#Calculating minimum solvent required from a ternary diagram free#
Further, the WR layer requires less cost in free energy to insert proteins, while the OR layer is an efficient host for all types of lipids present, independent of their intrinsic hydrophilicity. Both layers are nanostructured close to a critical point. These two layers are in equilibrium via exchange through the meniscus, i.e., water, chloroform as well as ethanol chemical potentials are the same in the two phases. By adding a sufficient amount of water, a biphasic system is then spontaneously formed leading to the partitioning of proteins, carbohydrates and phospholipids into the WR-rich upper-layer (essentially water–methanol) and non-polar components such as most lipids into a hydrophobic, chloroform-rich lower layer. In this region of the phase diagram, no structuring appears and predictions of models based on random phase approximation such as COSMO-RS are expected to be good. The sample of biological origin is mixed with water, methanol and chloroform in solvent ratios to form a monophasic ternary solvent mixture. This ensures that all biomolecules, independent of their volume and “hydrophilicity”, are solubilized either in the OR or in WR coexisting phases. The crucial point in the second step is the partial miscibility of the chloroform in the WR phase and the water in the OR phase. The standard of these solvent mixtures is chloroform–methanol, described for the first time more than 50 years ago by Folch and Bligh and Dyer (B & D) and cited more than 50,000 times in the “experimental” section of papers describing the analytics of lipids and proteins extracted and separated with high efficiency form micro-organisms. In the second step, the initially extracted mixture of all biomolecules is separated into two phases: an organic-rich phase (OR) containing the total lipids and a water-rich phase (WR) containing others compounds (sugars, proteins, etc.). The first step is a solid–liquid extraction from an initial amorphous gel state, followed by a second liquid–liquid partitioning step. The safest and nowadays most popular way to ensure that all of the cellular lipids are extracted is to employ a ternary solvent composition including a polar as well as a non-polar solvent. Estimation of the total lipids content in a microorganism sample is crucial for various kinds of applications such as biodiesel, nutritional supplements, cosmetic, etc. They can be divided according to the polarity of their head groups: neutral lipids (acylglycerols, free fatty acids, sterols, sterols esters, waxes and hydrophobic pigments) which are synthetized by the cells to store energy, and polar lipids (phospholipids, glycolipids, polysaccharides and proteins) which are the matrix of the cellular membrane. Lipids from microorganisms’ matrices such as microalgae or yeasts are hydrophobic molecules which are soluble in many organic solvents. Moreover, using these bio-sourced solvents as an alternative system is shown to be as effective as the classical system in terms of the yield of lipids extracted from microorganism tissues, independently of their apparent hydrophilicity. Based on high performance thin-layer chromatography (HPTLC) to obtain the distribution of lipids classes, and gas chromatography coupled with a flame ionisation detector (GC/FID) to obtain fatty acid profiles, this greener solvents pair is found to be almost as effective as the classic methanol–chloroform couple in terms of efficiency and selectivity of lipids and non-lipid material. For this purpose, we consider similar points in the ternary phase diagrams of water/methanol/chloroform and water/ethanol/ethyl acetate, both in the monophasic mixtures and in the liquid–liquid miscibility gap. We confirm this by performing solid–liquid extraction of yeast ( Yarrowia lipolytica IFP29) and subsequent liquid–liquid partition-the two steps of routine extraction. Based on the Conductor-like Screening MOdel for realistic Solvatation (COSMO-RS), we select ethanol and ethyl acetate as being potentially suitable for the substitution of methanol and chloroform. Bligh and Dyer (B & D) or Folch procedures for the extraction and separation of lipids from microorganisms and biological tissues using chloroform/methanol/water have been used tens of thousands of times and are “gold standards” for the analysis of extracted lipids.