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TRANSCRIPT
Johannsen
5-1
Fundamentals and applications
of chromatography
WS 2004/05
PD Dr. rer. nat. habil. Monika Johannsen
Johannsen
5-2
Contents:
1 Introduction
2 Fundamentals of linear chromatography
3 Fundamentals of nonlinear chromatography
4 Adsorption isotherms
5 Process design
6 Equipment
7 Applications
Fundamentals and applications of chromatography
Johannsen
5-4
Way to solve a chromatographic separation problem
1) in analytical scale
! method development (stationary and mobile phase combination, temperature, and in SFC: pressure)
! determination of thermodynamic data (solubility, adsorption isotherms)
2) selection of preparative mode and simulation of separation
3) in preparative scale
! separation of diluted feed
! increase productivity by increasing the injected amount of substances
! productive separation
Johannsen
5-5
Selection of stationary phase for LC
Molecular weight of sample <1000, soluble in organic solvent
Normal phase
Reversed phase
Reversed phase
Reversed phase
Reversed phase with pH control
Reversed phase: Ion pair
chromatography
Reversed phase with
alkaline eluent
BasicIon
formingNon-ionic
Non-polar
Polar
[Chrombook, Merck]
Johannsen
5-6
Selection of stationary phase for LC
Molecular weight of sample <1000, soluble in water
Reversed phase
Reversed phase
Reversed phase with pH control
Reversed phase:
Ion pairing
Mono-, Disaccharides
Ion forming
Non-ionic
Strong acids or bases
[Chrombook, Merck]
Johannsen
5-7
Selection of stationary phase for LC
Molecular weight of sample >1000, soluble in water
Reversed phase
Hydrophobic interaction
(HIC)
Gel permeation (GPC, SEC)
Reversed phase
Saccharides Proteins, peptidesNon-ionic Nucleic
acids
Ion exchange
(Bio-)Affinity
Ion exchange
Normal phase
Gel permeation (GPC, SEC)
Ion exchange
[Chrombook, Merck]
Johannsen
5-8
Unmodified NP material:
! Al2O3 from Bayerit (Al(OH)3) or Böhmit (AlO(OH))
! Florisil - natural Mg-Al-silicate
! SiO2 from silica sand (silica gel) after pulping with HF
! Polymers on polystyrene-di-vinylbenzene basis
Normal phase (NP-) chromatography
Johannsen
5-9
Active sites in normal phase (NP-) chromatography
! covalent bonded hydroxyl groups (but: no RP-phases possible)
! combined retention by hydroxyl groups and Lewis centres
! pH stable from 2 -13
O O
AlO
Al
Lewis
basic
AlO
Al
OO
OHH2O
H
δ+
δ-
Alumina:
O O
AlO
Al
δ+
δ-
OH
chemisorbed wateracidic
Johannsen
5-10
SiO
O O OO
Si
O
Si
O
O O
OSi
Fe
OO
OO
O
O
O Al
H
H
Si
H
strong medium
weakvery strong
O
H
OO
Active sites in normal phase (NP-) chromatography
Silica gel:
Johannsen
5-11
Mobile phase: n-hexane or acetonitrile/MetOH:
Elution order: o, m, p-nitroaniline
H
O
H N2 NO
2
O
H H
O
H N2
NO2
O
H H
O
H N2
NO2 O
H
Retention mechanism in (NP-) chromatography
p-nitroaniline o-nitroanilinem-nitroaniline
Johannsen
5-12
Eluotropic series of solvents
Eluotropic series:
! a relative ranking of LC solvents ranging from non-polar to very polar properties in order of their eluting power
! polarity effects are due, e.g., to dielectric constant, dipole moment and hydrophobic-hydrophilic properties
Johannsen
5-13
Eluotropic series of solvents
n-hexanecyclohexene
toluenebenzene
diethyletherchloroform
dichloromethane1,2-dichloroethane
acetoneethylacetateacetonitrilepropanolethanol
methanolacetic acid
water
Incr
easi
ng p
olar
ity
On alumina:
Johannsen
5-14
Eluotropic elution on alumina
Start with 100% benzene then add acetonitrile
gradientelution
Benzene: ε°=0.32
Acetonitrile: ε°=0.65
isocraticelution
12 3
4
12 3 4
12 3 4
Johannsen
5-15
Common stationary phases (polar)
1. Diol [R = (CH2)2OCH2CH(OH)CH2OH ]
2. Cyano/Nitrile [R = (CH2)3CN ]
3. Amino [R = (CH2)3NH2 ]
! similar separations as with SiO2
! shorter equilibration times
! suitable for gradient elution
! usable for NP- and RP-chromatography
NP-chromatography with chemically bonded phases
Johannsen
5-16
SiO
O O OO
Si
O
Si
O O
OSi
O O
OO
OO
H
H
Si
HO
H
Si Si
Si
(CH )2 n
Cl
CH 3
CH 3
CH 3 Si
(CH )2 3
R
CH 3
CH 3
Cl
R= CN, Phenyl, NO , NH
2 2
SiO
O O OO
Si
O
Si
O O
OSi
O O
OO
OO
H
H
Si
O
Si Si
Si
(CH )2 n
CH 3
CH 3
CH 3
Si
(CH )2 3
R
CH 3
CH 3
Cl
R= CN, Phenyl, NO , NH
2 2
-H Cl-H
Chemically bonded silicas chromatography
silica backbone
spacer, e.g., propyl
R: end group
Johannsen
5-17
! stationary phase less polar than mobile phase
! mobile phase water or solvent mixtures with water
! the less polar the sample is the higher is retention
! C18 (octadecylsilan – “ODS“, RP18) phases most frequently used
Common stationary phases (non-polar)
1. Octadecyl, C18 [R = (CH2)17CH3 ]
2. Octyl, C8 [R = (CH2)7CH3 ]
3. Phenyl [R = (CH2)3C6H5 ]
Reversed Phase (RP-) chromatography
Johannsen
5-18
O OH
OH
O
Si
CH 3
CH 3
O
HO
Si
CH 3
H3C
O
HCH
3Si
CH 3
CH 3
H3C
OH
H3C H
3C
Mobile phase: H2O or H2O / ACN
Stat. phase: C-18
Reversed Phase (RP-) chromatography
Johannsen
5-19
! for separation of mixtures of acids, bases and neutral compounds
! RP phases used as stationary phases
! counterion influences separation factor
! separations mostly in buffered systems
! alternative to ion exchange chromatography
for acids ⇒ quaternary amines as counterion
for bases ⇒ alcyl and aryl sulfonate as counterion
Ion pair chromatography
Johannsen
5-20
O OH
O HH
O
Si
CH 3
CH 3
O
HO
Si
CH 3
H3 C
O
HCH 3Si
CH 3
CH 3H3 C H3 C
counterion+
Sample ion -
+sample
-
[
[paircounterion+
Stat. Phase: C18Mobile Phase:H2O oder H2O / AcN
Ion pair chromatography
Johannsen
5-21
! for separation of ionic organic and inorganic compounds
! analyses of chloride, nitrate, nitrite und sulfate
! separation of amino acids
! active sites fixed on organic resin (e.g. polystyrene-di-vinylbenzene)
! anion exchange: quaternary ammonium groups ( NR3+)
! cation exchange: sulfonic acid or carbonic acid groups ( SO3-)
Ion exchange chromatography
Johannsen
5-22
Stationary phase – cross linked polymer
CH CH2
vinyl benzene
CH CH2
CH CH2
di-vinyl benzene
! vinyl benzene ⇒ polystyrene
! 1 - 16% divinyl benzene as cross linker links polystyrene chains together (amount changes pore size and rigidity)
Johannsen
5-23
Stationary phase – cross linked polymer
CH CH2 CHCH CH2
CH CH2 CHCH CH2
CH CH2
R
Polystyrene-di-vinylbenzene:
cation exchange R = SO3-
anion exchange R = NR3+
Johannsen
5-24
! competition between sample and ions from the mobile phase
! separation influenced by type of ion exchanger, pH value, ionic strength and counterion of the mobile phase
NR3
+
Cl -
sample -
sample -
Cl -
NR3
+
SO3
-
sample + Na+
sample + SO3
-Na+
Ion exchange chromatography
Anion exchange Cation exchange
Johannsen
5-25
! Gel permeation chromatography - GPC (organic mob. phase)
! Gel filtration chromatography – GFC / Size exclusion chromatography - SEC (aqueous mob. phase)
! classification after molecular size (no adsorptive interactions with stat. Phase)
! elution volume only dependent on molecular size
! the smaller the molecule the higher the usable pore volume
! for separation between to molecules at least 10% difference in molecular mass required
Molecular exclusion chromatography
Johannsen
5-26
tR e t e n t i o n
Sig
nal
Molecular exclusion chromatography
! stationary phase contains small pores that analytes can diffuse into (depending upon size)
! larger molecules cannot fit into pores so they elute faster than smaller molecules
! pore size determines range of MW which can be separated
! exclusion limit: smallest molecule which can not fit into the pores; any larger molecule will have same VR
Johannsen
5-27
! stationary phase typically a cross-linked polymer, e.g. :
– Sephadex (glucose/glycerol polymer)– Bio-Gel (polyacrylamide gel)
! separation of complex mixtures (low molecular compounds from biological matrices)
! determination of distribution of molecular mass in quality control
! characterization of origin biological samples
! preparative isolation of single proteins
Molecular exclusion chromatography
Johannsen
5-28
! For analytical or preparative separation of complex biological molecules
! Very powerful method of purification in biology
! Selective separation of compounds or classes of compounds (nucleotides, proteins, enzymes, hormones, glycopeptides)
! makes use of tight, specific complex (highly selective):– antigen/antibody – enzyme/substrate
! Support from silica gel, glass, polystyrene or cellulose
(Bio-)Affinity chromatography
Johannsen
5-29
Ligand
sample
δ+δ−
δ+δ+δ−
δ− δ+ δ−
+δ+−
δ−
δ− δ+δ+ δ−
δ− δ+
δ+−
δ−+
δ+ δ−
δ+δ−
Stronger affinity pH change excessive ions
Elution by
(Bio-)Affinity chromatography
Support
Johannsen
5-30
Method development
Optimisation of:
! stationary phase
! mobile phase (liquid or supercritical)
! mobile phase modifier (e.g.alcanol)
! temperature
! pressure/density (in SFC)
Optimisation criteria:
separation factor / peak resolution, solubility """" productivity
cycc
Feedspecific tV
mPR⋅
=
Johannsen
5-31
! Reduction of organic solvents (→ no/less solvent recovery)
! complete separation of product from solvent by decrease in density(→ no further concentration step, high product purity)
! low operating temperature (suitable for separation of temperaturesensitive substances)
! high diffusion coefficient → high number of theor. plates/low viscosity→ high flow rates → high productivity
! selectivity variation by change in P/ρ and T
! increase of limited solubilities by modifier adding
Preparative SFC - Advantages
Johannsen
5-32
Polar substances in SFC
Methods for increasing migrations rate of medium polar substances:
1) Increase polarity of mobile phase (different fluid or modifier)
2) Increase density
3) Increase temperature
4) Decrease polarity of stationary phase
SFC with CO2 is normal phase chromatography !!!
Johannsen
5-33
Criteria- separation factor- peak resolution- retention factor
Optimization- stationary phase- mobile phase- modifier- mod. concentration- temperature- pressure
Method development in SFC
2,0 2,5 3,0 3,5 4,0 4,5 5,0
1,14
1,16
1,18
1,20
1,22
1,24
1,26
1,28
1,30 30°C 40°C 50°C 60°C 70°C 80°C
(α−/α3)-tocopherol; 22 MPa
Sep
arat
ion
fact
or [-
]
Modifier concentration [vol.-%]
Separation of tocochromanols by SFC on Kromasil
Johannsen
5-34
Criteria- separation factor- peak resolution- retention factor
Optimization- stationary phase- mobile phase- modifier- mod. concentration- temperature- pressure
Method development in SFC
30 40 50 60 70 805
6
7
8
9
10
11
12
13α-tocopherol, 2 % 2-propanol
15 MPa 19 MPa 22 MPa
Ret
entio
n fa
ctor
[-]
Temperature [°C]
Separation of tocochromanols by SFC on Kromasil
Johannsen
5-35
Design of SMB processes
Bel
adun
g q
Konzentration c
0 2 4 6 8-400
-200
0
200
400
600
800
1000
Retentionszeit [min]
Det
ekto
rsig
nal [
mAU
]Measurements Simulation I
4 6 80,0
0,2
0,4
0,6
0,8
1,0
Retentionszeit [min]
Det
ekto
rsig
nal [
mAU
]
m2
m3
Simulation II
Operating point
Production!
Johannsen
5-36
Optimization SMB-SFCInfluence of the feed concentration on productivity and solvent consumption
System:α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol, Lcolumn = 14.4 cm; 2/2/2/2 configuration
0 2 4 6 8 10 12 14 16 18 20 220
5
10
15
20
25
30
35
40
45
feed
flow
rate
[g/m
in]
feed concentration [mg/ml]
0 2 4 6 8 10 12 14 16 18 20 220
5
10
15
20
25
prod
uctiv
ity [g
Toco
/(l*h
)]
concentration [mg/ml]0 2 4 6 8 10 12 14 16 18 20 22
0
5
10
15
20
25
400
600
800
1000
1200
1400
1600
400
600
800
1000
1200
1400
1600
solvent consumption
productivity
solv
ent c
onsu
mpt
ion
[gC
O2 /
gTo
co]
Johannsen
5-37
4 6 8 10 12 14 165
10
15
20
25
30
35
40
45
50
55
solv
ent c
onsu
mpt
ion
[gC
O2/g
Toco
]
Prod
uctiv
ity [g
Toco
/(l*h
)]
length of column [cm]
550
600
650
700
750
solvent consumption
productivity
Optimization SMB-SFCInfluence of column lengthSystem: α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa;
5 wt.-% 2-propanol, 2/2/2/2 configuration; cfeed = 6 mg/ml
Johannsen
5-38
0 10 20 30 40 50 60
0,0
0,5
1,0
1,5
2,0
feed concentration cfeed= 6 mg/ml
Exp α-Tocopherol Exp δ-Tocopherol Sim α-Tocopherol Sim δ-Tocopherol
conc
entra
tion
[mg/
ml]
column length [cm]
Experimental results SMB-SFC
Comparison simulation – experimental results
System: α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol, Lc = 7.5 cm; 2/2/2/2 configuration
Johannsen
5-39
Optimization Batch-SFC Influence of column length
System: α- and δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol; umax = 0.55 cm/sec
10 15 20 25 30 35 40 45 50 55
0
20
40
60
80
100
column length [cm]
max
imum
of V
inj [m
l]
10 15 20 25 30 35 40 45 50 55 6070
80
90
100
110
120
130
prod
uctiv
ity [g
/(l*h
)]
column length [cm]
Johannsen
5-40
Comparison SMB - SFC and Batch-SFC
# System: α- and δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol; umax = 0.55 cm/sec
# Purity: Both components > 99 %
120.8620400.07220Elution
58.3495150.1306(sum: 36)
SMB(6 columns)
productivity[gToco/lSP⋅h]
CO2 consumption[gco2/ gToco]
cfeed
[mg/ml]mstat.phase
[kg] Lcolumn
[cm]
# higher specific productivity of the Batch SFC (factor 2)
# higher solvent consumption of the Batch SFC (factor 1,3)
Johannsen
5-41
Maximal loading in chromatography
Surface area of silica gel at 300 m2/g can adsorb 3 x 1019 molecules of
a size of 1 nm2 and a molecular mass of 150-250 g/mol.
These have a total mass of around 10 mg.
Loading: max. 3%!
Productivity for example with 60 injections per hour: 1800g/kgSGh
Rule of thumb: 10 – 30 g Feed per kg silica gel
Johannsen
5-42
Production costs
Influence of production capacity on relative production costs of EPA from fish oil
[Lembke, 1998]