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EXTRACTION: solvent partition

EXTRACTION: solvent partition. TIPS 1. aq. MeOH extracts. crude extract. hexane. hexane-solubles. polar residues. ethyl ether. wax, lipid, fatty acid…. Choose proper solvent. polar residues. ethyl ether-solubles. less polar more. ethyl ether methanol water. ethyl acetate.

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EXTRACTION: solvent partition

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  1. EXTRACTION: solvent partition TIPS 1 aq. MeOH extracts crude extract hexane hexane-solubles polar residues ethyl ether wax, lipid, fatty acid… Choose proper solvent. polar residues ethyl ether-solubles less polar more ethyl ether methanol water ethyl acetate carotenoids, terpenoids, chlorophylls… ethyl acetate-solubles polar residues 1-butanol polar terpenoids, polyphenols… water-solubles 1-butanol-solubles glycosides… sugars, amino acids… Weigh residues after solvent removal of an aliquot of each fraction. Store each fraction in cool and dark place as a solution. (warm and mix well before use) Keep in mind that solvolysis, ester exchange, etc. could occur in solutions. Separation is not perfect. Fractions may contain materials with unexpected polarity. activity fraction active principle other materials purity weight basis ‘n’ basis hexane 1 g 1 g 50%higher lower ethyl ether 9 g 21 g 30% lower higher

  2. EXTRACTION: acid-base separation is not perfect TIPS 2 aq. MeOH extracts Organic solvent extract dil. HCl Aqueous phase Organic phase NaHCO3 aq 1. NaOH aq 2. extraction Organic phase Organic phase Aqueous phase Na2CO3 aq Alkaloides, amines… 1. HCl 2. extraction Acid extraction can be omitted due to low possibility of basic constituents. Organic phase Organic phase Aqueous phase Neutral molecules Organic acids… 1. HCl 2. extraction Organic phase Phenolic compounds… Fractions may contain materials with unexpected character. Low polar hydrophobic acids or phenols may remain in organic phase even after alkaline extraction. Caution. Prolonged standing, agitating or warming raises pH of NaHCO3 solution. NaOH Na2CO3 NaHCO3 pH (0.1M) 13.0 11.6 8.4 CO32- + H2O + CO2 2 HCO3-

  3. TIPS 3 EXTRACTION EFFICIENCY “extraction of desired material with EtOAc from aqueous solution” H2O/EtOAc H2O/EtOAc 10 g material 10 g of undesired impurity 1:9 (partition ratio) 9:1 purity yield 100 9.0 g (90%) 100 1.0 g (90%) EtOAc (mL) H2O (mL) 100 1.0 g 100 9.0 g 200 9.5 g (95%) 200 1.8 g (84%) 100 0.5 g 100 8.2 g 100*2 9.9 g (99%) 100*2 1.9 g (84%) 100 0.1 g 100 8.1 g 50*2 9.7 g (97%) 50*2 1.0 g (91%) 100 0.3 g 100 9.0 g 25*2 9.1 g (91%) 25*2 0.5 g (95%) 100 0.9 g 100 9.5 g

  4. TIPS 4 EXTRACTION: Is EtOAc the best choice? ATTENTION EtOAc is a reactive ester hydrolyzable with water to give EtOH and AcOH. Major impurity coming from EtOAc extraction is water. EtOAc (100 mL) can dissolve 3 mL of water. EtOAc (10 mL) can be dissolved in 100 mL of water. DESICCANT SLOW Na2SO4 + 10 H2O Na2SO4・10 H2O (under 33 ℃) fw 142 10*18 322 Removal of H2O (3 mL) needs 2.4 g of Na2SO4. FAST MgSO4 + 7 H2O MgSO4・7 H2O (under 48 ℃) fw 120 7*18 246 Removal of H2O (3 mL) needs 2.9 g of MgSO4.

  5. SOLVENT: What you should know when using. (1) TIPS 5 Hexane: LD50 30 mg/kg, maximal permissible concentration (MPC). 50 ppm. Strong chronic toxicity is recently found. Replace with pentane or heptane (but expensive). In most case cheaper petroleum ether can work as well. Benzene: LD50 3800 mg/kg, MPC. 10 ppm. Carcinogenic. Do not use at all. Toluene: LD50 5000 mg/kg, MPC. 200 ppm. Replacement of benzene. Azeotropic mixture with pyridine or acetic acid is useful. But, do not remove by water pump. Chloroform: LD50 800 mg/kg, MPC. 10 ppm. Easily degrades to produce HCl by heat, light etc. Containing approx. 1% ethanol as a stabilizer. Distill before recrystalizing or chromatographic use . Do not remove by water pump. Dichloromethane: LD50 167 mg/kg, MPC. 100 ppm. Similar polarity and boiling point to ethyl ether. Do not remove by water pump. Ethyl ether: LD50 1700 mg/kg, MPC. 400 ppm. Always keep two crucial properties in mind. Easily produces explosive hydroperoxides. Never distill to dryness. Contains a phenolic stabilizer. THF and diisopropyl ether are more susceptible to the oxidation and thus more dangerous. Higher vapor pressure than most of other everyday solvents and thus highly flamable. Keep out of open flame. Ethyl acetate: LD50 11000 mg/kg, MPC. 400 ppm. Major impurities are ethanol and acetic acid. Ester exchange may occur.

  6. SOLVENT: What you should know when using. (2) TIPS 6 Acetone: LD50 9750 mg/kg, MPC. 200 ppm. Hydrophilic and lipophilic nature. Caution. Acetone solutions are easily absorbed in skin. Self-condensation gives dimers which may appear in evaporation residues. Methanol: LD50 13000 mg/kg, MPC. 200 ppm. Causes methyl ester artifacts when using as extraction or chromatographic solvent. In that case, try with ethanol. Frequently used as a polar part of mixed solvents. Precise quantification should be needed. Ethanol: LD50 7060 mg/kg, MPC. 1000 ppm. Gives annoying NMR signals when contaminated. Pyridine: LD50 891 mg/kg, MPC. 5 ppm. Toxic unpleasant odor. Use under the hood. Highly hygroscopic. Desiccate before use. Evaporate effectively as an azeotropic mixture with toluene. Acetic acid: LD50 3310 mg/kg, MPC. 10 ppm. Strong irritant. Avoid contact with skin. Freezes at low temperature (mp 16 ℃). Removable with toluene, but less effective than formic acid (FA). If use as an acidifying reagent in chromatography, carefully remove before FD-MS (FA, TFA as well). DMSO: LD50 17500 mg/kg. Non-protic polar solvent. Dissolves many compounds including inorganic salts. Hard to remove by evaporation due to high bp. (189 ℃). Relatively low toxicity in living body. Water: Can be contaminated from wide range of environment. May ruin many experiments. Use of dry solvent is recommended in most case.

  7. TIPS 7 zeotrope vs. azeotrope bp (C) EtOAc 77 zeotrope with water water 100 EtOAc / water (92:8) 71 acetic acid, acetone, methanol not being co-distilled zeotrope temp bp azeotrope being co-distilled lower boiling point azeotrope with water (%) A (%) 100 0 0 B (%) 100 acetonitrile (16) ethanol (4) benzene (9) hexane (6) 1-butanol (43) pyridine (42) chloroform (4) toluene (20) formic acid / toluene (50:50) acetic acid / toluene (32:68) bp (C) 101 111 86 118 111 104

  8. EXTRACTION-FRACTIONATION: Concept of ‘n’ basis TIPS 8

  9. EXTRACTION-FRACTIONATION: ‘n’ basis vs. weight basis TIPS 9

  10. CHROMATOGRAPHY: basic principle TIPS 10

  11. CHROMATOGRAPHY: column c. TIPS 11

  12. CHROMATOGRAPHY: 2 TIPS 12

  13. CHROMATOGRAPHY: 3 TIPS 13

  14. CHROMATOGRAPHY: 4 TIPS 14

  15. CHROMATOGRAPHY: ５ TIPS 15

  16. LAB SAFETY TIPS 16

  17. NMR: calculation of net weight in the mixture TIPS 99 aAmw A (g) = Y aAmw+ bBmw+ cCmw+… c% Total Y (g) (mixture of A, B, C…) b% C a% B A

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