1. EXTRACTION: solvent partition crude extract hexane-solubles ethyl ether-solubles ethyl acetate-solubles 1-butanol-solubles polar residues water-solubles 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. ethyl ether 1-butanol ethyl acetate hexane wax, lipid, fatty acid… carotenoids, terpenoids, chlorophylls… polar terpenoids, polyphenols… glycosides… Separation is not perfect. Fractions may contain materials with unexpected polarity. 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 activity Choose proper solvent. ethyl ether methanol water less polar more aq. MeOH extracts TIPS 1

2. EXTRACTION: acid-base separation is not perfect Organic solvent extract Aqueous phase Organic phase Neutral molecules NaHCO 3 aq Na 2 CO 3 aq dil. HCl Alkaloides, amines… Organic acids… Phenolic compounds… Fractions may contain materials with unexpected character. CO 3 2- + H 2 O + CO 2 aq. MeOH extracts 1. HCl 2. extraction Organic phase Aqueous phase 1. HCl 2. extraction Organic phase Aqueous phase 1. NaOH aq 2. extraction Organic phase Acid extraction can be omitted due to low possibility of basic constituents. Caution. Prolonged standing, agitating or warming raises pH of NaHCO 3 solution. 2 HCO 3 - Low polar hydrophobic acids or phenols may remain in organic phase even after alkaline extraction. NaOH Na 2 CO 3 NaHCO 3 pH (0.1M) 13.0 11.6 8.4 TIPS 2

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

4. Na 2 SO 4 + 10 H 2 O Na 2 SO 4 ・ 10 H 2 O 14210*18322fw (under 33 ℃ ) Major impurity coming from EtOAc extraction is water. EXTRACTION: Is EtOAc the best choice? EtOAc (100 mL) can dissolve 3 mL of water. EtOAc (10 mL) can be dissolved in 100 mL of water. Removal of H 2 O (3 mL) needs 2.4 g of Na 2 SO 4. SLOW MgSO 4 + 7 H 2 O MgSO 4 ・ 7 H 2 O 1207*18246fw (under 48 ℃ ) Removal of H 2 O (3 mL) needs 2.9 g of MgSO 4. FAST EtOAc is a reactive ester hydrolyzable with water to give EtOH and AcOH. ATTENTION DESICCANT TIPS 4

5. SOLVENT: What you should know when using. (1) Hexane : LD 50 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 : LD 50 3800 mg/kg, MPC. 10 ppm. Carcinogenic. Do not use at all. Toluene : LD 50 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 : LD 50 800 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. Ethyl ether : LD 50 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. Dichloromethane : LD 50 167 mg/kg, MPC. 100 ppm. Similar polarity and boiling point to ethyl ether. Do not remove by water pump. Ethyl acetate : LD 50 11000 mg/kg, MPC. 400 ppm. Major impurities are ethanol and acetic acid. Ester exchange may occur. TIPS 5

6. SOLVENT: What you should know when using. (2) Acetone : LD 50 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 : LD 50 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 : LD 50 7060 mg/kg, MPC. 1000 ppm. Gives annoying NMR signals when contaminated. Pyridine : LD 50 891 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. DMSO : LD 50 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. Acetic acid : LD 50 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). Water : Can be contaminated from wide range of environment. May ruin many experiments. Use of dry solvent is recommended in most case. TIPS 6

7. zeotrope vs. azeotrope EtOAc water bp (C) 77 100 EtOAc / water (92:8)71 azeotrope chloroform (4) ethanol (4) benzene (9)hexane (6) toluene (20) pyridine (42) acetic acid, acetone, methanol 1-butanol (43) acetonitrile (16) lower boiling point azeotrope with water (%) zeotrope formic acid / toluene (50:50) 86101111 acetic acid / toluene (32:68) 104118111bp (C) 0 0 100 A (%) B (%) bp TIPS 7 temp being co-distilled not being co-distilled zeotrope with water

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

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

10. TIPS 10 CHROMATOGRAPHY: basic principle

11. TIPS 11 CHROMATOGRAPHY: column c.

12. TIPS 12 CHROMATOGRAPHY: 2

13. TIPS 13 CHROMATOGRAPHY: 3

14. TIPS 14 CHROMATOGRAPHY: 4

15. TIPS 15 CHROMATOGRAPHY: ５

16. TIPS 16 LAB SAFETY

17. a% b% c% A B C Total Y (g) (mixture of A, B, C…) aA mw + bB mw + cC mw +… aA mw YA (g) = TIPS 99 NMR: calculation of net weight in the mixture