Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection

A new method for the separation and quantification of two hydrophobic lung surfactant proteins (SPs) is described. It is based on size-exclusion chromatography using the apolar stationary phase butyl silicagel with a pore size of 30 nm and isocratic elution with chloroform, methanol and trifluoroacetic acid. The samples were prepared from sheep lung lavage fluid by centrifugation and fractional extraction with butanol and chloroform–methanol. The chromatograms show three peaks in the elution order SP-B, SP-C and lipids. A small peak ahead of SP-B, which disappeared after reduction with 2-mercaptoethanol, was oligomeric SP-B. The response of the evaporative light-scattering detector was non-linear. For preparative high-performance liquid chromatography ultraviolet detection at 279 nm is recommended.