EXTRACTION: solvent partition

crude extract

hexane-solubles

ethyl ether-solubles

ethyl acetate-solubles

1-butanol-solubles

polar residues

polar residues

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’ basishexane 1 g 1 g 50% higher lowerethyl ether 9 g 21 g 30% lower higher

activity

cell

surface

vacuole

oil droplet

wax

glycosides

terpenoids, phenols

hydrophobic

hydrophilic

lipid

Choose proper solvent.

ethyl ethermethanolwater

lesspolar more

aq. MeOHextracts

TIPS 1

EXTRACTION: acid-base separation is not perfect

Organic solvent extract

Aqueous phase

Organic phase

Organic phase

Organic phase

Neutral molecules

NaHCO3 aq

Na2CO3 aq

dil. HCl

Alkaloides, amines…

Organic acids…

Phenolic compounds…

Fractions may contain materials with unexpected character.

CO32- + H2O + CO2

aq. MeOHextracts

1. HCl2. extraction

Organic phase

Aqueous phase

1. HCl2. extraction

Organic phase

Aqueous phase

1. NaOH aq2. extraction

Organic phase

Acid extraction can be omitted due to low possibility of basic constituents.

Caution. Prolonged standing, agitating or warming raises pH of NaHCO3 solution.

2 HCO3-

Low polar hydrophobic acids or phenols may remain in organic phase even after alkaline extraction.

OHO2N

NO2

CH3 CO2H>acidity

NaOH Na2CO3 NaHCO3

pH (0.1M) 13.0 11.6 8.4

TIPS 2

EXTRACTION EFFICIENCY

“extraction of desired material with EtOAc from aqueous solution”

H2O/EtOAc1:9 (partition ratio)

EtOAc (mL)H2O (mL)

100100

9.0 g1.0 g

(90%)yield

200100

9.5 g0.5 g

(95%)

100*2100

9.9 g0.1 g

(99%)

50*2100

9.7 g0.3 g

(97%)

25*2100

9.1 g0.9 g

(91%)

H2O/EtOAc9:1

100100

1.0 g9.0 g

(90%)

purity

200100

1.8 g8.2 g

(84%)

100*2100

1.9 g8.1 g

(84%)

50*2100

1.0 g9.0 g

(91%)

25*2100

0.5 g9.5 g

(95%)

10 g of undesired impurity

10 g material

TIPS 3

Na2SO4 + 10 H2O Na2SO4 ・ 10 H2O

142 10*18 322fw

(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 H2O (3 mL) needs 2.4 g of Na2SO4.

SLOW

MgSO4 + 7 H2O MgSO4 ・ 7 H2O

120 7*18 246fw

(under 48 ) ℃

Removal of H2O (3 mL) needs 2.9 g of MgSO4.

FAST

EtOAc is a reactive ester hydrolyzable with water to give EtOH and AcOH.

ATTENTION

DESICCANT

TIPS 4

SOLVENT: What you should know when using. (1)

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.

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.

Dichloromethane: LD50 167 mg/kg, MPC. 100 ppm. Similar polarity and boiling point to ethyl ether. Do not remove by water pump.

Ethyl acetate: LD50 11000 mg/kg, MPC. 400 ppm. Major impurities are ethanol and acetic acid. Ester exchange may occur.

TIPS 5

SOLVENT: What you should know when using. (2)

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.

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.

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).

Water:Can be contaminated from wide range of environment. May ruin many experiments. Use of dry solvent is recommended in most case.

TIPS 6

zeotrope vs. azeotrope

EtOAcwater

bp (C)

77100

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 pointazeotrope with water (%)

zeotrope

formic acid / toluene (50:50)86101 111

acetic acid / toluene (32:68)104118 111bp (C)

00100

100A (%)

B (%)

bp

TIPS 7

temp

being co-distilled

not being co-distilled

zeotrope with water

plant material (10 g)

extraction

methanol solution

partition

water phaseEtOAc phase

methanol

fractionation

100 mL 100 mL 100 mL

100 mL 100 mL 100 mL 100 mL 100 mL 100 mL

active

inactive

constituents

+

activeinactive

Activity of the flasks is the same.

1 n 1 n 1 n

1 n 1 n 1 n 1 n 1 n 1 neluates

EXTRACTION-FRACTIONATION: Concept of ‘n’ basis

TIPS 8

TIPS 9

fractionation

100 mL

100 mL 100 mL 100 mL 100 mL 100 mL 100 mL

1 n

1 n 1 n 1 n 1 n 1 n 1 neluates

dry weight base

n base highhighlow

lowrelative activity

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

TIPS 10CHROMATOGRAPHY: basic principle

Si OH

SiOH

OH

OH

Si O

SiO

O

OSi

Si

Si

Si

S

S

O

O

O

O

OO

N

N

H3C

CH2OH

CH3

CH2OH

CH3CH3

OG O

GO

OOG

O

OO

O

G

O

O

O

O

OO

OH

O

OH

absorption (normal phase) partition (reverse phase)

ion exchange

gel filtration

silica gel ODS

LH20

HO

HO

OH

OH

HO

H

HO

H

HO

H

HO

HMeOH

MeOH

Me

HO

Cl

Cl Cl

Cl

Cl Cl

MeHO

Cl

ClCl

Dowex

HO

TIPS 11CHROMATOGRAPHY: column c.

Si OH

SiOH

OH

OH

silica gel

Normal phase column chromatography

Deactivated with waterIf reproducible results are needed, dry (100 deg, 2-3 hr), add water (5-10% w/w) and shake well in a stoppered flask.

H

HO

H

HO

Vapors are also adsorbed.Keep closed and away from volatile matters.

Silica gel is acidic.Thus, acid-labile substances may decompose during chromatography.In that case, try with Florisil or neutral alumina.

Silica gel chromatography is easy, familiar and friendly, but not perfect.Always keep its merit and demerit in your mind.

Sample recovery is in some case poor due to irreversible adsorption.

Slower elution is worse than useless. 50-100 mL/hr for 2 cm i.d. class 250-500 mL/hr for 5 cm i.d. classDo not use fine (300 mesh) gel for a tall column.

TIPS 12CHROMATOGRAPHY: 2silica gel column chromatography

Gel surface must be carefully flattened.

Do not fully open a cock when packing gel.

Good gel packing and sample charge assure good separation.

Samples with low solubility in the first eluent should be coated on Celite powder and then put onto the column.Avoid using higher polar solvent when sample application.

To know the end of elution, put one drop on a glass joint and observe it. Solvent with no solute never leaves any traces.

TIPS 13CHROMATOGRAPHY: 3silica gel column chromatography

Suitable thickness-hight ratio depends on purpose.

2 cm

3 cm2

30 cm

4 cm

12 cm2

60 cm

4.2 cm

13.5 cm2

23 cm

scale size

This works well.

1.1 cm

1 cm2

20 cm20 x 20 x 0.05 cm

TLC plate

45 g

360 g

155 g

10 g

0.5 g4 g 4 g

0.1 g

TIPS 14CHROMATOGRAPHY: 4

silica gel column chromatography

Elution does not proceed linearly.

TIPS 15CHROMATOGRAPHY: ５column chromatography

1 cm2

10 cm

scale size

5 g

0.5 g 1

9

1

9

2 cm2

5 cm5 g

0.5 g

BEFORE ELUTION

DURING ELUTION

Watch!

TIPS 16LAB SAFETY

TLC plate disposable pipette ampule tube

a%

b%

c%

A

B

C

Total Y (g)(mixture of A, B, C…)

aAmw+ bBmw+ cCmw+…

aAmwYA (g) =

TIPS 99NMR: calculation of net weight in the mixture