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.
Phase Diagram of Carbon
Dioxide

Supercriti
Liqui cal
Solid
d Fluid
Pressur

Critical
Gas
Point
e

31.3 oC, 1072 psi

Triple Point

Temperature
 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
-1
Tc (°C) Pc(atm) c(g mL )
CO2 31.3 72.9 0.47
N2O 36.5 72.5 0.45
SF2 45.5 37.1 0.74
NH3 132.5 112.5 0.24
H2O 374 227 0.34
n-C4H10 152 37.5 0.23
n-C6H12 197 33.3 0.23
Xe 16.6 58.4 1.10
CCl2F2 112 40.7 0.56
CHF3 25.9 46.9 0.52

Source: “SFE and Its use in Chromatographic Sample Preparation” Ed. S.

Advantages of CO2 for SFE

 Low critical parameters

 Inert, Non-toxic, Nonflammable, Non-
corrosive
 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 Modifie
Pump r sample cell
(high
Pump in heated
pressure) chamber
CO2
Liquid Carbon Restrictor
Dioxide
(requires a dip CO2
tube)

Trap
Solid or
Liquid
Advantages of SFE over Solvent
Extraction

 Faster

Results in minutes rather than hours
 Selectivity results in cleaner extracts
 Low Critical Parameters

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
SC-CO2
diffuses matrix, SC-CO2 +
dissolves and resolves dissolved
High Pressure analyte from the matrix analyte to
Liquid the trap
CO2

Heat
 Trapping

Gaseous Gaseous
CO2 CO2
Trapping Solid
CO2 is changing
•High Surface Area
from a SF (2 ml/min)
•Adequate Amount
to an expanded gas
(1 L/min)
Trapping Liquid
•Analyte no longer
soluble •High Surface Tension
•Mechanical movement •Analyte is Soluble
of analyte due to the •Low Volatility
rapid expansion requires •Pressurized
the use of trapping •Cryogenically Cooled
material
SFE - Method Development

 Pressure

Increase pressure increases density = increase
in solubilizing power.
 Temperature

Increase temperature may decreases density =
decrease in solubilizing power (ie CO2 at 100
bar)
 Extraction Time
 Flow Rate
 Fluid Composition

Co-solvents/modifiers

Reactant Additive
 Static vs. Dynamic Extraction
Why Use Modifiers?
Analytes that have polar components require the use of a co-
solvent

Triglycerides Phospholipids

CH2OCOR1 CH2OCOR1
R2COOCH R2COOCH O
CH2OCOR3 CH2OPOR3
O-
R 1, 2 & 3 groups are long R 1 & 2 groups are long chain
chain hydrocarbons hydrocarbons (nonpolar),
(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 Time Allotment

Sample
Process.
61% Collection 6%
Analysis 6%

Data
Management
27%
Routine and Novel Applications of
Analytical SFE
SFE for Research
Environmental SFE Applications

• Matrices • Target Analytes

 Soil  TPH
 Tissue  PAH
 Clay  PCBs
 Sandy Loam
 Pesticides
 Sludge
 Dibenzofurans
 River Sediment

 Dioxins
Marine Sediment
 Fly Ash
 Incinerator Ash
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 German Magnolia Tansy

roseus chamomile grandiflora
Chamomile Ginger Peppermint Thyme
flowerheads
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 Dog feed/rodent feed

derivatives
Atovaquone Rat feed

Fluconazole Animal feed

Propanolol, Tamoxifen, ZM 95527, Rodent diet

169369
 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, Aqueous
pseudoephedrine
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, Hair Brewer, et.al.

benzoylecgonine,
codeine and morphine
 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