AUTOMATIC SOXHLET EXTRACTION
The method described by Soxhlet in 1879 is the most commonly used example of a semi-continuous method applied to extraction of lipids from foods. According to the Soxhlet's procedure, oil and fat from solid material are extracted by repeated washing (percolation) with an organic solvent, usually hexane or petroleum ether, under reflux in a special glassware.
||In this method the sample is
dried, ground into small particles and placed in a porous cellulose
thimble. The thimble is placed in an extraction chamber (2), which is
suspended above a flask containing the solvent (1) and below a condenser
(4). The flask is heated and the solvent evaporates and moves up into
the condenser where it is converted into a liquid that trickles into the
extraction chamber containing the sample. The extraction chamber is
designed so that when the solvent surrounding the sample exceeds a
certain level it overflows and trickles back down into the boiling
flask. At the end of the extraction process, which lasts a few hours,
the flask containing the solvent and lipid is removed. In some device a
funnel (3) allows to recover the solvent at the end of the extraction
after closing a stopcock between the funnel and the extraction chamber.
The solvent in the flask (1) is then evaporated and the mass of
the remaining lipid is
measured. The percentage of lipid in the initial sample can then be
Despite disadvantages of this procedure (poor extraction of polar lipids, long time involved, large volumes of solvents, hazards of boiling solvents), several methods involving automatic solvent extraction were described. Different automated or semi-automated extraction instruments may be found on the market.
Several solvent extraction systems based on the Soxhlet device are on the market to allow fast and safe determination of total lipids in food, soil, ....
As an example, FOSS has launched several types of "Soxtec Systems" including automated or semi-automated analyzers, which extract lipids rapidly and accurately.
These instruments perform boiling, rinsing and solvent recovery. Similarly, Soxtherm extractors from Gerhardt GmbH was developed to reduce extraction times.
The sample to be analyzed is weighed into cellulose thimbles and inserted in the extraction device. Except diethyl ether, all solvents may be used (about 15 ml per sample), with a 75% recovery of the solvent after the extraction which is completed in 30 to 60 min, depending on the application.
Another device by ViscoALPHA enables the user to have a 2, 4 or 6 place system in 2 versions (micro or macro). An electronic unit can control and monitor up to 4 extraction units individually.
Compact and simple systems with one to six samples are sold by Behr Labor-Technic GmbH.
The XT10 Extractor by ANKOM
provides an Official Procedure (AOCS Am
5-04) for efficient and economical solvent extractions of various sample types.
It allows batch processing for the extraction
of up to fifteen samples at a time and up to 100 samples per day. A process
temperature of 90°C accelerates extraction kinetics, reducing most extraction
times to under 40 minutes. Samples are placed
into filter bags which prevent sample transfer error. The XT10 automatically
recycles solvent and makes it ready for reuse. Solvent is manually added after
each extraction. The XT15
Extractor provides a totally automatic operation for up to 150 samples.
The Büchi Extraction System B-811 is an automated system which can be used to perform an extraction according to the original Soxhlet principle. Four different extraction methods are possible without making any changes to the unit : Soxhlet standard, Soxhlet warm, hot extraction and continuous extraction. The system has an inert gas supply to avoid oxidation during extraction and to accelerate the evaporation and drying process even with high boiling point solvents (up to 150°C). Several application notes may be downloaded from the Büchi site.
A comparison of different extraction methods for total lipid quantification in meat and meat products was reported (Perez-Palacios T et al., Food Chem 2008, 110, 1025). The Soxhlet method with previous acid hydrolysis had the same efficiency as that of the method described by Folch.
A microwave-assisted Soxhlet extraction of seed oil (sunflower, soybean, rape) was described using a cellulose cartridge placed into a quartz extraction vessel inserted in a modified Microdigest 301 device (Prolabo). This procedure is slightly different from that described for the extraction of dry materials. Despite a 3 h analysis, the time reduction and the lack of need for seed grinding makes this procedure a suitable competitor of the previously described methods (Garcia-Ayuso LE et al., Anal Chem 1998, 70, 2626; Garcia-Ayuso LE et al., Chromatographia 2000, 52, 103). Comparative experiments have shown that no significant differences between the extract obtained by the Folch reference method without fat alterations and the microwave-assisted Soxhlet extraction were detected. These results demonstrated the applicability of that method for the extraction step in routine analysis of a great quantity of food samples (Ruiz-Jimenez J et al., Anal Chim Acta 2004, 525, 159).
This extraction technique, called microwave-assisted Soxhlet extraction, uses two sources of energy, namely microwaves, applied on the extraction chamber of a modified Soxhlet, and electrical heating applied on the distillation flask. This system has been used for the determination of oil content and fatty acid composition of various biological materials and foodstuffs. To overcome some limitations of the analytical process (water content), a new and convenient process was designed and developed (Virot M et al., J Chromatogr A 2007, 1174, 138; Virot M et al., J Chromatogr A 2008, 1196-1197, 57).
A modification of that extraction procedure was proposed taking into account the use of a "green solvent", limonene, instead of hexane (Virot M et al., J Chromatogr A 2008, 1196-1197, 147). The proposed method is effective and valuable since no significant difference was obtained when using hexane or limonene for the extraction of oleaginous seeds.
A critical evaluation of various solvents systems used for the quantification of microalgal fatty acids with lyophilized samples has been reported (McNichol J et al., Lipids 2012, 47, 195).
A dynamic ultrasound-assisted
extraction method prior to the gravimetric determination of total fat content in
bakery products was proposed (Ruiz-Jimenez J et al., Anal Chim Acta 2004,
Samples were inserted in a stainless steel extraction chamber drained by the extraction fluid (hexane). The tube was immersed in a water bath in which an ultrasonic probe (Branson 450 sonifier) was immersed.
After testing several parameters, the most efficient approaches was an open system where fresh extractant was pumped in forward-and-back steps. Two grams of sample were optimally extracted with 25 ml of hexane for a time much shorter than conventional Soxhlet extraction. Comparisons with the Folch reference method have shown no significant differences when food samples were extracted (Ruiz-Jimenez J et al., Anal Chim Acta 2004, 525, 159).
ROBOT AUTOMATED TOTAL LIPID EXTRACTION
A novel automated chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots has been developed (Löfgren L et al., J Lipid Res 2012, 53, 1690). In only 60 min, 96 samples can be automatically extracted with results similar or better to what is obtained using the Folch's reference method.
The new procedure includes an initial one-phase extraction of plasma into 300 ml butanol/methanol mixture (3/1) followed by a two phase extraction into 300 ml heptane/ethyl acetate (3/1) using 300 ml 1% acetic acid as buffer.
This method involves low-toxic solvents and has been shown to have high extraction recoveries for all the investigated lipids, thus, it could replace the old chloroform-based methods and could become the method of choice for the modern lipid laboratory. For lipidomics applications, this automated process seems extremely valuable to reduce time-consuming multiple extraction procedures.