Supercritical Fluid Chromatography SFC Chromatographic Fundamentals Practical Verification of SFC...
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Transcript of Supercritical Fluid Chromatography SFC Chromatographic Fundamentals Practical Verification of SFC...
Supercritical Fluid Chromatography SFC
Chromatographic Fundamentals
Practical Verification of SFC
Theoretical Description of SFC / Scale-up
SFC on a Preparative Scale: Examples Prostaglandins, Tocopherols DHA / DPA, Phytol
On-line Analysis with SFC
Continuous Chromatography: SMB
Chapter 8
Chromatography with Supercritical Fluids
.
Mode of Operation: Elution chromatography
Elution Chromatography: A Chromatogram
Mass transport high
Solvent power high
Schoenmakers, Uunk 1987
Different Mobile Phases
Composition Trade name Application
Polysiloxane
R, R':
separation according tomolecular weight
100 % methyl OV-1, SE-3095 % methyl, 5 % phenyl OV-3, SE-5294 % methyl, 1 % vinyl, 5 % phenyl SE-5425 % cyanopropyl, 50 % methyl,25 % phenyl
OV-225
polyethylene glycol( CH2 CH2 O )n
Carbowax 20 M separation according to po-larity
SFC: Stationary Phases
Separation of aromatic hydrocarbons with different gases as mobile phase. Aromatic hydrocarbons: 1= benzene; 2 = naphthalene; 3 = fluorene; 4 = anthracene;5 = pyrene. Gases: a = carbon dioxide (CO2); b = nitrous oxide (N2O); c = propane (C3H8); d =propylene (C3H6); Column: 30 x 4.6 mm, unmodified silica gel. Initial pressure 12 MPa; Temperature296.15 K; Flow rate 670 cm3/min at STP (after Pickel /23/).
SFC: Different Gases as Mobile Phase
1 = caffeine;2 = theophylline;3 = theobromine;4 = xanthine(Randall 1984).
Variation of capacity ratios ofpolycyclic aromatic compounds due to modifier concentration (1.4-dioxane) in the mobilephase (n-pentane).P at column outlet 3.6 MPa;T = 513.15 K(Leyendecker et al. 1986).
SFC: Different Modifiers
Variation of retention times with temperatureof polycyclic aromatic components in n-butane at 4.5 MPa.1 = naphthalene; 2 = anthracene; 3 = pyrene; 4 = chrysene (Klesper and Leyendecker 1986).
Variation of retention timesof chrysene with pressure.Mobile phase n-butane(Klesper, Leyendecker 1986).
SFC: Influence of Pressure and Temperature
SFC: Pressure And Density Programming
Overloading by volume
Analytical injection
Overloading by concentration
Con
cent
ratio
n
Time
Chromatograms For Different Amounts of Injection
Adsorption Isotherms And Corresponding Chromatograms
SFC: Flow Scheme of Apparatus
Elution Chromatography: A Chromatogram
.// imismmr nntttk
,e
m
s K
V
VKk
tm = residence time in the mobile phasetr = retention time of the solutek' = capacity ratio = volumetric phase ratio Vs / Vm
Vs = the volume of the stationary phase, to,.Vm = the volume of the mobile phase
,e
e
m
se
K
n
nKk
./andand smesssmmm vvvnVvnV
.s
me
ms
sme v
vK
Vv
VvKk
e = molar phase ratiov = molar volume of a phaseV = total volume of a phase
Capacity Ratio
Capacity factors of paraffinesas a function of density(after Mollerup et al. /18/).
Capacity Factors
.
2exp
2
1
/
2
n
nv
nKVV
Fc i
ism
iim
with n = number of stages for p:
Chromatographic Separation
Maximum of the peak: ;nvi
Number of theoretical plates:
;/ ism
i KVV
Vvn
Points of inflection:
nnvnnv rightilefti ,, and
Points of intersection with the base line:
;21and21 ,, nnvnnv rightilefti
Chromatographic Separation
Width of peak:
.4 nb
Time at which the peak maximum appears
.or/1 iiiriiir nktnkt
Number of equilibrium stages
.4
2
i
iri b
tn
Chromatographic Separation
Chromatographic Separation
./ jiji kks Selectivity
.
2
ji
rirjji bb
ttR
Resolution
Resolution of two peaks of similar compounds
.1
1
4
2/1
k
k
s
snR
ij
ijij
Chromatographic Separation
.1
116
2
2
k
k
s
sRn
ij
ijij
Chromatographic Separation
,1
822 2
2
uDk
dk
u
DdH
isi
Fiimps
Van Deemter
Chromatographic Separation
Height of theoretical stage Hs
for SFC and HPLCfor packed columnswith different particle diameters(after Gere et al.)
Chromatographic Separation
SFC Analytical Scale, hp
Influence of temperature
20 MPa; mobile phase:CO2/methanol (5.3 wt.%);column: 125 x 4 mm; 5 m LiChrosorb Si 60.
Preparative separation
Chromatograms of fractions
Upnmoor
1992
Separation of Prostaglandins
Shapes of peaks under overloading conditionsChromatograms of -tocopherol mixture under overloading conditionsUpnmoor, Brunner, 1992
Separation of Tocopherols
Influence of modifier concentration
Solutions of -tocopherol in chloroform.Injected volume: 10 ml;mobile phase: CO2/methanol;15 MPa; 293 K; column: 125 x 4 mm;5 m LiChrosorb Si 60.Upnmoor 1992
Separation of Tocopherols
82
84
86
88
90
92
94
96
98
0 1 2 3 4 5 6 7
250 x 4.6 pS 250 x 8.0 pS
specific productivity DHA [mg/cm3 h]
Are
a D
HA
G
C [
%]
1mg DHA/(h,cm3) * 500 ml = 0,5 g DHA/h
Some kg DHA: Fully automatized plant !
RF=0,842
Productivity: DHA / DPA Separation by SFC
Dynamic axial compressed SFC column;
Dimensions:ID = 30 mm, length of packing: 0 to 190 (type I), 0 to 450 mm (type II) Pmax 400 bar, Tmax 200 °C.
SMB- Plant: Separation Columns
SFC, Preparative Scale
Rotating column Rotating ports
Continuous Chromatography
ExtractA + D
RaffinateB + D
FeedA + B + D
Desorbent D
Zone 1Purification of Adsorbent
Zone 3Enrichment of B
Zone 4 Purification of Desorbent
Zone 2Enrichment of A
True Moving Bed (TMB) Process
Principle of Simulated Countercurrent Separation
Mazzotti, ETH-Z
ExtractA+D
RaffinateB+D
FeedA+B
DesorbensD
Concentration A, B
Simulated Moving Bed-Process
Gottschall: PREP 95
Performance SMB vs Elution (99.5 % Purity)
Preparative SMB-Plant
Depta, 2000
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,60
10
20
30
40
50
concentration [mg/ml]
q [
mg
iso
mer/m
l stat
ion
ary
ph
ase]
Measurements
20
22
24
26
28
30
32
34
dq/dc
dq
/dc
0,0 0,5 1,0 1,5 2,0 2,5 3,00
20
40
60
80
100
120
dq/dc
concentration [mg/ml]
q [
mg
iso
mer/m
l stat
ion
ary
ph
ase]
Measurements
30
32
34
36
38
40
42
44
dq
/dc
Adsorption isotherms for Phytol cis- and trans- isomer (black lines) and derivatives (red lines). 225 bar, 40 °C, 1.8 mass% isopropanol as modifier.
Isotherms exhibit a point of inflection for each isomer.
221
21s cbcb1
)cb2b(cqq
Adsorption Isotherms
3 4 5 6 7 8 90,0
0,2
0,4
0,6
0,8
1,0
1,2feed concentration:
2 mg/ml 5 mg/ml 10 mg/ml 20 mg/ml 50 mg/ml
conc
entr
atio
n [m
g/m
l]
retention time [min]
Experimental and simulated phytol chromatogramssymbols: experimental data; lines: simulations.
Batch-Simulations
Model: equilibrium, axially dispersed plug flow with variable velocity of mobile phase,
Pressure drop: Ergun equation,
Properties of mobile phase (CO2) calculated with equation of state.
t
q
z
cD
z
uc
z
cu
t
c iiapi
ii
1
02
2
SMB process modeled with four key parameters: the net flow ratios mj:
Ruthven, Storti.
)1()1( totalcolumn
totalcolumnshiftSMB
zone
solid
solidTMBzone
zone V
VtQ
Q
QQm
SMB-Simulation
SMB- SFC: Volume-flow is a function of column length.Therefore, net flow ratios are not constant in each zone.
)1(_*
totalcolumn
totalcolumnshift
phasemobileSMBzone
zone V
VtQm
New parameter:
Representation of SMB-SFC process in a (m2*-m3
*)-plane,
solution of mass balance equations with finite difference method [Kniep et al.], adapted to variable velocity of mobile phase.
The algorithm is fast enough to calculate the region of complete separation in the (m2
*-m3*)-plane numerically, taking into account:
• any type of isotherm equation
• axial dispersion
• number of used columns
• change in mobile phase density
SMB-Simulation
242526272829303132333424
26
28
30
32
34
36
242526272829303132333424
26
28
30
32
34
36
operating point
raffinate (cis-isomer) pure
extract (trans-isomer) pureraffinate +extract pure
black triangles:infinite dilution situation and infinite number of theoretical plates same parameter set
as operating point in figure 5
Region of complete separation for phytol Cfeed=5.0 mg/ml 230 bar, no pressure drop, columns: 2/2/2/2; 300 plates per column
Columns: 1/1/1/1; 1000 plates per column
SMB-Simulation: Phytol Separation
242526272829303132333424
26
28
30
32
34
36
242526272829303132333424
26
28
30
32
34
36
operating point
raffinate (cis-isomer) pure
extract (trans-isomer) pureraffinate +extract pure
black triangles:infinite dilution situation and infinite number of theoretical plates same parameter set
as operating point in figure 5
Region of complete separation for phytol Cfeed=5.0 mg/ml 230 bar, no pressure drop, columns: 2/2/2/2; 300 plates per column
Columns: 1/1/1/1; 1000 plates per column
SMB-Simulation: Phytol Separation
20 25 30
20
25
30
35
20 25 30
20
25
30
35
Influence of pressure drop:
raffinate (cis-isomer) pure
extract (trans-isomer) pureraffinate +extract pure
Region of complete separation for phytol, infinite dilution, columns: 2/2/2/2; 300 plates per column, 230 bar, no pressure drop
Same as in left figure but calculations with pressure drop
Pressure drop leads to a shift of the complete separation region to lower values of m2
* and m3*
SMB-Simulation: Phytol Separation
1 2 3 4 5 6 7 80
1
2
Run N
Extract FeedRaffinate
1 2 3 4 5 6 7 80
1
Run M
1 2 3 4 5 6 7 80,0
0,5
1,0
1,5
2,0
2,5
Run O
ExtractFeedRaffinate Extract FeedRaffinate
7 8 9 10 117
8
9
10
11
m3
m2
low concentration in Feedlinear Adsorption isothermIdeal model
1 23
Experimental Results of Ibuprofen Separation
-10 0 10 20 30 40 50 60 70 80
0123456789
101112
peak
are
a [m
V*m
in]
conc
entr
atio
n [g
/l]
length [cm]
Sim S(+) Sim R(-) Exp S(+) Exp R(-)
0
2
4
6
raffinateextract
140 mgRacemate/min; 2/2/3/1 configuration
Separation of Ibuprofen
Verunreinigungen PhytolisomereConditions of separation:
240 bar, 50°C,column 4 x 250 mm packed withLiChrospher 100 (Silica),flow 2,56 g carbon dioxide / min,modifier 3 wt.- % EtOH,productivity 45 mg/(ml, h).
17mg pur
0,85 mg in Hexan
OH
CH3
CH3
CH3
CH3H H CH3
Phytol
• Diterpene-alcohol,• Intermediate for vitamin E, K1• esterified lipophilic compound
of chlorophyll