Asymmetric flow field-flow fractionation of liposomes: 2. Concentration detection and adsorptive loss phenomena

The applicability of different concentration detection methods for online quantification of liposomes upon asymmetric flow field-flow fractionation was investigated. Filter-extruded egg phosphatidylcholine liposomes of different size were used. Online quantification using a differential refractive index (dRI) detector was found feasible for relatively high sample loads in the magnitude of 100 microg lipid (under the chosen fractionation conditions). UV-Vis detection of the turbidity of liposomes was ruled out as online detection method because turbidity increases with particle size and the signal is not only concentration but also particle-size dependent. Staining of liposomes by Rhodamine phosphatidylethanolamine or Sudan Red and subsequent online UV-Vis detection at the absorption maximum of the dye enabled quantification with much higher sensitivity than dRI detection. Furthermore analyte loss and carry-over phenomena upon repeated injection of varying liposome sample loads were studied using regenerated cellulose (RC) membranes as accumulation wall. It could be shown that RC membranes are prone to adsorption in case of very small sample loads (0.5 microg). This effect may be overcome by pre-saturation of the membrane with sample loads of at least 2 microg. For higher sample loads adsorptive losses play a minor role. Recovery from pre-saturated membranes reached approximately 100% and carry-over was found negligible.