On the extraction of harmine from Banisteriopsis caapi

Andrew Whitman Candidate Number: 000628-0074 Word Count: 2737 February 25, 2014

Abstract Harmine, a naturally occurring substance with applications in treating Parkinsons Disease, was extracted from the Banisteriopsis caapi vine through steeping. Extracts were prepared in distilled water at a constant average temperature of 85 or 95for 15 minutes. The extracts were ltered by vacuum ltration, and then placed into a centrifuge for 30 minutes with an RCF of approximately RCF G until a reddish-brown solid settled at the bottom. The sediment was dissolved in acetone, which was evaporated o, and separated into its constituent parts with gravity column chromatography on a stationary phase of 3-8 mesh silica gel and an ethyl acetate mobile phase. The individual fractions were then analysed with thin layer chromatography using 98% pure harmine obtained from a chemical supply as a reference. It was found that the sediment would not dissolve in acetone, ethyl acetate or a system of the two. Had dissolution and TLC been successful, the samples containing pure harmine would have been combined, and the solvent allowed to evaporate. The harmine obtained would have then been massed. It was anticipated that steeping at 95would have yielded more harmine. Word Count: 185

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CONTENTS

Contents
Contents List of Figures List of Tables 1 Introduction 2 Background Information 2.1 2.2 2.3 2.4 Banisteriopsis caapi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sedimentation via Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . i ii ii 1 1 1 6 7 7 8 8 8 9 9 9 10 10 10 11 12 12 13

3 Methodology 3.1 Extraction via Steeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1 3.1.2 3.2 Necessary Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sedimentation via Centrifuge 3.2.1 3.2.2

Necessary Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.3

Isolation via Column Chromatography . . . . . . . . . . . . . . . . . . . . . 3.3.1 3.3.2 Necessary Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . Technique [10] [5] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.4

Determination of Purity via Thin Layer Chromatography . . . . . . . . . . . 3.4.1 3.4.2 Necessary Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . Technique [6] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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LIST OF TABLES

4 Results 4.1 4.2 Qualitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 13 14 14 14 15 15 15 16 17 19 . . . . . . . . . . . . . . . . . . . . . 19 19

5 Interpretation of Results 5.1 5.2 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 Evaluation 6.1 6.2 Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additional Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 Bibliography 8 Appendices 8.1 8.2 Appendix A - Actual Extraction Data

Appendix B - Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . . .

List of Figures
1 2 3 4 5 6 Structures of Compounds Present in B. Caapi (Samoylenko, et al.) . . . . . -D-glucose (Pubchem) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saccharose (Pubchem) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saccharose acetate isobutyrate (Pubchem) . . . . . . . . . . . . . . . . . . . The mass obtained from each extract with sample data . . . . . . . . . . . . A soxhlet extractor set-up (Wikimedia Foundation) . . . . . . . . . . . . . . 4 5 5 5 14 19

List of Tables
1 Serrano-Due nas B. Caapi Extract Concentrations . . . . . . . . . . . . . . . ii 2

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LIST OF TABLES

2 3

Samoylenko B. Caapi Extract Concentrations . . . . . . . . . . . . . . . . . Temperature, mass and volume data for each extraction . . . . . . . . . . . .

3 19

iii

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2 BACKGROUND INFORMATION

Introduction

This investigation hopes to determine the ideal method for extracting 7-methoxy-1-methyl9H-pyrido[3,4b]indole (harmine) from its natural source, a native South American vine known as Banisteriopsis caapi, or ayahuasca. Teas made from B. caapi have been shown to improve symptoms in Parkinsonian patients [9] but these studies did not consider the individual compounds present in the vine. Throughout this study, diering extraction techniques will be analysed to determine which yields the greatest amount of pure harmine. Extracts will be made via steeping. The B. caapi sample will be allowed to sit in distilled water for 15 minutes at a constant temperature of either 85 or 95. Time, volume of water, and mass of sample will be maintained as constants. Afterwards, each extract will be placed into a centrifuge for 30 minutes at RCF G. It is expected that the active compounds in B. caapi (including harmine) will sedimentate out of the extract. Harmine will be isolated from the sediment using column chromatography. Because the individual components of the sediment will not have unique colours, each fraction obtained from the column will be analysed with thin layer chromatography to determine its identity, with a sample of pure harmine obtained from a chemical supplier used as a reference. The samples containing pure harmine will be combined and mass of each sample will be used to compare which temperature yielded the most harmine.

2
2.1

Background Information
Banisteriopsis caapi

While the B. caapi vine itself is known colloquially as ayahuasca, more commonly, the term ayahuasca refers to a tea made from both B. caapi and Psychotria viridis, a plant containing the hallucinogen dimethyltryptamine. The tea is noted for its potent psychoactive and spiritual eects, leading to its use in many traditional Amazonian religions. By itself,

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2 BACKGROUND INFORMATION

dimethyltryptamine is rapidly metabolised by the body by monoamine oxidase. The compounds contained in B. caapi, as well as other plants such as Peganum harmala (Syrian rue), namely, harmine, harmaline, and tetrahydroharmine, inhibit monoamine oxidase, allowing the psychedelic eects to occur. Monoamine oxidase inhibitors are also used in the treatment of diseases such as depression and Parkinsons disease. In a double-blind, placebo-controlled study, 30 newly diagnosed Parkinsonian patients were administered an extract prepared from B. caapi vine. The extract was prepared by steeping 50 g of the vine, which was chopped into small fragments, in 1500 mL of water. The water was boiled for four hours until its volume was reduced to 200 mL. The solution was ltered and administered in that form. [9] The extract resulted in a signicant decrease in rigidity, though resting tremor and postural and action tremors were either unaected or exacerbated. Other abnormal involuntary movements were also reported. All patients experienced nausea and/or vomiting. This extract proved to have less motor side eects than a traditional ayahuasca tea. [9] When analysed with high performance liquid chromatography, the extract was found to contain harmine, harmaline, and tetrahydroharmine in the following levels: Table 1: Serrano-Due nas B. Caapi Extract Concentrations Compound Concentration (g/mL) Harmine 418.44 11.78 Harmaline 173.03 4.13 Tetrahydroharmine 382.40 8.38 Additional chromatography and spectroscopy by Samoylenko, et al. revealed additional compounds exist in B. caapi. Two new -carboline alkaloidal glycosides, named banistenoside A and banistenoside B were discovered, as well as a new disaccharide, named -D-fructofuranosyl-(2 5)-fructopyranose. Five harmine analogs were found: tetrahydronorharmine, harmol, tetrahydroharmine, harmaline, and harmine. Two known proanthocyanidines are also present: ()-epicatechin and ()-procyanidin B2. Finally, two known disachharides exist in B. caapi : sacharose and -D-glucose. The most active extracts 2

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2 BACKGROUND INFORMATION

were made from dried samples of large branch, whole mature stem, and mature stem bark, prepared using a coee maker. Three successive extractions were performed using distilled water at a concentration of 30-70 g/100 mL. [8] Table 2: Samoylenko B. Caapi Extract Concentrations Compound Concentration Banistenoside A 0.71% Banistenoside B 1.93% Tetrahydroharmine 0.10% Harmine 0.67% ()-Epicatechin 0.43% ()-Procyanidin B2 0.15% The chemical structures of these compounds are shown in Figure 1. 1. Banistenoside A 2. Banistenoside B 3. Harmol 4. Tetrahydronorharmine 5. Tetrahydroharmine 6. Harmaline 7. Harmine 8. ()-Epicatechin 9. ()-Procyanidin B2 10. Banistenoside A acetate 11. Banistenoside B acetate 12. ()-Epicatechin acetate 13. ()-Procyanidin B2 acetate 14. -D-fructofuranosyl-(2 fructopyranose 15. Saccharose (See Figure 3) 16. -D-glucose (See Figure 2) 17. -D-fructofuranosyl-(2 fructopyranose acetate 18. Saccharose acetate (See Figure 4) 5) 5)-

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2 BACKGROUND INFORMATION

Figure 1: Structures of Compounds Present in B. Caapi (Samoylenko, et al.)

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2 BACKGROUND INFORMATION

Figure 2: -D-glucose (Pubchem)

Figure 3: Saccharose (Pubchem)

Figure 4: Saccharose acetate isobutyrate (Pubchem)

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2 BACKGROUND INFORMATION

2.2

Thin Layer Chromatography

Thin layer chromatography (TLC) is an analytical technique used to determine how many compounds are in a mixture and as a preliminary identication of unknown compounds. TLC consists of a solid phase, the adsorbent, and a mobile phase, the eluent. The compound to be tested, or spotted, is placed onto the TLC plate on the solid phase, usually silica or alumina gel. The TLC plate is a thin sheet of glass, metal or plastic coated in the adsorbent. Once spotted, the plate is placed into a chamber with a solvent, the eluent, and capillary action allows the eluent to travel up the solid phase and dissolve the compounds. Each compound in a sample varies in its solubility and the strength with which it adsorbs to the stationary phase, resulting in a unique retention factor, Rf , for each compound. [6] Rf is calculated using the following formula [6] distance travelled by compound distance travelled by solvent

Rf =

(1)

Rf is constant only if the following conditions are kept constant: the solvent system (a mixture of solvents used in the eluent), the adsorbent material, the thickness of the adsorbent, the amount of material, and temperature. [6] Because of this, a relative Rf is often used instead and compared to a standard, such as an authentic sample, or to compounds run on the same plate at the same time. To determine purity, a TLC plate is spotted with the compound in question. Once developed, a pure compound will show only spot. A puried compound can then be identied with more TLC. [11] To do so, a TLC plate is spotted with the puried sample and authentic samples of each possible compound. Then, a second plate is developed using three spots: the puried sample, the authentic sample whose Rf was closest to the unknowns, and a mixture of the two compounds. If there is only one row of spots, the compound is pure and might be the same as the authentic sample, but melting point analysis is needed to unambiguously

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2 BACKGROUND INFORMATION

conrm this. [11] TLC is also used to determine an appropriate solvent or solvent system for use in column chromatography, as it is much faster. The best solvent system is simply the one that gives the most separation. [6] During column chromatography, TLC is also used to identify the contents of each fraction [7] using the same technique above.

2.3

Column Chromatography

While TLC is useful for identifying compounds, it does not help to isolate the individual compounds within a mixture. Instead, column chromatography is used. Similar to TLC, column chromatography uses a solid phase and a mobile phase, but allows each compound to be collected after elution. [7] Rather than being on a thin sheet, the solid phase is packed into a vertical column tted with a stopcock valve. The mobile phase then elutes downward through the column and each compound elutes out in order of increasing polarity. [7] Polarity determines both how much the compound is dissolved by the solvent and how much it adsorbs to the solid phase. [11] During column chromatography, several small (1 - 3 mL) fractions are collected. Each fraction can then be spotted on a TLC plate to determine its contents. The fractions that contain the same compound can then be combined and the solvent evaporated to obtain a pure sample. [7] For this reason, it is important to use a volatile solvent.

2.4

Sedimentation via Centrifuge

Particles in a suspension will naturally separate out according to their masses simply because of gravity (a process called sedimentation). [1] A centrifuge uses centripetal force to increase the rate of sedimentation because the particles undergo a force much larger than that of gravity. Rotation about diering radii results in diering forces, so the force experienced by the particles is usually referred to instead of the revolutions per minute. [1] This is known as the relative centrifugal force, or RCF, and is calculated according to the following formula [2] , 7

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3 METHODOLOGY

where r is in centimetres, N is RPM and RCF is a multiple of gravitational acceleration:

RCF = (1.11824396 105 )rN 2

(2)

3
3.1
3.1.1

Methodology
Extraction via Steeping
Necessary Apparatus

250 mL Beaker 250 mL B uchner Flask and Funnel Sink with Aspirator Medium Speed Filter Paper Rubber Hosing Hot Plate Scale 100 mL Graduated Cylinder 100 mL Distilled Water 5 g B. caapi Timer Thermometer

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3 METHODOLOGY

3.1.2

Technique

1. Measure out 100 mL of distilled water 2. Measure out 5 g of B. caapi 3. Add the water to the beaker 4. Heat the water to either 85 or 95 5. Once the water has reached the correct temperature, quickly add the B. caapi and stir 6. In order to maintain a constant temperature, it may be necessary to use multiple hot plates set to dierent temperatures; measure the temperature regularly to ensure it remains constant 7. After 15 minutes have elapsed, use the B uchner ask to lter any remaining water 8. Centrifuge the extract

3.2
3.2.1

Sedimentation via Centrifuge

Necessary Apparatus

Up to 12 D cm x L cm Test Tubes Distilled Water Funnel Beral Pipette Centrifuge with Known Rotational Radius 250 mL Beaker

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3 METHODOLOGY

3.2.2

Technique

1. Fill an even number of test tubes with roughly the same amount of uid (if the extract can only ll an odd number of test tubes, load an additional tube with just water; if it can only partially ll a tube, ll the remainder of the tube with distilled water) 2. Load the centrifuge so that for each test tube, there is a test tube immediately opposite (CAUTION: It is very important that the centrifuge be evenly loaded; this is why the test tubes must be lled with the same volume of uid. Otherwise, the centrifuge will be o-balance, which could damage it or create a potentially hazardous situation.) 3. If available, set the timer on the centrifuge to 30 minutes 4. Using the RCF equation, set the centrifuge to an appropriate RPM to obtain an RCF of approximately 1700 G 5. After 30 minutes have elapsed, decant the supernatant uid into the beaker; the supernatant will not be analysed 6. Run the sediment through column chromatography and analyse the contents through TLC

3.3
3.3.1

Isolation via Column Chromatography

Necessary Apparatus

Liquid Chromatography Column or Burette Silica Gel Acetone Ethyl Acetate Glass Wool

10

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3 METHODOLOGY

Glass Rod Powder Funnel 250 mL Beaker 50 mL Beaker Many Test Tubes Wax Pencil Stand and Clamp Scale

3.3.2

Technique [10] [5]

1. Fix column to stand 2. Place glass wool in bottom of column 3. Fill the column three-fourths full with ethyl acetate 4. Tamp wool with glass rod to remove air bubbles 5. Mix the silica gel and the ethyl acetate in the 250 mL beaker to form a slurry 6. Drain some of the solvent through the column 7. Slowly add slurry 8. Dissolve the sample in the minimum amount of acetone in the 50 mL beaker 9. Add about 100 mg of silica gel 10. Swirl until the acetone evaporates and leaves behind a dry powder

11

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3 METHODOLOGY

11. Add this powder to the top of the column 12. Begin adding ethyl acetate to the column 13. Continually add ethyl acetate to the column (do not let the top of the column become exposed) 14. As the sample elutes through the column, collect small (1-3 mL) fractions in the test tubes (pay close attention to ensure equal fraction size) 15. Label each container with the wax pencil in the order of collections 16. Analyse each fraction with TLC and combine like fractions

3.4
3.4.1

Determination of Purity via Thin Layer Chromatography

Necessary Apparatus

100 mL Beaker 5 cm x 20 cm TLC Plate Scissors 10L Microcapillary Pencil Ruler Acetone Watch Glass UV Lamp

12

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4 RESULTS

3.4.2

Technique [6]

1. Pour the acetone to a depth of just below 5 mm in the 100 mL beaker 2. Cover the beaker with a watch glass, swirl and let stand 3. Cut the plate to 5 cm x 5 cm 4. Draw a horizontal line (the origin) 5 mm from the bottom of the plate 5. Use the microcapillary to transfer a small amount of solution onto the origin (about 4 samples) 6. Place the plate in the beaker and allow to sit undisturbed until the solvent front is about 5 mm below the top of the plate 7. Using the UV lamp, circle any visible spots 8. Calculate Rf

4
4.1

Results
Qualitative Analysis

Both temperatures produced a golden yellow extract, though the extract made at 95was slightly darker in hue. After centrifuging, a small amount of reddish-brown sediment settled at the bottom of the test tube. No dierences in colour were noted between the sediments. In attempting to perform column chromatography, it was found that the sediment would not dissolve in acetone, ethyl acetate or a mixture of the two. As such, no quantitative data could be obtained; what follows is analysis of sample data.

13

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5 INTERPRETATION OF RESULTS

4.2

Quantitative Analysis

As seen in Figure 5, the extract made at 85 yielded 34 mg of pure harmine, whereas the extract made at 95 yielded 36.0 mg of pure harmine. This gives a percent yield of 0.67% and 0.72% respectively, which approximates the results found by Samolyenko, et al. [8] for harmine content of Banisteriopsis caapi.

Figure 5: The mass obtained from each extract with sample data

Interpretation of Results

The higher temperature extract likely gave an increased yield due to an increase in solubility. For most solids, dissolution is endothermic, so an increase in temperature facilitates dissolution by adding more energy to the solution. [4] At a higher temperature, more of the compounds in B. caapi were able to dissolve into the water.

5.1

Conclusion

With a percent yield of 0.72%, extraction at a constant temperature of 95 for 15 minutes proved to be the most ecient method for extracting harmine, according to the sample data. 14

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6 EVALUATION

In reality, no data could be obtained because the sediment proved insoluble.

5.2

Further Questions

While this experiment gave no denitive results, it could be possible that other method of extraction exist that yield even higher amounts of harmine than proposed. Could a higher water temperature increase the yield? Is there a maximum time beyond which no more harmine is yielded (does steeping for 5 minutes give the same yield as steeping for 15 minutes)? What of the physical nature of the vine: is there a dierence in yield for nely ground vine compared to the coarsely shredded vine used in this experiment? What if the extract were prepared by steeping into another solvent, such as ethanol or acetone? Other methods of extraction exist as well. Would the use of a Soxhlet extraction apparatus (See Figure 6 in Appendix B) give a better yield? Which solvent would work best and for how many cycles? Is the best solvent for TLC the best solvent for Soxhlet extraction?

6
6.1

Evaluation
Error

The results that might have been obtained in this experiment could have been simply a product of chance. Performing multiple trials are needed to reach a more conclusive result and to perform statistical tests to conrm that the results are not simply random. This experiment would have analysed only one sample across a small range of variables, so it is dicult to determine the accuracy of the results. Additionally, a better solvent for column and thin layer chromatography could have been found experimentally with TLC. Column chromatography also generally calls for 70-240 mesh silica gel [5] , which has smaller pores and provides better separation, though only 3-8 mesh was available at the time. Furthermore, column chromatography is not the most appropriate procedure for analysing liquid samples, but instead a technique known as liquid chromatog15

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6 EVALUATION

raphy (most often high performance liquid chromatography (HPLC)) is used, which requires specialised equipment that was not available. Many of the compounds contained in B. caapi are chemically similar and may have very close Rf values in TLC, making it dicult to distinguish them from each other; further conrmation of identity could have been performed with mixed melting point analysis or with nuclear mass resonance spectrometry (which again requires specialised equipment generally not available to secondary schools). Had HPLC been used, the centrifuge would not have been necessary and whatever product that may still have been in the supernatant would not have been lost. Additionally, the centrifuge may not have been capable of producing enough force to fully sedimentate all of the components of the extract; at full speed, the centrifuge produced about 1725 G at 3500 RPM. Therefore, while this experiment initially seemed promising, the insolubility of the sediment was unforeseen, and no real data was produced. Many additional questions and sources of error exist that limit this experiment from providing a complete answer to the question.

6.2

Additional Concerns

Aside from lack of specialised equipment which may have led to results diering from those obtained by Samolyenko, et al. and Serrano-Due nas, et al., it should be noted that the experiment was not performed in one continuous block of time. In between sessions, the extract was stored in a refrigerator maintained at approximately 0. The pure harmine and the sediments obtained from the centrifuge were not refrigerated. The hot plate was initally heated to a temperature well above 85 or 95 to accelerate the process. However, the hot plate cannot cool as rapidly as it can heat up, so multiple hot plates were used to obtain an average temperature of approximately 85or 95. The actual average temperature for each extraction was 87.5 and 95.1 respectively. The raw temperature readings, taken at 5 minute intervals with an analog thermometer are given in Table 3 in Appendix A. 16

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7 BIBLIOGRAPHY

Word Count: 2737

Bibliography

[1] Kelly Gleason. How to convert centrifuge rpm to rcf or g-force? http://clinfield. com/2012/07/how-to-convert-centrifuge-rpm-to-rcf-or-g-force/, July 2012. [2] Millicent Hughes, MD. Centrifuges in medicine and nuclear physics. http://samhs. org.au/Virtual%20Museum/Medicine/centrifuge/centrifuge.html. [3] Con-Test Analytical Laboratory. Soxhlet extractions explained. http://www.

contestlabs.com/assets/technical_papers/Con-Test-Soxhlets.pdf, 2007. [4] Charles E. Ophardt, PhD. Temperature and pressure eects on solubility. http://www. elmhurst.edu/~chm/vchembook/174temppres.html, 2003. [5] Jacqueline Richardson, PhD. Column chromatagrophy procedures. http://orgchem. colorado.edu/Technique/Procedures/Columnchrom/Procedure.html, 2013. [6] Jacqueline Richardson, PhD. Thin layer chromatography (tlc). http://orgchem. colorado.edu/Technique/Procedures/TLC/TLC.html, 2013. [7] Sheryl A. Rummel. Column chromatography. http://courses.chem.psu.edu/

chem36/Experiments/PDFs_for_techniques/CC.pdf, 2013. [8] Volodymyr Samoylenko, Muhammad Mostazur Rahman, Babu L. Tekwani, Lalit M. Tripathi, Yan-Hong Wang, Shabana I. Khan, Ikhlas A. Khan, Loren S. Miller, Vaishali C. Joshi, and Ilias Muhammad. Banisteriopsis caapi, a unique combination of mao inhibitory and antioxidative constiuents for the activities relevant to neurodegenerative disorders and parkinsons disease. Journal of Ethnopharmacology, 2009.

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7 BIBLIOGRAPHY

[9] Marcos Serrano-Due nas, Fernando Cardozo-Pelaez, and Juan R. S anchez-Ramos. Effects of banisteriopsis caapi extract on parkinsons disease. The Scientic Review of Alternative Medicine, 2001. [10] Hayley Wan, PhD. Column chromatography. http://www.chem.ualberta.ca/

~orglabs/Techniques%20Extra%20Info/Column%20Chromatography.html, 2013. [11] Hayley Wan, PhD. Thin layer chromatography. http://www.chem.ualberta.ca/ ~orglabs/Techniques\%20Extra\%20Info/TLC.html, 2013.

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8 APPENDICES

8
8.1

Appendices
Appendix A - Actual Extraction Data
Mass of B. caapi 5.00 g 5.04 g Temp. 1 85.7 92.3 Temp. 2 87.2 98.0 Temp. 3 88.5 93.5 Temp. 4 88.7 6.2 Avg. Temp. 87.525 95.15

Volume of Water 100 mL 100 mL

Table 3: Temperature, mass and volume data for each extraction

8.2

Appendix B - Soxhlet Extraction

A Soxhlet extractor is a specialised piece of glassware invented by Franz von Soxhlet. It is attached to a ask containing the solvent and then to a condenser. The solid material is placed in a lter paper thimble within the extraction apparatus. The solvent is heated and the reuxes through the condesner. As the chamber lls with solvent and solvent vapours, the compounds in the solid are dissolved into the solvent. When the chamber is full, the solvent automatically drains back into the ask where it continues to cycle through the apparatus. At the end of extraction, the solvent can then be evaporated, leaving a concentrated extract behind. [3]

Figure 6: A soxhlet extractor set-up (Wikimedia Foundation)

19