lecture vak 02-6007 may 17, 2005 johannes...
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Separation by ChromatographyLecture VAK 02-6007
May 17, 2005
Johannes Ranke
Separation by Chromatography – p.1/7
1D Separation methods
1
1
2
2
Electrophoresis
Separation by Chromatography – p.2/7
1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
Separation by Chromatography – p.2/7
1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
1
1
2
2
Membrane separation
Separation by Chromatography – p.2/7
1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
1
1
2
2
Membrane separation
Separation according to
Kinetic properties
Equilibrium distribution
Combinations thereof
Separation by Chromatography – p.2/7
Elution techniques
A + B
Separation by Chromatography – p.3/7
Elution techniques
A
B
Separation by Chromatography – p.3/7
Elution techniques
A
B
Separation by Chromatography – p.3/7
Elution techniques
Time [min]
Det
ecto
r si
gnal
0 2 4 6 8 10
A
B
Separation by Chromatography – p.3/7
Equilibrium based separation
tM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
= K · VSVM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
= K · VSVM
tM = F · VM
tR = F · VR
k′ = nSnM=
tStM=
tR−tMtM=
VR−VMVM
Separation by Chromatography – p.4/7
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
= K · VSVM
tM = F · VM
tR = F · VR
k′ = nSnM=
tStM=
tR−tMtM=
VR−VMVM
VR ≈ VM + K · VS
Separation by Chromatography – p.4/7
Equilibrium constant
Ki =cS
cM
Ki = exp(−∆G0i /RT) = exp(−
∆H0i − T∆S0
i
RT)
If ∆H0i ≈ 0, then
Ki ≈ exp(−∆S0i /R),
i.e. Ki is independent from temperature.
Separation by Chromatography – p.5/7
Equilibrium constant
Ki =cS
cM
Ki = exp(−∆G0i /RT) = exp(−
∆H0i − T∆S0
i
RT)
If ∆H0i ≈ 0, then
Ki ≈ exp(−∆S0i /R),
i.e. Ki is independent from temperature.
Separation by Chromatography – p.5/7
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
Dispersion in columns
van-Deemter equation:
H = A + B/v + C v
Eddy diffusionA = 2λdR
Longitudonal diffusionB = 2ΨDM
Lateral diffusion, disequilibriumC = K1 · R · (1 − R) · d2
f/DS + K2/DM
Separation by Chromatography – p.8/7
Dispersion in columns
van-Deemter equation:
H = A + B/v + C v
Eddy diffusionA = 2λdR
Longitudonal diffusionB = 2ΨDM
Lateral diffusion, disequilibriumC = K1 · R · (1 − R) · d2
f/DS + K2/DM
Separation by Chromatography – p.8/7
Dispersion in columns
van-Deemter equation:
H = A + B/v + C v
Eddy diffusionA = 2λdR
Longitudonal diffusionB = 2ΨDM
Lateral diffusion, disequilibriumC = K1 · R · (1 − R) · d2
f/DS + K2/DM
Separation by Chromatography – p.8/7
Van Deemter plot
http://www.chromatography-online.org/Dispersion/Van-Deemter-Equation/rs49.html
Separation by Chromatography – p.9/7
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
Separation by Chromatography – p.10/7
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
N = ( tRσ )2
Separation by Chromatography – p.10/7
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
N = ( tRσ )2
N = 16 · ( tRwb
)2
Separation by Chromatography – p.10/7
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
N = ( tRσ )2
N = 16 · ( tRwb
)2
N = 8 · ln 2 · ( tRw0.5
)2
� �
w0.5
Separation by Chromatography – p.10/7
Column performance
N = 16 · ( tRwb
)2
Neff = 16 · ( tR−t0wb
)2
H = LN
H is the Height Equivalent of a Theoretical Plate (HETP).
Separation by Chromatography – p.11/7
Column performance
N = 16 · ( tRwb
)2
Neff = 16 · ( tR−t0wb
)2
H = LN
H is the Height Equivalent of a Theoretical Plate (HETP).
Separation by Chromatography – p.11/7
Column performance
N = 16 · ( tRwb
)2
Neff = 16 · ( tR−t0wb
)2
H = LN
H is the Height Equivalent of a Theoretical Plate (HETP).
Separation by Chromatography – p.11/7
Column performance
H N nsample
[mm] [s−1]
TLC 0.7 1 µg - mgHPTLC 0.02 50 20 ng - mgColumn 20 0.0005 mg - kgHPLC 0.01 400 ng - mgpacked GC 0.5 100 µg - gcapillary GC 0.25 400 pg - µg
Separation by Chromatography – p.12/7
Column performance
Resolution RS =tR,1−tR,2
0.5·(wb,1+wb,2)
Separation by Chromatography – p.13/7
Column performance
Resolution RS =tR,1−tR,2
0.5·(wb,1+wb,2)
Separation factor α =k′2k′1=
tR,2−t0tR,1−t0
=K2K1
Separation by Chromatography – p.13/7
Column performance
Resolution RS =tR,1−tR,2
0.5·(wb,1+wb,2)
Separation factor α =k′2k′1=
tR,2−t0tR,1−t0
=K2K1
RS =√
Neff4α−1α
Separation by Chromatography – p.13/7