Liquid-Liquid and Solid-Liquid Extraction

Jennifer Driskell
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 CHEM 213B.102 | TA: Omair Khan Original Submission: February 19, 2011 Resubmission: March 24, 2011 Abstract Liquid-liquid extraction techniques were used to extract an acid, base and neutral from an unknown mixture. The technique was done on a microscale level using test tubes and pipets to separate and extract the layers of interest. Solid-liquid and liquid-liquid extraction were used to extract caffeine from tea leaves. The caffeine was extracted on a macroscale level using beakers and separatory funnels. The crude caffeine was sublimed to obtain a pure caffeine sample. For identification of the compounds extracted (unknowns and caffeine), melting point values were obtained using a Mel-Temp apparatus. Two of the compounds isolated from the unknown mixture were also analyzed using a 60 MHz 1H NMR. Introduction Whether making coffee, isolating a natural product or separating compounds in solution, the underlying technique being applied is extraction. Extraction is the process of pulling one compound away from or out of another compound.1 It is widely used both in everyday life and the laboratory. Two common examples of extraction are liquid-liquid extraction and solid-liquid extraction. A Pittsburg laboratory demonstrated liquid-liquid extraction when the scientists were able to isolate urea from a fluorous-amine scavenger.2 In their reaction, they prepared urea by mixing an amine (limiting reagent) with an excess of isocyanate. Once the reaction was

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complete they added an excess of fluorous amine quenching reagent to change the un-reacted isocyanate into a fluorous urea. The liquid-liquid extraction was then used to separate the organic urea product from the fluorous urea and amine.2 An example of solid-liquid extraction is brewing coffee. Solid-liquid extraction has two phases a solid phase, the coffee beans, and a liquid phase, the water. The water is able to dissolve small, polar molecules, like caffeine, away from the coffee beans due to similar molecular interactions. 1 Liquid-liquid extraction works using solubility differences of the compounds being separated. 2 Manipulation of a compound can cause it to move into a desired layer allowing it to be extracted. Determining which layer the compound resides or how much of it will be there can be calculated using, the distribution coefficient, K.1 The distribution coefficient is the distribution of the solute, in the solvent, at equilibrium. A small distribution coefficient means that not all of the solute resides in the organic layer. Multiple extractions might be necessary to completely extract the entire compound.1 The distribution coefficient is used in choosing acid-base reactions to manipulate compounds into the desired layer. Acid-base reactions use functional groups to manipulate the solubility of the molecule. By deprotonating or protonating a functional group we can affect the pH, which in turn will change the distribution coefficient, pulling molecules into the water layer. An acid is a compound that will donate a hydrogen ion to a compound capable of accepting a hydrogen ion. A base is a compound that has the ability to accept a hydrogen ion from a hydrogen donor.3 Compounds with similar functional groups, such as carboxylic acids, will not be separated by this kind of manipulation because they will both move into the same layer. Neutral compounds are unique in that they will not be affected by the acid-base reactions because they do not lose or gain protons.1 Scheme 1 provides a flow chart to give two examples of acid-base reactions.

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Hydrochloric acid, a strong acid, donates its hydrogen to the base, 3-aminoacetophenone. The conjugate acid of the base now has a positive charge allowing it to be pulled into the water layer. After extraction of the water layer, containing the conjugative acid, the addition of a strong base, K2CO3, will deprotonate the conjugative acid returning it to the original base. By deprotonating the hydroxyl group on the benzoic acid with a weak base, NaHCO3, a negative charge is created on the oxygen atom pulling it into the water layer. After extraction of the water layer, with the conjugative base, the addition of a strong acid, HCl, will protonate the oxygen atom leaving the original uncharged acid.3 The addition of acids and bases can help to extract one compound from another, however, it does not guarantee purity of the compound. A simple and inexpensive purification technique that can be used to remove impurities in a compound is sublimation.1 Sublimation is the direct phase change from a solid to a gas, without passing through a liquid phase, at a particular pressure. This experiment sublimed caffeine to obtain pure crystals from the crude caffeine extracted from tea leaves using solid-liquid and liquid-liquid extraction techniques. At atmospheric pressure, the impurities in the crude product had lower sublimation points than caffeine meaning they would have to pass through the liquid phase before becoming gaseous.1 In order for the impurities to sublime they would need a lower pressure. Caffeine on the other hand had a higher sublimation point and was able to become gaseous instead of liquid at atmospheric pressure. When the gas vapors touched the walls of the test tube they were cooled. The cooler temperature caused the phase change from the gas back into a solid this time without the impurities.1 In this experiment, three compounds were isolated from a mixture using acid-base reactions and liquid-liquid extraction techniques. The compounds were then identified using a 60 MHz 1H Nuclear Magnetic Resonance Analysis (NMR) and melting points were obtained

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using a Mel-temp apparatus. The second part to this experiment was the extraction of caffeine from tea leaves, this involved solid-liquid extraction followed by liquid-liquid extraction as the first step of purification. The caffeine was finally purified using sublimation and the crystals were identified using melting points obtained from a Mel-temp apparatus. Results and Discussion Isolation of Compounds from Unknown 34 The isolation of Unknown 34 can be easily followed using Scheme 1 to keep track of which acids and bases are being added and which layer the compound resides after the reaction. The first step was to isolate the three unknowns in the mixture this was done according to the procedure found in the experimental section. After isolation two 60 MHz 1H NMR analyses were preformed, one on the isolated amine and one on the isolated neutral. NMR number one was used for both analyses using d-chloroform as the solvent.

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Scheme 1 Flow Chart for Isolation of Unknown 34

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This is a visual diagram that will assist following the acid and base reactions that occurred when isolating compounds from the Unknown 34 mixture. The mixture came with a base, an acid and a neutral compound that had to first be isolated before they could be identified.

The NMR analysis of the neutral compound, Figure 1, assisted in determining that the neutral compound was fluorenone. This spectral analysis contains a lot of contamination which can be contributed to the other compounds not being completely extracted. Two of the contamination peaks can be attributed to the base and one attributed to the acid. There was one contamination peak that could not be accounted for as coming from the acid or base in particular. The theoretical shift and splitting was unknown for this particular contamination. The NMR disproved the compound 1, 4-Dimethoxybenzene because it did not display a methyl hydrogen

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peak that would be apparent around 3.5 ppm. All of the peaks of importance are jumbled closely together, so specific splitting patterns were not determined. The peaks can all be found in the 78 ppm range which was the appropriate range for aromatic hydrogens. This information can be viewed in Table 1. The NMR obtained for the amine compound, Figure 2, assisted in determining that the compound isolated was 3-Aminoacetophenone. All six peaks of interest are accounted for on the spectrum and displayed in Table 2. The spectrum disproves 4Aminoacetophenone because there was no Para splitting of the aromatic hydrogens. The only problem with the NMR of the base was incorrect integration ratios. A possible explanation for the incorrect ratios could be when the spectrum was normalized to account for the hydrogens in the amino group, Hf. It is possible that the machine did not record two hydrogens instead it viewed them as one. When that peak was normalized to two it added an additional hydrogen to the other integrated peaks. After subtracting one hydrogen from the other integrated peaks the numbers produced were expected values for 3-Aminoacetophenone. The actual shift for hydrogen A was higher than the theoretical shift. This is possibly due to the electronegative oxygen atom interacting with the methyl hydrogens. Hydrogen E did not have the theoretical splitting of a triplet. This hydrogen might have been able to interact with the amino group resulting in more splitting than what would normally be expected. Peaks pertaining to hydrogen B, C, and D are all within the circle and although not easy to determine they did in-fact have correct splitting.

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Figure 1 1H NMR Spectral of Neutral Compound

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This was the compound remaining after the extraction of both the acid and base. The likelihood of contamination would be greatest since the other compounds were extracted, leaving this one in solution. The spectrum has been annotated for fluorenone and contamination has been marked and identified in Table 1.

Table 1 1H NMR Spectral Data for the Neutral Compound This is the tabulated description of results from the neutral NRM analysis, Figure 1. The peaks of interest are closely packed around 7.5 ppm; the other peaks listed are noted as contamination. This contamination was contributed to the base and acid not being completely extracted from the neutral.

Hydrogen A B C D Contamination Methyl from Base Contamination unknown Contamination Aromatic from Base Contamination Aromatic from Acid

Theoretical Shift (ppm) 6.5-8.5 6.5-8.5 6.5-8.5 6.5-8.5 0.7-1.3 Unknown 6.5-8.5 6.5-8.5

Actual Shift (ppm) ~ 7.5 ~7.5 ~7.5 ~ 7.5 2.5 5.9 ~6.8 ~8.1

Theoretical Splitting doublet triplet triplet doublet singlet unknown triplet doublet

Actual Splitting unknown unknown unknown unknown singlet singlet multiplet multiplet

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Figure 2 NMR Spectral of Amine Compound

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This was the first compound extracted after isolation involving HCl. The chance of contamination for this molecule was low and no contamination peaks were observed on the spectrum. The printout has been annotated for 3Aminoacetophenone with all hydrogens accounted for and displayed in Table 2.

Table 2 NMR Spectral Data for the Amine Compound This is the tabulated description of results from the amine NRM analysis. The peaks of interest are labeled with letters corresponding to Figure 2. All of the expected peaks are visible and most theoretical splitting was observed. The biggest problem was the incorrect integration ratios.

Hydrogen A B C D E F

Theoretical Shift (ppm) 0.7-1.3 6.5-8.5 6.5-8.5 6.5-8.5 6.5-8.5 0.5-4.0

Actual Shift (ppm) 2.5 ~6.860 ~7.26-7.31 ~6.90-7.22 ~ 6.80 3.81

Theoretical Splitting singlet singlet doublet triplet triplet Singlet

Actual Splitting singlet singlet doublet triplet multiplet Singlet

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Melting points were obtained for all three compounds isolated from Unknown 34 and compared to literature values from Sigma-Aldrich. Melting points help determine a compound based on the range and comparison to literature values. The range will be considerably lower and broad due to impurities in the sample. The melting point data for Unknown 34 can be found in Table 3. The melting point range of fluorenone was considerably off compared to the SigmaAldrich values obtained. The NMR had contamination peaks which indicated impurities helping to explain the low melting point. The amine had a melting point range close to the expected value of 3-Aminoacetophenone helping to verify the NMR results. The melting point was slightly lower than normal, this could be contributed to the Mel-temp used not being completely accurate. The acid was the only compound isolated that did not have an NMR analysis preformed. The identity of this compound was completely derived from the melting point value obtained from the Mel-temp apparatus. The melting point range was higher than typically associated with benzoic acid. However, 3-toluic acid melts lower than benzoic acid therefore it would be safe to conclude that the isolated acid compound from Unknown 34 was benzoic acid. The higher melting point range could be associated with the sample not being completely dry allowing the water to interact with the molecule affecting the melting point. Melting points assist in identification of a compound but percent recoveries are calculated to determine how much the compound was obtained after the extraction.
Table 3 Melting Point Data for Unknown 34 The melting point data was obtained using Mel-temps and are listed along with the expected temperature values gathered from Sigma-Aldrich. Fluorenone was very low most of which can be contributed to contamination. The base and acid are relatively close to their literature values.

Compound Neutral- Fluorenone Amine- 3'-Aminoacetophenone Acid- Benzoic Acid

Melting Temperature C 47-51 94-96 126-128

Sigma-Aldrich Temperature (C) Values 4 80-83 97 121

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Percent recoveries were calculated for each of the compounds isolated from Unknown 34. Equation 1 shows the calculation for percent recovery and Table 4 has the calculated values for each percent recovery of the isolated unknowns. The percent recovery for the amine was above the possible 100%. The first reason for this high percent recovery would be that the mixture of Unknown 34 did not contain exactly 100 mg of each of the three compounds. This would result in the wrong original starting weight of the compound causing the calculation to be incorrect. A second possibility could be the measurements were inaccurate, resulting in faulty numbers. The amine was the only compound greater than 100% recovery; the acid had a low percent recovery at 13%. Reasons for this being so low are numerous; one reason could be that not all of the acid was extracted from the neutral. A second possibility was that crystals were removed along with the boiling stick and glass stirring rod that were needed to mix and boil the solution. Crystals could also have been lost on the final step of removal from the Hirsch funnel. The acid and base demonstrated the two extremes of percent recovery, however, the neutral, had a good percent recovery at 81%. This number was not accurate due to the NMR analysis and melting point values obtained demonstrating contamination. The contamination can be contributed to acid and base compounds that were not extracted completely remaining in the neutral crystals when dried. The extra crystals from the acid and base would increase the final weight of the neutral and not give an accurate value recovered.
Table 4 Percent Recovery Data for Compounds Isolated from the Unknown Percent recoveries for each of the compounds isolated from Unknown 34 are presented in the table. The theoretical weight was obtained by assuming one third of each compound was used in the mixture since 300 mg was initially weighed out. The percent recoveries were calculated using Equation 1.

Compound Neutral- Fluorenone Amine- 3'-Aminoacetophenone Acid- Benzoic Acid

Theoretical Weight (g) 0.1 0.1 0.1

Weight of Crystals (g) 0.081 0.116 0.013

Percent Recovery % 81 116 13

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Equation 1 Percent Recovery

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Isolation of Caffeine Isolation of caffeine from tea leaves worked using solid-liquid extraction because the tea leaves are made up of many compounds including cellulose, proteins and lipids that cannot dissolve into the liquid water phase. When the tea leaves were placed in the boiling water to soak, the caffeine compound dissolved out of the leaves and entered the liquid which was decanted and used for the experiment. Although solid-liquid extraction worked to extract caffeine from the tea leaves, it also extracted any other compound that was able to dissolve in the water. Liquid-liquid extraction was used as a complementary extraction to obtain only caffeine from the solution. In the liquid-liquid extraction, dichloromethane (DCM) was used to separate caffeine from the many other water soluble compounds that also dissolved from the tea leaves. After isolation the caffeine was sublimed to obtain pure crystals from the crude caffeine product. The caffeine extracted from tea contained many impurities even after liquid-liquid extraction. These impurities had lower sublimation points than the pure caffeine. Since caffeine has a higher sublimation point it was able to change from a solid to a gas before the impurities had a chance to change phases. The sublimation works by allowing the caffeine to directly change from a solid to a gas at atmospheric pressure without becoming liquid. Once the gas molecules interacted with the cool walls of the test tube the lower temperature caused the gas to solidify forming long, delicate, white crystals. The pure caffeine crystals were weighed to determine the percent recovery and tested for purity with a melting point, this data can be found in Table 5. The literature melting point range for caffeine is 234-236C, the melting point for the extracted caffeine was 226-233C.4 The slight melting point depression could be due in part to the Mel-temp used not being completely

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accurate. It could also be that the crystals were still wet and the water interfered with the melting temperature. Although the melting point was off, it fell into the accepted literature values when the standard deviation, of +/- 5 was applied. To determine how much caffeine was extracted from the tea the percent recovery was calculated, using Equation 1, and found to be very low. This was probably due to the repeated transfer of the compound from the beaker to the flask to the test tube, along the way product was lost. Another possibility was that during the sublimation the caffeine could have been evaporated out of the test tube resulting in loss of product. The data for caffeine can be found in Table 5.
Table 5 Caffeine Data The theoretical yield for caffeine was determined by multiplying the amount of caffeine in one tea bag by three (55 mg x 3) since the experiment used three tea bags. The percent recovery was calculated using Equation 1. The melting point values were obtained from the Mel-temp and the expected values were obtained from Sigma-Aldrich.

Compound Caffeine

Theoretical Weight (g) 0.165

Weight of Percent Melting Sigma-Aldrich crystals (g) Recovery % Point C Value C 4 0.012 7.27 226-230 234-236

The Utility of the Solid-Liquid and Liquid-Liquid Isolation Methods To assess the solid-liquid and liquid-liquid isolation methods, it would be best to look at the percent recoveries that were calculated from the data. For the isolation of the three unknown compounds from Unknown 34, it can be stated that the liquid-liquid extraction technique was not great. Table 4 shows the results for percent recoveries and none of the values were good. The best value, the neutral, was not accurate due to evidence of contamination. For the isolation of caffeine from tea leaves, the effectiveness of the solid-liquid and liquid-liquid extraction was not good; the percent recovery was only 7%. These isolation methods worked to isolate the compounds but need to be improved upon to be able to gather a better recovery of the product.

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This experiment used liquid-liquid extraction to isolate a base, acid and neutral from an unknown. The identities were determined using NMR analyses and melting points. The compounds were determined to be 3-Aminoacetophenone, benzoic acid and fluorenone. Percent recoveries were calculated for each isolated compound and were: 116% (base), 13% (acid) and 81% (neutral). Solid-liquid and liquid-liquid extraction were both used to isolate caffeine from tea leaves. The solid-liquid extraction isolated the caffeine from the leaves into the water and the liquid-liquid extraction isolated the caffeine from the water. The caffeine sublimed in the test tube forming long, white needles on the inside of the tube which were used to determine the melting point. The sublimed crystals were determined to be caffeine due to the melting point of 226-230C. The percent recovery of caffeine was low at about 7%. The experiment obtained results that were expected, but can be improved upon. For future experiments, the isolation of the unknowns should be done on a larger scale so that the separatory funnel can be used to separate the two layers, this will provide more accurate extraction compared to pipets. For the caffeine procedure, trying a couple of different experiments with different types of tea leaves and coffee beans to determine how much caffeine is extracted from each type would be interesting. This experiment is practical in that most people use these simple techniques everyday without actually knowing the science behind what they are drinking. Experimental Section Procedure 1: Microscale separation of basic, acidic, and neutral substances The mixture of Unknown 34 (300 mg) was weighed out and put into a test tube which was dissolved in ether (2 mL).

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Section A Separation of the Basic Component by Extraction with Acid: 5% aqueous HCl (1.0 mL) was added to the test tube containing the unknown and ether. Once layers separated, after mixing, the bottom layer was extracted into a second test tube. Water (.25 mL) was mixed with the remaining solution in the original test tube and the bottom layer was extracted into the second test tube. Ether (6 drops) was added to the second test tube and allowed to evaporate in the hood. Section B Separation of the Acid Component by Extraction with Base: 10% aqueous sodium bicarbonate (1.0 mL) was mixed into the original test tube now containing the acid and neutral. Once the layers separated, the bottom layer was extracted into a third test tube. Sodium bicarbonate (6 drops) was mixed to tube one, after separation the bottom layer was extracted into the third test tube. The third tube was backwashed with ether (6 drops) and allowed to evaporate in the hood. Section C Isolation of the Neutral Component: Saturated NaCl (1 mL) was mixed to the original test tube now containing just the neutral. Once the layers separated, the bottom aqueous layer was extracted into a beaker. Anhydrous sodium sulfate (about 0.75 mL) was added to the first test tube, which was corked to shake and vent for 10 minutes. The ether solution was then decanted from the drying agent into a fourth test tube. After drying, the crystals were weighed (0.081 g, 81% recovery). A melting point of 47-51C was obtained and an NMR was conducted. This data can be found in Figure 1 and Tables 1, 3 and 4. Section D Isolation of the Amine Component: 50% potassium carbonate (4 drops) was mixed to tube two and the pH was tested using litmus paper. The tube was corked, to shake and vent for one minute, and then placed in an ice bath. The cold solution of crystals was added to a clean 100 mL beaker and allowed to dry. Once dry the crystals were weighed (0.116 g, 116%

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recovery). A melting point of 94-96C was obtained and an NMR was conducted. This data can be found in Figure 2 and Tables 2, 3 and 4. Section E Isolation of the Carboxylic Acid Component: Concentrated HCl (.128 mL, 0.0015) was mixed to tube three and the pH was tested using litmus paper. The calculation for the amount of HCl added can be seen in Equation 2. The tube was placed in an ice bath to form crystals, after which the liquid was extracted and discarded. Distilled water (about 1.0 mL) was added to the tube and placed in a sand bath to dissolve the crystals. After cooling in an ice bath, a Hirsch Funnel was used to collect the crystals. Once dry the crystals weight was obtained (0.013g, 13% recovery) and a melting point of 126-128C was recorded. This data can be found in Tables 3 and 4. Procedure 2: Isolation of Caffeine from Tea Three tea bags were allowed to soak in boiling water (75 mL) for 10 minutes and then the liquid was decanted into a 125 mL Erlenmeyer flask. When the solution cooled it was poured into a separatory funnel with dichloromethane (15 mL). The mixture was swirled and vented for two minutes and then allowed to separate. The bottom layer was extracted into a clean flask. The solution was dried and formed yellowish/green crystals. DCM was added to bring the crystals into solution to be transferred to a test tube. The test tube was placed in a sand bath and the DCM was boiled off. The contents of the tube were allowed to sublime for 30 minutes. Long, white needles formed on the inside of the tube. After cooling and collecting the crystals they were weighed (0.012 g, 7.27% recovery) and a melting point of 226-230C was obtained. This data can be found in Table 5.
Equation 2 Amount of HCl Needed for Acidification

Experiment 5 Extraction References

1 Linclau,

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Bruno; Sing, Ashvani K.; Curran, Dennis P. Journal of Organic Chemistry. 1999, 64

(8), 2835-2842.
2

Minard, B.; Masters, K.M.; Halmi, T.O.; Williamson, K.L. Lab Guide for Chemistry 203 and 213B, 2010-2011, 111-138.

Brown, Theodore L.; LeMay, H. Eugene Jr.; Bursten, Bruce E.; Murphy, Catherine J. Chemistry The Central Science, 11ed. 2009: Pearson Prentice Hall, 129-130.

Sigma-Aldrich. Sigma-Aldrich Co, 2011. February 1, 2011- February 14, 2011. http://www.sigmaaldrich.com/united-states.html.

https://cms.psu.edu/section/default.asp?id=MRG%2D110110%2D102159%2DSAD270, Addendum to the Lab Guide, Chem 213 and Chem 213B, spring 2011, Experiment 5.