Extração de Soxhlet de Biomarcadores Lipídicos de Sedimentos
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Fonte: Laboratório de Jeff Salacup – Universidade de Massachusetts Amherst
Todo laboratório precisa de padrões que rastreiem o desempenho, a precisão e a precisão de seus instrumentos ao longo do tempo para garantir que uma medição feita hoje seja a mesma de uma medição feita daqui a um ano(Figura 1). Como as normas devem testar o desempenho dos instrumentos durante um longo período de tempo, grandes volumes das normas são frequentemente exigidos. Muitos padrões químicos podem ser adquiridos de empresas científicas de varejo, como Sigma-Aldrich e Fisher. No entanto, alguns compostos que ocorrem na natureza e que são relevantes para estudos paleoclimáticos ainda não foram isolados e purificados para compra. Portanto, esses compostos precisam ser extraídos de amostras naturais, e devido aos grandes volumes de padrões exigidos, grandes volumes de sedimentos precisam ser extraídos. As extrações de solventes aceleradas (Dionex) e as extrações de sônica não são apropriadas para a extração de grandes volumes de sedimentos. Nestas circunstâncias, uma extração de Soxhlet é usada.
Figura 1. Esquema mostrando como o padrão químico rastreia o desempenho de um instrumento através do tempo. A linha tracejada representa uma relação 1:1 entre o valor aceito e medido (no instrumento) de uma variável. Cada estrela é uma medição semanal do padrão químico. Estrelas verdes representam padrões que são precisos. As estrelas vermelhas refletem aquelas que não são precisas indicando que o instrumento requer manutenção corretiva.
Principles
Procedure
Results
At the end of extraction, a total lipid extract (TLE) for the sample is produced. The round-bottomed flask contains the extractable organic matter from the sediment sample. This TLE can now be analyzed, and its chemical constituents identified and quantified.
Applications and Summary
The extract from the marine mud contains compounds called alkenones, which are used in paleoceanography. Alkenones are long-chained alkyl-ketones produced by certain classes of haptophyte algae that live in the sunlit surface ocean3 (Figure 3). The two most common alkenones are 37 carbon atoms long and have two or three double bonds in them. The haptophytes adjust the ratio of these two alkenones in their cells according to the temperature of the water they live in. The ratio of the two alkenones defines the Uk'37 ratio:
Equation 1) Uk'37 = (C37:2) / (C37:2 + C37:3) 4,5
Culture6,7 and core-top sediment8 calibration studies led to the development of the Uk'37 Index as a quantitative SST proxy. In this work we use:
Equation 2) Uk'37 = 0.034(SST) + 0.039; ±1.4 °C from 0 to 28 °C [culture-based7]
Alkenones are preserved in sediments dating as far back as the Early Eocene (~56 million years ago)9. Knowing the distribution of alkenones in a sediment core through time relates information on the evolution of sea surface temperature at that location. However, it's necessary to first make sure the instrument accurately and precisely measures the ratio of the two alkenones, and that is why standards are needed.
Figure 3. Alkenones with 2 (C37:2) and 3 (C37:3) double bonds (left) are produced by certain haptophyte algae that live in the sunlit surface ocean (right). (Photo courtesy of Tim I. Eglinton, Woods Hole Oceanographic Institution)
References
- Jensen, W. B. The Origin of the Soxhlet Extractor J Chem Ed. 84, 1913-1914, (2007).
- Levey, M. Chemistry and Technology in Ancient Mesopotamia, Elsevier. 33-34, (1959).
- Conte, M. H., Thompson, A., Eglinton, G. Primary production of lipid biomarker compounds by Emiliania huxleyi: results from an experimental mesocosm study in fjords of southern Norway, Sarsia, 79, 319-332 (1994).
- Brassell, S. C., Eglinton, G., Marlowe, I. T., Pflaumann, U., Sarnthein, M. Molecular Stratigraphy – a New Tool for Climatic Assessment, Nature, 320 (6058), 129-133 (1986).
- Herbert, T. D. Alkenone paleotemperature determinations, in Treatise in Marine Geochemistry, edited by H. Elderfield, Elsevier 391-432 (2003).
- Prahl, F. G., Wakeham S. G., Calibration of Unsaturation Patterns in Long-Chain Ketone Compositions for Paleotemperature Assessment, Nature, 330(6146), 367-369 (1987).
- Prahl, F. G., Muehlhausen, L. A., Zahnle, D. L. Further evaluation of long-chain alkenones as indicators of paleoceanographic conditions, Geochimica et Cosmochimica Acta, 52(9), 2303-2310 (1988).
- Müller, P. J. et al. Calibration of the alkenone paleotemperature index U37K′ based on core-tops from the eastern South Atlantic and the global ocean (60°N-60°S), Geochimica et Cosmochimica Acta, 62(10), 1757-1772 (1998).
- Marlowe, I. T. et al. Long-chain Alkenones and Alkyl Alkenoates and the Fossil Coccolith Record of Marine-sediments, Chem Geol, 88(3-4), 349-375 (1990).
Transcript
Soxhlet extraction is a method of isolating compounds, such as lipids, from a large amount of solid material with a relatively small volume of solvent.
Many of the compounds relevant to paleoclimatic studies are not available to purchase from retail scientific companies. Standards of these compounds must therefore be prepared from natural samples.
Large quantities of standard are needed to assess the performance of an instrument over time. To obtain a suitable amount of a biomarker for standard preparation, a large volume of sediment must be extracted.
The Soxhlet extractor, invented in the 1870’s by Franz von Soxhlet, allows automated, batch extraction from a solid, increasing the overall efficiency while using a small amount of solvent.
This video is part of a series on lipid extraction, purification, and analysis from sediments. It will illustrate Soxhlet extraction of lipid biomarkers from marine sediment for use in paleothermometry and will introduce a few other applications of Soxhlet extraction in Earth science and chemistry.
A typical assembly uses a round-bottomed flask, a cold water condenser, and the Soxhlet apparatus itself. The solid to be extracted is placed in a thimble in the central chamber of the apparatus. The extraction is aided by the addition of energy in the form of heat, known as refluxing. The solvent vapor rises through the distillation path in the Soxhlet apparatus to the condenser. Upon condensing, the solvent collects in the chamber, dissolving some of the organic material in the thimble. As the chamber fills, the siphon fills as well. When the siphon is full, the solution flows back into the flask. The solution level never exceeds the top of the thimble, so no solid enters the flask.
The lipid extract continually collects in the flask, whereas the solvent becomes part of the next extraction cycle. Thus, the cycle can repeat indefinitely without loss of solvent.
The conservation of the solvent, the continuous nature of the extraction, and the ability to accommodate large sample sizes makes Soxhlet extraction ideal for isolating organic compounds from large portions of insoluble material.
Now that you understand the principles of Soxhlet extraction, let’s go through a procedure for Soxhlet extraction of lipid biomarkers from sediment.
For this experiment, a sample of excess marine sediment from a coring expedition is used. The sample will be freeze-dried, crushed, and homogenized. For more instruction, please reference this collection’s video on Extraction by Sonication.
To prepare for the extraction, first make a 9:1 solution of dichloromethane to methanol. This solution will be used as the extraction solvent and to wash the glassware and laboratory instruments.
To remove organic contaminants, combust the round-bottomed flask, Soxhlet apparatus, glass fiber thimble, and weighing tins for 6 h at 550 °C. Wash a round-bottomed flask the DCM-methanol solution. Once ready to set up the extraction, rinse a laboratory spatula and five to ten boiling chips with the DCM-methanol solution.
To begin constructing the extraction assembly, set up a heating mantle in a fume hood. Obtain a condenser, a support stand to secure the round bottom flask, and the Soxhlet apparatus.
Tare a combusted weighing tin. With the solvent-rinsed spatula, transfer approximately 50 g of sample to the weighing tin and record the mass. Load the material into the combusted glass fiber thimble.
Next, fill the combusted and rinsed round-bottomed flask slightly more than half full of the DCM-methanol solution. Add the washed boiling chips and place the round-bottomed flask in the heating mantle.
Then, place the sample thimble open end up in the chamber of the Soxhlet apparatus. Connect the apparatus to the round-bottomed flask and clamp the apparatus in place.
Secure the condenser to the top of the Soxhlet apparatus. Connect the cold water line to the lower port of the condenser with a hose clamp or zip tie. Connect the outlet line to the upper port of the condenser and route it to the drain.
Turn on the water to the condenser and verify the flow path. Then, turn on the heating mantle and heat the solvent to reflux.
As the solvent begins condensing, ensure that the condensate is dripping into the chamber and that the extract is siphoned into the round-bottomed flask. The solvent should stay at a low boil throughout the extraction.
Monitor the extraction process and the condenser water flow until the extraction is complete. Then, stop the extraction by turning off the heating mantle. Once the extract has cooled, remove the condenser and Soxhlet apparatus. Finally, seal the round-bottomed flask containing the total lipid extract and store for further processing.
Soxhlet extraction is often used for chemical analysis of a solid sample, and can also be used for reagent preparation and purification.
Soxhlet extraction can be used to detect the presence of polychlorinated biphenyl compounds, or PCBs, in the environment. The transfer efficiency of PCBs from prey fish to predator fish was measured to gain more information about the health risks to humans and wildlife from eating contaminated fish. Soxhlet extraction of fish tissue allows preparation of samples for gas chromatography and mass spectrometry.
Compounds to be introduced to the environment in large quantities are analyzed for the presence of PCBs. Biochar is a byproduct of pyrolysis of organic matter that, when added to soil, may improve soil quality and take up pollutants. Validation of biochar production methods for widespread use includes Soxhlet extraction to test for the presence of PCBs by gas chromatography.
Soxhlet extraction can also be used to purify a solid by extraction of unwanted compounds. Long-chain fatty acids were selectively removed from tomato skins by stepwise extraction to yield the wax-free tomato cuticle. The stepwise extraction was performed with multiple solvents of varying polarities in succession. This not only provided comprehensive wax removal from the tomato skin, but allowed isolation of individual wax moieties based on solubility characteristics as well.
You’ve just watched JoVE’s introduction to Soxhlet extraction of lipid biomarkers from geological archive sediments. You should now be familiar with the principles behind Soxhlet extraction, the procedure for Soxhlet extraction of a sediment sample, and some examples of how Soxhlet extraction may be used for analytical purposes.
Thanks for watching!