Supercritical Fluid Extraction SFE for the production of natural products by 8d7gUqg

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									Thomas P. Geanacou   01/12/09


Supercritical fluids (Supercritical Fluid Extraction and Chromatography a.k,a, SFE/SFC) can be
used to extract analytes from samples. 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. For these reasons supercritical fluid CO2 is the reagent used to extract
caffeine from coffee. They can also be used to extract essential oils from other plants.

The properties of supercritical fluids also provide some advantages for analytical extractions.
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 and temperature, or adding modifiers to the supercritical fluid. A common
modifier is methanol (typically 1-10%) which increases the polarity of supercritical CO2.

 The Use of Supercritical Fluid Extraction and Supercritical Fluid Chromatography for the Separation
                                 of Natural and Man-Made Products

First discovered in 1879, supercritical fluids have been used for extraction applications since the
1950’s. The 1980’s saw an increase in their use as a mobile phase for analytical separations. The
work was mainly capillary scale (GC type) work, but some packed column (LC type)
applications were developed. Since then, the expected growth in the technique has not taken
place but the availability of new programmable pumps and an electronic back pressure regulator
opens the door for future development.1

Over the years, many companies have responded to the growing emphasis on reducing chemical
waste by offering an alternative to traditional HPLC with a full line of “green” SFC/SFE
products. The reduction in the use of organic solvents has cost, health and safety benefits as well
as faster, cleaner sample recovery during experimental procedures. The benefits of using
supercritical fluids are their liquid-like densities offering higher solubility and increased column
loading. They have low viscosity and are highly diffuse enabling faster separation and extraction.

Before SFE/SFC (and often today) most herbs were extracted with a mix of 50/50 water and ethanol.
If this was not sufficient , then the compounds must be extracted in more exotic solvents like chloroform
or ethyl ether. Moderation is the key. Temperatures in the range of 100 – 150oF are usually ideal.
The solvents are usually distilled under gentle heat, low vacuum , or both.

Supercritical Fluid Extraction (SFE) is the process of separating one component (the extractant)
from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is usually
from a solid matrix, but can also be from liquids. SFE can be used as a sample preparation step

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for analytical purposes, or on a larger scale, to either strip unwanted material from a product (e.g.
decaffeination) or collect a desired product (e.g. essential oils). Carbon dioxide (CO2) is the most
used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol.
Extraction conditions for supercritical CO2 are above the critical temperature of 31°C and critical
pressure of 74 bar. Addition of modifiers may slightly alter this. The discussion below will
mainly refer to extraction with CO2, except where specified.2

SFE is an alternative to liquid extraction using solvents such as hexane or dichloromethane.
There will always be some residual solvent left in the extract and matrix, and there is always
some level of environmental contamination from their use. In contrast, carbon dioxide is easy to
remove simply by reducing the pressure, leaving almost no trace, and it is also environmentally
benign. The use of SFE with CO2 is approved by the Soil Association for organic products4. The
CO2 used is largely a by- product of industrial processes or brewing, and its use in SFE does not
cause any extra emissions.

The properties of a supercritical fluid can be altered by varying the pressure and temperature,
allowing selective extraction. For example, volatile oils can be extracted from a plant with low
pressures (100 bar), whereas liquid extraction can remove lipids. Lipids can be removed using
pure CO2 at higher pressures, and then phospholipids can be removed by adding ethanol to the

Extraction is a diffusion-based process, with the solvent required to diffuse into the matrix, and
the extracted material to diffuse out of the matrix into the solvent. Diffusivities are much faster
in supercritical fluids than in liquids, and therefore extraction can occur faster. Also, there is no
surface tension and viscosities are much lower than in liquids, so the solvent can penetrate into
small pores within the matrix inaccessible to liquids.

The requirement for high pressures increases the cost compared to conventional liquid
extraction, so SFE is only feasable where there are significant advantages. Carbon dioxide itself
is non-polar, and has somewhat limited dissolving power, so cannot always be used as a solvent
on its own, particularly for polar solutes. The use of modifiers increases the range of materials
which can be extracted. Food grade modifiers such as ethanol can often be used, and can also
help in the collection of the extracted material, but reduces some of the benefits of using a
solvent which is gaseous at room temperature.1

PhytoMyco Research Corporation (Greenville, NC) performs their extractions the following
way: First, the plants are harvested fresh, air dried, and extracted by SFE with proprietary
solvents. For the complete extraction of the secondary metabolites from the ground-up tissues,
the samples are cleaned up by passing then through a polyamide gel to remove unwanted tannins
and other assay interfering materials. All these plants have been taxonomically identified, so that
when a 'lead' is generated, a quick search can be performed to obtain information on the plant. In
addition, other in-house ethnopharmaceutical databases are available for dereplication purposes.
Some of the plants extracted can be found in Table 1.

Table 1.

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  Acalypha rhomboidea             Elymus virginicus              Nepeta cataria
  Achillea depressa               Erigeron strigosus             Oenothera laciniata
  Ginger essential oil            Eupatorium rotundifolium       Onoclea sensibilis
  Ammi visnaga                    Filago arvensis                Paliurus spina-christi
  Angelica sylvestris seed        Galinsoga ciliata              Ranunculus sardous
  Baccharis halimifolia           Glechoma hederacea             Rhus copallina
  Borago officinalis              Hosta seiboldiana              Sabatia angularis
  Briza minor                     Inula helenium root            Salvia aethiopis
  Calamintha sylvatica            Juncus compressus              Tagetes minuta
  Callicarpa americana            Kniphofia uvaria               Tanacetum vulgare
  Centaurea diffusa               Lactuca sativa                 Verbascum thapsus
  Dactylis glomerata              Lavandula latifolia            Vicia grandiflora
  Deschampsia cespitosa           Majorana hortensis             Zelkova

The clean extracts were fractionated using solid phase extraction (SPE). Each extract has been
fractionated into ten semi-purified portions and apportioned into 96 well microtiter plates, freeze-
dried, and stored at -20 0C. Each fraction contains 1-4 compounds, very enriched and clean (based
on HPLC analysis of randomly selected plant fractions). These fractions contain low molecular
weight compounds (ranging from 200-600 DA) that are ideal for targets such as transcription and
other mechanisms based assays. Over 40,000 semi-purified phytochemicals have been generated
from plant extracts. The dried fractions in microtiter plates are readily available for screening
purposes and can be easily dissolved in DMSO. Dilution plates can also be prepared easily for high
throughput screening. 3

  Averica Discovery Inc. (Worcester, MA) is a client-driven specialist analytical

 laboratory offering natural compounds,small molecule purification and structure verification for
chiral and other challenging compounds.

Averica provides confidence in the drug discovery project results by addressing the need for pure
material. Their analytical and preparative scale equipment allows purification of lead material
needed for in vivo studies or candidate nomination. Using highly efficient Supercritical Fluid
Chromatography (SFC) technology, they excel at problematic separations like enantiomer

Averica provides separations for drug discovery project. They use a variety of approaches to purify and
characterize small molecule leads, but their core technology is Supercritical Fluid Chromatography

SFC is a highly efficient separations technology. Comparable to normal phase HPLC, but
providing much greater selectivity and speed, SFC is particularly important in difficult
separations - such as the resolution of enantiomers.

About 80% of pharmaceuticals are chiral, and within the last ten years the number of new drugs
approved as single enantiomers exceeds those approved as racemates by 3:1. Often one

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enantiomer is inactive or toxic, while the other has the desirable activity. The FDA requires
evaluation of each stereoisomer as part of the drug development process, but modern drug
discovery project teams often prefer to work with purified enantiomers as they work to
understand pharmacology of lead candidates prior to development.

Here is an example of a challenge Averica recently faced. A small, East Coast pharmaceutical
firm using outsourced chemistry resources to support active discovery programs with unique
biological targets needed help.

Business Needs were single enantiomer test articles in 20 gram quantities to be used as probes of
in vivo pharmacology. The technical challenge came after the preparative chromatography work
was under way. Averica recognized that the compound was slowly racemizing and oxidizing in
its free base form. The racemic starting material, a hydrochloride salt, had been stable and useful
in a previous round of pharmacology experiments.5

Although rapid forced degradation tests with the racemate had indicated no issues, the collected
eluent when each enantiomer began to turn yellow, show unidentified chromatographic peaks,
and to show some conversion to the other enantiomer. Averica developed a prcedure for rapid
conversion of the collected free base to the hydrochloride salt using HCL gas, and this procedure
was carried out every 8 hours dring the production run. The salt was isolated as a dry white
powder through a solvent exchange process developed by Averica, and repeated quality check
assays showed it to be as stable as the parent racemic. The planned in vivo assays could be
carried out on each isomer, allowing the lead series to continue to further development. The
planned in vivo assays could be carried out on each isomer, allowing the lead series to continue
to further development. Averica is also associated with the Geen Chemistry group. The Green
Chemistry Group, organizers of the upcoming SFC 2009 Conference in Philadelphia, PA, USA,
is a non–profit organization dedicated to the advancement of environmentally sustainable
chemical research and development throughout the world.6

Eden Labs LLC (Columbus, OH) describes their procedures this way: High pressure extraction is
the most effective and efficient way to extract valuable constituents from botanicals. The
simplest way to explain this process is that you take the plant material and put it in a pressure
vessel and pump a particular liquefied gas or liquid solvent through it at a specific pressure and

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The pressure forces the solvent into the cell walls of
the botanical and separates the desired constituent
rapidly. The process of separating the extract from
the solvent varies from one solvent to another. Eden
Labs has conducted in depth studies and trials with
all the most important solvents and designed
equipment which can utilize all of them. Carbon
Dioxide is the king of extraction solvents for
botanicals. It is an all natural product which leaves
no toxic residues behind. Its extraction properties
can be widely and precisely manipulated with subtle
changes in pressure and temperature.

It is inexpensive and widely available. The capability of processing botanicals skillfully with
CO2 gives a company an added edge of status and prestige.

There are two basic types of CO2 extraction. Low pressure cold extraction involves chilling CO2
to between 35-55 degrees F and pumping it through the plant material at between 800-1,500 psi.
Supercritical Fluid extraction involves heating the CO2 to above 87F and pumping it above
1,100 psi. Usually this work is done between 6,000-10,000 psi. Supercritical Fluid CO2 can best
be described as a dense fog whereas the first method described uses the CO2 in a dense liquid

Low pressure CO2 is often the best method for producing high quality botanical extracts. CO2
has a high loading rate in this state meaning that you will have to pump many volumes of CO2
through a given volume of botanical. The loading rate is typically 10-40 volumes. For this
reason, it is important to have a high flow pump and a CO2 recycle system unless wasting high
volumes of CO2 is not a concern.

Supercritical CO2 has a much faster loading rate 2-10 volumes and a wide range of uses. The
downside is that some extracts can be damaged by either the high pressure breaking molecular
ring structures or the fact that moisture in the botanicals can react with the CO2 and form
carbonic acid which can turn some oils rancid. Following proper procedures can avoid these

Eden Labs has pioneered a method of fractionating supercritical extracts so that constituents with
different molecular weights fall out in to different separators during extraction. Below is a
picture of a supercritical unit with this feature that was manufactured at a research lab at Loyalist
College in Belleville, Ontario.

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 The majority of extracts are made with ethanol and it is the most widely accepted method in use.
There are many ways of extracting with alcohol but high pressure is the best. It is faster, does a
more thorough job and requires less alcohol per volume of herb than other methods. It is a good
idea to purchase vacuum distillation equipment with your high pressure alcohol extractor so that
you can remove alcohol from the extract. High pressure alcohol equipment can also utilize
compressed water which is very effective in many cases.

Propane- There is a little known school of thought in the natural products industry which
believes that propane is the ultimate solvent for extracting botanicals. Eden Labs has tested this
theory thoroughly and they have come to the conclusion that there is something to it. Although
propane cannot be as widely manipulated through temperature and pressure as CO2, it produces
very similar results, sometimes better. It has an amazingly small loading ratio 1-4 volumes and it
can be recovered quickly. This means much faster production times. It leaves no toxic residues
and it is an all natural, organic solvent. The material data safety sheet, MSDS, says it is harmless
except for the fact that is flammable. Because it works at relatively low pressures, 80-150 psi, the
technology costs much less than a full supercritical CO2 system and can be very competitive in
terms of quality and speed of production.

The downside to propane is that it is highly flammable so precautions such as sparkless rooms
with powerful ventilation are a must. The fact that is not widely understood or accepted can also
be an issue.

Butane/Isobutane- In some cases where propane doesn't do the job, butane works better. It has all
the pros and cons of propane and requires identical equipment for utilization.

Dimethyl Ether- This is the ultimate extraction solvent. It strips everything out of plant material
almost instantly. All of the same equipment and precautions as propane should be used as it is
also highly flammable. Has a vapor pressure slightly above propane.

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R134a and other refrigerant gases- There has been a lot of talk in recent years about using R134a
and related gases in natural product isolation. Eden’s experience has shown that R134a has
similar extraction properties to low pressure CO2. It works better than anything for isolating
fragrance and perfume essences.

The downside is that it becomes highly toxic if overheated and there a number of conflicting
patent and intellectual property claims regarding its usage. Eden

In general terms, supercritical fluids have properties between those of a gas and a liquid. In Table
2 the critical properties are shown for some components, which are commonly used as
supercritical fluids.

                Table 2. Critical properties of various solvents (Reid et al, 1987)

                           Molecular            Critical             Critical          Critical
                            weight            temperature            pressure          density

                                g/mol                K              MPa (atm)            g/cm3

  Carbon dioxide
                                44.01             304.1             7.38 (72.8)          0.469

      Table 2.             Molecular            Critical             Critical          Critical
                            weight            temperature            pressure          density

    Water (H2O)                 18.02             647.3            22.12 (218.3)         0.348

  Methane (CH4)                 16.04             190.4             4.60 (45.4)          0.162

   Ethane (C2H6)                30.07             305.3             4.87 (48.1)          0.203

  Propane (C3H8)                44.09             369.8             4.25 (41.9)          0.217

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  Ethylene (C2H4)               28.05              282.4              5.04 (49.7)          0.215

 Propylene (C3H6)               42.08              364.9              4.60 (45.4)          0.232

Methanol (CH3OH)                32.04              512.6              8.09 (79.8)          0.272

 Ethanol (C2H5OH)               46.07              513.9              6.14 (60.6)          0.276

 Acetone (C3H6O)                58.08              508.1              4.70 (46.4)          0.278

Table 3. shows density, diffusivity and viscosity for typical liquids, gasses and supercritical

    Table3. Comparison of Gases, Supercritical Fluids and Liquids

                       Density (kg/m3) Viscosity (cP) Diffusivity (mm2 / s)

       Gases                     1             0.01                1-10

Supercritical Fluids       100-1000          0.05-0.1            0.01-0.1

      Liquids                   1000         0.5-1.0              0.001

In addition, there is no surface tension in a supercritical fluid, as there is no liquid / gas phase
boundary. By changing the pressure and temperature of the fluid, the properties can be “tuned”

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to be more liquid or more gas like. One of the most important properties is the solubility of
material in the fluid. Solubility in a supercritical fluid tends to increase with density of the fluid
(at constant temperature). Since density increases with pressure, then solubility also tends to
increase with pressure. The relationship with temperature is a little more complicated. At
constant density, solubility will increase with temperature. However, close to the critical point,
the density can drop sharply with a slight increase in temperature. Therefore, close to the critical
temperature, solubility often drops with increasing temperature, then rises again.

All supercritical fluids are completely miscible with each other so for a mixture a single phase
can be guaranteed if the critical point of the mixture is exceeded. The critical point of a binary
mixture can be estimated as critical temperatures and pressures of the two components,

       Tc(mix) = (mole fraction A) x TcA + (mole fraction B) x TcB.

For greater accuracy, the critical point can be calculated using equations of state as the Peng
Robinson, or othrer equations. Other properties, such as density, can also be calculated using
equations of state.

Now that we have a better understanding of how (SFE, SFC) works, let us investigate some of
the other uses. The advantages of supercritical fluid extraction (compared with liquid extraction)
are that it is relatively rapid because of the low viscosities and high diffusivities associated with
supercritical fluids. The extraction can be selective to some extent by controlling the density of
the medium and the extracted material is easily recovered by simply depressurising, allowing the
supercritical fluid to return to gas phase and evaporate leaving no or little solvent residues.
Carbon dioxide is the most common supercritical solvent. It is used on a large scale for the
decaffeination of green coffee beans, the extraction of hops for beer production,and the
production of essential oils and pharmaceutical products from plants.1 Few laboratory test
methods include the use of supercritical fluid extraction as an extraction method instead of using
traditional solvents. Averica, already mentioned, is one of the few to utilizie it.5

Supercritical fluid chromatography (SFC) can be used on an analytical scale, where it combines
many of the advantages of HPLC and GC. It can be used with non-volatile and thermally labile
analytes (unlike GC) and can be used with the universal flame ionization detector (unlike
HPLC), as well as producing narrower peaks due to rapid diffusion. In practice, the advantages
offered by SFC have not been sufficient to displace the widely used HPLC and GC, except in a
few cases such as chiral separations and analysis of high molecular weight hydrocarbons. For
manufacturing, efficient preparative simulated moving bed units are available.The purity of the
final products is very high, but the cost makes it suitable only for very high value materials such
as pharmaceuticals.

Changing the conditions of the reaction solvent can allow separation of phases for product
removal, or single phase for reaction. Rapid diffusion accelerates diffusion controlled reactions.
Temperature and pressure can tune the reaction down preferred pathways, (e.g. .to improve yield
of a particular chiral isomer.)There are also significant environmental benefits over conventional
organic solvents.

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Impregnation is essentially the converse of extraction. A substance is dissolved in the
supercritical fluid, the solution flowed past a solid substrate, and is deposited on or dissolves in
the substrate. Dyeing, which is readily carried out on polymer fibres such as polyester using
disperse (non-ionic) dyes, is a special case of this. Carbon dioxide also dissolves in many
polymers, considerably swelling and plasticising them and further accelerating the diffusion

The formation of small particles of a substance with a narrow size distribution is an important
process in the pharmaceutical and other industries. Supercritical fluids provide a number of ways
of achieving this by rapidly exceeding the saturation point of a solute by dilution,
depressurization or a combination of these. These processes occur faster in supercritical fluids
than in liquids, promoting nucleation or spinodal decomposition over crystal growth and yielding
very small and regularly sized particles.

Supercritical drying is a method of removing solvent without surface tension effects. As a liquid
dries, the surface tension drags on small structures within a solid, causing distortion and
shrinkage. Under supercritical conditions there is no surface tension, and the supercritical fluid
can be removed without distortion. Supercritical drying is used for manufacture of aerogels and
drying of delicate materials such as archeological samples and biological samples for electron

Supercritical water oxidation uses supercritical water to oxidise hazardous waste, eliminating
production of toxic combustion products which incinerating can produce.

The efficiency of a heat engine is ultimately dependent on the temperature difference between
heat source and sink (carnot cycle). To improvethe efficiency of power stations the operating
temperature must be raised. Using water as the coolant, this takes it into supercritical conditions.
Efficiencies can be raised from about 39% for sub critical operation to about 45% using current
technology.Supercritical water reactors (SCWRs) are promising advanced nuclear systems that
offer similar thermal efficiency gains. Carbon dioxide can also be used in supercritical cycle
nuclear plants, with similar efficiency gains.

Conversion of vegetable oil to biodiesel is via a transesterification reaction, where the
triglyceride is converted to the methyl ester plus glycerol. This is usually done using methanol
and caustic or acid catalysts, but can be achieved using supercritical methanol without a catalyst.
This has the advantage of allowing a greater range and water content of feedstocks (particularly
used cooking oil), the product does not need to be washed to remove catalyst, and is easier to
design as a continuous process.

Supramics, environmentally beneficial, low-cost substitutes for rigid thermoplastic and fired
ceramic, are made using supercritical carbon dioxide as a chemical reagent. The supercritical
carbon dioxide in these processes is reacted with the alkaline components of fully hardened
hydraulic cement or gypsum plaster to form various carbonates. The sole by-product is ultra-pure
water. Because supramics consume and sequester carbon as stable compounds in useful products,
they may serve to reduce carbon that would otherwise be released into the environment.

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Currently, only schemes isolating fossil CO2 from natural gas actually use carbon storage, (e.g.
Sleipner gas field), but there are many plans for future CCS schemes involving pre- or post-
combustion CO2. There is also the possibility to reduce the amount of CO2 in the atmosphere by
using biomass to generate power and sequestering the CO2 produced.

Supercritical carbon dioxide is also an important emerging natural refrigerant, being used in new,
low carbon solutions for domestic heat pumps. These systems are undergoing continuous
development with supercritical carbon dioxide heat pumps already being successfully marketed
in Asia. The EcoCute systems from Japan, developed by consortium of companies including
Mitsubishi, develop high temperature domestic water with small inputs of electric power by
moving heat into the system from their surroundings. Their success makes a future use in other
world regions possible.

Suprercritical fluids can be used to deposit functional nanostructured films and nanometer-sized
particles of metals onto surfaces. The gas-like surface tension, diffusivities, and viscosities
allows access to nano pores much smaller than can be accessed by liquids, and the liquid-like
solubilities allow much higher precursor concentrations than are typical in chemical vapour
deposition. This is crucial in developing more powerful electronic components, and metal
particles deposited in this way are also powerful catalysts for chemical synthesis and
electrochemical reactions.1


1 .http://www.








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