The separation and quantification of lipids by analytical chromatography is widely used in food and drink analysis, studies of tissues and cellular components, and compositional analysis of model lipid membranes, such as synthetic liposomes. A common approach is to use an evaporative light scattering detector (ELSD) to quantify lipids separated by high-performance liquid chromatography (HPLC). ELSD is preferred over the more common UV-visible detectors because most lipids are poor chromophores. In many HPLC-ELSD methods, volatile buffers, such as ammonium formate, ammonium acetate, and trifluoroacetic acid, are often added to the mobile phase to stabilize pH and facilitate preferred separation. Here, we present data demonstrating that the buffer additives themselves can cause the ELSD response to vary substantially for a variety of common lipids, an unintended and commonly overlooked effect for most users. In some cases, this variation can be rationalized as being due to the buffer additive forming charged pair adducts with the lipids during evaporation (a mass-amplification effect), or otherwise altering the dry particle size and shape. In other cases, the variation can be attributed to alterations in peak symmetry and width, to which ELSD is particularly sensitive as a non-linear detector. Our results underscore the necessity to optimize buffer conditions when quantifying lipids using HPLC-ELSD, and serve as a guide to find favorable conditions. We also discuss the application of a buffer-optimized HPLC-ELSD method to analyze lipid composition variance and yield during the preparation of synthetic liposomes, specifically investigating the role of preparation method and starting lipid composition. We anticipate that these results will be informative to those who use synthetic liposomes in a variety of applications.

Commonly overlooked effects of mobile phase buffers on HPLC-ELSD response for lipid quantification

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