Transcript EXTRACTION

EXTRACTION: solvent partition
aq. MeOH
extracts
TIPS 1
crude extract
hexane
hexane-solubles
wax, lipid, fatty acid…
wax
lipid
vacuole glycosides
oil droplet terpenoids, phenols
surface
polar residues
ethyl ether
ethyl ether-solubles
carotenoids, terpenoids, chlorophylls…
cell
polar residues
ethyl acetate
ethyl acetate-solubles
polar terpenoids, polyphenols…
hydrophilic
hydrophobic
Choose proper solvent.
less ethyl ether
methanol
polar
more water
polar residues
1-butanol
1-butanol-solubles
water-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
1g
1g
50%
higher
lower
ethyl ether
9g
21 g
30%
lower
higher
EXTRACTION: acid-base separation is not perfect
aq. MeOH
extracts
TIPS 2
Organic solvent extract
dil. HCl
Aqueous phase
Organic phase
1. NaOH aq
NaHCO3 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 Organic phase
Organic phase
Aqueous phase
of basic constituents.
Neutral molecules
1. HCl
Organic acids…
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.
2 HCO3-
CO32- + H2O + CO2
O2N
NO2
OH
>
CH3 CO2H
acidity
NaOH Na2CO3 NaHCO3
pH (0.1M) 13.0
11.6
8.4
TIPS 3
EXTRACTION EFFICIENCY
“extraction of desired material with EtOAc from aqueous solution”
H2O/EtOAc 10 g material
1:9 (partition ratio)
EtOAc (mL) 100
H2O (mL) 100
200
100
H2O/EtOAc 10 g of undesired
impurity
9:1
purity
yield
9.0 g (90%)
100
1.0 g (90%)
1.0 g
100
9.0 g
9.5 g (95%)
0.5 g
200
100
1.8 g (84%)
8.2 g
100*2 9.9 g (99%)
100
0.1 g
100*2 1.9 g (84%)
100
8.1 g
50*2
100
9.7 g (97%)
0.3 g
50*2
100
1.0 g (91%)
9.0 g
25*2
100
9.1 g (91%)
0.9 g
25*2
100
0.5 g (95%)
9.5 g
EXTRACTION: Is EtOAc the best choice?
TIPS 4
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
(under 48 ℃)
MgSO4 + 7 H2O
MgSO4・7 H2O
fw 120
7*18
246
Removal of H2O (3 mL) needs 2.9 g of MgSO4.
SOLVENT: What you should know when using. (1) TIPS
Hexane: LD50 30 mg/kg, maximal permissible concentration (MPC). 50 ppm.
5
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.
SOLVENT: What you should know when using. (2)
Acetone: LD50 9750 mg/kg, MPC. 200 ppm.
TIPS 6
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.
TIPS 7
zeotrope vs. azeotrope
bp (C)
EtOAc
77
water
100
EtOAc / water (92:8) 71
temp
A (%) 0
B (%) 100
zeotrope with water
acetic acid, acetone,
methanol
zeotrope not being co-distilled
azeotrope being co-distilled
bp
100
0
lower boiling point
azeotrope with water (%)
acetonitrile (16)
benzene (9)
1-butanol (43)
chloroform (4)
formic acid / toluene (50:50)
bp (C)
101
111
86
ethanol (4)
hexane (6)
pyridine (42)
toluene (20)
acetic acid / toluene (32:68)
118
111
104
EXTRACTION-FRACTIONATION:
Concept of ‘n’ basis
extraction
methanol
TIPS 8
partition
1n
1n
+
1n
plant material (10 g)
active
methanol solution
100 mL
EtOAc phase
100 mL
water phase
100 mL
inactive
inactive
fractionation
Activity of the flasks
is the same.
constituents
eluates
1n
100 mL
active
1n
1n
1n
1n
1n
100 mL
100 mL
100 mL
100 mL
100 mL
EXTRACTION-FRACTIONATION:
‘n’ basis vs. weight basis
TIPS 9
1n
fractionation
100 mL
eluates
1n
100 mL
1n
1n
1n
1n
1n
100 mL
100 mL
100 mL
100 mL
100 mL
n base
dry weight base
low
high
high
low
relative activity
CHROMATOGRAPHY: basic principle
Cl
Me
HO
Cl
Cl
Cl
Cl
Me
Cl
Me
HO
HO
OH
TIPS 10
Cl
Me
Cl
OH
H
OH
O
O
OH
Si O Si
OH
Si
OH
Si
O
O Si
Si
silica gel
ODS
partition (reverse phase)
absorption (normal phase)
O
O
G
H3C
CH3
N
CH2OH
O
O
O
G
O
O
O
OH
O
CH3
N CH3
CH2OH
O
G
O
O
Dowex
ion exchange
H
H
H
HO
O
S O
O
H
Si
Si OH
O
OH
Cl
HO
O
O
S
O
H
OH
O
G
O
O
O
LH20
gel filtration
O
O
H
H
CHROMATOGRAPHY: column c.
TIPS 11
Normal phase column chromatography
Silica gel chromatography is easy, familiar and friendly, but not perfect.
Always keep its merit and demerit in your mind.
OH
Si OH
OH
Si
OH
silica gel
H
HO
H
HO
Deactivated with water
If reproducible results are needed, dry (100
deg, 2-3 hr), add water (5-10% w/w) and
shake well in a stoppered flask.
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.
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. class
Do not use fine (300 mesh) gel for a tall column.
CHROMATOGRAPHY: 2
silica gel column chromatography
TIPS 12
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.
Gel surface must be carefully flattened.
Do not fully open a cock when packing gel.
To know the end of elution, put one drop on a glass joint
and observe it. Solvent with no solute never leaves any
traces.
CHROMATOGRAPHY: 3
TIPS 13
silica gel column chromatography
Suitable thickness-hight ratio depends on purpose.
13.5 cm2
12 cm2
3 cm2
2 cm
0.5 g
4g
45 g
4.2 cm
4 cm
4g
155 g
30 cm
360 g
60 cm
23 cm
This works well.
1 cm2
1.1 cm
TLC plate
scale size
0.1 g
20 x 20 x 0.05 cm
20 cm
10 g
CHROMATOGRAPHY: 4
silica gel column chromatography
Elution does not proceed linearly.
TIPS 14
CHROMATOGRAPHY: ５
TIPS 15
column chromatography
1 cm2
2 cm2
0.5 g
1
0.5 g
1
Watch!
5g
BEFORE ELUTION
9
5g
9
10 cm
scale size
DURING ELUTION
5 cm
LAB SAFETY
TLC plate
TIPS 16
disposable pipette
ampule tube
NMR: calculation of net weight in the mixture
aAmw
A (g) =
aAmw+ bBmw+ cCmw+…
TIPS 99
Y
c%
Total Y (g)
(mixture of A, B, C…)
b%
C
a%
B
A