purchased from either Supelco, Bellefonte, PA or Research Products International Corp., Elk Grove Village, IL. Each sample gave a single spot on thin-layer chromatography and charring the plate. All solvents used for HPLC, obtained from either Fisher Chemical Co. (Fairlawn, NJ) or Burdick and Jackson, Inc. (Muskegan, MI) were of HPLC grade quality and were degassed by boiling briefly before use. The HPLC analysis was performed with a Waters Associates (Milford, MA) Model 6000 solvent delivery system and a Model U-6K injector. The chromatographic column was a (30 cm X 4 mm) stainless tube prepacked with either p-Bondapak-C18material (Waters Assoc.) or slurry packed with Nucleosil-5CIS 5-pm particles (Macherey-Nagel, Diiren, Germany) with the aid of a Micromeritics Instrument Corp. The detection was with a Laboratory Data Control (Riviera Beach, FL) variable wavelength spectromonitor
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