Liquid Extraction: Folch
KeywordsLipid Class Biphasic System Volumetric Ratio Cell Biological Process Lower Chloroform Phase
Folch’s extraction procedure is one of the most popular methods for isolating lipids from biological samples. It takes advantage of the biphasic solvent system consisting of chloroform/methanol/water in a volumetric ratio of 8:4:3 (v/v/v).
Folch’s method is one of the major contributions to the field of lipid biochemistry by the Catalan biochemist and prominent scientist in the field of neurochemistry Jordi Folch-Pi (1911–1979) (Lees and Pope 2001). Prior to Folch’s method there was no effective way to quantitatively isolate lipids from biological tissue samples. The standard method for lipid extraction from tissues until Folch was Bloor’s method that applied a number of successive extractions using ethanol, ether, and chloroform and/or petrolether (Bloor 1928). This method was very time consuming and the resulting lipid extracts were not free from non-lipid contaminants. Folch, driven by his interest in neurochemistry, developed his famous extraction method in the course to characterize all structural components in the brain. With the limited methods available to him, he was able to identify the structure of phosphatidylserine (Folch and Schneider 1941; Folch 1941, 1948), isolate mono-, di-, and triphosphoinositides (Folch 1949; Folch and Woolley 1942), characterize gangliosides (Folch et al. 1951a), and describe myelin (Folch and Lees 1951). In 1951 Folch et al. published the first protocol based on the chloroform/methanol solvent system that included an excessive washing step with 10 volume equivalents of water (Folch et al. 1951b). In 1957 Folch and coworkers showed that only 0.2 volume equivalents of water are sufficient for washing the lipid-containing organic phase from non-lipid contaminants (Folch et al. 1957). Folch’s approach enabled, for the first time, the ability to generate pure lipid extracts from tissue in a direct extraction step that also could be performed at 0 °C to preserve labile lipid molecules from degradation. The final protocol published in 1957 became very popular and was cited approximately 45,000 times by 2014 making it the 9th most cited research paper of all time and a golden standard for lipid extraction methods (Noorden et al. 2014).
The basic idea behind this extraction procedure was to homogenize a tissue sample (Folch and Lees 1951; Sperry and Brand 1955) in a mixture of chloroform and methanol in volumetric ratios of 2:1. The initial extraction step results in non-extractable residue and one liquid phase containing a wide range of small biomolecules. Important for the success of the method is the usage of methanol as polar component in the extraction mixture to improve the solubilization of lipid molecules from cell membranes and cell compartments (Iverson et al. 2001). When water (or buffer) is added to the chloroform/methanol mixture, a new aqueous phase is formed. The final volumetric ratio of chloroform, methanol, and water of Folch’s method is 8:4:3 (v/v/v). In a state of equilibrium, the upper phase has the composition 3:48:47 (v/v/v) and the lower phase comprises 86:14:1 (v/v/v) (Folch et al. 1957). Because the upper phase is predominantly composed of water and methanol, hydrophilic components and salts are enriched here, whereas the lower phase, being predominantly chloroform, retains lipids. Afterward, the crude lipid-containing phase is washed with water (or buffer) to minimize the amount of salts, non-lipid and hydrophilic components.
c 1 A : Concentration of component 1 in phase A
c 1 B : Concentration of component 1 in phase B
A part of Folch’s success (and derived approaches) lies in the phase behavior of the chloroform/methanol/water mixture that has a low aggregate-forming capacity in the initial extraction step and after phase separation. In return, diffusion processes, mostly defined by the speed to reach equilibrium state, are optimal. However, it is obvious that biological samples are inhomogeneous and several interactions within the sample suspension will hinder free diffusion.
From this perspective one has to consider adjusting the homogenization procedure (e.g., mechanical degradation of dried tissue (Hansen et al. 2015), mechanical homogenization of wet tissue in buffer (Ragab et al. 2015), or digestion of tissue (Ruiz-López et al. 2003) to minimize the diffusion distances and enable large interaction surfaces at the smallest possible particle size. In this regard, the specific biological matrix has to be considered in order to effectively adjust homogenisation procedure, volume ratios, incubation time, and number of extraction repetition and/or washings steps.
In Folch’s original method, the crude extract is filtered to separate precipitated non-extractable residues. In modern lipidomics workflows, this step is often omitted because of practical aspects in the microscale extraction as well as throughput and to avoid contamination, which interfere with highly sensitive LC-MS instruments. Afterward, 0.2 equivalents (of the total volume of the chloroform/methanol mixture) of water are added to the suspension to induce phase separation. An upper water/methanol phase separates from the lower chloroform phase in a volume ratio 40:60 (v/v) (Fig. 1). The most popular way to support phase separation is centrifugation at 1000 g for 5–10 min (Folch et al. 1957; Matyash et al. 2008; Patterson et al. 2015). The lower (organic) phase has to be carefully collected by penetrating the non-extractable residues at the interphase to minimize the collection of particular material. That is especially important for performing subsequent analysis by LC-MS to avoid clogging capillaries and valves.
Only high-quality chloroform should be used because degradation of chloroform under air yields phosgene and hydrochloric acid, which represents a potential health risk and chemically degrades lipids. Utilizing only chloroform with ethanol stabilizer is highly recommended (Maudens et al. 2007; Turk 1998). A simple quality check is to measure the pH of the water phase above chloroform, which should be neutral or mildly acidic.
Folch’s lipid extraction method is very effective for isolating a broad range of phospholipids and neutral lipids from biological specimen. The recovery for most prominent lipid classes like PC, SM, PE, TAG, and DAG is reported above 90 %. Lipid classes with more polar and partially negative charged headgroups (at physiological pH), like PI, PG, and PA, as well as lyso-lipids, have lower recoveries of around 60–70 % (Matyash et al. 2008; Patterson et al. 2015; Byeon et al. 2012). The most famous derivative method is the Bligh and Dyer procedure (Bligh and Dyer 1959) see Chapter Liquid Extraction: Bligh and Dyer.
- Bloor WR. The determination of small amounts of lipid in blood plasma. J Biol Chem. 1928;77(1):53–73.Google Scholar
- Folch J. The isolation of phosphatidyl serine from brain cephalin, and identification of the serine component. J Biol Chem. 1941;139(2):973–4.Google Scholar
- Folch J, Schneider HA. An amino acid constituent of ox brain cephalin. J Biol Chem. 1941;137(1):51–62.Google Scholar
- Folch J, Woolley DW. Inositol, a constituent of a brain phosphatide. J Biol Chem. 1942;142(2):963–4.Google Scholar
- Lees MB, Pope A. Jordi Folch-Pi. Biographical memoirs, vol. 79. National Academy of Sciences of the United States of America; 2001. p. 135–56, ISBN: 0-309-7572-6.Google Scholar
- Ragab S, Abd Elghaffar S, El-Metwally T, Badr G, Mahmoud M, Omar H. Effect of a high fat, high sucrose diet on the promotion of non-alcoholic fatty liver disease in male rats: the ameliorative role of three natural compounds. Lipids Health Dis. 2015;14(1):83.CrossRefPubMedPubMedCentralGoogle Scholar