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