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May 2017
Chromatography seminar
BÜCHI Labortechnik GmbH
May 2017
Agenda
Principles of chromatography · Retention time
· Resolution
· van Deemter equatation
· From TLC to column chromatography
· Optimization of TLC plates
· How to transfer results from TLC to a column
May 2017
Büchi: Solution for your synthesis
Design and
preparationSynthesis Evaporation Drying
Quality
control
Chromatography
May 2017
Büchi: Solution for extraction
May 2017
Chromatography
Chromatography is a method of separating and identifying the components of a
complex mixture by differential movement through a two-phase system, in which
the movement is effected by a flow of a liquid (mobile phase) through an adsorbent
(stationary phase).
Preparative Chromatography is the process of using liquid chromatography to
isolate a sufficient amount of material for other experimental or functional
purposes.
Preparative Chromatography can be separated into three major segments;
traditionally differentiated by the pressure range:
A low pressure Traditional Flash segment ( 0 -15 bar)
A medium pressure MPLC segment (20 – 50 bar)
A high pressure Prep HPLC segment (up to 200 – 300 bar)
Definitions:
May 2017
Chromatography – Goal
Source: www.kunstaufräumen.ch
May 2017
Source: www.kunstaufräumen.ch
Chromatography – Goal
May 2017
A separation technique, based on an equilibrium
between 2 phases
• Stationary phase
(column packing)
• Mobile phase
(solvent)
How does it work?Stationary and mobile phase
May 2017
Adsorption and Desorption
Adsorption: Binding of a molecule
Desorption: Detaching of a molecule
Adsorption
Desorption
May 2017
Mechanism of separation
Different
Adsorption/Desorption
charateristic
Adsorption
strength
Desorption
strength
May 2017
Stationary phases
- Normal phase:
polare silica gele
- Reversed phase:
modified silica gele
May 2017
Normal phase
Si
O
O
OHO
Si
Si
Si
O
O
OH
OH
Si O
Si O
Reversed Phase
Si
O
O
O
Si
Si
Si
O
O
OH
Si O
Si O
O
O
Si
CH3
(CH2)n CH
3
Stationary phases
May 2017
Stationary phases
May 2017
Mobile Phase: Eluotropic serie
Polarity Viscosity
Refractive Index
Dielectricity Constant
n-Pentane 0.00 0.24 1.358 1.84
n-Hexane 0.01 0.33 1.375 1.88
Isooctane 0.01 0.50 1.391 1.94
Tetracarbon 0.02 0.97 1.466 2.24
Di-isopropyl-ether 0.28 0.37 1.368 3.88
Toluene 0.29 0.59 1.496 2.38
n-Propylchloride 0.30 0.35 1.389 7.7/
Benzene 0.32 0.65 1.501 2.28
Ethylbromide 0.37 0.39 1.421 9.34
Di-Ethylether 0.38 0.23 1.353 4.33
Chloroform 0.40 0.57 1.443 4.80
The solvent strength is the property of displacing a substance from
the stationary phase.
May 2017
Mobile Phase: Parameter of solvents
relevant characteristic of the mobile phase
• Dipole-parameter
• Proton-aczeptor-parameter
• Proton-donor-parameter
Selectivity
May 2017
Mobile PhaseGroups of solvents with similar separation properties
IIsopropyl ether
Diethyl etherTriethyl amine
VIDioxane
Ethyl acetate
AcetoneAcetonitril
VIIToluene
Benzene
IVAcetic acidFormamide
VDichloromethane
1,2-Dichloroethane
IIPropanol
EthanolMethanol
VIIIChloroform
IIITetrahydrofuran
Pyridin
Dimethylforamide
May 2017
H-
Acceptor
DipoleH-
Donor
TolueneVII
V
Dichloro-methane
I
Diethyl-ether
II
Ethanol
IV
Aceticacid
III
Tetra-hydrofuran
VIII
Chloroform
VI
Ethylacetate
Selectivity triangle
May 2017
Influence of the selectivity in TLC
TLC: Silica Si60 Detection: UV lamp 254 nm
Tetrahydrofuran
Mix.
Dichloromethane
Mix.
Chloroform
Mix.
CH3
O
NH
May 2017
Influence of the solvent strength in TLC
THF/Hexan 1:1
Mix.
Si= 1.0
Tetrahydrofuran
Mix.
Si= 2.0
TLC: Silica Si60 Detection: UV 254 nm
CH3
O
NH
TLC 1 TLC 2
May 2017
Selectivity vs solvent strength
Selectivity interacts the distance of the components
Solvent strength interacts the moving velocity
For good results selectivity and solvent strength have
to be carefully adapted to the separation problem!
May 2017
Terms in thin layer chromatography (TLC)
Solvent front
Start position
D0
D1
D2
D3
Solute distanceMobile phase distance
D1
D0
Rf =
Rf =
Retention factor Rf
May 2017
Calculation of Rf-values (TLC)
D0 = 7,4 cm
D1 = 6,6 cm
D2 = 3,6 cm
D3 = 1,5 cm
89.04.7
6.61
Rf
49.04.7
6.32
Rf
20.04.7
5.13
Rf
May 2017
General terms in a chromatogram (1)
t0 = Dead time
tR = Retention time
H100% = Peak height
H50% = 50% of the total
peak height
H10% = 10 % of the total
peak height0 1 2 3 4 5 6
Time (min)
H100%
H50%
H10%
t0
tR
May 2017
0 1 2 3 4 5 6
Time (min)
H100%
H50%
H10%
t
0
t
R
General terms in a chromatogram (2)
b0,5 = Peak width at
half higth
a b
b0,
5
Base line
a = Peak width at 10%
of peak higth,
front part
b = Peak width at 10%
of peak higth,
back part
May 2017
General terms in a chromatogram (3)
Number of plates N: Number of theoretical equlibria of a substancebetween the stationary phase and the mobile phaseduring passage through the column
Chromatogram: Record of all detector signals during achromatographic separation
Eluent: Mobile phase, solvent
Flow rate u: Rate of flow of solvent through the column, in mm/s
Fronting: Modification of the shape of the front peak flank
Tailing: Modification of the shape of the rear peak flank
May 2017
The resolution
0 2 4 6 8 10 12 14
Time (min.)
)2(5,0)1(5,0
12177.1
bb
ttR RR
O
O
O
O
CH3
CH3
O
O
O
O
C
H2
CH
2
CH
2
C
H2
C
H2
CH
2
CH3
CH3
O
O
O
OC
H2
CH
2
CH3
CH3
CH3
The resolution is a measure
for the separating power of
a columnR
May 2017
Theoretical plates
Theoretical distance for
1 adsorption/desorption
step = 1 theoretical plate
Adsorption Desorption
2
5,0
54.5
bR
tN
(N = Number of theoretical plates)
May 2017
Plate height
2
5,0
54.5
bR
tN
Number of theoretical plates N:C
olu
mn
len
gth
L
Plate height H (or HETP):
N
LH
Height equivalent of a theoretical plate
May 2017
Retention factor
0
1
D
DRf
Theoretical plates
2
5,0
55.5
b
tN R
Resolution
)2(5,0)1(5,0
12177,1
bb
ttR RR
Peak symmetry
a
bIS ..
Linear flow rate
26
4
d
Fu m
Summary
May 2017
Effects on column efficiency
Particle
size
Flow rate
Loading
May 2017
Flow rate (u)
Pla
te h
eig
ht
(H
)Effect of the particle size (1)
udp
udpH
16
63
2
The plate height H and
hense the number of
theoretical plates N of
a column are strongly
determined by the
particle size dp
H0
Van Deemter curve1)
1) Simplified by Halasz et al., Z Anal.Chem. 277 (1975)257
May 2017
0
100
200
300
400
500
600
700
800
0 0,5 1 1,5 2 2,5 3
Linear flow rate (mm/s)
Pla
te h
eig
ht
H (
mic
ron
)
Effect of the flow rateDependence of the plate height H on the linear flow rate for
adsorbents of different particle size
The column efficiency
shows greater dependence
on the flow rate in the case
of coarse adsorbents than
in case of fine particle sizes
dp = 100
dp = 60
dp = 50
dp = 30
May 2017
Loading (B)
Pla
te h
eig
ht
(H
)Effect of the loading (1)Dependence of the plate height H on the loading B
High loading increases
the plate height and is
therefore lowering the
column efficiency
B0
Some facts:
Loading B0 200 g / g SiO2
Analyt. loadings: B < B0
Prep. loadings: B > B0
Good prep. separations
up to 30 mg / g silica gele
May 2017
How to optimize?
Rf1= 0.49
Rf2= 0.30
Rf3= 0.14
Rf1= 0.97
Rf2= 0.84
Rf3= 0.73
May 2017
What can we optimize?
Solvent 1 Solvent 2 Solvent 3
Optimize the selectivity
May 2017
H-
Acceptor
DipoleH-
Donor
TolueneVII
V
Dichloro-methane
I
Diethyl-ether
II
Ethanol
IV
Aceticacid
III
Tetra-hydrofuran
VIII
Chloroform
VI
Ethylacetate
Selectivity triangle
May 2017
Link between TLC and column? (1)
TLC Column
0
1
D
DRf
1'
'
141
2
2
k
kNR
Calculate the required theoretical plates of the column
2
2
20,1
'1
1'4
k
kNR
May 2017
Link between TLC and column? (2)
TLC Column
0
1
D
DRf
Calculate the required theoretical plates of the column?
2
2
20,1
'1
1'4
k
kNR
11
' fR
k
1
2
'
'
k
k
May 2017
Terms in the formulae
1'
'
141
2
2
k
kNR
R = Resolution, measure for
the separation of
2 components (distance
between 2 peaks)
N = Number of theor. plates
NR1,0 = Number of plates for
a resolution of 1.0
= Separation factor
(selectivity)
k = Capacity factor
(solvent strength)
2
2
20,1
'1
1'4
k
kNR
May 2017
Rule for optimization
2
2
20,1
'1
1'4
k
kNR
Rf1= 0.49
Rf2= 0.30
k1 = 1.04
k2 = 2.33
= 2.24
N = 106
Rf1= 0.97
Rf2= 0.84
k1 = 0.03
k2 = 0.19
= 6.33
N = 885
The capacity factor k
affects the required
column efficiency to a
greater extend than the
separation factor .
Optimum k values are 1...5(Rf values between 0.2 and 0.5)
Optimum values are 1.2
(Rf differences 0.05)
May 2017
Evaluation of the mobile phaseSample: Crude reaction mixture
Tetrahydrofuran
Ethanol
Ethylacetate
Chloroform
Dichloromethane
Diethylether
Step 1
Different solvents,
diluted 1:1 with
hexane
Step 2
solvent , &
diluted 1:3 with
hexane
May 2017
Appoint the parameters for preparative
separation on the column
Selectivity:
Diethylether
()
Adsorbent:
Silica Gel Si 60(same as TLC)
Solvent strength:
15% Diethylether
in Hexane( 1/3 of TLC, k)
Column:
15 x 230 mm
( 20 g silica gel
for 600 mg sample,
loading
30mg/g)Delivery 10 ml/min
(linear flow rate
1,25 mm/s)
May 2017
BÜCHI Labortechnik GmbH
Thank you for your attention!