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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