Polarity gradient issues in differential centrifugation of complex lipid mixtures

Since phospholipids are one of the most important classes of natural regulators of physiological processes, the search for methods allowing their analytical and preparative separation is of considerable interest. The aim of this study is to reveal principles underlying the differential centrifugation of complex lipid mixtures on the example of phospholipids. The current study uses calculation methods for estimating the Hansen solubility parameters for phospholipids, as well as instrumental research methods comprising tensometry (density measurement), dynamic laser light scattering (particle size evaluation) and IR spectroscopy with Fourier transformation. These methods were applied to samples of phospholipids isolated from calf brain tissue. The affinity of the studied phospholipids to individual solvent layers was characterised. As a result of the work performed, a mathematical model of the process of differential centrifugation of phospholipid complex mixtures in the gradient of solvent polarity is proposed on the basis of the Nernst-Brunner equations and main centrifugation equation. The established optimal parameters of the centrifugation process comprise 1500 rpm for 15 minutes at a temperature of 2–4 °С. IR spectral analysis revealed significant differences between the substances of each solvent layer, which are due to the separation of the phospholipid classes between the layers of solvents. The necessity for further research into new combinations of solvents contributing to a more specific separation of the mixture and aimed at increasing the separation efficiency of phospholipid complex mixtures, is justified. The volume modification of solvent layers and determination of its ratio with the volume of the introduced suspension also appear to be relevant further research directions. Given the simplicity and low cost of the hardware, the developed method can be easily scaled to industrial conditions for the production of biologically active substances for inclusion in food products.

phospholipids, differential centrifugation, polarity gradient, Hansen parameters

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