6-Supercritical Fluid Chromatography SFC

Supercritical Fluid
Chromatography
Aurelia Leyva and Trang Duong
Chem 230
11/30/2010
1
Chapter 33
Supercritical-Fluid
Chromatography
Supercritical-fluid chromatography (SFC), in
which the mobile phase is a supercritical
fluid, is a hybrid of gas and liquid
chromatography that combines some of the
best features of each. For certain
applications, it appears to be clearly
superior to both gas-liquid and highperformance liquid chromatography.
Important Properties of Supercritical

Fluids
A supercritical fluid is formed whenever a
substance is heated above its critical
temperature. At the critical temperature, a
substance can no longer be condensed into
its liquid state through the application of
pressure.
The physical properties of a substance in
the
supercritical-fluid
state
can
be
remarkably different from the same
Supercritical Fluid
Substance at a T
and P above critical
point
Effuse through
solids like a gas and
dissolve materials
like a liquid
Close to critical
point, small
changes in T or P
results in large
changes in density
W.W. Christie and Lipid Technology
T/P Phase Diagram
www.che.tohoku.ac.jp
Supercritical fluids have solvent power

similar to a light hydrocarbon for most
solutes. However, fluorinated compounds
are often more soluble in scCO 2 than in
hydrocarbons.
Solublity increases with increasing density
(i.e. with increasing pressure).
Supercritical fluids can have solvating

powers similar to organic solvents, but
with higher diffusivities, lower viscosity,
and lower surface tension.
The solvating power can be adjusted by
changing the pressure or temperature, or
adding modifiers to the supercritical fluid.
A common modifier is methanol (typically
1-10%) which increases the polarity of
supercritical CO2.
SCF Extractions
The main advantages of using
supercritical fluids for extractions is that
they are inexpensive, contaminant free,
and less costly to dispose of safely than
organic solvents.
Supercritical fluid CO2 is used to extract
caffeine from coffee.
Flavours for brewing are extracted from
hops using SCF extraction.
Analytes easily recovered

Equilibrate at low temp and atmospheric P
CO2 is most commonly used
Good for thermally unstable species
SFC Instrumentation
Solvent delivery system
Injector
Column/Column Oven
Restrictor
Detector
Data System
SFC Apparatus
1. Mobile phase (CO2) 5. Column

2. Pump
6. Detector
3. Injection System
7.Chromatogram
4. Oven
http://www.cee.vt.edu/ewr/environmental/teach/smprimer/sfc/sfc.html
1
3
Schematic Diagram of a Packed Column

SFC
14
Restrictor
Back pressure regulator is very important
or gas P will drop in column
May let SFC convert to gas before
detector
Solvent Delivery System

Maintains precise mobile phase flow
(1 to 10 L/min {OT} or 1 to 10
mL/min {Packed}).
Aids in the control of the system
pressure (up to 60 Mpa).
Moves mobile phase in the liquid
state under pressure through the
injector & into the column.
SCF Chromatography
High pressures are used so that the SCF is
much more dense (102-103 x) than a gas
Thus it has better solvating properties
but still not as dense as a liquid
For chromatography we want:
low viscosity coefficient (gas)
Low diffusion coefficient (liquid)
The values of these for SCFs are intermediate
High MW compounds that cant be separated by
GC can be analyzed by SFC.
Compound Tc C
31.3 72.9
CO2
C2H4
9.9
Pc atm
0.96
50.5
N2O
36.5
72.5
NH3
132.5 112.5
--0.94
0.40
n-C5
196.6 33.3
0.51
n-C4
152.0 37.5
0.50
CCl2F2
CHF3
111.8
25.9
d*
40.7
46.9
1.12
----
Supercritical Fluid
Chromatography (SFC)
Property
Density (g/cm 3 )
Diffusion coefficient
(cm 2 /s)
Viscosity (G Cm -1 s -1 )
Gas (STP)
(0.6-2) x 10 -3
(1-4) x 10 -1
SCF
0.2-0.5
10 -3 x 10 - 4
Liquid
0.6-2
(0.2-2) x 10 -5
(1-4) x 10 - 4
(1-3) x 10 - 4
(0.2-3) x 10 -2
Hewlett-Packard developed and manufactured the SFC instrument in 1982
Combinations of desirable characteristics from both GC and HPLC
Used for compounds:

Highly polar
Large MW
Too thermally labile for GC
Compounds undetectable for HPLC detector
Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011
2
4
Types of Injectors
a. Loop injection
-Low pressure feed pump needed to fill
the loop
-Mostly for preliminary tests of column
performance and elution parameters
b. In-line injection
-Flexibility for changing injected volume
-High-pressure pump required to inject
feed solution
-Injected stream dissolved in eluent flow
c. In-column injection
-Permits injection of the feed solution
directly onto the column
-No dilutions required
Journal of Liquid Chromatography & Related Technologies, 28: 12331252, 2005
2
5
25
Injectors
Typical HPLC design injectors for
packed columns.
Split/Splitless valve injector (0.01 to
0.05 L injections) for open tubular
columns.
Timed - split injector (0.01 to 0.05 L
injections) for open tubular columns.
Injector Volumes
Open tubular columns
Injection volumes >96nL
Greater volume affects resolution
Packed columns
Injection volumes >1uL
2
7
Column
Open Tubular
- Smaller column diameter more efficient
- Efficiency decreases at high density (decrease u)
* Decrease column diameter
* Increase T (increase diffusion, solubility, volatility)
- Minimal pressure drop effect
J.W. King, H.H. Hill, and M.L. Lee. 1993
2
8
28
Column
Packed
- Packing reduces permeability and creates flow resistance, L <25cm
- Efficiency increases with increasing density (Dm decreases)
- Smaller particle size = smaller H, but increase pressure drop, and decrease
permeability
- Large P drop effect (density decreases, solubility decreases along column length)
2
9
Column
Retention Time
-Packed columns are more

efficient with higher density
analytes.
Packed capillary
- Lower volumetric flow rate = more compatible with mass flow
detector, greater sensitivity in conc. detector, easier sample
transfer in multi-D system, higher permeability-longer column
3
0
Stationary Phase
Same as those for GC and LC, with some modification.
Silica/Alumina
Useful for non-polar compounds
Lead to irreversible adsorption of some polar solutes
Bonded to provide less adsorptive packing material
Octyl, octadecyl, cyanoalkyl, aminoalkyl, diolakyl
Need organic modifiers to elute analytes
Widely used polar S Phase
Polysiloxanes-- stable, flexible Si--O bond lead to good diffusion.
Substituted with chemical groups for selective interaction with analyte
Polymethylsiloxanes--increase efficiency in separating closely related polar
analytes
Cyanopropyl polysiloxanes-useful for compounds with COOH
3
1
Stationary phase
Columns similar to those of HPLC.
Capillary SFC organic films bonded to
capillaries.
Because SCFs have low viscosities can
use very long columns.
Get high resolution in a reasonable time.
SFC Columns
Open tubular (derived from GC)
- Large # theoretical plates (~X500)
- Easier to control pressure (low P
drop)
Packed (derived from HPLC)
- Faster analysis
- Higher flow rates
- Higher sample capacity
Open Tubular Columns

Smaller than GC capillary columns,
typically 50 m i.d., 10 to 20 m in
length
MP must be more stable due to
extreme conditions of supercritical
fluids
Packed Columns
Similar to HPLC columns (10, 5, or 3
m porous particles)
Silica based chemically bonded
phases
Typically 10 cm long X 4.6 mm i.d
Mobile Phase
Neat Fluid
CO2 low Tc, low toxicity, nonflammability
N2O high degree of nonideality,
permits high fluid density when
compressed
Isopropyl/diethyl ether separation
of polynuclear aromatics and
polymers.
Hydrocarbon limited use due to high
flammability
n-Pentane separation of oligomers
Flourocarbon low Tc, unique
selectivity for certain solutes
3
6
36
Mobile Phase
Mix Fluid: Addition of polar organic modifier
-Enhance solubility of polar analyte
-Reduce retention volume
-Eliminate strong interaction between absorptive site and polar solute
-Will modify polarity of eluent (asso. with and polarity index)
-k and affected by modifier identity and concentration
Caution when using

mixed fluids to
assure that the
components are
miscible over the
range of T and P
used.
3
7
SFC and Retention

Retention dependent on temperature,
pressure, mobile phase density, and
composition of the stationary and mobile
phase.
Complex interactions and not easily
predictable.
For supercritical fluids
- solvating properties similar to liquids
- viscosity closer to gases
Solvating power density
Temperature/Pressure Effects
At lower P, > T, < solubility
At higher P, > T, > solubility
-> T, Pv of solute > solute solubility
-< fluid density < solubilizing power
> T, < solvent
>P, > solvent
Supercritical CO2 Density

P (MPa)
T (oC)
(g/cm3)
7.3
40
0.22
7.3
80
0.14
7.3
120
0.12
40
40
0.96
40
80
40
120
0.82
0.70
Solvent Programming
Programming is very useful in controlling
solvent strength.
Variations in P (density), T, and mobile
phase composition.
Density programming is most widely
used (not simple relationship, T & P).
-> density, > solubility, < retention
- Combined T & P programming to
control and thereby solubility and
diffusion
SFC Mobile Phases

Generally non-polar compounds with low
to moderate critical properties
- CO2, N2O, ethane, pentane
Normal phase type separations
- non-polar mp and low polarity sp
(substrate + amino, diol, or cyano
groups)
Elution = function of molecular mass &
Carbon Dioxide: SFC Solvent

Low Tc
- operating T as low as 40oC
Moderate Pc and c of 0.448g/cm3
- reach high with P < 40 MPa
Safe to use
nontoxic,
nonflammable,
noncorrosive, inert
Detector compatible
Wide range
CO2 as mobile phase
Commonly used
Transmits UV
Colourless
Odourless
Non-toxic
Relatively cheap
Suitable Tc T is generally kept ~10o
above the Tc.
Other SFC Solvents

Nitrous Oxide - Similar in solvating and
separations properties to CO2
Alkanes - less safe and not as detector
compatible than CO2
- better solvent characteristics for nonpolar solutes
Halocarbons, xenon, etc. - specialty
applications only
More polar solvents for highly polar &
high molecular weight compounds
Solvent Modifiers
Add organic modifiers to > solvent
strength
- methanol
- isopropanol
- dichloromethane
- THF
- acetonitrile
Mobile phase has solvating power which

can be adjusted Can vary composition (organic additives),
T and P.
Rapid equilibration times.
Lower T in SFC than GC good if
thermally unstable.
Solvates polymers
More environmentally friendly no organic
solvents to dispose of or recycle
Detectors
Can use either GC or LC detectors with adaptations
-LC detector: in close cell, fluid maintain under pressure, but cooled liquid
-GC detector: in open cell, fluid decompressed to gas
Ambient Pressure Ionization
FID : organics
hydrogen-atmosphere FID: organometallics
thermionic detector: N, P containing compounds
ECD: Halogenated/electronegative compounds
photoionization detector: organics with less than 10.2eV ionization potential
ion mobility detector: high electron/proton affinity compounds
Optical
UVD
FT-IR
Flourescence detector
FPD (flame photometric detector): S, P containing
compounds
CD (chemiluminescence detector): S, and easily
oxidizable compounds
Element selective plasma emission detector
LSD (light scattering detector)
Vacuum
MS with (CI, EI, CE, API ionizer)
4
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Detectors
Most any detector used in GC or
HPLC can be used.
FID and UV detectors commonly
used.
Coupled Detectors
- MS
- FTIR
Detectors
UV absorption and FID are most common
UV absorption with packed columns
FID with capillary SFC
SFC Comparison
5
2
SFC Comparison
5
4
SFC Separations
SFC is a hybrid of gas and liquid
chromatography that combines some of
the best features of each
As in HPLC, variation of the mobile phase
composition affects separation
In SFC, mobile phase affinity for the
analyte is a function of mobile phase
density
Density is controlled by controlling system
pressure
Highly polar samples are not easy to
handle (high critical parameters & high
SFC Advantages vs HPLC

Supercritical fluids have low viscosities
- faster analysis (5 to 10 X faster)
- less pressure drop across the
column
- the use of open tubular
columns is
feasible
Column lengths from 10 to 20 m are
used
Can be used with a wide range of
sensitive detectors
Resolving power is ~5X that of HPLC
SFC Advantages vs GC
Can analyze non-volatile, polar, or
adsorptive
solutes
without
derivatization.
Can
analyze
compounds.
thermally
labile
Can analyze solutes of much higher

molecular weight.
SFC Advantages
Provides rapid separations without the use of organic
solvents
Uses environmentally conscious technology
Faster separation process
Decrease in resistance to mass transfer in column
High resolution at lower temperature

Low viscosity
High diffusion coefficient
Greater diffusibility
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9
SFC Disadvantages
SCF is a recent chromatography technique that requires
development in the following areas:
Method development
Hardware development
Pump design
Injection methodology
Expensive technology
Pressure & Temperature
Cleaning is time consuming
Ind. Eng. Chem. Res. 2000, 39, 4531-4535
6
0
Applications
Fossil Fuels and Hydrocarbons

Agrichemicals
Drugs and Pharmaceuticals
Polymers
Explosives and Propellants
Lipids
Carbohydrates
Industrial Chemicals
Foods and Flavors
Natural Products
Metal Chelates and Organometallic Compounds
Enantiomers
6
1
Separation of polyaromatic
hydrocarbons in coal tars
Gradient of 50
100% acrylonitrile
(CH2CHCN) over 40
minutes
GC: T gradient, 80C to 250C over

the 50-minute retention time
SFC
A pressure ramp is
used in the solvent,
such that solvent
density rose from
0.225g/L to 0.7 g/ml
over the retention
period of 120
minutes.
Agrichemical Application
Pesticide detection
GC conditions:
15m x 200pm
Chrysene
Open tubular column

0.15 q film thickness
Temp. program from 40 to
200C at l0C/min, then
4C/min to 240C
SFC conditions:
10m x 50pm
Open tubular column
0.15 pm film thickness
CO2 100C
Density program from 0.55 to
GC
SFC
0.70g/mL at 0.005g/mL per

min after a 10-min
J. W. KING, H. H. HILL, AND M. L. LEE
6
6
Natural Products Application
Cholesterol content of a fish oil
Content of capsule was emptied

into injection solvent n-hexane
The oil-laden solution was

directly injected into SFC &
separated using density program
Two peaks were eluted with in

program density interval 0.400.42 g/mL
Found by spiking the above
solution with known amounts
of alpha-tocopherol (vitamin
E) and cholesterol
6
Journal of Chromatographic Science, Vol. 28, January 1990 7
Comparison of HPLC to SFC
Metabolized Camazepam by HPLC

(Rat Liver
Microsomes,
37
C, 90 min)
M5, TMZ
Column: Zorbax SB-C18, 150 x 4.6 mm

Mobile phase: ACN - 0.02 M phosphate
buffer pH 7 ( 40 : 60 V/V )
Flow rate: 1.2 ml/min
Detection: 230 nm
CMZ
Camazepam(CMZ)
Norcamazepam(NCMZ)
Temazepam(TMZ)
M4, M5, M9' metabolites
7.571
3.921
1.223
1.325
1.478
13.058
6.160
HPLC Analysis
M 4
M9'
NCMZ
0.0
Time (min)
68
15.0
Metabolized Camezapam by SFC

(rat
liver
mAU
microsomes,
37
C, 90 min)
CMZ
SFC Analysis
Column: Lichrosphere NH 2 , 250 x 4.6 mm
Mobile phase: 13% ethanol in CO 2 for 4 min,
then 3%/min to 30%
Flow rate: 2.5 ml/min
Outlet pressure: 150 bar
Oven temperature: 30 C
Detection: 227 nm
400
350
300
M5
250
Camazepam
(CMZ)
Norcamazepam
(NCMZ)
Temazepam (TMZ)
M4, M5, M7, M9, M9' metabolites
200
150
100
M4
NCMZ
50
M9'
TMZ
M7
M9
0
1
Time (min)
69
8
Identification of Metabolites using UV Spectra
M4
3.703
2.316
CMZ
2.941
M5
mAU
400
350
300
250
200
150
100
50
0
Time
Norm.
400
Spectrum of M4
Spectrum of M5
Spectrum of CMZ
350
300
250
200
150
100
50
0
200 225 250 275 300 325 350 375
Time (min)
70
CMZ
4.461
mAU
400
350
300
250
200
150
150
100
0
2.316
Identification of Metabolites Using UV Spectra
Time (min)
Norm.
Spectrum
Spectrum
of the peak at 4.461 min

of CMZ
400
350
300
CMZ
250
200
150
100
50
0
200 225 250 275 300 325 350 375
71
Effect of Organic modifier
Tricyclic Antidepressants
6.Buclizine
7. Benactyzine
OH
CH
CH
2
C(CH )
3 3
CCOCH2CH2N(C2H 5) 2
8. Hydroxyzine
Cl
CH
9.Perphenazine
Cl
CH2CH2OCH 2CH2OH
10.Thioridazine
CH 3
CH2CH2CH2
N
N
Cl
CH2CH2OH
N
CH2CH 2
SCH 3
N
S
S
72
Effect of Organic modifier
Packed Column Analysis of Tricyclic

Antidepressants
(9.5 ppm each; 5
3
mAU
15
l injected)
1.
2.
3.
4.
5.
Conditions:
Column : 4.6 x 250 mm Lichrosphere Cyanopropyl (5 m)
Mobile phase: CO 2 + 10% MeOH + 0.5% Isopropylamine
Flow rate: 3 ml/min
Column temperature: 50 C
1
Detector: UV - 254 nm
Amitriptyline
Imipramine
Nortriptyline
Desipramine
Protriptyline
4
5
10
2
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Time
4.00
4.50
5.00
5.50
6.00
(min)
10% Methanol
73
6.50
7.00
20% Methanol
Packed Column Analysis of

Tricyclic Antidepressants
(9.5 ppm each; 5 ul injected)
mAU
25
20
Conditions:
Column : 4.6 x 250 mm Lichrosphere Cyanopropyl (5 u m)
Mobile phase: CO 2 + 20% MeOH + 0.5% Isopropylamine
Flow rate: 3 ml/min
1
Column temperature: 50 C
2
Detector: UV - 254 nm
15
1.
2.
3.
4.
5.
Amitriptyline
Imipramine
Nortriptyline
Desipramine
Protriptyline
10
0
0.50
1.00
1.50
2.00
Time (min)
2.50
3.00
74
3.50
Same columns used in HPLC can be used in SFC
Steroids
H 3C
OH
H3C
CH 3
OH
CH 3
H3C
C=O
HO
CH 3
Testosterone
Estradiol
CH 2 O H
H3C O
Progesterone
H3 C
C=O
OH
CH 3
HO
CH 2 O H
Estrone
HO
H3C
H3 C
Cortisone
C=O
OH
OH
CH 3
CH 3
OH
OH
CH 3
Hydrocortisone
HO
Estriol
CH 3
17 Methyltestosterone
75
Packed Column Analysis of Steroids on

Different Silica Columns
2.1 x 200 mm Hypersil
60 C, 3 mL/min, 250 bar, 6% methanol
R es p o n se
215 nm
Progesterone, Methyltestosterone, Testosterone
Estrone, Estradiol, Cortisone, Hydrocortisone,
Estriol
2.1 x 250 mm Lichrosphere Si-60

70 C, 2.5 mL/min, 200 bar, 20% methanol
210 nm
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Time
2.00
2.20
2.40
2.60
2.80
3.00
(min)
76
3.20
3.40
Packed Column SFC for Pharmaceutical

Applications
Needs:
Product quality assurance
Fast method development
Chiral Purity Analysis
Complementary techniques
1.06% -l-
High throughput
220 nm
Key Applications:
Chiral compounds
Excipients
Drugs
Drug/metabolites
carbobenzyloxy-d,l-alanine
1.76% -d4.0
4.50
5.0
5.5
Time (min)
220 nm
6.0
6.5
250 x 4.6 mm Chiralcel-OD

20% ETOH(+0.2% TPA)/CO2 1ml/min
77
Effect of Additive on Chiral Separation

of Propranolol by Packed Column SFC
mAU
60
50
40
without additive
100 ppm R,S-propranolol

Chiralcel OD 4.6 x 250 mm, 10
30% methanol in CO 2
2 ml/min
200 bar outlet pressure
30 C oven temperature
m particles
30
20
10
0
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
Time (min)
78
Effect of Additive on Chiral Separation

of Propranolol by Packed Column SFC
100 ppm R,S-propranolol
150
Chiralcel OD 4.6 x 250 mm, 10 m particles

30% methanol with 0.5% isopropylamine in CO 2
2 ml/min
200 bar outlet pressure
30 C oven temperature
with additive
100
50
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
Time (min)
79
7.00
7.50
Fast Chiral Separation of Propranalol

by Packed Column SFC
mAU
300
250
200
250x 4.6mm,10
m Chiracel OD
40 C, 250 bar, 4 mL/min

30% (Methanol + 0.5%
Tripropylamine)
150
100
Rs = 3.5
50
Time(min)
80
References
M. Perrut. Supercritical Fluid Applications: Industrial Developments

and Economic Issues. Ind. Eng. Chem. Res., 39, 12, 2000
J. W. King. Applications of Capillary Supercritical Fluid

Chromatography-Supercritical Fluid Extractions to Natural Products.
Journal of Chromatographic Science, 28, 1990
W. Majewski, E. Valery, O. Ludemann-Hombourger. Principle and

Applications of Supercritical Fluid Chromatography. Journal of Liquid
Chromatography & Related Technologies, 28, 12331252, 2005
H. S. Bhatt, G. F. Patel, N. V. Vekariya1, S. K. Jadav. Super critical fluid

chromatography-an overview. Journal of Pharmacy Research,
2(10),1606-16011, 2009
J. W. King, H. H. Hill, M. L. Lee. Analytical Supercritical Fluid

Chromatography and Extraction. Ind. Eng. Chem. Res., Vol. 39, No. 12,
1993
8
1
Capillary SFC
capillary SFC can be used to separate the analytes of interest from a

relatively nonvolatile sample matrix without resorting to sample
preparation prior to chromatography.
capillary SFC include the separation of reaction products from higher

molecular weight starting materials and the deformulation of commercial
products.
Capillary SFC, when coupled with micro-scale SFE also permits the
characterization of small samples, such as portions of single seeds and
extractables from single, live insects.
Journal of Chromatographic Science, Vol.
28, January, 1990
82
8
2
PROBLEMS
What controls the pressure in SFC?
Why is a modifier fluid needed?
To help elute polar analytes
List one advantage of SFC compared to HPLC and GC?
The restrictor
can analyze high MW, non-volatile compound, higher resolution compare to GC (see slide
17)
Compare the plate number (N) between an open tubular (Ds = 1x10-6cm2/s,
dc = 50um, df = 0.25um, L = 10m, u = 5.8cm/s) and packed column (ko =
0.5, dp = 5um, L = 0.1m, u = 2.1cm/s). k=2, and Dm = 2x10-4cm2/s.
OT (N) = 19000, Packed (N)= 9100
8
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Important Info Web pages
http://www.google.com/imgres?
imgurl=http://www.chem.leeds.ac.uk/People/CMR/images/scco24.jpg&imgrefurl=http://www.chem.leeds.ac.uk/People/CMR/criticalpics.ht
ml&usg=__y5nA5mu0N3cnYx3cqlUrb7dKik=&h=294&w=308&sz=54&hl=en&start=3&zoom=1&um=1&itbs=1&tbnid=chFV9_BtWriMMM:&tbnh=112&tbnw=117&prev=/im
ages%3Fq%3Dsupercritical%2Bfluid%26um%3D1%26hl%3Den%26sa%3DN%26tbs%3Disch:1
http://en.wikipedia.org/wiki/Supercritical_fluid
8
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8
5
Supercritical Fluid
Chromatography
Combines some advantages of liquid
chromatography with the high efficiency
of gas chromatography.
Instrumentation and Operating Variables

Instruments for supercritical-fluid chromatography
are similar in design to high-performance liquid
chromatography.
Columns
Both packed columns and open tubular columns are
used in supercritical fluid chromatography. Packed
columns have the advantages of greater efficiency
per unit time and the capability of handling larger
sample volumes. Packed columns for SFC can be
much longer than those for HPLC, providing well
over 100,000 plates. Because of the low viscosity of
supercritical media, columns can be much longer
than those used in liquid chromatography, and
column lengths of 10 to 20 m and inside diameters of
50 or 100 m are common.
Mobile Phases
The most widely used mobile phase for
supercritical-fluid chromatography is carbon
dioxide. It is an excellent solvent for a variety
of nonpolar organic molecules. In addition, it
transmits in the ultraviolet and is odorless,
nontoxic, readily available, and remarkably
inexpensive
relative
to
other
chromatographic
solvents.
Its
critical
temperature of 31oC and its pressure of 73
atm at the critical temperature permit a wide
selection of temperatures and pressures
without exceeding the operating limits of
modern
high-performance
liquid
Detectors
A major advantage of supercritical-fluid
chromatography is that the sensitive and
universal
detectors
of
gas-liquid
chromatography are applicable to this
technique as well. For example, the
convenient flame ionization detector of gasliquid chromatography can be applied by
simply allowing the supercritical carrier to
expand through a restrictor and into a
hydrogen flame, where ions formed from the
analytes are collected at biased electrodes,
giving rise to an electrical current.
Supercritical-Fluid Chromatography
versus Other Column Methods
Several physical properties of supercritical
fluids are intermediate between the properties
of gases and liquids. As a consequence, this
new type of chromatography combines some
of the characteristics of both gas and liquid
chromatography.
Thus,
like
gas
chromatography,
supercritical-fluid
chromatography is inherently faster than liquid
chromatography because of the lower
viscosity and higher diffusion rates in the
mobile phase.
Applications
It is applicable to a class of compounds that
is not readily amenable to either gas-liquid or
liquid chromatography. These compounds
include species that are nonvolatile or
thermally unstable and, in addition, contain
no chromophoric groups that can be used for
photometric detection. Separation of these
compounds is possible with supercritical-fluid
chromatography at temperatures below 100
o
C; furthermore, detection is readily carried
out by means of the highly sensitive flame
ionization detector.
CHAPTER 29
Supercritical Fluid
Chromatography
The mobile phase is a supercritical fluid
(a fluid above its critical T and critical
pressure)
Supercritical fluid properties (density,
viscosity, and refractive index) vary with
T&P
Supercritical Fluids
At temperatures and pressures above its critical
temperature and pressure (critical point), a
substance is called a supercritical fluid. The
critical temperature is the temperature above
which a distinct liquid phase cannot exist. The
vapor pressure at its critical temperature is its
critical pressure.
Where supercritical fluids exist: The forces from
the kinetic energy of the molecules exceeds the
forces from condensing influence of the
intermolecular forces, so no distinct liquid phase
SFC Mobile Phases

Mobile phases should have critical
parameters that are easily reached using
chromatographic pumps and ovens
common
to
currently
used
instrumentation.
Advantages of supercritical fluids over
carrier gasses and liquid mobile phases
are in its solubility properties, physical
properties, and detector compatibility.
Injectors
Typical HPLC design injectors for
packed columns.
Split/Splitless valve injector (0.01 to
0.05 L injections) for open tubular
columns.
Timed - split injector (0.01 to 0.05 L
injections) for open tubular columns.
Chapter 8
Chromatography with Supercritical Fluids
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
Mode of Operation: Elution chromatography
Elution Chromatography: A Chromatogram
Different Mobile Phases

Mass transport high
Solvent power
high
Schoenmakers, Uunk 1987
SFC: Stationary Phases

Composition
Trade name
Polysiloxane
R, R':
100 % methyl
95 % methyl, 5 % phenyl
94 % methyl, 1 % vinyl, 5 % phenyl
25 % cyanopropyl, 50 % methyl,
25 % phenyl
polyethylene glycol
( CH2 CH2 O )n
Application
separation according
molecular weight
to
OV-1, SE-30
OV-3, SE-52
SE-54
OV-225
Carbowax 20 M
separation according to polarity
SFC: Different Gases as Mobile Phase
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; Temperature
296.15 K; Flow rate 670 cm 3/min at STP (after Pickel /23/).
SFC: Different Modifiers

1 = caffeine;
2 = theophylline;
3 = theobromine;
4 = xanthine
(Randall 1984).
Variation of capacity ratios of

polycyclic aromatic compounds
due to modifier concentration
(1.4-dioxane) in the mobile
phase (n-pentane).
P at column outlet 3.6 MPa;
T = 513.15 K
(Leyendecker et al. 1986).
SFC: Influence of Pressure and Temperature
Variation of retention times

of chrysene with pressure.
Mobile phase n-butane
(Klesper, Leyendecker 1986).
Variation of retention times with temperature

of polycyclic aromatic components in n-butane at 4.5 MPa.
1 = naphthalene; 2 = anthracene; 3 = pyrene; 4 = chrysene
(Klesper and Leyendecker 1986).
SFC: Pressure And Density Programming
Chromatograms For Different Amounts of Injection
Concentration
Analytical
injection
Overloading by
volume
Overloading by
concentration
Time
Adsorption Isotherms And Corresponding Chromatograms
SFC: Flow Scheme of Apparatus
Elution Chromatography: A Chromatogram
Capacity Ratio
k tr tm / tm nis / nim .
tm = residence time in the mobile phase
tr = retention time of the solute
k' = capacity ratio
= volumetric phase ratio Vs / Vm
Vs = the volume of the stationary phase, to,.
Vm = the volume of the mobile phase
Vs K e
k K
,
Vm
n s K e
k K e
,
nm e
Vm nm vm
and Vs ns v s and e vm / v s .
e = molar phase ratio

v = molar volume of a phase
V = total volume of a phase
k K e
vm Vs K e vm
.
v s Vm
vs
Capacity Factors
Capacity factors of paraffines
as a function of density
(after Mollerup et al. /18/).
Chromatographic Separation
with n = number of
stages for p:
Fi
cim
Vm Vs / K i
vi n
1
exp
2n
2 n
Maximum of the peak:
Number of theoretical plates:
vi n ;
V
n vi
;
Vm Vs / K i
Points of inflection:
vi,left n n and vi,right n n

Points of intersection with the base line:
vi,left n 1 2 n and vi,right n 1 2 n ;
Width of peak:
b 4 n.
Time at which the peak maximum appears
tr i 1 / ki ni or tr i ki ni .
Number of equilibrium stages
4 tr i
.
ni
bi
Selectivity
si j ki / k j .
Resolution
Ri j
2 trj tri
bi b j
Resolution of two peaks of similar compounds
n1 / 2 sij 1 k
Rij
.
4 sij 1 k
sij 1 k

n 16 R
.
s 1 k
ij
2
ij
2
8
k
d
2 Dim
i F
Hs 2 d p
2
u,
u
1 ki Dis
Van Deemter
Height of theoretical stage Hs
for SFC and HPLC
for packed columns
with different particle diameters
(after Gere et al.)
SFC Analytical Scale, hp
Separation of Prostaglandins
Preparative separation
Influence of temperature
Upnmoor
20 MPa; mobile phase:
CO2/methanol (5.3 wt.%);
column: 125 x 4 mm;
5 m LiChrosorb Si 60.
1992
Chromatograms of fractions
Separation of Tocopherols
Shapes of peaks under overloading conditions
Chromatograms of -tocopherol mixture

under overloading conditions
Upnmoor, Brunner, 1992
Separation of Tocopherols
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
Influence of modifier concentration
Productivity: DHA / DPA Separation by SFC

250 x 4.6 pS
250 x 8.0 pS
98
Area DHA GC [%]
96
94
92
90
RF=0,842
88
1mg DHA/(h,cm3 ) * 500 ml = 0,5 g DHA/h
86
84
82
specific productivity DHA [mg/cm h]
Some kg DHA: Fully automatized plant !
SMB- Plant: Separation Columns
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.
SFC, Preparative Scale
Continuous Chromatography
Rotating column
Rotating ports
True Moving Bed (TMB) Process
Desorbent D
Extract
A+ D
Feed
A+B+D
Raffinate
B+D
Zone 1
Purification of Adsorbent
Zone 2
Enrichment of A
Zone 3
Enrichment of B
Zone 4
Purification of Desorbent
Principle of Simulated Countercurrent Separation
Mazzotti, ETH-Z
Simulated Moving Bed-Process

Concentration A, B
Desorbens
D
Extract
A+D
Feed
A+B
Raffinate
B+D
Performance SMB vs Elution (99.5 % Purity)
Gottschall: PREP 95
Preparative SMB-Plant
Depta, 2000
Adsorption Isotherms
Isotherms exhibit a point of inflection for each isomer.
c(b1 2b 2c)
q qs
1 b1c b 2c 2
50
30
30
28
20
26
24
10
Measurements
0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
concentration [mg/ml]
22
1,4
20
1,6
42
100
80
dq/dc
40
38
60
36
40
34
Measurements
20
0
0,0
0,5
1,0
1,5
2,0
2,5
Adsorption isotherms for Phytol cis- and transisomer (black lines) and
derivatives (red lines). 225 bar, 40 C, 1.8 mass% isopropanol as modifier.
32
3,0
30
dq/dc
32
dq/dc
q [mgisomer/mlstationary phase]
40
44
120
dq/dc
q [mgisomer/mlstationary phase]
34
Batch-Simulations
feed concentration:
2 mg/ml
5 mg/ml
10 mg/ml
20 mg/ml
50 mg/ml
1,2
1,0
0,8
0,6
0,4
0,2
0,0
retention time [min]

Experimental and simulated phytol chromatogramssymbols: experimental data;
lines: simulations.
SMB-Simulation
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.
ci
ci
ci 1 qi
u
0
u
ci
Dap 2
t
z
z
z
t
2
SMB process modeled with four key parameters: the net flow ratios mj:
Ruthven, Storti.
mzone
TMB
SMB shift
Qzone
Qsolid Qzone
t Vcolumn total
Qsolid (1 )
Vcolumn (1 total )
SMB-Simulation
SMB- SFC: Volume-flow is a function of column length.
Therefore, net flow ratios are not constant in each zone.
New parameter:
m*zone
SMB
Qzone
mobile _ phaset shift Vcolumn total
Vcolumn (1 total )
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: Phytol Separation

black triangles:infinite dilution
situation and infinite number of
theoretical plates
36
34
36
34
operating point
32
30
28
26
same parameter set

as operating point in
figure 5
raffinate (cisisomer) pure

extract (transisomer) pure
raffinate +
extract pure
24
24 25 26 27 28 29 30 31 32 33 34
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
32
30
28
26
24
24 25 26 27 28 29 30 31 32 33 34
Columns: 1/1/1/1;
1000 plates per column

black triangles:infinite dilution
situation and infinite number of
theoretical plates
36
34
36
34
operating point
32
30
28
26
same parameter set

as operating point in
figure 5

raffinate +
extract pure
24
24 25 26 27 28 29 30 31 32 33 34

phytol Cfeed=5.0 mg/ml 230 bar, no
pressure drop, columns: 2/2/2/2; 300
plates per column
32
30
28
26
24
24 25 26 27 28 29 30 31 32 33 34
Columns: 1/1/1/1;
1000 plates per column

Influence of pressure drop:
35
35
30
30
25

raffinate +
extract pure
20
20
25
30

phytol, infinite dilution, columns:
2/2/2/2; 300 plates per column,
230 bar, no pressure drop
25
20
20
25
30
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*
Experimental Results of Ibuprofen Separation

11
10
m3
low concentration in Feed

linear Adsorption isotherm
Ideal model
8
7
Run O
2,5
1 2
9
m2
10
11
Run M
Run N
2,0
1,5
1,0
0,5
0,0
0
1
Extract Feed Raffinate
Extract
Feed Raffinate
Extract Feed Raffinate
Separation of Ibuprofen
12
11
10
9
8
7
6
5
4
3
2
1
0
-10
Sim S(+)
Sim R(-)
Exp S(+)
Exp R(-)
0
0
10
20
extract
30
40
50
length [cm]
60
70
raffinate
80
peak area [mV*min]
concentration [g/l]
140 mgRacemate/min; 2/2/3/1 configuration
Phytol
Diterpene-alcohol,
Intermediate for vitamin E, K1
esterified lipophilic compound
of chlorophyll
CH3 H
CH3
CH3
OH
CH3
CH3
17mg pur
0,85 mg in Hexan
Conditions of separation:
240 bar, 50C,
column 4 x 250 mm packed with
LiChrospher 100 (Silica),
flow 2,56 g carbon dioxide / min,
modifier 3 wt.- % EtOH,
productivity 45 mg/(ml, h).
Verunreinigungen
Phytolisomere
144
A Mettler-Toledo Company
Breaking the Purification

Bottleneck
BERGER
SFC
HPLC
145
Supercritical fluid chromatography

Supercritical fluid is used as the mobile phase.
SFC combines advantages of gas and liquid chromatography.
Critical temperature exists in which a substance can no longer be converted into
the liquid phase even if higher pressures are applied.
The vapour pressure belonging to the critical point is the critical pressure.
The fluid is in a supercritical state close to the critical point and has
characteristics which lie somewhere between those of gases and liquids.
Characteristics particularly important for chromatography on the critical point are
the density, the viscosity, and the diffusion coefficient.
146
Typically supercritical fluids are used at densities ranging from 0.1 to 0.8 of
their liquid density and 100 - 1000 times greater than that of a gas at ambient
temperature.
Pressures range from 50 atm to 500 atm.
Diffusion coefficients of supercritical CO2 varies between 10-4 and 10-3 cm2s1
.
Liquids typically have diffusivities of less than 10-5 cm2s-1.
The viscosities of supercritical fluids mirror their diffusivities and are typically
10 - 100 times lower than for liquids.
Supercritical fluids are not only used for chromatography but

also for supercritical fluid extraction.
1) caffeine-free coffee can be produced by the
extraction
from coffee
2)cigarettes with a low nicotine content can be produced in the
extractive separation of nicotine from tobacco
147
Instrumentation
Precise temperature setting must take place in the column.
This is achieved with a thermostatted column oven as in GC.
To achieve subambient temperatures cooling with an auxiliary CO2 tank is
employed- Usually to ~ -10 to -50C.
Pressure in system must be precisely monitored since the density of the
supercritical fluid alters with the pressure and pressure differences lead directly
to a change in the capacity factors.
Higher pressure provides greater density.
This causes elution power of the mobile phase to increase and shorter retention
times are observed.
Pressure programming in SFC as a gradient in the same way we are familiar
with it for temperature in GC or the composition of the mobile phase in HPLC.
148
The range of solvating power of practical supercritical fluids for SFC is of primary
importance, and ultimately defines the limits of the application.
The solubility of the analytes typically increases with density, and a maximum rate
of increase in solubility with pressure is generally observed near the critical pressure
where the rate of the increase of density with pressure is the greatest.
Increase pressure--> Increase density---> should decrease k
Increase temperature--> Decrease density --> should increase k to a certain
temperature until other variables start playing predominant role.
149
Stationary Phases
Stationary phase can be either a packed column or be set up in a capillary.
Packed columns are typical HPLC columns
In fused silica capillaries the stationary phase is applied in the form of
siloxanes as liquids or chemically bonded to the inner wall.
The usual measurement of the capillaries are lengths of between 10 and 20 m
with inner diameter of between 0.05 and 10 mm and film densities of 0.05 to 1
m.
150
Mobile Phases
Carbon dioxide is most frequently used as the mobile phase.
Cheap, nonpoisonous, odorless and does not absorb in the UV range up to 190
nm.
The critical CO2 data are such that the temperature and the pressure can be
varied over relatively wide ranges.
Organic modifiers are often employed such as methanol, ethanol, IPA, n-propanol,
dioxane.
The addition of these modifiers however change the critical point of the system.
Typically at even low levels of organic modifier the supercritical region is traversed.
That is because the addition of the modifier shifts the critical point to higher
temperatures and pressures.
151
Detectors
FID detection
Coupling of mass spectrometry is considerably easier to achieve by
comparisons to LC and has been used.
UV, IR, Fluorescence, Flame photometric, TCD and ECD can also be
employed.
- DAD for purity assessment
- MS to verify presence of target
- NCD to quantify on nitrogen content
-FTIR
152
Effect of temperature on retention factor
At constant pressure solute retention in SFC depends on temperature in a very

characteristic manner.
Example: Ref N.Wu, J.Medina, J.Bradshaw, M.Lee, J.Microcolumn
Separations, 12(8) 454-461 (2000)
At moderate pressures (150 atm) increasing the temperature from ambient to
critical (Tc,31.3oC), k values decrease and reach a minimum which resulted
from an increase in solute solubility with temperature in liquid carbon dioxide.
After passing Tc, k values increase significantly.
153
154
Effect of temperature on retention factor

1- The density of CO2 and solvent power decreases as the temperature
increases, which results in an increase in k.
2- The vapor pressure of the analyte also increases at higher temperature,
leading to an increase in solubility and thus a decrease in k.
The second effect over compensates the first around 100oC(1/T=0.0028),
resulting in a maximum in retention factor.
At temperatures higher than 100oC , the second effect becomes more
pronounced and k values decrease with increasing temperature.
At high pressure the decrease in density with increasing temperature is not
as important as the effect of temperature on solvating power. Therefore
plots of k vs. 1/T are usually more flat. The two variables effects may
compensate each other.
Results indicate that low temperature is favored for fast chiral separation in
SFC, at which both low retention factor and high selectivity can be obtained.
155
156
Effect of temperature on efficiency

Efficiency generally increases with an increase in temperature
1) Mobile phase diffusivity increase with temperature at a constant pressure and
increases N.
2) Decreased density at higher temperatures lead to a drop in mobile phase
viscosity which increases N.
3) Retention factor increases which will increase N.
R.Stringham, J.Blackwell, Anal.Chem., 1996, 68, 2179-2185
However it has been seen that efficiency has increased with temperature to a point and then
levels off. Then as the critical temperature is transversed column efficiency declines. This is
attributed to the adsorption of solvent to the stationary phase. The presence of this adsorbed
layer would slow the kinetics of binding and hence a decline in column efficiency.
157
158
Effect of temperature on resolution
N 1 k2
R s

4 k2 1
Resolution depends on column efficiency, solute retention and selectivity.
In SFC we can vary the variables to obtain the desired resolution.
Ex. Increase the temperature will
* Decrease the CO2 density
* May increase or decrease the retention factor
* Vapor pressure of the analyte also increases
* Increases rate of diffusion (increased efficiency)
Depending on what variable/variables predominate the Rs could improve or not.
159
Low temperature is beneficial because chiral selectivity usually increases with

decreasing temperature
SFC often provides higher resolution per unit time than LC because mobile phases have
low viscosities and small mass transfer resistance which lead to higher optimum linear
velocities.
160
Effect of pressure
Pressure has a little effect on the selectivity

Pressure affects linear velocity, efficiency, and retention factor.
Therefore resolution depends on the effective pressure.
Retention factor decreases with increasing pressure in SFC since the density of CO2 and
solvent power increases.
At increasing pressures mobile phases have greater viscosities and greater mass transfer
resistance which may lead to decreased efficiency.
161
162
163
Breaking the
Purification
Bottleneck
What is Packed Column SFC?

A separation technique similar to HPLC
Mostly same components as HPLC
Almost always uses carbon dioxide as the
main component in the mobile phase
Almost always uses binary or ternary
mixtures as mobile phase
Usually perform gradient elution changing the
composition of the mobile phase
164
Schematic Diagram of a Packed Column SFC

Breaking the
Purification
Bottleneck
165
Practical Advantages of
SFC
SFC is Normal Phase

complimentary to reversed phase HPLC
Diffusion is much Faster
higher speed/throughput-more samples/day
more rapid re-equilibration-shorter cycle time
Non-Linear Solvent Strength-first small addition of
polar additives dramatically increases solvent
strength
Viscosity is Much Lower
lower pressure drop, higher flows, or longer
columns possible
166
What Compounds Can SFC

Separate?
Any solute soluble in methanol or a less polar
organic solvent will elute in SFC.
Strong organic acids and bases require a
modifier and an additive in the mobile phase.
Most salts of organic acids and bases elute.
Small lipophylic peptides elute
With carbon dioxide based fluids, molecular
weights up to 15,000
167
Vanomycin is Not Very Soluble in MeOH o

in MeOH/CO2 Mixtures
Conditions: 2.5 ml/min [MeOH +0.4% ibam] in CO2,

35C, 120 bar, 4.6 x 150 mm, 5m Zymor CN
Limits of SFC
Peptides with Very Limited Solubility
in Methanol Yield Square Topped Peaks
80% modifier
Which Indicates Limited
Solubility in the Mobile Phase
Not
to
Scale
40%
60%
0.5
1.5
Retention Time, minutes
2.5
3.5
4.5
4
168
min
Some Peptides Elute Just

Fine
Limits of SFC
Separation of a Small Pepetide:

Actinomycin D
mAU
800 mAU
1400
Spectrum
1200
UV
Absorbance
At 240 nm
205-500 nm
1000
800
25% Methanol + 0.1% TFA in CO2

2.5 ml/min, 35C, 120 bar
4.6 x 150 mm, 5m Zymor CN
600
400
200
250
300
350
400
0.5
1.5
2.5
Retention Time, minutes
3.5
4
169
450
nm
What Compounds Will NOT

Elute in SFC?
Any solutes requiring an aqueous
environment
any solutes requiring a buffered or ionic
aqueous environment
I.E., Biomolecules such as Proteins
will NOT elute
inorganic salts will not elute
With carbon dioxide, few solutes over 15,000
MW elute
170
Sample Matrix?
Usually solution in organic solvent
methanol or less polar allows larger
injections
can use DMSO
small (i.e., 0.5-1 L) aqueous samples
direct
often reduced sample prep because SFC is
compatible with both lipids and polar
molecules
pills dissolved with excipients, etc. present
171
extracts from natural products
Why Carbon Dioxide?

Miscible with much more polar solvents-wide range
of solvent strength available.
program composition from 0 to 100% organic
modifier
Low operating temperatures (i.e., 30-50C)
Compatible with most HPLC and GC Detectors
Relatively safe
product of human respiration
can use with the FID
easy disposal
low purchase price
Lack of a superior alternative
172
Polarity Range of Solute Families

hydrocarbons
PAH's
ethers
esters
aldehydes
and ketones
alcohols
phenols
acids
monofunctional
fatty acids
hydroxy acids
polyacids
tertiary
anilines
benzylamines
sulfonamides
phenylureas
sulfonylureas
triazines
carbamates
organochlorine
organophosphorus
primary amines
amines
secondary
amines
Polarity Rangesof Stationary Phases

C 18 < C 8
phenyl
CN
silica
NH 2
diol
SA
Appropriate Mobile PhaseComposition

pure
carbon
dioxide
CO 2 +
methanol
CO 2 + methanol
additives
173
Instrumentation
Berger Instruments dual pump SFC with extended
flow range to 10 mls/min.
Autosampler: 1,2, 4 ml or microtiter plate accessory,
partial to full loop, multiple solvent, or sample
washes, up to 2.5 ml syringe, cooling option
Diode array detector, or variable wavelength UV
Solvent switching valve, column switching valve
available
ECD, NPD, FID GC like detectors available
Full function Chemstation for instrument control, data
acquisition, data analysis, report generation
174
THE CHROMATOGRAPHY DILEMMA

Breaking the
Purification
Bottleneck
Gas Chromatography (GC)

Old, well established (30 years)
>250,000 units in place; >500,000 sold; ~20,000 units/yr.
currently sold
Fast, high resolution, wide range of sensitive
detectors
Inexpensive @ $10K-$30K/unit
Inexpensive to operate
Non-polluting
APPLICABLE TO ~10% OF ALL MOLECULES
175

(cont.)
High Pressure Liquid Chromatography (HPLC)

Well established (20 years)
>100,000 units in place; >250,000 sold; ~20,000 units/yr.
currently sold
Slow, moderate resolution, limited range of

insensitive detectors
Relatively expensive @ $25K-$60K/unit
Expensive to operate
Polluting
176

(cont.)
Supercritical Fluid Chromatography (SFC)

Relatively new (5-10 years)
>1,000 units in place; >2,000 sold; ~200 units/yr. currently sold
3-5 times faster than LC
2-3 times better resolution than LC
Uses both GC and LC detectors
Hardware is priced 1.3 times LC pricing
Inexpensive to operate
Non-polluting
177
Berger SFC Separation of Antidepressants

5 ml/min 2.5-55% {MeOH + DMEA] in CO2 at 30%/min

35C, 100 bar, 2 l injected
Column: 4.6 x 150 mm, 5m (ES Industries CN-BD Column)
Antidepressants, 2 L 20 ppm each
Amitriptyline
Imipramine
Nortriptyline
Desipramine
Protryptyline
UV
Absorbance
At 220 nm
0.25
0.5
0.75
1.0
1.25
1.5
1.75
178 minutes
Retention Time,
min
2.0
The Analytical SFC Opportunity

Breaking the
Purification
Bottleneck
Pharmaceutical
Chiral Drugs
CombiChem/High Throughput Screening
Linked closely to the success of PrepSFC, SFC/MS
and quantitative detection
Petrochemical
Aromatics in Diesel Fuel
Other ASTM methods awaiting approval
Agricultural and Other Chemical Products
179
SUPERCRITICAL FLUID
CHROMATOGRAPHY (SFC)
SFC is a hybrid of HPLC and GC

Uses HPLC like equipment
Detector is usually a flame ionization detector, although
mass selective, and spectrophotmetric detectors can be
used
Useful for compounds that

Are non volatile or thermally unstable and
Cannot be used with spectophotometric or electrochemical
detectors
Properties of supercritical fluids

Compound
CO2
NH3
H2O
CH3OH
C2H5OC2H5
Critical Temp.
(oC)
31.3
132.3
374.4
240.5
193.6
Critical Press.
(atm)
72.9
111.3
226.8
78.9
36.3
Critical Dens.
(g/mL)
0.448
0.24
0.344
0.272
0.276
Supercritical Fluid Chromatography-I

Instrumentation
Similar to HPLC as pressures are like that of HPLC
Need an oven like GC to keep temperature above critical
temperature of mobile phase
A restrictor is required at the end of the column to maintain
the column
pressure above that of the critical pressure
of the mobile phase
Instead of temperature programming used in GC, pressure
programming, which increases super critical fluid density, is
used to modify the
solvent power of the mobile phase to
control retention times
Columns
Wall coated open tubular columns made from fused silica

Usually chemically bonded liquid stationary phase
0.05-0.10 mm ID
Film thickness: 0.05-1.0 mm
10-60 m long
Packed HPLC columns can also be used

-II
Mobile phase used most is CO or CO with some dissolved methanol
which
increases the solubility of polar compounds
Nontoxic
Low TC and PC
Easily volatilized at the end of the separation, leaving solute analytes in the
gas phase for flame ionization detection
The FID does not respond to CO2, whereas the mass spec does
Comparison to HPLC and GC: based on the fact that supercritical

fluids have properties intermediate between gases and liquids
Faster elutions than HPLC because the viscosity of mobile phase less than
liquids allowing for higher flow rates
Lower band broadening than HPLC but greater than GC because the
diffusion coefficient of solutes in supercritical fluids is intermediate
between that found for gases and liquids
The overall result is that resolution is greater than HPLC but less than GC
Speed of analyses is greater than HPLC but less than GC
Some figures of merit by example

See Fig. 27-3 in Skoog, et al.
At m = 0.6 cm/s, SFC gives HETP of 0.013 mm, HPLC3gives 0.039 mm

This means zone broadening is reduced by
HETPminimum is 0.15 cm/s for HPLC, 0.6 cm/s for SFC

This means the same resolution can be obtained in one-forth the time
See Fig. 27-4 in Skoog, et al.
SCF-III
One disadvantage of SFC is that selectivity cannot be modified
in the same way as with HPLC by using gradient elution. This
is important for large, nonvolatile molecules
Applications: see Figs. 27-6, 27-7, 27-8 in Skoog, et al.
Capillary Zone Electrophoresis
Electrophoresis is a separation that is produced by differential
migration of species in an electric field
In pure electrophoresis, this is not a chromatographic separation
because no partitioning of dissolved analytes occurs between a mobile
and stationary phase
It is generally applied to ionic substances, although some new
techniques allow separation of neutral molecules by combining
electrophoresis with chromatography d e t e c t o r
H ig h V o lt a g e
S u p p ly
2 0 -3 0 k V
F u s e d S ilic a
C a p illa r y
5 0 -10 0 cm
15 -10 0 m I D
B oth b eakers
f ille d w it h a
buffer

6-Supercritical Fluid Chromatography SFC

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6-Supercritical Fluid Chromatography SFC

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Supercritical Fluid

Important Properties of Supercritical

W.W. Christie and Lipid Technology

T/P Phase Diagram

Supercritical fluids have solvent power

Supercritical fluids can have solvating

Analytes easily recovered

1. Mobile phase (CO2) 5. Column

Schematic Diagram of a Packed Column

Solvent Delivery System

Hewlett-Packard developed and manufactured the SFC instrument in 1982

Combinations of desirable characteristics from both GC and HPLC

Used for compounds:

Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

J.W. King, H.H. Hill, and M.L. Lee. 1993

J.W. King, H.H. Hill, and M.L. Lee. 1993

-Packed columns are more

Open Tubular Columns

J.W. King, H.H. Hill, and M.L. Lee. 1993

Caution when using

SFC and Retention

Supercritical CO2 Density

SFC Mobile Phases

Carbon Dioxide: SFC Solvent

CO2 as mobile phase

Other SFC Solvents

Mobile phase has solvating power which

Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

J.W. King, H.H. Hill, and M.L. Lee. 1993

SFC Advantages vs HPLC

Can analyze solutes of much higher

High resolution at lower temperature

Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

Cleaning is time consuming

Ind. Eng. Chem. Res. 2000, 39, 4531-4535

Fossil Fuels and Hydrocarbons

GC: T gradient, 80C to 250C over

Open tubular column

0.70g/mL at 0.005g/mL per

Natural Products Application

Cholesterol content of a fish oil

Content of capsule was emptied

The oil-laden solution was

Two peaks were eluted with in

Comparison of HPLC to SFC

Metabolized Camazepam by HPLC

Column: Zorbax SB-C18, 150 x 4.6 mm

Metabolized Camezapam by SFC

Identification of Metabolites using UV Spectra

Identification of Metabolites Using UV Spectra

of the peak at 4.461 min

Effect of Organic modifier

Effect of Organic modifier

Packed Column Analysis of Tricyclic

Packed Column Analysis of

Same columns used in HPLC can be used in SFC

Packed Column Analysis of Steroids on

2.1 x 250 mm Lichrosphere Si-60

Packed Column SFC for Pharmaceutical

Chiral Purity Analysis

250 x 4.6 mm Chiralcel-OD

Effect of Additive on Chiral Separation

Representation of SMB-SFC process in a (m2-m3)-plane,