![Page 1: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/1.jpg)
Separation by ChromatographyLecture VAK 02-6007
May 17, 2005
Johannes Ranke
Separation by Chromatography – p.1/7
![Page 2: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/2.jpg)
1D Separation methods
1
1
2
2
Electrophoresis
Separation by Chromatography – p.2/7
![Page 3: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/3.jpg)
1D Separation methods
1
1
2
2
Electrophoresis
1
1
2
2
Chromatography
Separation by Chromatography – p.2/7
![Page 4: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/4.jpg)
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
![Page 5: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/5.jpg)
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
![Page 6: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/6.jpg)
Elution techniques
A + B
Separation by Chromatography – p.3/7
![Page 7: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/7.jpg)
Elution techniques
A
B
Separation by Chromatography – p.3/7
![Page 8: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/8.jpg)
Elution techniques
A
B
Separation by Chromatography – p.3/7
![Page 9: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/9.jpg)
Elution techniques
Time [min]
Det
ecto
r si
gnal
0 2 4 6 8 10
A
B
Separation by Chromatography – p.3/7
![Page 10: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/10.jpg)
Equilibrium based separation
tM
Separation by Chromatography – p.4/7
![Page 11: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/11.jpg)
Equilibrium based separation
tMtR
Separation by Chromatography – p.4/7
![Page 12: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/12.jpg)
Equilibrium based separation
tMtR
k′ = nSnM
Separation by Chromatography – p.4/7
![Page 13: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/13.jpg)
Equilibrium based separation
tMtR
k′ = nSnM=
tStM
Separation by Chromatography – p.4/7
![Page 14: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/14.jpg)
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
Separation by Chromatography – p.4/7
![Page 15: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/15.jpg)
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
Separation by Chromatography – p.4/7
![Page 16: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/16.jpg)
Equilibrium based separation
tMtR
k′ = nSnM=
tStM=
tR−tMtM
k′ = cS·VScM·VM
= K · VSVM
Separation by Chromatography – p.4/7
![Page 17: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/17.jpg)
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
![Page 18: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/18.jpg)
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
![Page 19: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/19.jpg)
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
![Page 20: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/20.jpg)
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
![Page 21: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/21.jpg)
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
![Page 22: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/22.jpg)
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
![Page 23: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/23.jpg)
Peak shapes
Isotherm
Signal shape
Retention time
cS
cMA
ttR
Vi
Separation by Chromatography – p.6/7
![Page 24: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/24.jpg)
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
![Page 25: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/25.jpg)
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
![Page 26: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/26.jpg)
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
![Page 27: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/27.jpg)
Peak dispersion
Injection
Dispersion in connecting tubes/capillaries
Dispersion in columns
Dispersion caused by signal detection
Separation by Chromatography – p.7/7
![Page 28: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/28.jpg)
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
![Page 29: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/29.jpg)
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
![Page 30: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/30.jpg)
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
![Page 31: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/31.jpg)
Van Deemter plot
http://www.chromatography-online.org/Dispersion/Van-Deemter-Equation/rs49.html
Separation by Chromatography – p.9/7
![Page 32: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/32.jpg)
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
Separation by Chromatography – p.10/7
![Page 33: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/33.jpg)
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
N = ( tRσ )2
Separation by Chromatography – p.10/7
![Page 34: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/34.jpg)
Column performance
t
S
tR
� �
2σ� �
wb = 4σ
N = ( tRσ )2
N = 16 · ( tRwb
)2
Separation by Chromatography – p.10/7
![Page 35: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/35.jpg)
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
![Page 36: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/36.jpg)
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
![Page 37: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/37.jpg)
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
![Page 38: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/38.jpg)
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
![Page 39: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/39.jpg)
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
![Page 40: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/40.jpg)
Column performance
Resolution RS =tR,1−tR,2
0.5·(wb,1+wb,2)
Separation by Chromatography – p.13/7
![Page 41: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/41.jpg)
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
![Page 42: Lecture VAK 02-6007 May 17, 2005 Johannes Rankechem.uft.uni-bremen.de/pdf/separation_ranke_2005_05_17.pdf · Elution techniques A B Separation by Chromatography – p.3/7. Elution](https://reader034.vdocument.in/reader034/viewer/2022050415/5f8b945349520072b7136b52/html5/thumbnails/42.jpg)
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