WO2004026430A2

WO2004026430A2 - Solvent extraction method and apparatus

Info

Publication number: WO2004026430A2
Application number: PCT/GB2003/004105
Authority: WO
Inventors: Mazin Nicola
Original assignee: Advanced Phytonics Limited
Priority date: 2002-09-18
Filing date: 2003-09-17
Publication date: 2004-04-01
Also published as: GB2393720A; AU2003267617A1; AU2003267617A8; GB0321797D0; WO2004026430A3; GB0221667D0

Abstract

Apparatus for carrying out a fractional extraction comprises an extraction column (2) in which a biomass to be extracted is tightly packed.In use, liquified gas passes from vessel (4) and, optionally, a co-solvent passes from vessel (14) into the material within the column (2) and percolates through the material to extract compounds therefrom. The concentration of the co-solvent in the solvent formulation which passes through the column, the temperature or pressure of the solvent formulation and the temperature of the biomass may be varied in order to affect the composition of material extracted. Individual aliquots of material may be collected, at varying times, in vessels (20, 22 and 24).

Description

Extraction

This invention relates to extraction and particularly, although not exclusively, relates to the extraction of components from a biomass for example a botanical material .

Most botanical materials contain a complex spectrum of chemical compounds . These are often present in a great number and a wide variety of chemical types and properties. It is often the case that compounds that are associated with fragrance or flavour are non-polar, volatile molecules. They are frequently described as "essential oils" and often, either individually or synergistically, the molecules comprising essential oils exhibit certain bioactivities. Examples of some of the most commonly occurring volatile molecules are the various structural isomers of oxygen derivatives of open chain terpenes or high alcohols such as linalool, citronellol, geraniol and fernesol.

In addition to essential oils, many plants contain a variety of other types of compounds that also exhibit pharmacological activity such as antioxidant, antiseptic, antifungal, cytotoxic and enzyme inhibition properties. Frequently, these compounds are non-volatile liquids or crystalline solids and often they exhibit a degree of polar characteristics and, in some cases, they may be hydrophilic. In terms of chemical structure, these compounds may be hydroaromatic compounds such as cycloterpenes and sesquiterpenes . Typical examples of these are thymol, carvacrol, rosmarenic acid, carnosol and carnosic acid and diosphenol. Some plants may even contain dihydric phenols, such as catechol (o- dihydroxybenzene) present in the acacia catechu. These compounds may be highly polar and substantially water- soluble .

There are several well known and highly developed processes for the extraction of flavour, fragrance and pharmacologically active compounds from natural ra material. The most commonly used of these processes suffer the drawback of being capable of either non-selective and indiscriminate extraction of a complex cocktail of chemicals, or selective extraction of only one specific type of chemicals whilst the rest are sacrificed. Thus, known processes tend to produce extracts of limited use; or extract only small amounts of useful compounds contained within raw materials; or the extracts themselves need to be processed further to be commercially useful . For example, solvent extraction is characterised as a high yield, low selectivity process. In the process, the biomass is soaked, stirred or washed, as a slurry, with an organic solvent to remove the soluble components by extraction or mass transfer. In order to maximise yield, solvent extraction is often carried out at elevated temperature. The type of solvent used is chosen for its particular solvating properties in relation to the compounds of interes . Most commonly used solvents are the petrochemicals such as hexane and the more polar alcohols such as ethanol and methanol, ketones such as acetone, dichloromethane, ethyl acetate and others.

Steam distillation is still the most widely practised process for the extraction of essential oils from botanical biomasses . Steam is employed to carry the volatile compounds from the biomass. Although economical and simple to operate, steam distillation has the drawback of being unsuitable for the extraction of non-volatile compound; only the volatiles are recovered. Furthermore, due to the high temperatures required to generate steam, molecules that are very volatile are often not captured and those that are thermally unstable are often sacrificed due to degradation.

More recently, the use of liquefied gases as an alternative technology for the extraction of compounds from biomasses has been gaining prominence. For example, US 5512285 (Advanced Phytonics Limited) describes the use of a wide range of non-chlorinated fluorinated hydrocarbon solvents for example solvents containing carbon, hydrogen and fluorine atoms only such as tetrafluoroethane and heptafluoropropane. US 6224847B1 (Imperial Chemical Industries) describes the use of tetrafluoroethane in combination with a co-solvent selected from an alkane or hydrocarbon ether. WO00/43471 (Naturol) and O02/36232 (Ineos Fluor Holdings) describe the use of iodotrifluoromethane and heptafluoropropane respectively as extraction solvents.

Carbon dioxide is the most widely used supercritical fluid for extractions. However, extractions must be operated at high pressures which result in the need for complex and expensive apparatus which is also costly to run. Consequently, the use of carbon dioxide in extractions tends to be limited to high value products and small scale production or situations where intensive equipment utilisation is possible such as toll or contract operations. In many of the abovementioned known extraction processes a mass of material is subjected to solvent extraction in a first step to produce a first extract . Subsequently, in a second step which follows the first step, the first extract is further treated, for example extracted, to isolate predominantly or preferentially a desired material. However, such multiple step processes are time consuming and expensive. Furthermore, subjecting some extracts to further processes is undesirable since it may result in contamination, loss or damage of the desired materials.

A further known process for extracting antioxidants and essential oils from plants of Lamiaceae species is described in O01/26472 (Kemin) . In the process, in a first step, an organic solvent is used to prepare a coarse extract of antioxidants and essential oils from the plant material. The extract is then mixed with a surfactant and the mixture treated in a wipe film or rolled film evaporator to produce a fraction predominantly containing the essential oils of the plant material and a fraction predominantly containing antioxidant compounds thereof. Disadvantageously, however, the use of a surfactant may contaminate the fractions and the temperature used in the evaporation may damage the extracted material . Furthermore, since the process uses an additional step and apparatus it may increase the time of production of the desired extracts.

It is an object of the present invention to address problems associated with extraction and particularly, although not exclusively, to provide an extraction method which may be improved over known methods and/or which allows in situ fractionation of extracted materials.

According to a first aspect of the invention, there is provided a method of extracting components from a mass of material, the method comprising:

(a) arranging a mass of material in a receptacle between an inlet and an outlet of the receptacle, said mass of material including components to be extracted as constituent parts;

(b) selecting a first set of conditions for undertaking an extraction; (c) in accordance with said first set of conditions, passing a solvent formulation via said inlet into the receptacle, through the mass of material and out of the receptacle via said outlet; (d) altering the conditions for extracting components from said mass of material by moving towards a second set of conditions, via an intermediate set of conditions, and undertaking extractions of said mass of material under said intermediate set of conditions and said second set of conditions; (e) wherein said first, said intermediate and said second sets of conditions differ in at least one variable selected from: (i) the physical state of the solvent formulation; (ii) the physical state of said mass of material ; and (iii) a chemical property of the solvent formulation.

Advantageously, the method allows extracts to be collected at intervals as the conditions for the extraction are changed on moving from said first to said second sets of conditions. The extracts collected are found to differ in dependence on the conditions. Furthermore, the process provides a means whereby extracts may be produced which differ from extracts produced batchwise by slurrying a mass of material and a solvent formulation as in the prior art. This is believed to be a consequence of the process whereby the solvent formulation is passed from said inlet, through the mass of material and out of the receptacle via said outlet after which it is suitably collected rather than the solvent formulation charged with the components extracted being re-circulated through the mass of material numerous times as would happen if the mass of material and solvent are tumbled or stirred together. As a results, the mass of material closest to the inlet (hereinafter, a "first band of said mass") is initially contacted with "virgin" solvent (i.e. solvent which is not charged with any components extracted) under a first set of conditions and the solvent extracts material from said first band of material. The solvent, charged with components from the first band, passes towards the outlet, and becomes charged with components removed from second, third, fourth etc bands of material downstream of the first band. After a time, new virgin solvent which contacts the first band under the first set of conditions is unable to extract any further components from the first band, so the second band immediately downstream of the first band is, effectively, contacted with virgin solvent. In turn each band of material (including a band closest to the outlet of the receptacle) may effectively be contacted with virgin solvent. However, when the conditions for the extraction are changed as described, the solvent formulation under, for example, said intermediate conditions is able to extract further components from the first band (but those extracted components will differ from the components extracted under the first set of conditions) and the components extracted may be collected, suitably separately from the components extracted under the first set of conditions. The extraction of the second, third, fourth etc. bands will then take place as described above for the first set of conditions. It should be appreciated that the discussion above uses the term "band" to aid the explanation given; however, it is believed unlikely that discrete bands will be formed but that the depletion of the mass of material under the conditions used will occur gradually or moving through the mass of material towards the outlet.

Said components to be extracted are preferably produced in a natural process in said mass of material and/or are naturally occurring in said mass of material. Said mass of material is preferably a biomass. Said mass of material may comprise a fermentation mixture. Said mass of material may be a botanical biomass. Said mass of material may comprise bark, leaves, flowers or seeds. Said mass of material may comprise fermentation mycelia, moulds or algae.

Components extracted in the method may be essential oils, flavours, fragrances, biologically active compounds, pesticides, neutraceuticals, pharmaceuticals or a precursor of any of the aforesaid. Examples of pesticides are insecticides such as pyrethroids . Examples of pharmaceuticals include, antimicrobials, antifungals, antivirals, .mood modifiers, enzymes inhibitors, anti- emetics and anti-cancer agents. Examples of neutraceuticals include dietary supplements such as antioxidants and vitamins .

Said mass of material may include a mixture of at least two, components and the method preferably includes the step of preparing separate extracts of said mass of material wherein the extracts differ from one another in terms of the concentration of components extracted. Thus, one extract may include more of a particular component compared to the level of the same component in another extract. Preferably, at least three, more preferably at least four, especially at least five, separate extracts are prepared wherein each extract differs from each other extract in terms of the concentration of a component extracted.

Said mass of material suitably includes a mixture of at least three, preferably at least four, more preferably at least five, different components which may be extracted in the method.

Said mass of material arranged in said receptacle may be in any suitable form. The form is suitably selected to optimise extraction of components therefrom. Thus, naturally occurring material may be processed to adjust its form for use in the process. For example, solid material may be comminuted and such comminuted material may be arranged in the receptacle. In preferred embodiments, the mass of material arranged in the receptacle is in a finely divided form.

The method of the first aspect may involve selecting a said mass of material which includes components to be extracted and packing the material into free space in said receptacle so that said material extends over a length of at least 5cm, preferably at least 20cm, more preferably at least 40cm in said receptacle, preferably in a column. Said mass of material preferably extends to an upper region of the receptacle and a lower region thereof and, suitably, only the mass of material is present in the receptacle between said ^' upper and lower regions . Only after said mass of material has been packed into said receptacle so that it extends over said length described is a solvent formulation passed into said receptacle under said first conditions as described.

Said mass of material may be packed into said receptacle at a density of at least 0.25 g/cm³, preferably at least 0.30 g/cm³, more preferably at least 0.35 g/cm³, especially at least 0.40 g/cm³. The density of the bed may be achieved by use of a ram (or the like) to compress the mass of material . The mass of material is suitably substantially immovable when in position. The mass of material is substantially static during the flow of said solvent formulation therethrough.

Said solvent formulation may be passed through the mass of material at a rate of at least 0.02 ml/minute per gram of said mass of material in the receptacle. Said rate may be less than I l/minute per gram. The flow rate may be at least 0.5 BV/hour where ^λλBV" refers to the bed volume. Thus, the unit refers to the volume of said solvent formulation passing through the mass of material per hour divided by the volume taken up by the mass of material. The flow rate is preferably 10 BV/hour or less.

Said solvent formulation used in step (c) may include any solvent which is suitable for extracting components from a mass of material being treated in the method. Said solvent formulation used in step (c) preferably includes a first solvent selected from a Cι-₄ fluorinated hydrocarbon and a Cι-₄ hydrofluorocarbon ether.

A said hydrofluorocarbon ether preferably comprises one or more carbon, fluorine, hydrogen and oxygen atoms only. It may include up to 10, preferably up to 8, more preferably, up to 6, fluorine atoms. It preferably includes at least 2, more preferably at least 3 fluorine atoms. It is preferably aliphatic and/or saturated. An example of a hydrofluorocarbon ether is 1,1,1,2,2- pentafluorethyl methyl ether.

Preferably, the solvent formulation used in step (c) includes a first solvent which is a said Cι-₄ fluorinated hydrocarbon, rather than a said hydrofluorocarbon ether.

Said Cι-₄ fluorinated hydrocarbon is preferably non- chlorinated. Preferably, it comprises one or more carbon atoms, one or more fluorine atoms together with one or more other atoms selected from hydrogen atoms and iodine atoms. More preferably, it comprises one or more carbon, fluorine and hydrogen atoms only. Preferably, said fluorinated hydrocarbon is a Cχ._{Z l} more preferably a C₂-_3/ fluorinated hydrocarbon. Especially preferred is a C₂ fluorinated hydrocarbon.

Said fluorinated hydrocarbon may include 10 or fewer, suitably 8 or fewer, preferably 7 or fewer, more preferably 5 or fewer, especially 4 or fewer, fluorine atoms . Preferably, said fluorinated hydrocarbon includes at least 2, more preferably at least 3, fluorine atoms.

Said fluorinated hydrocarbon is preferably aliphatic. It is preferably saturated.

Said fluorinated hydrocarbon may have a boiling point at atmospheric pressure of less than 20°C, preferably less than 10°C, more preferably less than 0°C, especially less than -10°C. The boiling point may be greater than -90°C, preferably greater than -70°C, more preferably greater than -50°C, especially greater than -40°C.

Said solvent formulation may include a first solvent selected from iodotrifluoromethane, CF₃H (HFC-23, trifluσromethane) , CH₃F (HFC-41, fluoromethane) , CH₂F₂

(HFC-32, difluoromethane), CF₃CF₂H (HFC-125, pentafluoroethane) , CF₃CH₃ (HFC-143 A, 1,1,1- trifluoroethane), HCF₂CH₃ (HFC-152 A, 1, 1-difluoroethane) , CF₃CHFCF₃ (HFC-227 EA, 1, 1, 1, 2 , 3 , 3 , 3-heptafluoropropane) , CF₃CF₂CF₂H (HFC-227 CA, 1, 1, 1, 2 , 2 , 3 , 3-heptafluoropropane) , CF₃CH₂CF3 (HFC-236 FA, 1, 1, 1, 3, 3 , 3-hexafluoropropane) , CF₃CF₂CH3 (HFC-245 CB, 1, 1, 1, 2 , 2-pentafluoropropane) , CF₃CF₂CH₂F (HFC-236 CB, 1, 1, 1 , 2 , 2 , 3-hexafluoropropane) , HCF₂CF₂CF₂H (HFC-236 CA, 1, 1, 2 , 2, 3, 3-hexafluoropropane) , CF₃CHFCF₂H (HFC-236 EA, 1, 1, 1 , 2 , 3 , 3-hexafluoropropane) , and CH2FCF3 (HFC-134A, 1, 1, 1, 2-tetrafluoroethane) . Preferably, said solvent formulation includes a first solvent selected from iodotrifluoromethane, 1,1,1,2,3,3,3- heptafluoropropane (R-227 EA) , 1,1,1,2,2,3,3- heptafluoropropane (R-227CA) and 1,1,1,2- tetrafluoroethane .

More preferably, said solvent formulation includes a first solvent selected from 1,1,1,2,3,3,3- heptafluoropropane (R-227EA) and 1,1,1,2- tetrafluoroethane, with 1, 1,1, 2-tetrafluoroethane being especially preferred.

Said first solvent preferably has a purity of at least 98% w/w.

Said solvent formulation is preferably in a liquid state when contacted with said mass of material in the method. Said solvent formulation is preferably in a sub- critical state when contacted with said mass of material in the method.

Preferably, extractions under each of said first, second and intermediate sets of conditions include the use of a solvent formulation which includes a said first solvent of the type described above. Preferably, the identity of the first solvent used under each of said first, second and intermediate sets of conditions is the same.

Suitably, during passage of the solvent formulation through the mass of material in the receptacle, said solvent formulation becomes charged with one or more components extracted from said mass of material. Thus, suitably, solvent formulation (hereinafter "the charged solvent formulation") passing out of the receptacle via said outlet contains one or more components extracted. The method of the first aspect preferably includes the step of collecting the charged solvent formulation after it has passed out of the receptacle in step (c) . More preferably, the method includes the steps of collecting a plurality of, (preferably at least three, more preferably at least four, especially at least five) aliquots of charged solvent formulation. The collection of said charged solvent formulation is preferably carried out in evacuated collection means, suitable under vacuum. The aliquots are preferably collected in separate collection means, for example collection vessels. The or each collection vessel is preferably not open to atmospheric pressure during collection in the method. The or each collection vessel is preferably evacuated prior to collection of an aliquot. The or each collection vessel is preferably evacuated to a pressure of less than atmospheric pressure prior to the collection of an aliquot in the method. The aliquots are preferably collected at intervals during the method. The collection of one aliquot may be commenced after the conditions have been altered by moving towards a second set of conditions as described in step (d) . The collection of another aliquot may be commenced after the second set of conditions have been selected. The collection of said one aliquot is preferably terminated before the commencement of the collection of said another aliquot. The collection of an aliquot may be commenced after said first conditions have been selected and terminated prior to selection of the second set of conditions. By way of example, the commencement of collection of a first aliquot may be at least five minutes, preferably at least ten minutes (and preferably less than 5 hours) prior to the commencement of collection of a second aliquot; and the commencement of collection of a third aliquot may be at least five minutes, preferably at least ten minutes (and preferably less than 10 hours) after commencement of collection of the second aliquot; and suitably said first, second and third aliquots are collected successively with no aliquot collected in between them. The concentration in said first aliquot of a first component extracted from the mass of material in the method is preferably different compared to the concentration of the same said first component in said third aliquot. The concentration of the first component in the first aliquot is preferably greater than in the third aliquot. Similarly, the concentration of a second component extracted from the mass of material in the method is preferably greater in the third aliquot than in the first aliquot. The concentration (s) of the first component and suitably the second component in the second aliquot is/are suitably intermediate the concentrations of the respective components in the first and third aliquots . In some embodiments, separate aliquots may be collected in dependence upon data obtained by in-line physical, chemical or electrochemical analysis (e.g. using a UV flow cell or fluorescence) of solvent formulation after it has passed out of the receptacle.

At least three, preferably at least four aliquots are collected in the method. Preferably, at least one, more preferably at least two, especially at least three aliquots collected are kept separate from one another and not combined at any stage. Preferably, in the method, the solvent formulation initially passed into the receptacle via said inlet is not charged with any component extracted previously from the mass of material in the receptacle. Thus, preferably, the solvent formulation initially passed 5 into the receptacle is a virgin solvent formulation. Preferably, said solvent formulation (suitably said virgin solvent formulations described) is passed through the receptacle only once prior to collection of the charged solvent formulation. Thus, preferably, the one or more

10 components extracted from the mass of material which are carried by the charged solvent formulation are derived from a single passage of the solvent formulation through the receptacle. Preferably, the charged solvent formulation is not recirculated to the inlet of the

15 receptacle.

It should be appreciated from the above that the method allows a single mass of material in a receptacle to be treated with solvent formulations thereby to produce a

20. plurality of different aliquots which differ in their constitution of materials extracted from the mass of material. Thus, in a single step, in a single receptacle, a said mass of material may be extracted and the extract divided into separate fractions which differ from one

25 another. Thus, the mass of material can be extracted and fractionated in a single step thereby reducing the number of separate processes required compared to the number of processes often used to produce usable extracts in the prior art .

30

After the charged solvent formulation has been collected, the method preferably includes the step of isolating components extracted from the solvent formulation. This may include the step of separating a mass comprising one or more components extracted from at least part of the solvent formulation. For example, if the solvent formulation comprises more than one type of solvent, the most volatile solvent may initially be separated from the mass comprising the one or more components extracted. There may then remain the mass together with a less volatile solvent which was a part of said solvent formulation. The more volatile solvent (or the entirety of the solvent formulation if said formulation consists essentially of a single solvent) separated from the mass comprising the one or more components extracted may, after any necessary treatment (e.g. compression), be re-introduced into the inlet of the receptacle as required. It should be appreciated, however, that the solvent re-introduced will be substantially uncontaminated with any material previously extracted from the mass of material in the receptacle. In the event that the one or more components extracted are still associated with a solvent after removal of the most volatile solvent as described, the one or more components extracted may be isolated from any residual solvent (s) , as necessary, by standard techniques.

In the method, the conditions may be altered in any desired manner between said first and second sets of conditions. For example, the conditions may be altered step-wise. In this case, the first set of conditions may be maintained constant for a time, after which the conditions are changed to a second set of conditions. Alternatively, the conditions may be changed gradually by gradually changing a parameter for example as described in points (e) (i) , (ii) and (iii) of the first aspect. The rate of change of conditions may be constant or it may increase over time.

Preferably, at some stage in the method, the conditions for extracting components from said mass of material are changed without at the same time interrupting the passage of said solvent formulation into the receptacle. Preferably, the flow of said solvent formulation is uninterrupted at least from commencement of the method under said first set of conditions until the method is undertaken using said intermediate set of conditions. More preferably, the flow of said solvent formulation is substantially uninterrupted from commencement of the method under said first set of conditions until the method is undertaken under said second set of conditions.

As described in (e) (i) above, the physical state of the solvent formulation may be varied during extraction of said mass of material in the receptacle. The temperature or pressure of said solvent formulation may be varied. If the physical state of the solvent formulation is to be varied, it is preferred that the temperature of the solvent formulation is varied. For example, said first set of conditions may involve the solvent formulation being at a relatively low first temperature. The temperature may be raised so that said intermediate set of conditions involve the solvent formulation being at a second temperature, greater than the first temperature. The temperature may be further raised so that said second set of conditions involve the solvent formulation being at- a third temperature greater than the second temperature. The solvent formulation may, during the method, be contacted with the mass of material when it is at a temperature intermediate the first and second, and second and third, temperatures. Preferably, during the method, the mass of material is contacted with said solvent formulation when said formulation is at first, second, third and fourth respective different temperatures. Preferably, said mass of material is contacted with solvent formulation at at least five different temperatures during the method. Each of said first, second, third, fourth (and preferably fifth) different temperatures are within the range -10°C to 60°C. Preferably, during the method, the mass of material is contacted, at some stage, with said solvent formulation at a temperature of less than 10°C, preferably less than 5°C, more preferably less than 0°C. Preferably, during the method, the mass of material is contacted, at some stage, with said solvent formulation at a temperature of greater than 35°C, preferably greater than 40°C. Suitably, the difference between the temperature of the solvent formulation under said first set of conditions and the temperature of it under said second set of conditions is at least 10°C, preferably at least 15°C, more preferably at least 20°C, especially at least 30°C.

As described in (e) (ii) above, the physical state of the mass of material may be varied during extraction of the mass of material in the receptacle. Preferably, the temperature of the mass of material is varied. For example, said first set of conditions may involve the mass of material being subjected to a relatively low temperature, so that said mass is at a first temperature. The temperature may be raised so that said intermediate set of conditions involve the mass of material being subjected to a higher temperature so that said mass is at a second temperature, greater than said first temperature. The temperature may be further raised so that said second set of conditions involve the mass of material being subjected to a higher temperature still, so that said mass of material is at a third temperature, higher than said second temperature. Suitably, during the method, the mass of material is contacted with said solvent formulation when said mass of material is subjected to at least two, preferably at least three, more preferably at least four, especially at least five different temperatures. Each of the four, preferably five, different temperatures to which the mass of material is subjected are preferably in the range -10°C to 60°C.

In one embodiment, the physical state of the solvent formulation or the mass of material may be varied by application of ultransonic excitation, for example, the intensity and/or frequency of an ultrasonic source may be varied to vary the physical states. Alternatively, the intensity and/or. frequency of an ultrasonic source may be kept constant, but the source is arranged to facilitate the method by, for example, facilitating contact/passage of the solvent formulation. Preferably, however, no ultrasonic excitation of any of the types described is used in the method.

As described in (e) (iii) above, a chemical property of the solvent formulation may be varied during extraction of said mass of material in the receptacle. This may be achieved by including varying amounts of a selected modifier in said solvent formulation which is passed, via said first inlet, into the receptacle. For example, the first set of conditions may involve passing a said solvent formulation which includes a first weight per cent of said selected modifier via said inlet and into the receptacle. Said first weight per cent may be, and preferably is, substantially zero, so preferably under said first set of conditions said solvent formulation does not include said selected modifier. The amount of said selected modifier included in said solvent formulation may then be increased so that said intermediate set of conditions involve the solvent formulation including a second weight per cent of said selected modifier. The amount of said selected modifier in said solvent formulation may be increased further so that said second set of conditions involve the solvent formulation including a third weight per cent of said selected modifier. The solvent formulation may, during the method, include amounts of said selected modifier between the first and second weight per cents, and second and third weight per cents. Preferably, during the method, at least three, preferably at least four, more preferably at least five, solvent formulations which differ from one another in the amount of the same selected modifier therein, are passed via said inlet into the receptacle. The amount of said selected modifier in said solvent formulation may be increased step-wise or continuously in said method. The amount of said selected modifier may be varied from 0wt% up to 100wt% relative to the total weight of the solvent formulation passed into the receptacle. During the method 100wt% of a modifier may be used towards the end of the extraction of the mass of material to remove remaining extractables in the material . Said selected modifier may comprise any material which is capable of modifying the properties of the solvent formulation thereby to affect extracts obtained from the mass of material. Said selected modifier may affect the pH of the solvent formulation. Preferably, said selected modifier comprises a co-solvent. A said co-solvent may be selected from any of said first solvents referred to above so that said solvent formulation includes a said first solvent together with a co-solvent acting as a modifier and being a different solvent selected from said list of first solvents. Preferably, said solvent formulation includes a modifier selected from: a C₂-_ hydrocarbon such as an alkane or cycloalkane with alkanes such as ethane, n-propane, i-propane, n-butane and i-butane being especially preferred; and hydrocarbon ethers, particularly dialkylethers such as di ethylether, methylethylether and diethyl ether. In other embodiments, said modifier may be polar, for example having a dielectric constant, at 20°C, of greater than 5. Such modifier may be selected from: amides, especially N,N' -dialkylamides and alkylamides, with dimethylformamide and fo mamide being preferred; sulphoxides, especially dialkyl sulphoxides, with dimethylsulphoxide being preferred; alcohols, especially aliphatic alcohols for example alkanols, with methanol, ethanol, 1-propanol and 2-propanol being preferred; ketones, especially aliphatic ketones, for example dialkyl ketones, with acetone being especially preferred; organic acids, especially carboxylic acids with formic acid and acetic acid being preferred; carboxylic acid derivatives, for example anhydrides, with acetic anhydride being preferred; cyanide derivatives, for example hydrogen cyanide and alkyl cyanides, with methyl cyanide and liquefied anhydrous hydrogen cyanide being preferred; ammonia; sulphur containing molecules including sulphur dioxide, hydrogen sulphide and carbon disulphide; inorganic acids for example hydrogen halides with liquefied anhydrous hydrogen fluoride, chloride, bromide and iodide being preferred; nitro derivatives, for example nitroalkanes and nitroaryl compounds, with nitromethane and nitrobenzene being especially preferred.

Preferred modifiers include those described above having a dielectric constant at 20°C, of greater than 5.

Said solvent formulation used in the method may include a first modifier and a second modifier. The variation of the amounts of the second modifier may be as described above for said first modifier. Suitably, however, the amount of only a single modifier is varied during an entire treatment of a mass of material in the method.

Thus, preferably, said solvent formulation. used in the method described in (e) (iii) includes a said first solvent as described and varying amounts of a said modifier.

The method of the first aspect could involve adjusting more than one variable selected from those described in

(e) (i) , (ii) and (iii) above during treatment of a mass of material in said receptacle. Preferably, however, only one variable is adjusted. In one especially preferred embodiment, a chemical property of the solvent formulation is varied as described in (e) (iii) .

Said receptacle in which said mass of material is arranged is preferably a column. The column preferably has a. circular cross-section between its inlet and outlet. The column preferably has a substantially constant cross- sectional area and shape between its inlet and outlet. The column may have an inside diameter of at least 2cm, preferably at least 4 cm, more preferably at least 5cm. The diameter of the column may be less than 30cm, preferably less than 15cm. The length of the column between its inlet and outlet may be at least 40cm, preferably at least 50cm, more preferably at least 75cm, especially at least 100cm. The length of the column may be less than 500cm, preferably less than 400cm, more preferably less than 250cm. The column may have a length: inside diameter ratio of greater than 1:1; and preferably less than 100:1. The length: diameter ratio may be at least 10, preferably at least 20. The column may have a wall thickness midway between its upper end and lower end of at least 1mm, preferably at least 2mm, more preferably at least 5mm. The wall thickness may be less than 100mm, preferably less than 50mm. The column preferably has a circular cross-section over at least 50%, preferably 80% of its length. It preferably has a circular cross-section over substantially the entirety of its length. The length: inside diameter ratio may be less than 50:1. The column is preferably made out of metal, such as steel.

It has been found that if separate aliquots of charged solvent are collected in the manner described above at intervals but without there being an alteration of the conditions as described in (e) (i) , (ii) and (iii) (i.e. physical state or a chemical property of the solvent formulation and the physical state of said mass of material are not changed as described) , the aliquots also differ from one another in an interesting manner and this may be explained by virtue of the gradual contact of successive "bands" of the mass of material with virgin solvent as described above. Accordingly, in a second aspect of the invention, there is provided a method of extracting components from a mass of material, the method comprising:

(b) passing a solvent formulation via said inlet into the receptacle, through the mass of material and out of the receptacle via said outlet; and (c) collecting separate aliquots of said solvent formulation passing out of the receptacle.

At least three, preferably at least four, separate aliquots are collected. Preferably, at least one, more preferably at least two, aliquots collected are kept separate from one another and not combined at any stage.

Preferred features of the first aspect, for example insofar as they relate to the mass of material, said receptacle, said solvent formulation and the collection of aliquots, may be applied to said second aspect described.

The identity and other physical and chemical characteristics of the solvent formulation may be kept constant during the method. However, if desired conditions may be varied as described in (e) (i) , (ii) and

(iii) of the first aspect. According to a third aspect of the invention, there is provided apparatus for use in the method of the first and/or second aspects, the apparatus comprising:

(a) a receptacle for containing a mass of material including components to be extracted;

(b) solvent formulation delivery means for delivering a solvent formulation via an inlet of the receptacle, through the mass of material and out of the receptacle via an outlet thereof; and

(c) at least one of the following:

(i) first means for varying the physical state of the solvent formulation delivered via said solvent formulation delivery means; (ii) second means for varying the physical state of the mass of material in the receptacle; (iii) third means for varying a chemical property of the solvent formulation delivered via said solvent formulation delivery means .

Said apparatus preferably includes a said mass of material, suitably of a type described according to said first aspect, contained within said receptacle. Said mass of material is suitably packed in said receptacle. It is preferably substantially immovably arranged in the receptacle. Said receptacle is preferably a column.

Said solvent formulation delivery means preferably contains a said solvent formulation for use in the process. Preferably, the apparatus includes a said solvent formulation distributed through the mass of material in the receptacle. Said solvent formulation delivery means preferably includes a hold vessel which may contain a said solvent formulation, for example a first solvent (suitably as described according to the first aspect) , in liquefied form.

Said first means may comprise means for altering the temperature of solvent formulation prior to it being contacted with the mass of material.

Said second means may comprise means for altering the temperature of the mass of material in the receptacle. To this end, the receptacle may include associated heating and/or cooling means. The receptacle may be jacketed.

Said third means may comprise means for introducing a selected modifier into the receptacle. Preferably, said third means is arranged to delivery a modifier into a solvent formulation prior to contact of the solvent formulation with a mass of material in the receptacle. Where a hold vessel is provided for containing a said first solvent, said modifier may be arranged to be mixed with solvent from said hold vessel upstream of said receptacle.

Said apparatus preferably includes the features of (c) (iii) . It may include at least the features of (c) (i) and (c) (iii) . In some embodiments, the apparatus may include the features of (c) (i) , (ii) and (iii) .

Said apparatus preferably include collection means for collecting charged solvent formulation passing out of the receptacle. Said apparatus suitably include a plurality, preferably at least three, more preferably at least four, collection receptacles for collecting solvent formulation passing out of the receptacle. Preferably, the apparatus includes a conduit means extending from an outlet end of the receptacle. A plurality of branches preferably extend from said conduit means and are connected to respective collection receptacles. The apparatus preferably includes

^' means for directing the flow of solvent formulation out of the receptacle via said conduit means and into a selected said collection receptacle. Respective valve means may be associated with each branch for controlling the flow to respective collection receptacles.

Said apparatus preferably includes removal means for removing, for example evaporating, solvent from materials collected in said collection receptacles. Means may be provided for liquefying solvent evaporated. Means may be provided for re-introducing the liquefied solvent back into the receptacle in which the mass of material is contained.

Any feature of any aspect of any invention or embodiment described herein may be combined with any feature of any aspect of any other invention or embodiment described herein mutatis mutandis .

Specific embodiments of the invention will now be described, by way of example, with reference to the accompanying diagrammatic drawings, in which:

Figure .1 is a schematic representation of apparatus for carrying out extractions;

Figure 2 is a graph of temperature of extraction medium vs. time for the extraction of Example 1; Figure 3 is a graph of yield as a percentage of the total yield vs. the fraction number of collected eluate;

Figure 4 is a graph of yield as a percentage of the total yield vs. fraction number for a St. John's Wort extraction;

Figure 5 is a representation of an analysis of extracts of Example 2;

Figure 6 is a graph representing HPLC results; and

Figure 7 is a graph representing the amount of carnosic acid against extraction time.

The following is referred to hereinafter: R134a - refers to 1, 1,1, 2-tetrafluoroethane.

In general terms, fractional extraction of compounds from biomasses, for example plant material, fermentation mycelia, moulds and algae may be undertaken continuously. Apparatus for the extraction includes an upright extraction column. Material containing, as constituent parts, compounds to be extracted (e.g. the compounds may be formed in said material in a natural, for example biological and/or microbiological, process) is provided in a finely divided form and is tightly packed in the column so that an extraction medium is able to flow through the packed bed in an even fashion, substantially without forming preferential flow channels. To further ensure an even flow of the extraction medium through the packed bed, the top end and bottom end of the column may be fitted with flow distributors designed to produce a substantially uniform pressure drop across the cross-sectional area of the column. In the extraction, extraction medium is caused to flow downwardly through the column in a controlled, uniform and even fashion. Nonetheless, in some embodiments extraction medium may flow upwards in the column. The column eluate solution is collected in aliquots at predetermined time or volume intervals. As an alternative, the aliquots may be determined by monitoring the eluate solution through a flow cell fitted with an appropriate detection apparatus such as colour, optical properties, specific gravity, V or IR absorption and fluorescence.

In one embodiment of the process, the temperature of the extraction medium is changed during the elution step. Typically, the change made during the entire elution step may be wholly within the range from -25 °C to 100°C, preferably from -15°C to 75°C, more preferably from -10°C to 60°C. The temperature of the extraction medium may be raised in a stepwise fashion or a gradient fashion. The temperature change gradient may be linear or non-linear. The rate of temperature change may be pre-determined to ideally suit the purpose of the particular application.

In another embodiment of the process, the solvating properties or the polarity of the extraction medium may be gradually changed by use of a solvent mixture comprising a first solvent together with varying amounts of a co- solvent. The co-solvent may be introduced to the extraction medium in-line and in an increasing concentration or gradient fashion. The gradient of increasing concentration of the co-solvent may be stepwise or linear. Alternatively, the extractio medium may comprise a mixture of a Ci - C₄ HFC and one or more co- solvents. The composition of this mixture may be changed in a step-wise fashion or a linear gradient fashion during the elution step. The optimum rate of change of the composition of the solvent may be predetermined.

Further details of the apparatus used and examples of fractional extraction are provided below.

Referring to figure 1, apparatus for carrying out fractional extraction using a liquefied gas as extraction medium comprises an extraction column 2 in which material to be extracted is tightly packed. The column may be jacketed and include heating/cooling means for temperature control. Upstream of the column 2 is a hold vessel 4 for containing the liquefied gas. The vessel 4 is connected downstream, by pipework 6, to the upper end of the column 2 for transferring the extraction medium into the column 2. A liquid metering pump 8 is provided in pipework 6 for controlling the flow of extraction solvent to the column. Immediately upstream of the column, the pipework includes an in-line heat exchanger 10 arranged to heat (or cool) liquid prior to its passage into the column. Between the pump 8 and heat-exchanger 10, there is a modifier solvent supply pipe 12 which is arranged to deliver a modifier solvent from a storage vessel 14 into the pipework 6 so that it mixes with liquefied gas rom vessel 4. A liquid metering pump 16 in supply pipe 12 controls the flow of liquid within the pipe 12.

Downstream of the vessel 2 are shown three collection/evaporation vessels 20, 22, 24 although more such vessels would generally be provided for collecting more than three different aliquots. Each of the vessels 20, 22, 24 includes an inlet pipe 26 and an outlet pipe 28 each having associated control valves 30. The vessels 20, 22, 24 are arranged to communicate with column 2 via pipeline 32 which is connected to the bottom of the column. A monitoring device 34 is arranged to monitor and/or analyse fluid flowing in pipeline 32. Downstream of pipeline 32 is a pipeline 36 which communicates with vessel 4 and includes an associated gas compressor 38 for liquefying gas prior to its passage back into the vessel 4.

The apparatus further includes any necessary in-line filters, one-way valves, flow control valves, pressure regulators and pressure release valves and instrumentation for reading temperature, pressure and pH to allow appropriate process control and safe operation of the apparatus .

In use, a vacuum pump (not shown) is operated to remove air from the apparatus after the material to be extracted has been packed into the column 2. Liquefied gas is then charged to the vessel 4 and co-solvent, if this is used, is charged into vessel 14. With any heating/cooling means of the apparatus appropriately set, liquid is passed from vessel 4 to the column 2. The liquid slowly percolates through the material in the column and extracts compounds from the material as it does so. Initially, the most soluble compounds included in the biomass are extracted preferentially and these are entrained with liquid as it passes from the column into pipeline 32. The liquid (and entrained extract) is then directed into vessel 20 by opening the appropriate valve.

After a period of time (which may be determined, in dependence upon an output from monitoring device 34) subsequent liquid passing out of the column is directed into vessel 22. Subsequently, it is directed into vessel 24 and later to other vessels (if provided) . Thus separate aliquots are collected in vessels 20, 22, 24 and the constitution of the extracts therein should differ, with compounds or compositions which are most soluble in liquid passing through the column being more concentrated in the vessels which initially are used for collection and less soluble compounds or compositions being more concentrated in collection vessels used later in the process .

The . constitution of extracts may also be affected by delivering a co-solvent from vessel 14 into pipeline 8 and mixing the co-solvent with liquid from vessel 4. The combined extraction solvent may then be adapted to extract preferentially certain compounds or compositions. The co- solvent may be delivered as described herein for manipulating the extraction of the biomass. Additionally and/or alternatively, the heat-exchanger 10 may be used to adjust the temperature of the extraction solvent thereby to control the nature of compounds or compositions preferentially extracted. Also, the temperature of the column itself (and thereby the biomass therein) may be adjusted as another means of affecting the nature of extracts .

After the extraction of the biomass has been completed (or prior to completion whilst extraction in the column 2 is ongoing) , the control valve to outlet pipe 28 of vessel

20 may be opened and compressor 38 operated to remove liquefied solvent from the vessel 20 and return it to vessel 4, leaving the compound (s) /composition (s) in vessel 20. This process may be repeated to isolate the different extracts in the respective vessels 20, 22, 24.

As will be appreciated from the examples which follow, the apparatus may be used to maximise the amount (s) of desired compound(s) /composition (s) in selected vessels, 20, 22, 24. This may obviate the need to undertake multiple separate extractions using separate and distinct extraction solvents. Furthermore, the apparatus can be, and preferably is, operated with a continuous, uninterrupted (but possibly and preferably changing) flow of extraction solvent through the column.

The following examples illustrate specific embodiments of use of an apparatus of the type described.

Example 1

Powdered root of Kava Kava (103g) was packed tightly into a jacketed stainless steel extraction column having dimensions of 2.3cm inside diameter and 60cm length. The apparatus was evacuated then R134a (1 litre) was charged into the jacketed hold vessel 4. Chilled ethylene glycol was recirculated through respective jackets surrounding the extraction column 2 and hold vessel 4 until a temperature of -1°C was attained. R134a was then passed through the packed bed of powdered root at a flow rate of 18ml/minute and the eluate collected in a separate vessel. Next, the elution temperature was increased in a linear gradient from -1°C to 41°C over a period of 2 hours and eluate samples were collected at regular intervals and analysed by reverse phase HPLC. The experimental conditions are summarised below:

Mass of Kava: 103.3 g Operating Temperature (mean) : See Figure 2 of the accompanying drawings. Vessel Pressure: 3-5 bar

Column Pressure 5-18 bar

Pump Pressure: 6-15 bar Flow Rate: 18 ml/ in

The HPLC conditions were as follows:

Wavelength: 220 nm

Column: C18, 4 μm, Jones Chromatography Flow Rate: 1.0 ml/min

Mobile Phase: Water 60%, isopropylacohol 20%,

Acetonitrile 19.9%, Acetic Acid 0.1%

Temperature : 20°C

Peak identification was carried out using Kavain standard, supplied by Phytochem

Figure 3 summarises results for the extraction and shows the fraction yield as a percentage of the total for each component for each eluate collected. The following points are noted:

66.4% of total Kavain yield is extracted in fractions 4,5,6 and 7

71.7% of total Mithysticin yield is extracted in fractions 6,7,8 and 9

79% of total Yangonin yield is extracted in fractions 10,11 and 12 Thus, it will be appreciated that the method provides a means of preparing a range of different solutions of extracts having individual components present at different concentrations. Furthermore, multiple separate extractions need not be carried out to produce the extracts once the extracts are produced in a continuous process by varying conditions used.

Example 2 (Pilot Scale Extraction)

Ground Kava Kava root (753.5 g) was tightly packed into a 5cm id/ 100cm length, jacketed stainless steel column. R134a (3 litres) was charged to the hold vessel and both column and hold vessel contents were chilled to 0°C. The column, fraction collection vessels and interconnecting pipes and valves were evacuated then pressure equilibrated to the vapour pressure of R134a. Column elution was carried out by passing liquefied R134a through the packed bed using a liquid flow pump whilst the extraction temperature of the packed bed was elevated using a stepped gradient from 0°C to 45°C over 510 minutes. Column eluates were collected in six fractions as shown in the table below. As each fraction was collected, the solvent was removed using the gas compressor and the resulting extraction was collected.

The following operating conditions were used: column pressure 12 barG fraction collection vessel pressure 8 barG R134a feed pump pressure 13 barG

The total yield was 61. Ig (equivalent to 8.1 wt% of the raw material) .

The six fractions collected exhibited differing physical characteristics in terms of form, colour and aroma. The table below summarises characteristics of the products .

Figure 5 represents the amounts of Kavain methsticin and yangonin in fractions isolated. Example 3 - gradient elution using co-solvent

Finely ground Hypericum perforatum, or St. John's wort, (120g) was packed tightly into a stainless steel extraction column having dimensions of 2.3cm inside diameter and 60cm length. The apparatus was evacuated, then R134a (2.5L) was charged into a jacketed hold vessel and methanol (1L) into a second hold vessel. Extraction was carried out by feeding a mixture of R134a and methanol through the packed bed and collecting the eluate collection/evaporation vessels. R 134a was continuously recycled by evaporating from the collection/evaporation vessels then recompressed into the hold vessel. The concentration of the methanol in the extraction solvent mixture commenced at 0% then increased in a stepped fashion up to a maximum of 20% v/v. The extraction flow rate was maintained at approximately 9 BV/hr, where BV refers to "bed volume" and temperature at 12°C. The extraction was carried out for a total of 13,5 minutes. 8 line samples were collected at regular intervals and analysed (see Figure 4) . Accumulated bulk extracts were recovered in three fractions as follows: Fraction A after 62 minutes, fractions B and C after further 35 minutes each.

Hypericin and pseudohypericin were identified for each sample by reverse phase HPLC analysis using the following method: Column: C18 Waters reverse phase Loop volume: 10 ml

Mobile Phase: MECN 50%, MeOH 30%, 0,1 M NH4AC 20% Flow Rate: 2 ml/min Wavelength: 590 nm

It was found from a visual assessment that the colour and form of the samples collected changed progressively in four distinct stages. Very, pale yellow oils were obtained initially, then pale yellow semi-solids, then brown oils and finally very dark brown gums.

Figure 4 illustrates the yield of each component in fractions collection from the extraction of Example 3 where "Hyp" in the key refers to hypericin; and "P.hyp" refers to pseudohypericin. The x-axis denotes fraction number and the y-axis shows yield in each fraction expressed as total for each of the two components . It will be appreciated from the graph that hypericin and pseudohypericin were substantially completely separated in the process .

Example 4 Finely ground Kava Kava (400g) was tightly packed into a stainless steel column having dimensions of 3.5cm inside diameter and 60cm length. The volume of the packed bed was 570ml. Using the apparatus and procedure described in previous examples, fractional extraction was carried out using HFC227ea (1, 1, 1, 2, 3,3, 3-heptafluoropropane by varying the extraction temperature from 2 to 48°C. The flow rate of the HFC227 ea was kept constant at about 39ml/min (2BV/hr) . It was found that the extracts could be fractionated as described above.

Example 5

Ground Echium Seed (239.9g) were tightly packed into a 35 mm inside diameter jacketed extraction column. HFC227 ea was pumped through the column at 93 ml/min with increasing temperature (detailed in table below) , with outlet back pressure of 12 barg, and collected, in a jacketed evaporator. The evaporator had a constant service side temperature of 30°C. Extract samples were collected at intervals as shown in the table. All samples collected were mobile oils. The distinct colour difference show fractionation was achieved.

Example 6

The method of Example 5 was used on ground Kava root (129.7g) with the conditoins/results as in the table below.

Samples were analysed by HPLC method: C18, Mobile: Water/MeCN/lPA/AcH (60:20:19.9:0.1), 0.85 ml/min. Sample in mobile @100 μl/ml. UV detection @ 254nm.

Figure 6 shows the relative distribution of a number of Kava lactones relative to time/ temperature.

Example 7

The method of Example 5 was used on powdered Kava root (134.5g) with HFC 236 (1, 1, 1, 3, 3, 3-hexafluoropropane) as the solvent at a flow rate of 46.7ml/min. A visual assessment of the samples showed them to be similar to those of Example 6, indicating that a similar degree of fractionation was obtained.

Example 8

In this example, HFC134a was used in a fractional extraction of Rosemary using a combined temperature and co-solvent gradient. Ground rosemary (133.4g) was packed tightly into a 35mm inside diameter jacketed extraction column. The extraction was carried out by pumping HFC134a through the column then into an evaporation vessel. Acetone/Methanol mixture (50:50) was introduced in the HFC stream as co solvent and the mixture pumped through the column at a total flow rate of 50 ml/min with increasing temperature and co-solvent concentration .(detailed in the table below) , with outlet backpressure of 12 barg. Extracts were collected in a jacketed evaporator. The evaporator had a constant service side temperature of 30 degrees Celsius. Samples of the residue from the evaporator were collected at the times detailed in the table.

The green solutions (from samples d to 1) were seen to get more bright green as the temperature/co-solvent increased. The samples obtained distinctly differed in terms of physical appearance and aroma indicating fractionation of the aroma compounds and the heavier phenols.

Example 9

In this example, HFC227 was used in fractional extraction of rosemary using a combined temperature and solvent gradient. The method used was similar to that outlined in Example 8, with the weight of ground rosemary used being 143.3g.

HFC227 was pumped through the column with increasing co- solvent (methanol/acetone (50:50)) concentration (detailed in table below) , with a total flow rate of 50ral/min. Samples of the residue from the evaporator were collected at the times detailed in the table. It was observed that fractions obtained exhibited gradual but distinct differences in colour and aroma.

Example 10

In this example HFC134a was used in the fractional extraction of Buchu Leaf using a combined temperature and solvent gradient . In the method powdered Buchu leaf (143g) was tightly packed into a jacketed column. HFC 134a was pumped through the column at 90ml/min, for 15min at -3°C. Co-solvent composed of acetone/methanol (50/50) was introduced to produce the gradient shown in the table. Extract samples were collected from the evaporation vessel at regular intervals as shown. The samples obtained had distinct differences in colour, consistency and aroma indicating a good degree of fractionation.

Example 11

133g of powdered rosemary leaves were packed into a jacketed extraction column (35mm i.d. x 610mm). Glass tubular packing was used to fill out excess volume at the inlet end. Extraction was carried out using a linear gradient of HFC134a with 50:50 acetone/methanol co-solvent at flow rate of 50 ml/min. A linear temperature gradient of -4 to 38 °C was applied. Extracts were collected in 30 minute fractions. All fractions observed for colour, consistency and aroma and were analysed for carnosic acid using HPLC.

Results: A total of 12 fractions of extract were collected. Fractions 1 and 2 were light yellow, mobile oils with a strong rosemary aroma which was more pronounced for Fraction 1. Fraction 3 had very faint aroma. Subsequent samples became gradually darker green in colour and all had no detectable rosemary aroma. The consistency of the fractions changed from light yellow mobile oil to denser gum to watery solution. HPLC analysis of the Fractions showed a peak at sample 6 and no carnosic acid present in Fractions 1 and 2. Figure 7 is a representation of the amount of carnosic acid against extraction time. A conclusion was made that this method may be optimised successfully to separate the anti-oxidant components present in rosemary from the essential oils .

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) , may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment (s) . The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. A method of extracting components from a mass of material, the method comprising:

(b) selecting a first set of conditions for undertaking an extraction;

(c) in accordance with said first set of conditions, passing a solvent formulation via said inlet into the receptacle, through the mass of material and out of the receptacle via said outlet;

(d) altering the conditions for extracting components from said mass of material by moving towards a second set of conditions, via an intermediate set of conditions, and undertaking extractions of said mass of material under said intermediate set of conditions and said second set of conditions; (e) wherein said first, said intermediate and said second sets of conditions differ in at least one variable selected from: (i) the physical state of the solvent formulation; (ii) the physical state of said mass of material ; and (iii) a chemical property of the solvent formulation.

2. A method of extracting components from a mass of material, the method comprising:

(a*) arranging a mass of material in a receptacle between an inlet and an outlet of the receptacle, said mass of material including components to be extracted as constituent parts;

(b*) passing a solvent formulation via said inlet into the receptacle, through the mass of material and out of the receptacle via said outlet; and

(c*) collecting separate aliquots of said solvent formulation passing out of the receptacle.

3. A method according to claim 2, the method including:

(B) selecting a first set of conditions for undertaking an extraction;

(C) in accordance with said first set of conditions, passing said solvent formulation via said inlet into the receptacle, through the mass of material and out of the receptacle via said outlet; (D) altering the conditions for extracting components from said mass of material by moving towards a second set of conditions, via an intermediate set of conditions, and undertaking extractions of said mass of material under said intermediate set of conditions and said second set of conditions;

(E) wherein said first, said intermediate and said second sets of conditions differ in at least one variable selected from: (i) the physical state of the solvent formulation; (ii) the physical state of said mass of material ; and (iii) a chemical property of the solvent formulation.

4. A method according to any preceding claim, wherein said mass of material is a biomass .

5. A method according to any preceding claim, wherein said mass of material includes a mixture of at least two components and the method includes the step of preparing separate extracts of said mass of material wherein the extracts differ from one another in terms of the concentration of components extracted.

6. A method according to any preceding claim, wherein said mass of material is a comminuted material.

7. A method according to any preceding claim, which includes selecting a said mass of material which includes components to be extracted and packing the material into free space in said receptacle so that said material extends over a length of at least 5cm in said receptacle.

8. A method according to any preceding claim, wherein said mass of material is packed into said receptacle at a density of at least 0.25g/cm³.

9. A method according to any preceding claim, wherein said solvent formulation is passed through the mass of material at a rate of at least 0.02 ml/minute per gram of said mass of material in said receptacle.

10. A method according to any preceding claim, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from a Cι-₄ fluorinated hydrocarbon and C_ι_₄ hydrofluorocarbon ether.

11. A method according to any preceding claim, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from a Cι-₄ fluorinated hydrocarbon having at least 3 fluorine atoms and a boiling point of greater than -40°C and less than 10°C, for example of greater than -40°C and less than 0°C.

12. A method according to any preceding claim, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from a C₂ to C₃ perfluorocarbon.

13. A method according to any of claims 1 to 11, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from iodotrifluoromethane, CF₃H (HFC-23, trifluoromethane) , CH₃F (HFC-41, fluoromethane) , CH₂F₂ (HFC-32, difluoromethane) , CF₃CF₂H (HFC-125, pentafluoroethane) , CF₃CH₃ (HFC-143 A, 1,1,1- trifluoroethane) , HCF₂CH₃ (HFC-152 A, 1, 1-difluoroethane) , CF₃CHFCF₃ (HFC-227 EA, 1, 1, 1 , 2 , 3 , 3 , 3-heptafluoropropane) , CF₃CF₂CF₂H (HFC-227 CA, 1, 1, 1, 2, 2, 3 , 3-heptafluoropropane) , CF₃CH₂CF₃ (HFC-236 FA, 1, 1 , 1, 3 , 3 , 3-hexafluoropropane) , CF₃CF₂CH₃ (HFC-245 CB, 1 , 1 , 1, 2 , 2-pentafluoropropane) , CF₃CF₂CH₂F (HFC-236 CB, 1 , 1 , 1, 2 , 2 , 3-hexafluoropropane) , HCF₂CF₂CF₂H (HFC-236 CA, 1 , 1, 2 , 2 , 3 , 3-hexafluoropropane) , CF₃CHFCF₂H (HFC-236 EA, 1, 1, 1, 2 , 3 , 3-hexaf luoropropane) , and CH₂FCF₃ (HFC-134A, 1, 1,1,2 -tetraf luoroethane) .

14. A method according to any if claims 1 to 11, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from CF₃CHFCF₃ (HFC-227 EA, 1, 1,1, 2, 3, 3, 3-heptaf luoropropane ) , CF₃CF₂CF₂H (HFC-227 CA, 1, 1, 1,2, 2, 3, 3-heptaf luoropropane ) , CF₃CH₂CF₃ (HFC-236 FA, 1,1, 1,3, 3, 3-hexaf luoropropane) , CF₃CF₂CH₃ (HFC-245 CB, 1, 1,1, 2, 2-pentaf luoropropane ) , CF₃CF₂CH₂F (HFC-236 CB, 1, 1,1, 2, 2, 3-hexaf luoropropane) , HCF₂CF₂CF₂H (HFC-236 CA, 1, 1,2, 2, 3, 3-hexaf luoropropane ) , CF₃CHFCF₂H (HFC-236 EA, 1,1, 1,2, 3, 3-hexaf luoropropane ) , and CH₂FCF₃ (HFC-134A, 1, 1, 1, 2 -tetraf luoroethane) .

15. A method according to any of claims 1 to 10, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from HFC-134a, HFC-245fa, HFC- 236ea and HFC-227ea.

16. A method according to any of claims 1 to 11, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from perfluorocarbons for example hexafluoroethane (R-116) and octafluoropropane (R-218) .

17. A method according to any of claims 1 to 11, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from hydrofluoromethanes, the hydrofluoroethanes and the hydro luoropropanes, for example, trifloromethane (R-23) , fluoromethane (R-41) , difluoromethane (R-32) , pentafluoroethane (R-125) , 1,1,1- trifluoroethane (R-143a) , 1, 1,2, 2-tetrafluoroethane (R- 134), 1,1, 1,2-tetrafluoroethane (R-134a) , 1,1- difluoroethane (R-152a) , 1, 1, 1,3,3-pentafluoropropane (R- 245fa) , 1,1, 2, 2, 3-pentafluoropropane (R-245ca) , 1,1,1,2,3- pentafluoropropane (R-245eb) , 1,1, 2, 3, 3-penta luoropropane (R-245ea) , 1, 1, 1, 2 , 3 , 3-hexafluoropropane (R-236ea) , 1,1,1,2,2,3- hexafluoropropane (R-236cb) , 1,1,1,3,3,3- hexafluoropropane (R-236fa) , 1,1,1,2,3,3,3- heptafluoropropane (R-227ea) and 1,1,1,2,2,3,3- heptafluoropropane (R-227ca) .

18. A method according to any of claims 1 to 10, wherein said solvent formulation used in step (c) or (C) includes a first solvent which is HFC-134a.

19. A method according to any of claims 1 to 10, wherein said solvent formulation used in step (c) or (C) includes a first solvent which is HFC-227 ea.

20. A method according to any of claims 1 to 10, wherein said solvent formulation used in step (c) or (C) includes a first solvent selected from HFC-236ea.

21. A method according to any preceding claim, which includes the step of collecting a plurality of aliquots of charged solvent formulation.

22. A method according to claim 21, wherein said aliquots are collected in separate collection means wherein said collection means are not open to atmospheric pressure during collection in the method.

23. A method according to claim 21 or claim 22, wherein the collection of one aliquot is commenced after the conditions have been altered by moving towards a second set of conditions as described in step (d) or (D) ; and the collection of another aliquot is commenced after the second set of conditions have been selected.

24. A method according to any preceding claims, wherein at some stage in the method, the conditions for extracting components from said mass of material are changed without at the same time interrupting the passage of said solvent formulation into the receptacle.

25. A method according to any preceding claim, wherein in (e) (i) or (E) (i) the temperature or pressure of said solvent formulation is varied.

26. A method according to any preceding claim, wherein in (e) (i) or (E) (i) , the temperature of the solvent formulation is varied.

27. A method according to any preceding claim, wherein said first set of conditions involves the solvent formulation being at a relatively low first temperature; said intermediate set of conditions involves the solvent formulation being at a second temperature, greater than the first temperature; and said second set of conditions involve the solvent formulation being at a third temperature greater than the second temperature.

28. A method according to any preceding claim, wherein, during the method, the mass of material is contacted with said solvent formulation when said formulation is at first, second, third and fourth r-espective different temperatures .

29. A method according to any preceding claim, wherein in (e) (ii) or (E) (ii) , the temperature of the mass of material is varied.

30. A method according to any preceding claim, wherein in

(e) (iii) or (E) (iii) a chemical property of the solvent formulation is varied by including varying amounts of a selected modifier in said solvent formulation which is passed, via said first inlet, into the receptacle.

31. A method according to any preceding claim, wherein, during the method, at least three solvent formulations which differ from one another in the amount of the same selected modifier therein, are passed via said inlet into the receptacle.

32. A method according to claim 30 or claim 31, wherein said selected modifier comprises a co-solvent.

33. A method according to any of claims 30 to 32, wherein said solvent formulation includes a modifier selected from: a C₂-₆ hydrocarbon; a hydrocarbon ether; and solvents having a dielectric constant, at 20°C, of greater than 5.

34. A method according to any preceding claim, which involves adjusting more than one variable selected from those described in e(i), (ii) or (iii); or E(i), (ii) , or (iii) .

35. A method according to any preceding claim, which involves adjusting both the physical state of the solvent formulation as described in (e) (i) and (E) (i) ; and a chemical property of the solvent formulation as described in (e) (iii) and (E) (iii) .

36. Apparatus for use in a method according to any preceding claim, the apparatus comprising:

(c) at least one of the following:

37. Apparatus according to claim 36, which includes a said mass of material contained within said receptacle.

38. Apparatus according to claim 36 or claim 37, wherein said solvent formulation delivery means contains a said solvent formulation for use in the process.

39. Apparatus according to any of claims 36 to 38, wherein said first means comprises means for altering the temperature of solvent formulation prior to it being contacted with the mass of material .

40. Apparatus according to any of claims 36 to 39, wherein said second means comprises means for altering the temperature of the mass of material in the receptacle.

41. Apparatus according to any of claims 36 to 40, wherein said third means comprises means for introducing a selected modifier into the receptacle.

42..Apparatus according to any of claims 36 to 41, which includes at least the features of both (c) (i) and (c) (iii) .

43. Apparatus according to any of claims 36 to 42, wherein the apparatus includes a conduit means extending from an outlet end of the receptacle, wherein a plurality of branches extend from said conduit means and are connected to respective collection receptacles; said apparatus including means for directing the flow of solvent formulation out of the receptacle via said conduit means and into a selected said collection receptacle.