Fish 110 Lab Postlab Discussion Experiment 1 and 2 PDF

Post-Lab Discussion: Experiment 1 and 2 1 Experiments Conducted Experiment 1: Solvent Extraction Experiment 2: Pigment Extraction 2 Expt. 1: Solvent Extraction Define: a. Extraction b. Liquid-liquid extraction; provide examples. c. S...

Post-Lab Discussion: Experiment 1 and 2 1 Experiments Conducted Experiment 1: Solvent Extraction Experiment 2: Pigment Extraction 2 Expt. 1: Solvent Extraction Define: a. Extraction b. Liquid-liquid extraction; provide examples. c. Solid-liquid extraction; provide examples. 3 Expt. 1: Solvent Extraction Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM 4 Expt. 1: Solvent Extraction Dissolving in Extraction Weighing of 5 mL H2O % yield and Kp using EtAC Samples and 5 mL determination and DCM EtOH Salicylic Acid Sucrose: Glucose + Fructose Benzoic acid HOC6H4COOH C12H22O11 C6H5COOH 5 Expt. 1: Solvent Extraction Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM Any observations after being dissolved in H2O and EtOH? 6 Expt. 1: Solvent Extraction (Above) Separation of aqueous (H2O) and organic (Ether, low density) solvents (Right) Separation of aqueous (H2O) and organic (DCM, high density) solvents 7 Expt. 1: Solvent Extraction Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM At microscale extraction process, two general steps were conducted: 1. mixing the two immiscible solutions, and 2. separating the two layers after the mixing process. evaporated 8 Expt. 1: Solvent Extraction Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM Where did the solutes (SA, BA, sucrose) dissolve in? evaporated 9 Expt. 1: Solvent Extraction Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM evaporated 10 Expt. 1: Solvent Extraction Density of common extraction solvents 11 Expt. 1: Solvent Extraction 1. Sample Preparation Dissolving in 5 Extraction Weighing of % yield and Kp mL H2O and 5 using EtAC Samples determination mL EtOH and DCM 12 Expt. 1: Solvent Extraction Sample calculation Suppose 50 mg of compound A in 10 mL aqueous solution is extracted using 10 mL t-butyl methyl ether at room temperature. After extraction, 75.6% was extracted. Questions: a. How much is the extracted compound in mg? in g? b. Solve for what remains in the aqueous solution. c. Solve for Kp and interpret the generated value. 13  Expt. 1: Solvent Extraction Partition/Distribution coefficient X = solute Organic phase “partitioning” Aqueous phase determines the appropriate solvent in LLE KP is not the same as yield. KP is just a ratio. Separation of different phases in liquid-liquid extraction (Chemlibre Texts: Overview of Extraction_ 14  Expt. 1: Solvent Extraction Starting material: 4.60 g crude pigment KP value (Hexane/Water) system? Organic phase 3.12 g (Hexane) Aqueous phase (Water) 1.48 g Yield (organic)? Yield (aqueous)? 15  Expt. 1: Solvent Extraction Starting material: 4.60 g crude pigment 3.79 g 0.55 g 0.81 g 4.05 g KP value (Diethyl ether/Water)? KP value (DCM/Water)? Yield (DEE)? Yield (DCM)? 16  Expt. 1: Solvent Extraction Organic Solvent is Multiple Extraction Heavier than Water separation of an organic solvent (DCM) denser (1.33 g/mL) than aqueous/water solvent Reference: Nichols, L. 2023. Organic Chemistry Lab Techniques. Open Education Resource (OER) LibreTexts Project (Book 17 Link)  Expt. 1: Solvent Extraction Organic Solvent is Lighter than Water 3. Multiple Extraction separation of an organic solvent (ether) lighter (0.713 g/mL) than aqueous/water solvent Reference: Nichols, L. 2023. Organic Chemistry Lab Techniques. Open Education Resource (OER) LibreTexts Project (Book 18 Link) Expt. 1: Solvent Extraction Solubility Properties SA has more polar components, why is it less soluble in water? Slightly (low) soluble to water Highly soluble Slightly (low) soluble to water 3.44 mg/mL (25 °C) to water 2.48 mg/mL (25 °C) 1.7 g/L (0 °C) ∼2,000 mg/mL 4.14 mg/mL (40 °C) 56.31 g/L (100 °C) (25 °C) 17.41 mg/mL (75 °C) 77.79 mg/mL (100 °C 19 Expt. 1: Solvent Extraction Solubility Properties Solubility curve of acids, alcohols, and hydrocarbons. (From Kamm, O. Qualitative Organic Analysis, 2nd ed.; Wiley: New York, 1932.) 20 Expt. 1: Solvent Extraction Solubility Properties 1.00 g/mL Boiling Point: 100°C Slightly soluble in water 8.7 g/100 mL (20 °C) Boiling Point: 77.1°C Slightly soluble in water 17.5 mg/mL (25 °C) 15.8 mg/mL (30 °C) 5.2 mg/mL (60 °C) Miscible in water Boiling Point: 39.6°C Boiling Point: 78.23°C 21 Expt. 1: Solvent Extraction Solubility Properties 22 Expt. 1: Solvent Extraction Solubility Properties Solubility (w) of salicylic acid as a function of temperature in: water, ethanol, ▲ carbon tetrachloride (CCl4), ethyl acetate, ○ xylene (dimethylbenzene) The solubility of salicylic acid decreases in the order of (1 being the highest solubility): 1. Ethanol 2. Ethyl acetate 3. Carbon tetrachloride (CCl4) 4. Xylene (dimethylbenzene) 5. Water Shalmashi, A., & Eliassi, A. (2008). Solubility of Salicylic Acid in Water, Ethanol, Carbon Tetrachloride, Ethyl Acetate, and Xylene. Journal of Chemical & Engineering Data, 53(1), 199–200. 16.85°C 56.85°C 86.85°C https://doi.org/10.1021/je7004962 23 Expt. 1: Solvent Extraction Solubility Properties The low cohesive energy of the solvent may relate to high solubility in ethanol and ethyl acetate. In carbon tetrachloride (CCl4) and xylene (dimethylbenzene), the affinity for salicylic acid is reduced. The cohesive energy of water is high as substantiated through the high boiling point. Nordström, F. L., & Rasmuson, Å. C. (2006). Solubility and Melting Properties of Salicylic Acid. Journal of Chemical & Engineering Data, 51(5), 1668–1671. Salicylic acid extract crystallizes as https://doi.org/10.1021/je060134d needles after heating. 24  Residue after heating Expt. 1: Solvent Extraction Solubility Properties Salicylic acid exhibits a very low solubility in water as compared with organic solvents. The aromatic ring of the salicylic acid induces an increased structuring of the surrounding water which leads to an unfavorable decrease of entropy. Increased nucleation/ crystal formation at lower temperature. Right (molecular arrangement in the crystal lattice of salicylic acid) Reference: Khamar, D., Zeglinski, J., Mealey, D., & Rasmuson, Å. C. (2014). Investigating the Role of Solvent–Solute Interaction in Crystal Nucleation of Salicylic Acid from Organic Solvents. Journal of the American Chemical Society, 136(33), 11664–11673. https://doi.org/10.1021/ja503131w 25  Expt. 1: Solvent Extraction Solubility Properties 1. The solubility of benzoic acid increases as the temperature increases. 2. High to low in the order: N,N- dimethylacetamide > 3,6-dioxa-1- decanol > cyclohexanone > propanoic acid > thiacyclopentane dioxide > 1,4- dimethylbenzene (1,3-dim ethylbenzene,1,2-dimethylbenzene) > 1,2-ethanediol > deca hydronaphthalene > octane. Wang, Z., Xu, Z., Xu, X., Yang, A., Luo, W., & Luo, Y. (2020). Solubility of benzoic acid in twelve organic solvents: Experimental measurement and thermodynamic modeling. The Journal of Chemical Thermodynamics, 150, 106234. https://doi.org/10.1016/j.jct.2020.106234 26 Expt. 1: Solvent Extraction Solubility Properties (Solubility (w) of BA in seven pure solvents— water, methanol, ethanol, acetonitrile, dichloromethane, toluene, and ethyl acetate. 1. The solubility of benzoic acid increased with temperature (at any given point). 2. Order of solubility: ethanol > methanol > acetonitrile > ethyl acetate > dichloromethane > toluene > water. 3. Solubility of substances with larger polarities in polar solvents is generally Zhang, X., Chen, J., Hu, J., Liu, M., Cai, Z., Xu, Y., & Sun, B. (2021). The solubilities of benzoic acid and its nitro- higher than that of sub-stances with lower derivatives, 3-nitro and 3,5-dinitrobenzoic acids. Journal of Chemical Research, 45, 174751982110586. polarity. https://doi.org/10.1177/17475198211058617 27 Expt. 1: Solvent Extraction Effect of temperature on solubility Solid solutes: 1. inc. temp, inc. solubility: more kinetics to break and form new ones. 2. Not true to all (CaCl₂, MgSO₄, NH₄NO₃): Le Chatelier’s principle. endothermic, where ΔH is positive: the product will absorb the excess heat, ^ sol. exothermic, where ΔH is negative: the reactants will reduce the excess heat; inhibitory ( sol.) Gases: 1. temp, sol.: more kinetic energy to escape from the solution Effects of Temperature and Pressure on Solubility. (n.d.). Retrieved September 22, 2024, from https://saylordotorg.github.io/text_general-chemistry-principles-patterns- and-applications-v1.0/s17-04-effects-of-temperature-and-pre.html 28 Expt. 1: Solvent Extraction  Troubleshooting common problems 1. There is only one (1) layer. A large amount of ethanol was added: CH3CH2OH (could bind having only 1 layer in a both polar and non-polar separatory funnel instead of 2 compounds) A wrong layer was added to the Add the aqueous layer to the separatory funnel. (organic solvent organic layer (or v/v). is added to the organic layer) Label solvents properly. Remove the remaining ethanol. 29  Expt. 1: Solvent Extraction  Troubleshooting: 2. An emulsion is formed. Emulsions are tiny droplets (bubbles) suspended between the aqueous (polar) and organic (non- polar layers) No distinct interface (“third layer”)  Density of each solvent similar or narrow difference: Ethyl acetate (0.90 g/mL): Diethyl ether (0.71 g/mL)  Presence of detergents (soap-like), emulsifying substances 30 Expt. 1: Solvent Extraction  Troubleshooting: 2. An emulsion is formed. A. An emulsion is formed. (3 layers) B. Ideal separation. “Salting out” Saturating the aqueous layer with inorganic salt; Addition of acid or base to produce salts or ions 31 Expt. 1: Solvent Extraction  Troubleshooting common problems 3. The interface cannot be seen. having too dark layers that obscure visualization of layers. A. (Dark) Interface of the solution. B. Visualization using flashlight. C. Tilting of the separatory funnel. 32 Expt. 1: Solvent Extraction In solvent extraction: 1. Extraction and solution solvent must be immiscible with each other (polar paired with non-polar solvents) 2. Extraction solvent must have a high distribution coefficient (Kp) for the component 3. The extraction solvent should be readily separated from the desired component after extraction, so it should have a low boiling point. 4. The extraction solvent must not react chemically with any component in the aqueous mixture being extracted. 5. The extraction solvent must not readily dissolve the substance to be extracted. 6. The extraction solvent should not be highly flammable or toxic; should also be relatively inexpensive. 33 Expt. 2: Pigment Extraction Extraction method Sample using acetone, Absorbance Pigment concentration methanol, and preparation reading determination ethanol Quartz cuvette is Why to need to centrifuge better. and dark incubation? 34 Expt. 2: Pigment Extraction Extraction method Sample using acetone, Absorbance Pigment methanol, and concentration preparation reading determination ethanol Spectrophotometer measures the intensity of light absorbed after it pass through the solution. - Beer-Lambert Law: A= ϵcl - Unique wv (abs) for a unique compound Johan, F., Jafri, M. Z., Lim, H. S., & Wan Maznah, W. O. (2014). Laboratory measurement: Chlorophyll-a concentration measurement with acetone method using spectrophotometer. 2014 IEEE International Conference CC BY-4.0; Heesung Shim via LibreTexts on Industrial Engineering and Engineering Management, 744–748. https://doi.org/10.1109/IEEM.2014.7058737 35 Expt. 2: Pigment Extraction Sample calculation Compound OrgChem (18.5 L mol⁻¹ cm⁻¹) has an absorbance of 1.35 at 750 nm in a 10-mm cuvette Questions: a. What is its molar absorptivity? b. Calculate c. 36 Expt. 2: Pigment Extraction Sample calculation A 0.08 mol 0.5 L-1 solution of compound OrgChem has an absorbance of 1.2 at 480 nm when measured using a 0.01- m-quartz-cuvette spectrophotometer. Questions: a. Calculate for ϵ 37  Expt. 2: Pigment Extraction Solvent extraction methods based on the solubility characteristics of organic substances in the solvents used in a particular separation procedure Organic phase polarity is due to the charge separation “partitioning” Aqueous phase partial ionic character Separation of different phases in liquid-liquid extraction miscible or soluble to water (Chemlibre Texts: Overview of Extraction_ 38 Expt. 2: Pigment Extraction dominant –OH group (polar) Ethanol forming H-bonds with water dominant alkyl group (non-polar) octanol is completely miscible with diethyl ether How about dominant alkyl group (non-polar) acetone? 39  Expt. 2: Pigment Extraction TIP: Five or more hydrocarbon chains, more soluble in nonpolar solvents Solubility curve of acids, alcohols, and hydrocarbons (From Kamm, O. Qualitative Organic Analysis, 2nd ed.; Wiley: New York, 1932.) 40  Expt. 2: Pigment Extraction The presence of more than one polar group in a compound will increase that compound’s solubility in water (and decrease its solubility in nonpolar solvents). Which compound is more soluble to water? 41 Expt. 2: Pigment Extraction soluble to water or not? phytol = tail Ester (R-O-R) 42 Expt. 2: Pigment Extraction Different types of chlorophylls rank based on abundance in nature. 43 Expt. 2: Pigment Extraction Phycocyanin soluble to water (polar) or not? Phycoerythrobilin accessory to the main chlorophyll pigments 44 Expt. 2: Pigment Extraction soluble to water or not? 45 Expt. 2: Pigment Extraction 46 Expt. 2: Pigment Extraction 47 Expt. 2: Pigment Extraction 48  Expt. 2: Pigment Extraction Pigment content determination (in acetone) 49 Expt. 1: Solvent Extraction Studies on pigment extraction Effects of different solvents on pigment extraction of two Pseudomonas sp. isolates (M1, MS2) 1. Methanol is more efficient in extracting the pigments followed by acetone. 2. Absorption maximum (400-600 nm) revealed that the pigments are carotenoids (β-carotene, torulene, and torularhodin). 3. DPPH reduction results showed that both of the isolates have antioxidant activities. Meenakshi, M., Rana, N., & Chauhan, A. (2018). Original article Extraction and characterization of biocolors from bacterial isolates of Pseudomonas sp. M1 and MS2. Annals of Phytomedicine: An International Journal, 7, 63–68. https://doi.org/10.21276/ap.2018.7.1.7 50 Expt. 1: Solvent Extraction Studies on pigment extraction Graphical representation of the absorbance at different wavelengths by the pigment of Rhodotorula. Inferences? Absorbance -peaks -wavelength range -potential phytochemical property? Joshi, V., Sharma, R., & Girdhar, A. (2013). Production and Evaluation of Biocolour (carotenoids) from Rhodotorula using Apple Pomace: Effect of Composition of different Nitrogen Sources and Methods of Cell Disruption. International Journal of Food and Fermentation Technology, 3, 127–134. https://doi.org/10.5958/2277-9396.2014.00340.7 450 500 51 650 400 600 Thank you. 52