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Lab scale isolation of compounds
by Flash Chromatography
• Properties of Silica Gel
• Using Thin Layer Chromatography (TLC) for
chromatographic method development
• Transfer from TLC to Flash Chromatography
Lab scale isolation of compounds by Flash Chromatography
• Applications
- Rapid and reliable transfer from TLC to Flash Chromatography
Dr. Michael Schulte
Merck Chimie S.A.S.
© Merck
Editor: Presentation name here
25/11/2005
Page 2
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Silica Production Process
Hydrosol
Na2SiO3
(water glass)
+ H2SO4
(oligosilicic acid,
orthosilicic acid)
Hydrogel
temperature control
Production Plant
control of
temperature and pH
drying temperature control
Xerogel
(raw silica powder,
particle size 0.5 - 6 mm)
Ground silica gel
(dp 5 - 500 µm)
Classified
silica gels
... the biggest chromatographic
silica gel plant in the world,
washing pH-control,
ion content
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 3
Merck Chimie S.A.S.
25/11/2005
Page 4
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Standardized Silica Gels
Production Plant
LiChrosorb Si60
LiChroprep Si 60
Three reactors for silica gel
production,
Silica Gel 60
+ Silica Gel for TLC
... Large scale production,
btach size: several tons
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 5
All from one Production Process
CONSTANT SELECTIVITY
Merck Chimie S.A.S.
25/11/2005
5, 10µm
15-25 µm
25-40 µm
15-40µm
40-63µm [9385]
below 63µm
63-100µm
63-200µm [7734]
200-500µm
Page 6
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Factors influencing Selectivity
and Usability
Separation Optimization
Form
irregular / spherical
Efficiency, Pressure Drop
Surface
Modification
Selectivity
Area / Pore Size
Loadability
Purity
Selectivity, Resolution
pH
Selectivity
Water Content
Selectivity
mechanical
Life Time
chemical (pH)
CIP - Conditions
Stability
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Resolution - the key parameter for
preparative chromatography
RS = (
k´
N
α −1
)⋅(
)⋅
α
1 + k´ 4
maximize through
sorbent screening
Page 7
Merck Chimie S.A.S.
25/11/2005
minimize - keep
cycle time
maximize - high
short
efficiency sorbent
Page 8
4
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Method Development –
using Thin Layer Chromatography
Method Development –
using Thin Layer Chromatography
•You have to consider three phases in TLC :
• Several parameters have to be considered when
developing a TLC method :
– Activity of the stationary phase
Gas phase
– Conditioning / preparation
• methanol / isopropanol (plate developing or dipping)
• drying (30 min @ 120 °C)
– Support (plastic, aluminium, glas)
Sample
– Binder
– Fluorescent indicator
– Manufacturer / lot reproducibility
Stationary phase
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Liquid phase
– Manufacturer / comparability with Flash / column chromatography
Page 9
Merck Chimie S.A.S.
25/11/2005
Page 10
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Method Development –
using Thin Layer Chromatography
Method Development –
using Thin Layer Chromatography
• The mobile phase should :
• Solvents classification: Snyder triangle
0.6
0.2
– Dissolve the sample
– Desorb the sample from the stationary phase
0.3
– Transport the sample over an acceptable distance
• The mobile phase should in general be :
proton donor
– Simple (maximum 4, better 2 components)
xd
xe
0.4
proton acceptor
– Non-toxic
– of chromatographic quality
0.5
0.3
– Not cause any secondary reactions
– Not demixing
0.6
0.2
– Of favourable low viscosity
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 11
Merck Chimie S.A.S.
25/11/2005
0.3
x
0.4
n
dipole interaction
0.6
0.2
0.7
Page 12
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TLC - equipment
TLC/HPLC - Conversion
Rf =
d oS
d of
ATTENTION : the migration
distance is measured from
the sample spot
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
k´DC =
• CAMAG – vario chamber parallel us of 6 different
solvents or solvent mixtures
1
−1
Rf
Page 13
Merck Chimie S.A.S.
25/11/2005
Page 14
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Thin Layer Chromatography (TLC)
as pilot technique
TLC/HPLC - Conversion
Some general rules:
Place a spot of
the sample on a
TLC-plate
• Adjust polarity to lower retention
values on TLC plates
Develop chromatogram with
mobile phases of different
elution strength
• Try to maintain the same relative
activity on the plate and on the column
• Do not use volatile mobile phase
components
Literature:
ε0
TLC as a pilot technique for transferring retention data to HPLC
W.Jost, H.-E.Hauck, F.Eisenbeiss, Kontakte 1984 (3), p.45-52
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 15
(elution
strength)
far too low
too low
perfect
too high
Literature: E.Stahl,
Chemiker-Ztg. 82 (1958) 323
Merck Chimie S.A.S.
25/11/2005
Page 16
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TLC-Approach 1 (Solvent Screening model
TLC/HPLC - Conversion
Apolar Compound
n-Heptane
0.01
0.38
Cyclohexane
0.04
0.56
0.57
0.58
Dioxane
THF
Etat
MTBE
0.38
0.82
0.88
0.95
IPA
EtOH
MeOH
ACN
ε0
Merck Chimie S.A.S.
25/11/2005
Editor: Presentation name here
1) Two series of neat solvents covering a broad polarity and selectivity range
Polar Compound
MTBE
Dichloromethane 0.42
© Merck
J.Dingenen, Janssen Pharmaceutica)
Run 1
MTBE
Toluene
CH2Cl2
CH3CN
EtAc
THF
Run 2
MeOH
EtOH
IPA
Acetic acid
2-propanone
2-butanone
2) Solvent series based on dichloromethane – methanol mixtures
Run 3
Lane
ε0
Page 17
Merck Chimie S.A.S.
N-Hexane 50%
CH2Cl2 50 %
1
25/11/2005
MeOH 2%
2
MeOH 5%
3
MeOH 10% MeOH 20% MeOH 50%
4
5
6
Page 18
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TLC-Approach 1 (Solvent Screening model
TLC-Approach 1 (Solvent Screening model
J.Dingenen, Janssen Pharmaceutica)
J.Dingenen, Janssen Pharmaceutica)
2) Solvent series based on dichloromethane – methanol mixtures
Good selectivity for one of the tested neat solvents
or for one of the dichloromethane – methanol mixtures
1)
Start experiments on the preparative chromatography column
2)
Good selectivity for one of the tested neat solvents but Rf values to high
Addition of an apolar solvent to adjust the Rf values between 0.10 and 0.35
(corresponding to a k` value between 1.85 and 9)
3)
Unsatisfactory resutls
On the basis of the observed spot shapes in the experiments with neat
solvents the most suitable solvent combinations (binary – ternary) are chosen
and further inverstigated
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 19
Merck Chimie S.A.S.
25/11/2005
Page 20
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TLC-Approach 1 (Solvent Screening model
TLC-Approach 1 (Solvent Screening model
J.Dingenen, Janssen Pharmaceutica)
J.Dingenen, Janssen Pharmaceutica)
Binary solvent mixtures: THF/acetone or Ethanol/Acetone
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Ternary solvent mixtures
Page 21
Merck Chimie S.A.S.
25/11/2005
Page 22
11
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TLC-Approach 2
TLC-Approach 2
(EtAc/Heptane-Gradient, Solvias)
(EtAc/Heptane-Gradient, Solvias)
TLC-Screening with
Ethyl acetate / n-hexane (1:4)
Transfer to LiChrospher Si 60, 12 µm packed into
Selfpacker columns 125 mm length and 25 or 50 mm I.D.
Exponential gradient: c( EtAc ) =
Segment 3
(Rf=0.60-0.90)
(e ( a⋅t / T ) − 1) ⋅ (1 − K )
+K
ea − 1
T=length of gradient(min), K=c(EtAc) at t=0, a=exponential gradient parameter, t=runtime
Segment 2
Rf
(Rf=0.25-0.60
a
K
T
Segment 1
(Rf=0.05-0.25)
0.05-0.25
7
0.15
8
0.25-0.60
6
0.09
6
0.60-0.90
8
0.05
6
Literature:
Transfer to preparative
normal phase silica,
according to segment
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Automated normal-phase preparative HPLC as a substitue for flash
chromatography in the synthetic research laboratory
P.Renold, E.Madero, Th.Maetzke, J.Chromatogr. 2001 (908), 143-148
Page 23
Merck Chimie S.A.S.
25/11/2005
Page 24
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Example for TLC method
development (Diastereomers + impurities)
TLC-Approach 2
(EtAc/Heptane-Gradient, Solvias)
ea / hex = 1:4
0.9
1.00
0.8
0.70 Bibenzyl
0.7
0.80
0.6
0.54 Dibutylphtalate
0.50 Acetophenone
0.5
0.43 Diéthylphtalate
0.4
%B(C)
0.60 Ethylbenzoate
0.60
G-1
100 % n-Heptan
95 % n-Heptan
5 % Ethylacetat
85 % n-Heptan
15 % Ethylacetat
80 % n-Heptan
20 % Ethylacetat
0.40
G-2
0.33 Diméthylphtalate
90 % n-Heptan
10 % Ethylacetat
G-3
0.3
0.20
0.22 Benzylalcool
0.2
12.7
12.1
11.5
10.9
9.7
9.1
8.5
7.9
7.3
6.7
6.1
5.5
4.9
4.3
10.3
0.05 Acetanilide
3.7
3.1
2.5
1.9
1.3
0.1
0.7
0.00
0.1
t
Ref1
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 25
Merck Chimie S.A.S.
25/11/2005
75 % n-Heptan
25 % Ethylacetat
Page 26
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Example for TLC method
development (Diastereomers + impurities)
Example for TLC method
development (Diastereomers + impurities)
Retention Time (min)
0
50
100
2
1,55
1,68
1,89
4
6
8
10
12
14
16
18
150
200
250
High
300
90 % n-Heptan
10 % Ethylacetat
5,04
95 % n-Heptan
5 % Ethylacetat
Intensity (mV)
2,62
3,08
7,85
100 % n-Heptan
HPLC
chromatogram
Retention
Low
TLC
chromatogram
85 % n-Heptan
15 % Ethylacetat
Merck Chimie S.A.S.
© Merck
80 % n-Heptan
20 % Ethylacetat
25/11/2005
Editor: Presentation name here
75 % n-Heptan
25 % Ethylacetat
90 % n-Heptan / 10 % Ethylacetat
Page 27
Merck Chimie S.A.S.
25/11/2005
Page 28
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Comparison of TLC and HPLC
Comparison of TLC and HPLC
Example 1: Triazines
TLC:
HPLCe:
Used stationary phases:
TLC
HPTLC KG 60
LiChrospher Si 60
HPTLC RP-8
LiChrospher 100 RP-8, LiChrospher 60 RP select B
HPTLC RP-18
LiChrospher 100 RP-18
HPTLC CN
LiChrospher 100 CN
HPTLC NH2
LiChrospher 100 NH2
HPTLC Diol
LiChrospher 100 Diol
Merck Chimie S.A.S.
© Merck
HPLC
25/11/2005
Editor: Presentation name here
Mobile phase:
Pesticide
Terbutryn
Prometryn
Terbuthylazin
Propazin
Sebuthylazin
Atrazin
Metribuzin
Simazin
Cyanazin
Hexazinon
Desethylatrazin
Metamitron
Desisopropylatrazin
Page 29
Merck Chimie S.A.S.
25/11/2005
HPTLC-Fertigplatte RP-8 F254s
LiChroCART® LiChrospher ® 100 RP-8 bzw.
LiChroCART ® LiChrospher ® 60 RP select B
Acetonitril/Water 60/40
hRf
19,3
21,4
27,1
30,0
30,0
35,7
45,7
45,7
47,1
52,1
55,0
60,7
61,4
K’P
4,18
3,67
2,69
2,33
2,33
1,80
1,19
1,19
1,12
0,92
0,82
0,65
0,63
K’S(RP-8P K’S(select B)
15,70
3,20
14,18
2,90
2,10
1,91
1,89
1,71
1,85
1,68
1,46
1,31
1,06
0,94
1,10
1,01
0,95
0,85
0,81
0,79
0,66
0,62
0,61
0,54
0,52
0,49
Page 30
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Comparison of TLC and HPLC
Comparison of TLC and HPLC
Example 1: Triazines
Example 1: Triazines
a1) RP-8 (without Terbutryn and Prometryn): r = 0,996; K’S = 0,11 + 0,75 K’P
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
a2) RP-select B: r = 0.996; K‘S = 0,03 + 0,75 K‘P
Page 31
Merck Chimie S.A.S.
25/11/2005
Page 32
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Comparison of TLC and HPLC
Comparison of TLC and HPLC
Transfer of a real sample (salad)
1
All
samples,
except
0,995
0,99
Phenoxycarbonic
acids
0,985
0,98
0,975
0,97
HPTLC-Chromatogramm
(λ = 200 nm)
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Silica
HPLC-Chromatogramm
(λ = 200 nm)
CN
Amino Diol
RP-8 RP-18
Real
sample
(Salad,
spiked)
Correlation coefficient between TLC and HPLC
Page 33
Merck Chimie S.A.S.
25/11/2005
Page 34
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Transfer to Flash
Chromatography
Transposition, example 1
• We now have a complete range of Flash Cartridges :
• In ideal conditions,
transposition from
TLC to Flash
Chromatography
should give such
results
800g
70g
90g
200g
300g
150g
200g
300g
400g
600g
2,5g
10g
15g
Merck Chimie S.A.S.
© Merck
25g
30g
25/11/2005
Editor: Presentation name here
Registered Trademarks
of Götec-Labortechnik
Page 35
Merck Chimie S.A.S.
25/11/2005
Page 36
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How to increase performance…
Particle size comparisons
• .. by using smaller particles, but:
Test silice Si60 40-63 µm
Test silice Si60 63-200 µm
Test silice Si60 15-40 µm
300000
Smaller granulometry Higher pressure !!!
250000
Ex : Back pressure in Cyclohexane 90 / 10 Dioxane
Bed height = 100 mm Flowrate = nominal
200000
150000
Si60 63-200 µm
Si60 40-63 µm
Si60 15-40 µm
0,4 bar
1,2 bar
4,3 bar
100000
50000
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Need for pressure stable flash cartridges
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 37
Merck Chimie S.A.S.
25/11/2005
Page 38
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Efficiency depending on particle
size
Efficiency depending on particle
size
• Comparison between 15-40, 25-40 spherical and 40-63 µm
• Comparison between 15-40, 25-40 spherical and 40-63 µm
Biotage 40-63 µm
Biotage 40-63 µm
LiChroprep 15-25 µm
Pharmprep CC 25-40 µm
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 39
Merck Chimie S.A.S.
25/11/2005
Page 40
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Difficult separations –
cis/trans-Phytol
Difficult separations –
Citrus-Oil
HPLC-Chromatogram
HPLC-Chromatogram
Biotage 40-63 µm
Merck Chimie S.A.S.
© Merck
Supervarioprep 15-40 µm
25/11/2005
Editor: Presentation name here
Biotage 40-63 µm
Page 41
Merck Chimie S.A.S.
25/11/2005
Supervarioprep 15-40 µm
Page 42
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Dry loading
Dry loading
• Dry loading :
Another advantage of pre-packed cartridges
• In order to obtain good transposition to Flash on basis of
TLC results(especially in Anti-Langmuir because of low
solubility in solvent) :
Check loading in TLC.
Use Dry loading.
– In TLC, the sample is always deposited as a "Dry loading".
– In Flash, it depends of the solubility of the sample in the eluent.
Our cartridges have special empty volume for this Dry
loading :
But… the sample does not always behave identically if
injected in solution, or as a Dry loading.
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 43
Merck Chimie S.A.S.
25/11/2005
Page 44
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Conclusion
Contact
• New Flash cartridges packed with Si60 15-40 µm offer :
• We remain at your service for any question regarding
Flash Chromatography :
– Easy transposition TLC / Flash.
– Very good purification efficiency.
– Products presentation
– Quicker purifications than Glass columns.
– Tests on your products
– Smaller fractions to evaporate.
– Purification strategy
– Secure working area (no glass breakage risk).
– Trainings
– Easy Dry loading directly on-column.
– Etc…
Thomas PONCET Process Separations
Merck Chimie SAS
[email protected]
Merck Chimie S.A.S.
© Merck
25/11/2005
Editor: Presentation name here
Page 45
Merck Chimie S.A.S.
25/11/2005
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