JoVE Science Education Chemistry Essentials of Organic Chemistry Solid-Liquid Extraction

Solid-Liquid Extraction

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1. Extraction of Adsorbed Organics from Soil

  1. Place 20 g of soil in a clean, dry wide-mouth Pyrex dish in a 50 °C oven and dry for a minimum of 12 h. After drying, remove the soil from the Pyrex dish and grind to a uniform powder using a mortar and pestle. Weigh 5.00 g of the soil and place it into a clean, dry round-bottom flask (100 mL in size). To the flask, add 15 mL of n-hexane. Place flask in an ultrasonic bath, and sonicate for 60 min.

2. Separation of Extract and Soil

  1. Prepare a Büchner funnel with analytical filter paper. Wet the filter paper with 1 mL of n-hexanes and begin vacuum filtration. Slowly pour the contents of the round-bottom flask over the filter paper. The Büchner flask now contains the n-hexanes with the organics extracted from the soil. The filter retains the stripped soil solids.

3. Clean up and Pre-concentration

  1. If the n-hexane solution is cloudy, there is residual water. To dry the n-hexane solution, add one small spatula of CaCl2. Swirl the solution and observe for a minimum of 15 min. If the solution is still cloudy and/or all of the CaCl2 is clumped, there is still water remaining, and step 3.1 should be repeated. If the solution is translucent and the CaCl2 is free flowing, then do not repeat step 3.1. Once a clear solution has been achieved, separate the hexanes from CaCl2 using gravity filtration. If the extract concentration is sufficient for detection, the filtered hexanes may be transferred to a clean, dry flask for storage and later analysis. If extract concentration is low relative to the limit of detection, transfer the filtered hexanes into a clean, dry three-necked round-bottom flask, 100 mL in size. Place a rubber stopper into the center neck of the flask, and a rubber septum over one of the other necks. Leave the third neck open. Pierce the rubber septum and introduce a nitrogen flow through the flask. The nitrogen should be flowing in the space above the solution, not bubbling through the solution. The extract can now be pre-concentrated by flowing nitrogen to evaporate excess solvent. The sample is now ready for analysis.

Extraction is a crucial separation technique in organic chemistry, used to separate components of a mixture based on their solubilities in two different phases that do not mix.

Extractions are performed between two phases. In the case of a liquid-liquid extraction, the dissolved solute is transferred from one liquid phase to another. Extractions are also performed with a liquid and solid phase, called solid-liquid extraction, where the solute is transferred from a solid phase to a liquid phase. A simple example of solid-liquid extraction is coffee brewing, which involves the mixing of solid coffee grounds with water. The coffee flavor compounds are extracted from the grounds into the water to form coffee. This video will illustrate the principles of extraction, and demonstrate solid-liquid extraction in the lab through the removal of organochloride residues from soil.

Extraction uses the property of solubility to transfer a solute from one phase to another. In order to perform an extraction, the solute must have a higher solubility in the second phase than in the original. In general, very nonpolar solutes will partition into an organic phase, while very polar solutes will partition into an aqueous phase. The choice of phases will depend on the solute of interest.

The two phases also must be immiscible. Immiscible solutions never mix and remain as separate phases, like oil and water. Miscible solutions are completely homogeneous after mixing.

In liquid-liquid extraction, a solute is separated between two liquid phases, typically aqueous and organic. This is often performed in a separatory funnel fitted with a stopcock at the bottom and stopper at the top.

In the simplest case, three components are involved: The solute, the carrier liquid, and the solvent. The initial mixture, containing the solute dissolved in the carrier liquid, is mixed with the solvent. Upon mixing, the solute is transferred from the carrier liquid to the solvent, as long as the solute is more soluble in the solvent than in the carrier liquid, and as long as the carrier liquid and solvent are immiscible. The denser solution settles to the bottom.

There are two resulting phases: the raffinate, containing the carrier liquid, and the extract, which contains the solute and the solvent. In reality, there is likely to be residue of each component in both phases. Solid-liquid extraction is similar to liquid-liquid extraction, except that the solute is dispersed in a solid matrix rather than in a carrier liquid. The solid phase, containing the solute, is dispersed in the solvent and mixed. The solute is extracted from the solid phase to the solvent, and the solid phase is then removed by filtration. Now that the principles of extraction have been outlined, the solid-liquid extraction technique will be demonstrated by performing the extraction in the laboratory.

In this experiment, soil samples were collected from a brownfield site, similar to this one in Sewickley, Pennsylvania. Brownfields, as defined by the U.S. EPA, are real property, where the expansion, redevelopment, or reuse may be complicated due to the potential presence of hazardous contaminants. The pollutant of interest in this case is an organochloride: the herbicide atrazine. Once a soil sample has been collected from the site of interest, transfer it into the laboratory.

Weigh out 10 g of the soil in a clean, dry, wide-mouth Pyrex dish. Put the dish into an oven to dry for at least 12 h. Once dry, grind the soil to a uniform powder with a mortar and pestle. Place 5 g of the ground soil into a clean, dry 100-mL round-bottom flask. Add 15 mL of hexane and loosely stopper the flask. Place it into an ultrasonic bath and sonicate for 60 min.

Prepare a Büchner funnel with analytical filter paper. Once sonication is complete, wet the paper with hexane and begin vacuum filtration. Slowly pour the sample over the filter paper. Rinse the residual solids from the flask with hexane and add it to the filter. The stripped soil remains on the filter, while the hexane and extracted organics collect in the flask.

If the hexane solution is cloudy, residual water is present. To dry the solution, add a small spatula of desiccant, such as calcium chloride. Stir the solution until the desiccant is dissolved, and observe the solution.

If the solution is still turbid or if the calcium chloride has aggregated, there is still water in the solution. Repeat the process until the solution is clear and the desiccant is free flowing.

Next, remove the calcium chloride by gravity filtration.

If the concentration of the compound of interest is below the limit of quantification, it must be concentrated. Transfer the filtered extract to a clean, dry 3-necked round-bottom flask. Stopper the center neck, and place a rubber septum over one of the other necks. The third is left open.

Pierce the septum and attach tubing to a nitrogen line. Begin flowing nitrogen through the flask. The gas should be flowing in the headspace above the solution, and not bubbling through it. The flowing gas evaporates the excess solvent. Allow the gas to flow until there is about 50% volume reduction.

Once the organic components of the soil are extracted and concentrated, they can be analyzed by gas chromatography.

The atrazine concentration can be calculated using atrazine standard concentrations. In this case, the approximate atrazine concentration in the brownfield site studied was 2 mg of atrazine per 1 kg of soil.

Solid-liquid extraction is used in a wide range of fields.

This technique can be used to understand the transfer of polychlorinated biphenyls, or PCBs, from fish. PCBs are man-made chlorinated hydrocarbons that have been banned by the EPA. PCBs do not readily decompose in the environment and tend to accumulate in fish.

In this experiment, prey fish containing PCBs were fed to predator fish. The predator fish were then collected and sacrificed. The fish tissue was ground in preparation for extraction.

The PCB in the fish tissue was extracted to an organic phase using a Soxhlet extractor. The Soxhlet extractor setup, composed of a round-bottom flask, condenser, and the Soxhlet apparatus, is frequently used to extract solutes that are poorly soluble in solvents. The Soxhlet extraction enables a small amount of solvent to be used with a large solid sample. The extract was then tested for PCB content using mass spectrometry.

Dry plant matter, called lignocellulose, is the most abundant raw material being researched for bio-derived fuels. However, the carbohydrates used as the fuel are trapped within the rigid plant matrix, called lignin.

When the carbohydrates are removed, the lignin matrix is typically disposed of as waste. However, in this experiment, waste lignin was examined as a fuel source. Solid-liquid extraction was utilized to separate the carbohydrate components from lignocellulose, leaving lignin behind. The lignin was then used for further fermentation experiments.

Solid-liquid extraction can also be used to measure the wax content in fruit skins. In this experiment, the wax content of tomato skins was analyzed.

Exhaustive dewaxing of dried tomato skins was completed using a Sohxlet apparatus, in order to fully remove the wax content in the skins. Tomato skins with wax removed were then further analyzed using nuclear magnetic resonance spectroscopy. This helped elucidate the composition and degradation of native and engineered fruits.

You've just watched JoVE's introduction to solid-liquid extraction. You should now have a better understanding of the extraction of solutes between solid and liquid phases.

Thanks for watching!

Overview

Source: Laboratory of Dr. Jay Deiner — City University of New York

Extraction is a crucial step in most chemical analyses. It entails removing the analyte from its sample matrix and passing it into the phase required for spectroscopic or chromatographic identification and quantification. When the sample is a solid and the required phase for analysis is a liquid, the process is called solid-liquid extraction. A simple and broadly applicable form of solid-liquid extraction entails combining the solid with a solvent in which the analyte is soluble. Through agitation, the analyte partitions into the liquid phase, which may then be separated from the solid through filtration. The choice of solvent must be made based on the solubility of the target analyte, and on the balance of cost, safety, and environmental concerns.

Cite this Video

JoVE Science Education Database. Essentials of Organic Chemistry. Solid-Liquid Extraction. JoVE, Cambridge, MA, (2017).

Principles

Extraction uses the property of solubility to transfer a solute from one phase to another phase. In order to perform an extraction, the solute must have a higher solubility in the second phase than in the original phase. In liquid-liquid extraction, a solute is separated between two liquid phases, typically an aqueous and an organic phase. In the simplest case, three components are involved: the solute, the carrier liquid, and the solvent. The initial mixture, containing the solute dissolved in the carrier liquid, is mixed with the solvent. Upon mixing, the solute is transferred from the carrier liquid to the solvent. The denser solution settles to the bottom. The location of the solute will depend on the properties of both liquids and the solute.

Solid-liquid extraction is similar to liquid-liquid extraction, except that the solute is dispersed in a solid matrix, rather than in a carrier liquid. The solid phase, containing the solute, is dispersed in the solvent and mixed. The solute is extracted from the solid phase to the solvent, and the solid phase is then removed by filtration.

In this video, an example of the solid-liquid extraction technique will be illustrated by showing the extraction of organochlorine residue from soil. The illustrated solid-liquid extraction entails combination of the sample with n-hexanes followed by ultrasonic agitation, filtration, removal of residual water by drying over CaCl2, and pre-concentration under flowing nitrogen. The as-prepared sample is then ready for analysis by a range of spectroscopic and chromatographic methods.

Procedure

1. Extraction of Adsorbed Organics from Soil

  1. Place 20 g of soil in a clean, dry wide-mouth Pyrex dish in a 50 °C oven and dry for a minimum of 12 h. After drying, remove the soil from the Pyrex dish and grind to a uniform powder using a mortar and pestle. Weigh 5.00 g of the soil and place it into a clean, dry round-bottom flask (100 mL in size). To the flask, add 15 mL of n-hexane. Place flask in an ultrasonic bath, and sonicate for 60 min.

2. Separation of Extract and Soil

  1. Prepare a Büchner funnel with analytical filter paper. Wet the filter paper with 1 mL of n-hexanes and begin vacuum filtration. Slowly pour the contents of the round-bottom flask over the filter paper. The Büchner flask now contains the n-hexanes with the organics extracted from the soil. The filter retains the stripped soil solids.

3. Clean up and Pre-concentration

  1. If the n-hexane solution is cloudy, there is residual water. To dry the n-hexane solution, add one small spatula of CaCl2. Swirl the solution and observe for a minimum of 15 min. If the solution is still cloudy and/or all of the CaCl2 is clumped, there is still water remaining, and step 3.1 should be repeated. If the solution is translucent and the CaCl2 is free flowing, then do not repeat step 3.1. Once a clear solution has been achieved, separate the hexanes from CaCl2 using gravity filtration. If the extract concentration is sufficient for detection, the filtered hexanes may be transferred to a clean, dry flask for storage and later analysis. If extract concentration is low relative to the limit of detection, transfer the filtered hexanes into a clean, dry three-necked round-bottom flask, 100 mL in size. Place a rubber stopper into the center neck of the flask, and a rubber septum over one of the other necks. Leave the third neck open. Pierce the rubber septum and introduce a nitrogen flow through the flask. The nitrogen should be flowing in the space above the solution, not bubbling through the solution. The extract can now be pre-concentrated by flowing nitrogen to evaporate excess solvent. The sample is now ready for analysis.

Extraction is a crucial separation technique in organic chemistry, used to separate components of a mixture based on their solubilities in two different phases that do not mix.

Extractions are performed between two phases. In the case of a liquid-liquid extraction, the dissolved solute is transferred from one liquid phase to another. Extractions are also performed with a liquid and solid phase, called solid-liquid extraction, where the solute is transferred from a solid phase to a liquid phase. A simple example of solid-liquid extraction is coffee brewing, which involves the mixing of solid coffee grounds with water. The coffee flavor compounds are extracted from the grounds into the water to form coffee. This video will illustrate the principles of extraction, and demonstrate solid-liquid extraction in the lab through the removal of organochloride residues from soil.

Extraction uses the property of solubility to transfer a solute from one phase to another. In order to perform an extraction, the solute must have a higher solubility in the second phase than in the original. In general, very nonpolar solutes will partition into an organic phase, while very polar solutes will partition into an aqueous phase. The choice of phases will depend on the solute of interest.

The two phases also must be immiscible. Immiscible solutions never mix and remain as separate phases, like oil and water. Miscible solutions are completely homogeneous after mixing.

In liquid-liquid extraction, a solute is separated between two liquid phases, typically aqueous and organic. This is often performed in a separatory funnel fitted with a stopcock at the bottom and stopper at the top.

In the simplest case, three components are involved: The solute, the carrier liquid, and the solvent. The initial mixture, containing the solute dissolved in the carrier liquid, is mixed with the solvent. Upon mixing, the solute is transferred from the carrier liquid to the solvent, as long as the solute is more soluble in the solvent than in the carrier liquid, and as long as the carrier liquid and solvent are immiscible. The denser solution settles to the bottom.

There are two resulting phases: the raffinate, containing the carrier liquid, and the extract, which contains the solute and the solvent. In reality, there is likely to be residue of each component in both phases. Solid-liquid extraction is similar to liquid-liquid extraction, except that the solute is dispersed in a solid matrix rather than in a carrier liquid. The solid phase, containing the solute, is dispersed in the solvent and mixed. The solute is extracted from the solid phase to the solvent, and the solid phase is then removed by filtration. Now that the principles of extraction have been outlined, the solid-liquid extraction technique will be demonstrated by performing the extraction in the laboratory.

In this experiment, soil samples were collected from a brownfield site, similar to this one in Sewickley, Pennsylvania. Brownfields, as defined by the U.S. EPA, are real property, where the expansion, redevelopment, or reuse may be complicated due to the potential presence of hazardous contaminants. The pollutant of interest in this case is an organochloride: the herbicide atrazine. Once a soil sample has been collected from the site of interest, transfer it into the laboratory.

Weigh out 10 g of the soil in a clean, dry, wide-mouth Pyrex dish. Put the dish into an oven to dry for at least 12 h. Once dry, grind the soil to a uniform powder with a mortar and pestle. Place 5 g of the ground soil into a clean, dry 100-mL round-bottom flask. Add 15 mL of hexane and loosely stopper the flask. Place it into an ultrasonic bath and sonicate for 60 min.

Prepare a Büchner funnel with analytical filter paper. Once sonication is complete, wet the paper with hexane and begin vacuum filtration. Slowly pour the sample over the filter paper. Rinse the residual solids from the flask with hexane and add it to the filter. The stripped soil remains on the filter, while the hexane and extracted organics collect in the flask.

If the hexane solution is cloudy, residual water is present. To dry the solution, add a small spatula of desiccant, such as calcium chloride. Stir the solution until the desiccant is dissolved, and observe the solution.

If the solution is still turbid or if the calcium chloride has aggregated, there is still water in the solution. Repeat the process until the solution is clear and the desiccant is free flowing.

Next, remove the calcium chloride by gravity filtration.

If the concentration of the compound of interest is below the limit of quantification, it must be concentrated. Transfer the filtered extract to a clean, dry 3-necked round-bottom flask. Stopper the center neck, and place a rubber septum over one of the other necks. The third is left open.

Pierce the septum and attach tubing to a nitrogen line. Begin flowing nitrogen through the flask. The gas should be flowing in the headspace above the solution, and not bubbling through it. The flowing gas evaporates the excess solvent. Allow the gas to flow until there is about 50% volume reduction.

Once the organic components of the soil are extracted and concentrated, they can be analyzed by gas chromatography.

The atrazine concentration can be calculated using atrazine standard concentrations. In this case, the approximate atrazine concentration in the brownfield site studied was 2 mg of atrazine per 1 kg of soil.

Solid-liquid extraction is used in a wide range of fields.

This technique can be used to understand the transfer of polychlorinated biphenyls, or PCBs, from fish. PCBs are man-made chlorinated hydrocarbons that have been banned by the EPA. PCBs do not readily decompose in the environment and tend to accumulate in fish.

In this experiment, prey fish containing PCBs were fed to predator fish. The predator fish were then collected and sacrificed. The fish tissue was ground in preparation for extraction.

The PCB in the fish tissue was extracted to an organic phase using a Soxhlet extractor. The Soxhlet extractor setup, composed of a round-bottom flask, condenser, and the Soxhlet apparatus, is frequently used to extract solutes that are poorly soluble in solvents. The Soxhlet extraction enables a small amount of solvent to be used with a large solid sample. The extract was then tested for PCB content using mass spectrometry.

Dry plant matter, called lignocellulose, is the most abundant raw material being researched for bio-derived fuels. However, the carbohydrates used as the fuel are trapped within the rigid plant matrix, called lignin.

When the carbohydrates are removed, the lignin matrix is typically disposed of as waste. However, in this experiment, waste lignin was examined as a fuel source. Solid-liquid extraction was utilized to separate the carbohydrate components from lignocellulose, leaving lignin behind. The lignin was then used for further fermentation experiments.

Solid-liquid extraction can also be used to measure the wax content in fruit skins. In this experiment, the wax content of tomato skins was analyzed.

Exhaustive dewaxing of dried tomato skins was completed using a Sohxlet apparatus, in order to fully remove the wax content in the skins. Tomato skins with wax removed were then further analyzed using nuclear magnetic resonance spectroscopy. This helped elucidate the composition and degradation of native and engineered fruits.

You've just watched JoVE's introduction to solid-liquid extraction. You should now have a better understanding of the extraction of solutes between solid and liquid phases.

Thanks for watching!

Results

A soil sample was collected from a Brownfield site similar to one in Sewickley Pennsylvania, as shown in Figure 1. Brownfields, as defined by the United States Environmental Protection Agency (U. S. EPA), are real property, where the expansion, redevelopment, or reuse may be complicated due to the potential presence of hazardous contaminants. The soil was collected from the Brownfield site using a soil sampler, as shown in Figure 2.

The pollutant of interest in this experiment was atrazine (Figure 3); a common organochloride herbicide. Once the organic components of the soil were extracted and concentrated, they were analyzed by gas chromatography with a flame ionization detector (GC-FID). The GC analysis was carried out using a Shimadzu 14A GC (detector: FID) equipped with split/splitless injector and a CBP-10 capillary column (30 m × 0.22 mm i.d.). The column temperature was first set at 150 °C and then programmed from 150 to 230 °C at a rate of 5 °C per min. The injector temperature was 250 °C and the detector temperature was 260 °C. Injections were performed with splitless mode. Helium carrier gas was used at a constant flow rate of 1 mL/min. The atrazine concentration was calculated using atrazine standard concentrations, as shown in Figure 4. In this case, the approximate atrazine concentration in the Brownfield site studied was 2 mg of atrazine per kg of soil.

Figure 1
Figure 1. Brownfield site in Sewickley, PA.

Figure 2
Figure 2. Contaminated soil collected using a soil sampler.

Figure 3
Figure 3. Chemical structure of the organochloride atrazine.

Figure 4
Figure 4. Gas chromatogram of soil sample with atrazine. Inset: atrazine standards.

Applications and Summary

The general solid-liquid extraction procedure is applicable to a range of fields from environmental monitoring (shown in this video) to cosmetics and food processing. The critical issue is to pick a solvent that effectively dissolves the analyte. With minimal changes in solvent, the sample preparation method in this video can be used to extract any of a broad range of semivolatile environmental contaminants that partition primarily on soils and sludges.

Examples of such semivolatiles include many harmful pollutants like pesticides, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). Because of the potential health effects of these molecules, identification and quantification of these species is of academic interest, and also widely practiced in the environmental consulting industry and in government agencies. The EPA maintains compendia of approved analytical and sampling methods to identify and quantify possible pollutants. The method shown in this video illustrates the basic principles contained in EPA method 3550C, which describes ultrasonic extraction of semivolatiles and nonvolatiles from solids.1 EPA method 3550C is one of the extraction methods referenced in EPA method 8081B, which describes GC analysis of organochlorine pesticides.2 Most of the EPA-approved method files are written with the assumption that the analyst has significant prior training. Thus, gaining familiarity with the basic characteristics of sample preparation aids in following the EPA methods.

The use of a Soxhlet apparatus can aid in the extraction of solutes that are poorly soluble in solvents. The setup consists of a round-bottom flask, a Soxhlet extractor, and a reflux condenser. This technique is demonstrated by the removal of PCBs from fish in order to examine the transfer of toxins between predator fish and prey fish.3 Additionally, this technique can be used to measure the wax content in fruit skins in order to understand the composition and degradation of native and engineered fruits.4 Finally, the extraction of carbohydrates from lignocellulose, or dry plant matter, can be accomplished using solid liquid extraction.5 When the carbohydrates are extracted, lignin is left behind. Both components can then be used for biofuel applications.

References

  1. US Environmental Protection Agency. Ultrasonic Extraction, Method 3550C. Washington: Government Printing Office (2007).
  2. US Environmental Protection Agency. Organochlorine pesticides by gas chromatography, Method 8081B. Washington: Government Printing Office (2007).
  3. Madenjian, C. P., Rediske, R. R., O'Keefe, J. P., David, S. R. Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey. J. Vis. Exp. (90), e51496, (2014).
  4. Chatterjee, S., Sarkar, S., Oktawiec, J., Mao, Z., Niitsoo, O., Stark, R. E. Isolation and Biophysical Study of Fruit Cuticles. J. Vis. Exp. (61), e3529, (2012).
  5. Mathews, S. L., Ayoub, A. S., Pawlak, J., Grunden, A. M. Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes. J. Vis. Exp. (82), e51373, (2013).

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