超臨界流體
萃取,層析及其他應用
Supercritical Fluid
Extraction, Chromatography
and Other Applications
林華經
引光生物科技有限公司
What is a Supercritical Fluid ?
When the pressure and temperature of a
substance is raised above its critical pressure
and critical temperature (the critical point) the
substance enters the supercritical state.
A Supercritical Fluid is a substance with both
gas- and liquid-like properties.
Critical Temperature and Pressure

The Critical Temperature (Tc) is the maximum
temperature at which a gas can be converted
to a liquid by increasing the pressure.

The Critical Pressure (Pc) is the maximum
pressure at which a liquid can be converted
to a gas by increasing the temperature.
Pressure
Phase Diagram of Carbon Dioxide
Solid
Liquid
Gas
Triple Point
Temperature
Supercritical
Fluid
Critical Point
31.3 oC, 1072 psi
Physical Properties of CO2
Phase
Gas
SCF
Liquid
Density
0.6–2.0 x 10-3
0.2 – 0.9
0.8 – 1.0
viscosity
0.5–3.5 x 10-4
2.0–9.9 x 10-4
0.3–2.4 x 10-2
Diffusivity
0.01 – 1.0
0.5–3.3 x 10-4
0.5–2.0 x 10-5
Advantages of Supercritical Fluids as
solvents

Solvating power related to density
(at constant T)

Gas-like mass transport properties

Facile penetration into porous material
Critical Parameters of Common
Fluids
Tc (°C)
c (g mL )
-1
Pc (atm)
CO2
31.3
72.9
0.47
N2 O
36.5
72.5
0.45
SF2
45.5
37.1
0.74
NH3
132.5
112.5
0.24
H2 O
374
227
0.34
n-C 4H10
152
37.5
0.23
n-C 6H12
197
33.3
0.23
Xe
16.6
58.4
1.10
CCl 2F2
112
40.7
0.56
CHF 3
25.9
46.9
0.52
Source: “SFE and Its use in Chromatographic Sample Preparation” Ed. S. Westwood. Chapter 1
Advantages of CO2 for SFE

Low critical parameters

Inert, Non-toxic, Nonflammable, Noncorrosive

Easily purified (inexpensive)

Nonpolar: dielectric constant similar to
hexane

Modifiers can be used to increase polarity
Supercritical Fluid Extraction (SFE)
Basic Theory
SFE System Components
CO2 Pump
(high
pressure)
Modifier
Pump
sample cell
in heated
chamber
CO2
Liquid Carbon
Dioxide
(requires a dip
tube)
Restrictor
CO2
Trap
Solid or
Liquid
Advantages of SFE over Solvent
Extraction

Faster



Selectivity results in cleaner extracts
Low Critical Parameters






Results in minutes rather than hours
Handling of thermally labile analytes
Non-hazardous solvents
Automated
Cost per test is lower
Limited or no solvent removal required
No solvent disposal costs
The Mechanism of SFE
A Three Step Process
1. Dissolving/Resolving analyte(s)
2. Sweeping the analyte(s) from the matrix
3. Trapping the analyte(s)
SFE Mechanism
High Pressure
Liquid
CO2
SC-CO2
diffuses matrix,
dissolves and resolves
analyte from the matrix
Heat
SC-CO2 +
dissolved
analyte to
the trap
Trapping
Gaseous
CO2
CO2 is changing
from a SF (2 ml/min)
to an expanded gas
(1 L/min)
•Analyte no longer
soluble
•Mechanical movement
of analyte due to the
rapid expansion requires
the use of trapping
material
Gaseous
CO2
Trapping Solid
•High Surface Area
•Adequate Amount
Trapping Liquid
•High Surface Tension
•Analyte is Soluble
•Low Volatility
•Pressurized
•Cryogenically Cooled
SFE - Method Development

Pressure


Temperature


Flow Rate
Fluid Composition



Increase temperature may decreases density = decrease in
solubilizing power (ie CO2 at 100 bar)
Extraction Time


Increase pressure increases density = increase in
solubilizing power.
Co-solvents/modifiers
Reactant Additive
Static vs. Dynamic Extraction
Why Use Modifiers?
Analytes that have polar components require the use of a cosolvent
Triglycerides
CH2OCOR1
R2COOCH
CH2OCOR3
Phospholipids
CH2OCOR1
R2COOCH
O
CH2OPOR3
OR 1, 2 & 3 groups are long
chain hydrocarbons
(nonpolar)
R 1 & 2 groups are long chain
hydrocarbons (nonpolar),
while R 3 contains
phosphorus and nitrogen and
is polar
Modifiers (Co-solvents) in SFE
Role of Modifiers in SFE
Changes in Solvent Polarity
 Interaction with Matrix
 Interaction with Analyte
Methods of Addition


Directly into extraction cell (spiking)
On line modifier addition (uses a second pump)
SFE vs. Traditional Sample Extraction Methods
Conclusion

SFE can be versatile, selective and faster

SFE reduces hazardous solvent use and cost

SFE can produce cleaner, more concentrated
extracts for post extraction analysis
Analyst’s
Sample
Process.
61%
Time Allotment
Collection 6%
Analysis 6%
Data
Management
27%
Routine and Novel Applications of
Analytical SFE
SFE for Research
Environmental SFE Applications
• Matrices









Soil
Tissue
Clay
Sandy Loam
Sludge
River Sediment
Marine Sediment
Fly Ash
Incinerator Ash
• Target Analytes
 TPH
 PAH
 PCBs
 Pesticides
 Dibenzofurans
 Dioxins
Approved Methods
Environmental
• US EPA 3560 - TPH in Soil:
Supercritical Fluid Extraction of Total
Recoverable Petroleum Hydrocarbons
• US EPA 3561 - PAH in Soil:
Supercritical Fluid Extraction of
Polyaromatic Hydrocarbons
• US EPA 3562 - PCB and OCP:
Supercritical Fluid Extraction of
Polychlorinated Biphenyls (PCBs) and Organochlorine
• US EPA 3545: Pressurized Fluid Extraction (PFE)
• USDOE STD-3013-99: Determination of Residual Water in Impure
Plutonium Oxides
• AOAC draft: SFE-GC/MS determination of pesticide residues in
non-fatty fruits and vegetables
Pharmaceutical/Natural Product
Applications
SFE of Natural Products -- Roger M. Smith
LC-GC International, Jan. 1996, 9-15
Catharanthus
roseus
German
chamomile
Magnolia
grandiflora
Tansy
Chamomile
flowerheads
Ginger
Peppermint
Thyme
Clove Oil
Kola nuts
Pimento
Turmeric
Dragon head
Lavender
Poppy seeds
Wheat germ oil
English yew
Lemon grass
Rosemary
Feverfew
Lemon peel
Savory
Microbial Natural Products
R. M. Smith, op. cit.
Organism
Extract
Agaricus species
Carboxylic and fatty acids
Beuveria nivea
Cyclosporin
Filamentous fungi
Polyunsaturated fatty acids
Flour, moldy bread, mushrooms
Ergosterol
Moldy bran
Sterol
Moldy grain
Aflatoxin
Extracts from Biomass
R. M. Smith, op. cit.
Microorganism
Extract
Actinomycete species
Mycolutein and luteoreticulin
Actinomycete species
Oligomycin A
Aspergillus fumigatus
Sydowinin B and epoxide
Bipolaris urochloae
Ophiobolin A
Penicillium expansum
Chaetoglobosin A
Penicillium sclerotium
(+)-Sclerotiorin
Streptomyces species
Elaiophylin
Summary
R. M. Smith, op. cit.
• Extracts typically cleaner than those
obtained with organic solvents.
• Mild conditions minimize degradation.
• SFE methods are faster than organic solvent
extractions.
Extraction of Pharmaceuticals Using Pressurized Carbon
Dioxide
J. R. Dean, S. Khundker, J. Pharm. & Biomed. Anal, 15 (1997) 875-886
• Recoveries from 81% - 95+%
• CO2 and CO2 with modifiers
• Generally faster than other methods with
better selectivity for target analytes.
• Preconcentration steps could be eliminated
in some cases.
• Liquid matrices required immobilization on
solid support or SPE cartridge.
Animal Feeds
J. R. Dean, S. Khundker, op. cit.
Analyte
Matrix
Menadione
Rat chow
Tipradane
Rodent diet
Hypolipidermic drug
Rat feed
Halogenated aromatic phenoxy
derivatives
Dog feed/rodent feed
Atovaquone
Rat feed
Fluconazole
Animal feed
Propanolol, Tamoxifen, ZM 95527,
169369
Rodent diet
Formulations
J. R. Dean, S. Khundker, op. cit.
Analyte
Matrix
Megesterol Acetate
Tablet
Felodipine
Tablet
Benzodiazipines (7)
Tablet/capsule
Caffeine,vanillin
Tablet
Vitamin A, E
Tablet
Retinol palmitate, tocopherol acetate
Ointment
Polymyxin B sulphate
Cream/Ointment
Acylvoir
Ointment
Sulfamethazole, trimethoprim
Septra infusion
Triamincinolone
Dermatological patches
Misoprostol
Hydroxypropyl methylcellulose
Biological Matrices
J. R. Dean, S. Khundker, op. cit.
Analyte
Matrix
Veterinary drugs (4)
Pig kidney
Nitrobenzamide residue
Liver
Codeine, morphine, ethyl morphine
Hair
Ketorolac, flavone
Plasma
Mebervine alcohol
Dog plasma
Morphine
Serum
Beudesonide
Plasma
Caffeine
Kola nuts
Taxanes
Yew tree needles
Chinese herbal medicines
Plants
Diosgenin
Tubers of Dioscorea nipponica
Taxol and baccatin III
Needles of Taxus cuspidata
Zingiber zerumet rhizomes
Plants
Mevinolin and hydroxy acid form
Fermentation broth
Phylloquinone
Soy protein and infant formula
Miscellaneous
J. R. Dean, S. Khundker, op. cit.
Analyte
Matrix
Triprolidine,
pseudoephedrine
Aqueous
Steroids (10)
Aqueous
Ibuprofen
Aqueous
Natural Materials Studied
M. J. Noh, et. al., op. cit.
Specific Name
Part Used
Lycium chinese
Fruit
Schizandre chinensis
Fruit
Citrus unshiu
Fruit bark
Angelica gigas
Root
Cornus officinalis
Fruit
Cnidium officinale
Rhizome
Ginko biloba
Leaf
Aralia cordata
Root
Evodia officinalis
Fruit
Crataegus pinnatifida
Fruit
Paeonia lactiflora
Root
Leonurus sibricus
All
Sophora japonica
Flower
Artemisia capillaris
All
Platago asiatica
Seed
Natural Materials Studied, contd.
M. J. Noh, et. al., op. cit.
Specific Name
Part Used
Ephedra sinica
All
Aconitum carmichaeli
Tuber
Scolopendra subspines
All
Paeonia suffruticosa
Root
Pueraria thunbergiana
Root
Polygala tenuifolia
Root
Coptis japonica
Rhizome
Astragalus membranaceus
Root
Eucommia ulmoides
Stem bark
Bupeuri falcatum
Root
Acanthopanax sessiliflorum
Bark
Epimedium koreaum
All
Morus alba
Root bark
Artium lappa
Fruit
Spirodela polyrhiza
All
Summary
M. J. Noh, et. al., op. cit
• For many materials, SFE yielded extracts with
higher bioactivity than LSE.
• SFE was found to be more selective than LSE
for target compounds.
• SFE conditions could be optimized to produce
maximum levels of bioactivity.
Drug Residues
Analyte
Matrix
Reference
Sulfamethazine
Swine Muscle Tissue
Cross, et.al.
Anabolic Steriods
Bovine Tissue (Muscle Houpalahti and Henion
and Liver)
Opiates
Hair, blood and tissue
Multiple Authors
Temazepam
Whole Blood
Scott and Oliver
Cocaine,
benzoylecgonine,
codeine and morphine
Hair
Brewer, et.al.
Study Summary
• Compared to a conventional SPE method,
the SFE method was more efficient and
gave cleaner extracts with recoveries above
80%
• K.S. Scott, J.S. Oliver, J. Anal.Toxicol. 21
(1997) 297.
Supercritical Fluid Chromatography
• SFC is a separation technique similar to
HPLC and GC where the mobile phase or
carrier gas is replaced by a supercritical
fluid
Limitations of GC and HPLC
GC Sample Limitations :
Volatility
Thermal stability
Low molecular weight
HPLC Analytical Limitations :
No universal detector
Low efficiency
Low resolution
SFC
Overcomes Limitations of GC and HPLC
•
•
•
•
•
•
Extends molecular weight range of GC
Lower operating temperature than GC
Faster separation time than HPLC
Higher separation efficiency than HPLC
Universal detector can be used, FID
Both packed (HPLC-type) and GC-type
columns can be used
Carbon Dioxide, CO2
has desirable properties as a SFC solvent
•
•
•
•
•
•
•
Inexpensive
Highly pure
Very low UV absorbance
NO FID background noise
Low critical pressure and temperature
Non-toxic
Supercritical CO2 behaves as a nonpolar solvent
such as heptane
• Polar organic modifiers can be mixed with CO2
for more polar samples
SFC Applications
Industrial :
•
•
•
•
•
•
•
Synthetic oligomers, polymers / additives
Surfactants (polyglycols)
Oligo / polysaccharides, sucrose polyesters
Pesticides
Isocyanates
Dyes
Waxes
SFC Applications
Biochemical :
•
•
•
•
•
Steroids
Prostaglandins
Fatty acids / lipids
Antibiotics
Drugs of abuse
SFC Applications
Fossil Fuels :
• Fractionation of petroleum and coal-derived
fluids
• Hydrocarbon group analysis
• Simulated distillation
Other Applications of
Supercritical Fluid
•
•
•
•
•
•
Supercritical Fluid Cleaning
Supercritical Fluid Drying
Supercritical Fluid Reactions
Micro Particles Formation
Supercritical Water Oxidation System
Others
Applications of Supercritical Fluid
Technologies in Taiwan
• IN THE PAST
Applications of Supercritical Fluid
Technologies in Taiwan
• AT PRESENT
Applications of Supercritical Fluid
Technologies in Taiwan
• IN THE FUTURE
Thanks For Your Attention !
•
林華經
•
•
•
國立清華大學化工系學士
國立清華大學生科所碩士
國立清華大學化工系博士班
•
•
友翔實業股份有限公司 儀器部經理
引光生物科技有限公司 研發部經理