Phase Diagram Lipid Class Liquid Extraction Curve Envelope Diluent Solution
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Liquid Extraction: Phase Diagram
Liquid extraction of lipids from biological samples has greater efficiencies through their better solubility of hydrocarbon chains in organic solvents. One of the most common extraction methods for lipids in biological systems is the use of chloroform and methanol in a ratio of 2:1 (v/v) previously reported by Bligh and Dyer (1959), Kofeler et al. (2012), and Folch et al. (1957). According to the Bligh-Dyer method, the solvent ratio of chloroform to methanol to water is 8:4:3 by volume, which provides the most efficient biphasic partitioning of lipids into the organic phase. Due to the solubility and polarity of the different lipid classes, modification of the Bligh and Dyer method using the chloroform-methanol extraction procedure improves the extraction efficiency. In addition to chloroform-methanol as the extraction solvents, isopropanol-hexane mixtures have also been reported for the extraction of prostaglandins (Folch et al. 1957). Although inexpensive, this extraction protocol provides an unfavorable toxic working environment for sample preparation and has poor extraction efficiency for gangliosides. In general, lipid extraction must be incorporated with organic solvents in lipidomics, but a single standard protocol for extracting all lipid classes has not been established.
Liquid-liquid extraction is a transferring process of a solute from one immiscible liquid phase to another liquid phase at equilibrium (Sunders and Horrocks 1984). When the two immiscible liquid phases are different in chemical properties, the distribution of the solute between two phases leads to a separation of the components according to their distribution or partition between the two phases. Generally, the liquid extraction procedure is performed with an aqueous solution and an organic solvent and is based on the transfer of a solute from one solvent to the other according to its solubility. If the solute of interest requires being separate from other solutes, and all solutes are partially soluble in solvent y, the solute of interest can be extracted by employing a second solvent phase, solvent x, in which the solute of interest has a higher solubility (Fig. 1). Liquid extraction provides the separation of substances selectively from a mixture as well as removing impurities from the solution.
Since Dahl’s introduction of ternary system phase diagrams in the process of fusion and crystallization, the use of a phase diagram has been reported in many applications (Kofeler et al. 2012). In liquid-liquid extraction, different solvent systems have been discussed according to the solubility of the solutes. When the combination of three components, i.e., the solute and two solvent systems, are at equilibrium, the distribution of the solute in extraction solvents can be calculated using a phase diagram. A phase diagram often represents a ternary system with the three apexes of the phase triangle representing the three components as 100 % pure solutions. As shown in Fig. 2, the three components in the solvent system are given as A, B, and C. At equilibrium, the mixtures to be separated must be inside the curve envelope, which allows biphasic separation. Point M represents a mixture of all three components, where XA, XB, and XC represent the mass fractions of each component, A, B, and C, at equilibrium, respectively. When two liquid phases are present, the composition of these phases is given by the positions of the ends of a tie line through the system point. The equilibrium tie lines shown in Fig. 2 are determined experimentally and the plait point is where the two phases are identical. When selecting extraction solvents, if phase C is chosen as the solute, phase B is chosen as the extraction solvent, and phase A is used as the diluent solvent (to remove impurities from the solute), the point of raffinate is the point of intercept of the curve envelope and the tie line toward the diluent solution. Likewise, the point of extract is the intercepting point of the curve envelope and the tie line close to the extraction solvent.
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