METHODS FOR THE EXTRACTION OF

METHODS FOR THE EXTRACTION OF METABOLITES FROM PLANT TISSUES A. Mohdaly1, I. Smetanska Key words: solvent extraction, superctitical fluid, liquid membrane, reverse micelle 1. INTRODUCTION Foods are commonplace and much taken for granted; however, their composition and structure are by no means simple. Extraction of one or more components from a complex mixture is a requirement for many operations in the food engineering and biotechnology industries. This technology is used for either to recover important food components, being a main processing stage for the production of certain food products (sugar, oils, proteins), to isolate desired components (antioxidants, flavors), or to remove contaminants and other undesirable components (alkaloids, cholesterol). Extraction methods rely on exploiting differences in physical or chemical properties of the mixture of components. Some of the more common properties involved in separation processes are particle or molecular size and shape, density, solubility and electrostatic charge. In some operations, more than one of these properties are involved. However, most of the processes involved are of a physical nature. The recent progress in extraction technology has been in the use of novel techniques for separation. In this chapter, we review the extraction methods as conventional solvent extraction, supercritical fluid extraction (SFE), aqueous two-phase extraction (ATPE), liquid membrane extraction, and reverse micelle extraction. They are applied in food processing from fundamental theory to optimum practical application through using the relevant equipment, solvents, and the appropriate methods of process optimization. 2. CONVENTIONAL SOLVENT EXTRACTION The art of solvent extraction has been practiced in one form or another since ancient times. It appears that prior to the 19th century solvent extraction was primarily used to isolate desired components such as perfumes and dyes from plant solids and other natural sources. Solvent extraction is the term used for liquid-liquid extraction as well as leaching since a solvent is used to preferentially separate one or more constituents from either a liquid or a solid mixture. The modern practice of liquid-liquid extraction has its roots in the middle to late 19th century when extraction became an important laboratory technique. Design of extraction systems and detailed selection of suitable equipment depends on the objective of the process and physical properties of the material to be extracted as well as of the obtained product. The solubilizing ability and selectivity of liquid solvents that are mainly based on water, hydrocarbons like hexane, or alcohol are used to leach or extract certain desired components from the applied source material, which is a naturally obtained solid in this case. Product quality and extraction efficiency depend very much on conditioning of the solid feed material ahead of solvent extraction. Posterior to extraction, the solvent has to be recovered from the product and the exhausted meal, taking care of the product quality and increasing environmental demands.

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Adel Mohdaly, and I. Smetanska, Methods of Food Biotechnology, Berlin University of Technology, Königin-LuiseStr.22 D-14195 Berlin, Germany, E-mail: [email protected] and [email protected]

A.Mohdaly and I. Smetanska

2.1. Solvent extraction principles Solvent extraction is one of the oldest methods of separation known and certainly dates back to prehistory. The science of solvent extraction has evolved accordingly over a long period of time and much progress has been made in the understanding of solvation and the properties of liquid mixtures used in extraction processes. The associated literature on phase behaviour is certainly extensive and, although representation of highly non-ideal mixtures is still problematic, many theoretical models have been successfully developed. Extensive databanks of pure component properties have grown to support such models in order to predict solvent performance in process applications. Today, even with the introduction of new separation technologies, solvent extraction remains one of the most widespread techniques operating on an industrial scale. Solvent extraction is a separation process which involves the removal of individual constituents from a mixture of solids or liquids upon addition of a solvent in which the original constituents have different solubilities. In liquid extraction, a liquid solvent, usually an immiscible liquid is used to separate miscible liquids by preferentially dissolving one of them. The extracted solute is separated from the solvent by distillation or evaporation. In leaching or solid extraction, a liquid solvent is used to dissolve soluble material from its mixture with an insoluble solid. The principle of solvent extraction is illustrated in Figure 1. The vessel (a separatory funnel) contains two layers of liquids, one that is generally water (S aq) and the other generally an organic solvent (Sorg). In the example shown, the organic solvent is lighter (i.e., has a lower density) than water, but the opposite situation is also possible. The solute A, which initially is dissolved in only one of the two liquids, eventually distributes between the two phases. When this distribution reaches equilibrium, the solute is at concentration [A]aq in the aqueous layer and at concentration [A]org in the organic layer. The distribution ratio of the solute is defined as the ratio of “the total analytical concentration of the substance in the organic phase to its total analytical concentration in the aqueous phase, usually measured at equilibrium” (2.1), irrespective of whether the organic phase is the lighter or heavier one. (2.1) D= [A]org / [A]aq If a second solute B is present, the distribution ratios for the various solutes are indicated by DA, DB, etc. If DB is different from DA, A and B can be separated from each other by (single or multistage) solvent extraction. D is also called the distribution coefficient or distribution factor; we here prefer the expression distribution ratio. For practical purposes, as in industrial applications, it is often more popular to use the percentage extraction %E (sometimes named the extraction factor), which is given by (2.2) %E=100D/(1+D) where D is the distribution ratio of the solute (or desired component). For D = 1, the solute is evenly distributed between the two phases. A requirement for practical use of solvent extraction is that a reasonable fraction (percentage) of the desired component is extracted in a single operation (or stage).

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Figure 1 A schematic representation of solvent extraction (liquid-liquid distribution). A solute A is distributed between the upper layer, for example an organic solvent, and the lower layer, an aqueous phase. (Michael Cox and Jan Rydberg. 2004) 2.2. Classification In the past, extraction or leaching was often divided into percolation and immersion methods, referring to whether the solid is completely submerged or the solvent is just trickled through a solid bed. In general, classification has become clearer since technical development has concentrated on a few different types of extractors, most of them working continuously as percolators. On the other hand, immersion leaching of solid-solvent slurries using agitators or screws has largely lost industrial relevance. Even though many of the former extractor types have nearly disappeared, they are still worth mentioning because older equipment may still be perfect for certain applications. Separation processes may be batch or continuous. A single separation process, for example a batch extraction, involves the contact of the solvent with the food. Initially concentration gradients are high and the rate of extraction is also high. The extraction rate falls exponentially and eventually an equilibrium state is achieved when the rate becomes zero. The extraction process may be accelerated by size reduction, inducing turbulence and increasing the extraction temperature. Equilibrium is achieved either when all the material has been extracted, in situations where the volume of solvent is well in excess of the solute or when the solvent becomes saturated with the solute, i.e. when the solubility limit has been achieved, when there is an excess of solute over the solvent. However, the attainment of equilibrium may take some considerable time. Batch reactions may operate far away from equilibrium or close to it. Equilibrium data is important in that it provides information on the best conditions that can be achieved at the prevailing conditions. Equilibrium data is usually determined at fixed conditions of temperature and pressure. Some important types of equilibrium data are: ♦ solubility data for extraction processes; ♦ vapour/liquid equilibrium data for fractional distillation; ♦ partition data for selective extraction processes; ♦ water sorption data for drying.

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Continuous equipment usually operates under steady state conditions, i.e. the driving force changes over the length of the equipment, but at any particular location it remains constant with time. However, when the equipment is first started, it may take some time to achieve steadystate. During this transition period it is said to be operating under unsteady state conditions. In continuous processes the flow may be either streamline or turbulent. Consideration should be taken of residence times and distribution of residence times within the separation process; the two extremes of behaviour are plug flow, with no distribution of residence times, through to a wellmixed situation, with an infinite distribution of residence times. Continuous processes may be single- or multiple-stage processes. The stages themselves may be discrete entities, for example a series of stirred tank reactors, or there may be many stages built into one unit of equipment, for example a distillation column or a screw extractor. The flow of the two streams can either be co-current or counter-current, although counter-current is normally favoured as it tends to give a more uniform driving force over the length of the reactor as well as a higher average driving force over the reactor. In some instances a combination of cocurrent and counter-current may be used; for example in hot air drying, the initial process is cocurrent to take advantage of the high initial driving rates, whereas the final drying is countercurrent to permit drying to a lower moisture content. 2.2.1. Liquid-Liquid extraction Liquid-liquid extraction is a process for separating the components of a liquid (the feed) by contact with a second liquid phase (the solvent). The process takes advantage of differences in the chemical properties of the feed components, such as differences in polarity and hydrophobic/hydrophilic character, to separate them. Stated more precisely, the transfer of components from one phase to the other is driven by a deviation from thermodynamic equilibrium, and the equilibrium state depends on the nature of the interactions between the feed components and the solvent phase. The potential for separating the feed components is determined by differences in these interactions. A diagram of a full basic process is given in Figure 2 to illustrate the common terminology. The incoming aqueous solution is called the feed. It is contacted with the (recycled) solvent phase in a mixer-settler unit. Here we do not indicate the exact type of unit, but only its function (extraction), as commonly is done. After extraction and separation of the phases, the depleted phase becomes the raffinate and the enriched solvent phase becomes the extract or loaded (or pregnant) solvent. The raffinate may undergo a solvent recovery stage to remove any entrained solvent before exiting the process. The extraction process is rarely specific so that other solutes may be co-extracted with the main component. These impurities may be removed with an aqueous scrub solution in a scrub stage producing a scrub extract and a scrub raffinate containing the impurities. The latter may return to the feed solution to maintain an overall water balance. The scrubbed extract is now contacted with another aqueous solution to strip or back-extract the desired component. The stripped solvent then may undergo some regeneration process to prepare the solvent phase for recycle. The loaded (pregnant) strip solution then is treated to remove the desired product and the strip solution is recycled. One of the important aspects of this flow sheet is that, wherever possible, liquid phases are recovered and recycled. This is important from both an economic and an environmental standpoint. Liquid-liquid extraction is used in food processing to either selectively recover valuable components of a natural product or to remove undesirable components from it. Extraction of flavours and essences from crude extracts of citrus oils and separation of monoglycerides and 4


lecithins from edible oils are examples of recovery of valuable components. Removal of fatty acids from edible oils, partitioning of the crude oils to obtain higher unsaturation are examples of the latter category. Decaffeination using liquid -liquid extraction is an example of value addition to the coffee and tea as well as recovery of valuable caffeine. Once the solvent has been removed from the extracted foods, some may be used directly (for example cooking oils) or they may be further processed by concentration and/or dehydration. Many extraction operations operate close to ambient temperature, but even when elevated temperatures are used to increase the rate of extraction, there is little damage caused by heat and the product quality is not significantly affected. The main types of solvents used for extraction are water, organic solvents or supercritical carbon dioxide.

Feed

Scrub raffinate

Extraction

Raffinate

Extracted/ Loaded solvent Scrub

Solvent recovery

Waste

Recycle solvent Scrub feed

Scrub extract Strip

Solvent regeneration

Stripped/Recycle solvent

Loaded/ Pregnant strip

Recycle Product recovery strip

Regenerant solution

Product Figure 2. Typical flowsheet of a Liquid-liquid extraction circuit 2.2.1.1. Selection of solvent Common industrial solvents generally are single-functionality organic solvents such as ketones, esters, alcohols, linear or branched aliphatic hydrocarbons, aromatic hydrocarbons, and so on; or water, which may be acidic or basic or mixed with water-soluble organic solvents. More complex solvents are sometimes used to obtain specific properties needed for a given application. Solvents are sometimes blended to obtain specific properties, another approach to achieving a multifunctional solvent with properties tailored for a given application. 5


The selection of solvent for extraction of food material is made on the basis of (1) solvent capacity, (2) selectivity, (3) chemical inertness, (4) thermophysical properties of the solvent such as density, viscosity, boiling point and latent heat of vaporization, (5) flammability, (6) toxicity, (7) cost, and (8) availability. In the case of liquid-liquid extraction, interfacial tension plays an important role. A low value of interfacial tension is desirable for getting a good dispersion with high interfacial area for extraction. However, a very low value will cause emulsion formation, which will create problems in phase separation. In the extraction of natural products some extracted material may also cause undesirable emulsification. The types of solvent used commercially to extract food components are shown in Table 1. Extraction using water (leaching) has obvious advantages of low cost and safety and is used to extract sugar, coffee and tea. Oils and fats require an organic solvent and as these are highly flammable, great care is needed in both operating procedures and to ensure that equipment is gas-tight and electrical apparatus is sparkproof. The toxicity is especially important in view of the extraction of food materials. The residual solvent concentration has to be within the specified limit. This reduces the number of acceptable solvents for extraction. Table 1.Solvents used to extract food components Food solvent Temperature (ºC) Decaffeinated coffee Supercritical carbon 30–50 (CO2) dioxide, water or methylene chloride Fish livers, meat byproducts Acetone or ethyl ether 30–50 Hop extract Supercritical carbon 90%) of hydrocarbons and very small amount of flavour-imparting citrals, the monoterpene aldehydes. These aldehydes, neral and geranial need to be separated from the terpene and sesquiterpene hydrocarbons. Ethanol separates the citrals from the insoluble hydrocarbons. A solvent pair consisting of aqueous methanol with n-pentane may be used to separate and concentrate the citrals into the alcohol layer using a rotating disc contactor. 2.2.1.3.3. Extraction of caffeine The liquid-liquid extraction of caffeine from the aqueous solution of coffee soluble is accomplished by using a chlorinated solvent such as dichloromethane. The first liquid- liquid extraction stage is maintained at high temperature (40-80°C). The extraction column may be a rotating disc contactor (RDC) or reciprocating plate contactor (Karr). The caffeine-rich dichloromethane stream is again contacted with water to back-extract the caffeine to the aqueous phase in a secondary extractor. The temperature is maintained at 20-25°C with a very high phase ratio of aqueous to organic phases. The dichloromethane stripped off is recycled back to the primary extractor and aqueous stream is used to obtain caffeine. The extractor is again an RDC or Karr unit. The conditions of operation vary depending upon whether coffee solubles are obtained from green beans or from roasted coffee beans. 2.2.2. Solid-liquid extraction Solid–liquid extraction or leaching is a separation process affected by a fluid involving the transfer of solutes from a solid matrix to a solvent. It is a widely used separation process for the following: 10


1) Extraction of edible oils from seeds, beans, nuts, rice bran, wheat germ, coconut and other sources. 2) Extraction of essential oils from flowers, leaves and seeds. 3) Extraction of sugar from sugarbeet and sugarcane. 4) Extraction of coffee and tea. 5) Extraction of fish meal. 6) Extraction of proteins in oilseed meals 7) Extraction of active ingredients from leaves, pods, seeds, flowers and barks e.g., extraction of tocopherols, etc. Solid– liquid extraction (or simply extraction) may also be used to remove undesirable contaminants and toxins present in foods and feeds. Solid–liquid extraction is easily automated, faster, and in general more efficient than liquid-liquid extraction. Owing to its widespread use in diverse industries, leaching is also known as lixiviation, percolation, infusion, elutriation, decantation, and settling. The two main operations in leaching are: (a) contact of liquid solvent with solid for transfer of solute from the solid to the solvent and (b) separation of the extract, from the residual solid. Other auxiliary operations are preparation of the solids for extraction and recovery of solute from the solvent by distillation or evaporation. 2.2.2.1. Preparation of solid material The mechanism of leaching may involve simple physical solution or solution due to chemical reaction. The rate controlling step in leaching may be the diffusion of solvent into the mass to be leached, diffusion of the solute into the solvent or diffusion of the extract solution out of inert material of solid. Whatever the mechanism, the leaching process is favoured by the reduction in size of the solid. The preparation of the material for extraction is to make the solute more accessible to the solvent by size reduction of solid. This gives increased surface area per unit volume of solids to be leached and reduced distance to be traversed within the solid by the solvent and the extract. The preparation of the material for extraction involves crushing, grinding, flaking or cutting into pieces or cosettes. Grinding to a very fine size may cause packing of solids during extraction such that free flow of solvent during extraction is impeded. In case of material with cellular structure, the cell rupture due to grinding may lead to extraction of undesirable components. 2.2.2.2. Selection of solvent and operating temperature The selection of solvent has been discussed in section 2.2.1.1 where the criteria are enumerated. Growing awareness of carcinogenic tendencies of certain solvents has restricted their use. Similarly, concentration of the residual solvent from the permitted category has also been fixed by the regulatory authorities. Desolventizing has to be carefully monitored to achieve the permissible residual solvent level. Normally n-hexane, short chain alcohols (methanol, ethanol), ketone (acetone), esters (ethyl acetate, n-butyl acetate), chlorinated hydrocarbons (methylene dichloride, ethylene dichloride) and liquid carbon dioxide have been used as solvents for leaching. In view of the very low maximum permissible limit of solvents such as chlorinated hydrocarbons in food materials and regulatory restrictions, the use of chlorinated solvents is discouraged. 2.2.2.3. Selection of operating temperature 11


Temperature plays an important role in solid extraction. Higher temperatures give higher solubility of solute in solvents, permitting higher rates of extraction. However, higher temperatures may also mean high solvent losses, extraction of some undesirable constituents, and damage to some sensitive components in the plant material. A compromise is necessary in the selection of operating temperature. 2.2.2.4. Solid-liquid extraction equipment The equipments are divided into two principal classes with respect to the solids handled. They are: (1) fixed bed contact and (2) dispersed contact. In the former, the solids are kept in the form of a fixed bed while in the latter the solids are dispersed by moving them in a liquid solvent. The fixed bed devices may have the solvent contacting done by three methods: (a) percolaton, (b) full immersion, and (c) intermittent drainage. The dispersed contact is usually affected by suitable agitation and is used for materials which disintegrate during leaching. Extractors are either single-stage or multi-stage static tanks or continuous extractors Single-stage extractors These are closed tanks, fitted with a mesh base to support the solid particles of food. Heated solvent percolates down through the particles and is collected below the mesh base, with or without recirculation. They are used to extract oils or to produce coffee or tea extracts. Although they have low capital and operating costs, single-stage extractors produce relatively dilute solutions which may require expensive solvent recovery systems for organic solvents or pollution control measures when water is used as the solvent. Multi-stage extractors These comprise a series of up to 15 tanks, each similar to single extractor, linked together so that solvent emerging from the base of one extractor is pumped countercurrently to the next in the series. These are used to produce oils, tea and coffee extracts and to extract sugar from beet. Figure 5 shows a battery of percolators for instant-coffee extraction. Vessel diameters range from 0.25 to 0.75 m. Typical capacities are around 1 t resulting in bed heights of 4.5–6 m. Applying vacuum to the vessel facilitates loading of the percolators. The ball valve at the top is opened and the grinded coffee enters the vessel. Grind sizes range from 3 to 5 mm, the result of a compromise between favoring mass transfer and limiting pressure drop. After being sufficiently leached, the spent coffee ground is discharged by “shooting” through the ball valve at the bottom of the vessel. Holdup is maintained at 30–35 min showing a tendency to shorter times. Continuous extractors There are a large number of designs of extractor, each of which may operate countercurrently and/or co-currently. For example, one design is an enclosed tank containing two vertical bucket elevators made from perforated buckets and linked to form a continuous ring. Fresh material is loaded into the descending buckets of one elevator and solvent is pumped in at the top to extract solutes co-currently. As the buckets then move upwards, fresh solvent is introduced at the top of the second elevator to extract solutes counter-currently. The solution collects at the base and is pumped to the top of the first elevator to extract more solute, or it is separated for further processing. Other designs of equipment employ perforated screw conveyors instead of bucket elevators. Some of the commonly used extractors are as follows: 12


♦ Boilman extractor, Blaw-Knox extractor (Moving bed perforated basket type percolation systems) ♦ Rotocel extractor (Multicompartment countercurrent percolation extractor) ♦ Kennedy extractor (Multichamber unit with impellers) ♦ De Smet extractor, Lurgi extractor (Endless belt percolation extractors) ♦ Bonotto extractor (Vertical plate continuous dispersed solid extractor) ♦ Hildebrandt extractor, DDS extractor (Total immersion screw conveyor extractor) ♦ Sherwin-Williams extractor, Allis Chalmers extractor (Multistage mixer-settler disperse contact systems) ♦ Dorr extractor (Multistage decantation system) ♦ Diffusion battery (Multibatch countercurrent extraction system)

Figure 5 Schematic of a percolator battery. Each percolator can be bypassed for discharging/charging as indicated only in the case of the second percolator. (Rudolf and Philip.2003.). 2.2.2.5. Applications 2.2.2.5.1. Extraction of tea and coffee solubles Extraction of dried, blended tea leaves with hot water is accomplished in 3 to 5 stages in a diffusion battery. Temperature for extraction is raised from 70°C in the initial stage to 90°C in the final stage using intermediate heat exchangers. The final solution usually contains 2.5-5% solids. For preparing instant tea, the solution is concentrated to about 50% solids in vacuum evaporators. The volatile aroma stripped off during this stage is condensed and blended back. The solution is dried by spray, freeze or vacuum belt dryers. In place of a diffusion battery, other extractors like Rotocel or continuous countercurrent multistage extraction system with split feed may be used. In the extraction of coffee solubles, ground and roasted coffee beans are extracted with hot water under pressure. The diffusion battery containing 5 to 10 percolators may be used. The 13


percolator may operate in a batch or semi-continuous mode with countercurrent flow of hot water. The extraction temperature is raised from 100°C in the initial stage to 180°C in the final stage. Intermediate heat exchangers are used for this purpose. The final solution contains 25-30% solids. This is evaporated and dried to get instant coffee. Decaffeination of tea and coffee may be done by using liquid-liquid extraction before drying. 2.2.2.5.2. Extraction of beet In the case of sugar beet extraction sucrose is present in the form of an aqueous solution (juice) in the cellular structure of the sugar beet the solvent within the solid matrix is identical to the external extraction medium. In all extraction systems, fresh cossettes are heated and subsequently extracted in a countercurrent of liquid and solid phases. The extraction equipment is fed at one end with fresh cossettes, and exhausted cossettes are removed at the other end. At this end, feed water is introduced and passes through the extractor countercurrent to the cossettes. An extraction plant is made up of (Figure 6).

Beet Beet

Beet slicing Cossettes Beet

Denaturation/scalding

Raw juice

Cossettes/juice mixture Countercurrent extraction

Fresh water

Exhausted cossettes Pulp pressing

Press water

Pressed pulp Figure 6 Block diagram of beet extraction process. (Pascal Christodoulou. 2003).

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2.2.2.5.3. Extraction of hops Figure 7 shows a flow diagram of a hop extraction plant using ethanol as solvent. Cone hops is fed to a double-deck Rotocel extractor with 16 compartments after being dried. The miscella drawn from the extractor is concentrated by passing a four-stage vacuum evaporator at gentle temperatures. Complete elimination of the alcohol is carried out in a posterior separation step. The spent hops discharged from the extractor is desolventized in a dryer and subsequently pelletized for animal feed application. Recovered ethanol-water mixture is adjusted to the desired composition by rectification and recycled. Compared to alternative CO 2 extraction, ethanol possesses little selectivity resulting in a product that contains almost the natural composition of extractables given by the feed material. 2.3. Safety Aspects Working with volatile and flammable solvents implies risks. During normal operation, a number of measures, such as the use of explosion-protected equipment, working at slight vacuum and continuous control of escaping gases by ignition detectors can for the most part guarantee safe handling. Thus, most accidents involving ignition of solvents or even explosions occur as a result of equipment failure. When the plant is shut down and vessels are opened for repair, strict safety guidelines might be missing and residual solvent vapors might come into contact with air, producing flammable mixtures. The U.S. National Fire Protection Association has formed a committee dedicated to safety of solvent extraction plants, which issued the following statement given in part: NFPA 36 Committee on Solvent Extraction Plants: from solvent recovery

hops

ethanol vacuum evaporation

wet milling

miscell

vapor

clarifying 40°C

to solvent recovery

ethanol +vapor 60°C

hot air

spent hops ethanol vapor to solvent recovery

80°C

60°C

Figure 7 Flow diagram of a hops extraction plant using ethanol as solvent. (Rudolf and Philip .2003) 15


Par. 5–8.3: Extractors, Desolventizers, Toasters, Dryers, Spent Flake Conveyers shall be of a design that minimizes the possibility of ignition of product deposits. Such equipment shall be protected by extinguishing systems using inert gas, steam, or a combination of the two, controlled from a safe remote location. Par. 5–8.1.7: The extractor shall be provided with means to remove solvent vapors so that the concentration of vapors inside the unit in the area where work is required can be maintained at or below 25% of the lower flammable limit, e.g., by a purge fan sized so that it changes the empty air volume of the vessel once every 3 min. 3. SUPERCRITICAL FLUID EXTRACTION There is an increasing public awareness of the health, environment and safety hazards associated with the use of organic solvents in food processing and the possible solvent contamination of the final products. The high cost of organic solvents and the increasingly stringent environmental regulations together with the new requirements of the medical and food industries for ultra-pure and high added value products have pointed out the need for the development of new and clean technologies for the processing of food products. Supercritical fluid extraction using carbon dioxide as a solvent has provided an excellent alternative to the use of chemical solvents. Supercritical fluid extraction (SFE) is the process of separating a mixture which is in solid state or in liquid state or in solid and liquid state by contacting it with a fluid maintained under conditions of temperature and pressure above its critical point. Significant milestones were reached with the establishment of the first commercial supercritical fluid process for decaffeination of green coffee beans, by Hag A.G. in Bremen, Germany, in 1978, and the liquid carbon dioxide process for extraction of hop flavours, by Carlton and United Breweries, (CUB) in Melbourne, Australia, in 1980. Both of these applications have been successful commercially, and have given rise to numerous variations and improvements which have also been developed to an industrial scale. Almost all food processing applications of supercritical fluid technology employ carbon dioxide as the solvent. Dense carbon dioxide is not only a powerful solvent for a wide range of compounds of interest in food processing, but it is also relatively inert, inexpensive, non toxic, non-flammable, recyclable, readily available in high purity, and leaves no residues. With a critical point at 31.1°C and 7.38 MPa, near-critical and supercritical carbon dioxide can be used at temperatures and pressures which are relatively safe, convenient and particularly appropriate for the extraction of a range of more volatile and/or heat-labile compounds. These properties are particularly significant when compared to the safety, toxicity and increasing expense of operation and regulatory constraints associated with some traditional food solvents, such as hexane and dichloromethane.

3.1. Major advantages of supercritical fluid extraction technique Because supercritical fluid extraction has several distinct properties, it is regarded as a promising alternative technique to conventional solvent extraction methods. Some of its major advantages are summarized as follows. l) Supercritical fluids (SFs) have relatively lower viscosity and higher diffusivity (the diffusivity for SFs is ~10_4 cm2 s_1 and for liquid solvents is ~10_5 cm2 s_1). Therefore, it can penetrate into 16


porous solid materials more effectively than liquid solvents and, consequently, it may render much faster mass transfer resulting in faster extractions. For instance, with comparable or better recoveries, the extraction time could be reduced from hours or days in a liquid–solid extraction to a few tens of minutes in SFE. 2) In SFE, a fresh fluid is continuously forced to flow through the samples; therefore, it can provide quantitative or complete extraction. 3) In SFE, the salvation power of the fluid can be manipulated by changing pressure (P) and/or temperature (T); therefore, it may achieve a remarkably high selectivity. This tunable solvation power of SFs is particularly useful for the extraction of complex samples such as plant materials. 4) Solutes dissolved in supercritical CO2 can be easily separated by depressurization. Therefore, SFE can eliminate the sample concentration process, which usually is time-consuming and often results in loss of volatile components. 5) SFE usually is performed at low temperatures, so it may be an ideal technique to study thermally labile compounds and may lead to the discovery of new natural compounds. For example, when SFE was used to extract ginger, many undesirable reactions such as hydrolysis, oxidation, degradation and rearrangement could be effectively prevented. Therefore, the difficulties for quality assessment in classical hydrodistillation could be avoided in SFE. 6) Compared with the 20–100 g of samples typically required in a liquid–solid methods, as little as 0.5–1.5 g of samples are needed in SFE methods. 7) SFE uses no or significantly less environmentally hostile organic solvents. A SFE method may need no or only a few milliliters of an organic solvent while in a liquid–solid extraction would require tens to hundreds of milliliters. 8) SFE may allow direct coupling with a chromatographic method, which can be a useful means to extract and directly quantify highly volatile compounds. 9) In large scale SFE processes, the fluid, usually CO2, can be recycled or reused thus minimizes waste generation. 10) SFE can be applied to systems of different scales, for instance, from analytical scale (less than a gram to a few grams of samples), to preparative scale (several hundred grams of samples), to pilot plant scale (kilograms of samples) and up to large industrial scale (tons of raw materials, such as SFE of coffee beans). In addition, there are no environmental risks and no fire hazards, when carbon dioxide is used as the solvent. 3.2. Extraction system A schematic diagram of a basic supercritical fluid extraction process is shown in Figure 8. As noted in the figure, the major equipment components are the extractor, separators, heat exchangers, pumps, and compressors. If desired, more than one extractor can be connected to the same system. The selection of equipment for a food processing system must ensure a sanitary and safe process. All surfaces, including seals and gaskets, in contact with process fluids and solids must be suitable for food processing and sterilization. Construction materials and fittings must be suitable for the pressure and temperature conditions required by the process. The solvent, i.e., CO2, is pumped into the system as a liquid or gas. If the solvent is pumped as a liquid, it will be cooled in a reservoir or it will be kept in liquid phase by cooling the pump head. Depending on the process requirements a system for entrainer addition can be incorporated into the system. The fluid is then pressurized and heated to the desired processing conditions. A solvent preheater can be installed into the system to avoid temperature fluctuations due to the solvent being pumped 17


into the system at a lower temperature. In the case of solid feed material processing, the matrix to be extracted will be packed into the extraction cell in a mesh basket or between frits to prevent its being carried from the cell during extraction. Following extraction, the pressure is reduced to precipitate the extract through a control valve. A high-pressure metering valve can control the flow rate of the fluid. The pressure of the system is controlled by the rate of pumping and a backpressure valve setting. A computer system can be used to control the extraction conditions. Choosing a continuous process rather than a batch or semicontinuous process may significantly improve the economics of an industrial scale process. Continuous transport of a large volume of solid feed, such as oilseeds, into and out of a high-pressure extractor is costly and difficult. However, advances in high-pressure technology may allow continuous feed of solid materials. Today an extractor design that allows intermittent loading and unloading of solid material through the lock-hopper vessels fitted below and above the extractor while it is pressurized is used in the coffee-decaffeinating plant in Houston. A portion of the solid is discharged to the bottom hopper, while fresh feed is simultaneously charged to the extractor from the top hopper.

Figure 8 Typical flow diagram for supercritical fluid extraction process. (Nurhan D. 2003). 1- CO2 tank controller 2- Valve 3- CO2 filter 4- Pressure regulator 5- Pressure gauge 6- Compressor 7- Ethanol reservoir

8- filter 9- ethanol pump 10- valve 11 extractor 12- extractor temperature controller 13- rupture disc 14- depressurization valve

15- temperature 16- cooling bath 17- collection tubes 18- silica trap 19- flow indicator 20- flow totalizer

In the meantime, SEF continuously passes through the extractor countercurrently relative to feed. Such an extractor design has several advantages. Sequencing these operations minimizes raw material feeding and vessel unloading times. The compression costs are lowered since feed loading and unloading are carried out simultaneously while maintaining pressure. Simulated 18


moving-bed technique has been developed in an effort to approach a countercurrent solid–fluid extraction process. In this method, a single fixed-bed column is subdivided into several zones. The apparent movement of the solid is achieved by switching between valves connected at the junctions between zones. The solid and extract withdrawal ports are periodically shifted in the direction of the solid movement and opposite to the fluid flow, thus simulating a countercurrent process. This technique has been applied to extraction and fractionation of tocopherol from oleic acid and fragrance and essential oil extraction. 3.3. The use of supercritical fluid extraction in food industry For the past three decades, the commercial application of supercritical fluid technology remained restricted to few products due to high investment costs and for being new and unfamiliar operation. With advances in process, equipment and product design and realization of the potentially profitable opportunities in the production of high added value products, industries are becoming more and more interested in supercritical fluid technology. The extraction is carried out in high-pressure equipment in batch or continuous manner. In both cases, the supercritical solvent is put in contact with the material from which a desirable product is to be separated. The supercritical solvent, now saturated with the extracted product, is expanded to atmospheric conditions and the solubilized product is recovered in the separation vessel permitting the recycle of the supercritical solvent for further use. Table 3 presents some of food-related applications of carbon dioxide extraction. Extraction with supercritical fluids is also a unit operation that could be employed for a variety of applications including the extraction and fractionation of edible fats and oils, purification of solid matrices, separation of tocopherols and other antioxidants, clean-up of herb medicines and food products from pesticides, detoxification of shellfish and concentration of fermentation broth, fruit juices, among others. Supercritical fluid extraction has proved effective in the separation of essential oils and its derivatives for use in the food, cosmetics, pharmaceutical and other related industries, producing high-quality essential oils with commercially more satisfactory compositions (lower monoterpenes) than obtained with conventional hydro-distillation. Alkaloids, organic compounds with bitter taste and toxic effects on animals and humans, but present therapeutic effects when applied in moderate doses, are found in many natural plants. Alkaloids such as caffeine, morphine, emetine, pilocarpine, among others, are the active components in a variety of stimulants and medicinal products and their recovery from natural plants is of great interest to the food, pharmaceutical, and cosmetic industries. Supercritical Carbon dioxide proved to be highly selective for caffeine prompting its use as the selected solvent in the commercial decaffeination of coffee and black tea. Recent investigations have demonstrated the potential exploration of solvent and anti-solvent properties of carbon dioxide in the recovery of alkaloids such as theophylline, theobromine and pilocarpine, among others. The association of high blood cholesterol levels with heart diseases or cancer is the motivating factor in recent works on the reduction of cholesterol levels in consumed meals that include meats, dairy products and eggs. Several methods including supercritical extraction have been proposed for the reduction of fat and cholesterol content in dairy products. Cholesterol was shown to be soluble in supercritical carbon dioxide and even more soluble in supercritical ethane. Extraction with supercritical fluids requires higher investment but can be highly selective and more suitable for food products. 19


Table 3. Examples of food extraction systems employing supercritical or near-critical carbon dioxide Process and food type Reference Process and food type 1- Extraction of fats and oils Fish Rice bran

Zosel (1978) Ramsey et al. (1991)

Soybean

List & Friedrich (1985)

Peanuts

Goodrum & Kilgo (1987)

Sunflower

Stahl et al. (1980)

Canola

Lee et al. (1986)

Rice, rice koji Beef

Taniguchi et al, (1987a) Chao et al. (1991)

Oats

Fors & Eriksson (1990)

Potato chips Corn germ Cottonseed

Hannigan (1981) List et al. (1984a) List et ul. (1984b)

5. Flavour/aroma extraction Hops Ambrette, Angelica, Aniseed, Anise Basil, Capsicum, Caraway, Cardamom, Carob, Carrot, Cassia, Celery, Cinnamon, Clove, Cocoa, Coffee, Coriander. Cumin. Cubeb, Fennel, Fenugreek, Ginger, ’ Juniper, Lovage, Mace, Marjoram, Nutmeg, Oregano, Parsley, Pepper, Pimento Rosemary, Sandalwood, Sage, Savoury, Thyme, Turmeric, Vanilla, Vetiver Onion Dried botino (Katsuobushi)

Egg yolk Krill Wheat germ Mustard Olive Cocoa Yeast extract

Froning et al. (1990) Yamaguchi et al. (1986) Taniguchi et al, (1985) Taniguchi et al. (1987b) Giovacchino et al. (1989) Rossi et al. (1989) Duwe et al. (1986)

Apple Passion fruit Wine Dried mushroom Citrus fruits Cheese Elderberry

Reference

Gardner ( 1993)

Moyler ( 1993) Sinha et al. (1992) Hamatani & Takahashi (1988) Bundschuh et al. (1988) Raasch & Knorr (1991) Camacini et al. (1989) Valle & Aguilera (1989) Temelli et al. (1988) Gmuer et al. (1986) Eberhardt & Pfannhauser (1985) 20


Biscuit wastes 2. Cholesterol extraction Beef Milkfat Dried egg yolk Fish 3. Fractionation of fats and oils Milkfat Rice bran oil Citrus oils Fatty acid esters Canola oil 4. Refining of fats and oils Deodorisation of soybean, palm kernel and peanut oils Deacidification of olive oil

Nakazono (1987)

Caraway, wormwood Lactones from milk fat Chao et al. (1991) Peppermint oil (Mohamed, et al., 2000) 6. Miscellaneous extractions (Bohac, 1998) Bixin pigment from annatto seeds Hardardottir & Kinsella ß-Carotene, lutein from leaf (1988) extracts Pesticides from contaminated senna leaves Arul et al. (1987) Antioxidants from sage extracts Saito et al. (1991) Antioxidants from Labiatae herbs Temelli et al. (1988) Fractionation of glucose/fructose mixtures Liong et al. (1992) Inactivation of orange juice pectinesterase Temelli ( 1992) Off-flavours from textured vegetable proteins Debittering of citrus juices Ziegler & Liaw (1993) Decaffeination of coffee, tea Zosel (1979) Brunetti et al. (1989)

Stahl et al. (1984) Rizvi et al. (1993) Goto et al. (1993) Degnan et al. (1991) Favati et al. (1988) Stahl et al. (1984) Djarmati et al. (1991) Nguyen et al. (1991) Bracey et al. (1991) Balaban et al. (1991) Sevenants (1987) Kimball (1987) Lack & Seidlitz (1993)

Concentration of alcohol and other Saito et al. (1987) fermentation products

De-oiling of soybean lecithin Heigel & Hueschens (1983) Deodorisation of rosemary extract Muehlnikel (1992) De-oiling of bleaching clays Waldmann & Eggers (1992) Extraction of tocopherols from Lee et al. (1991) soybean sludge Reconstructed from: Palmer and Ting, 1995

21


As ethane is much more expensive than CO2, the use of CO2/ethane and CO2/propane mixtures can be an attractive alternative for the removal of cholesterol from foods due to the compromise between higher ethane cost and better cholesterol removal efficiency. Cholesterol removal was also improved through the coupling of carbon dioxide extraction with an adsorption process operating at the same extraction conditions. The carbon dioxide extraction has also proved effective for the production of high quality cocoa butter from cocoa beans. Recent investigation point to the potential use of supercritical CO2 for microbial inactivation of foods and the implementation of an innovative technique for the sterilization of thermally and pressure sensitive materials. Supercritical fluid extraction was used for protein purification through the fractional precipitation of proteinalkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin and their mixtures from dimethylsulphoxide. Other investigations focused on coatings, semi-conductors and pharmaceuticals. More recently this technique has been employed for the encapsulation of micron size particles and the selective precipitation of products from reaction media. Finally it is important to mention that supercritical fluids are known to provide good reaction media due to their capacity to homogenize a reaction mixture, high diffusivity and controlled phase separations and distribution of products 3.4. Applications to food industry The last decade has seen a rapid growth in research activity in the area of SEF extraction. Although many glowing reviews and hundreds of publications have appeared covering a wide variety of applications, there are surprisingly few processes and plant operating on a commercial scale. It is possible that many of the early publications overstressed the potential advantages of SFs without addressing the limitations. It is certainly the case that many reports of applications do not differentiate between the extraction/identification of trace amounts of components and realistic quantities on which a process could be based. Without the necessary data it is often difficult or impossible to quantify the efficiency of a reported SEF extraction process in terms of mass transfer and throughput of Co 2. Against this background it is not surprising that many popular misconceptions abound regarding SFs. SEF extraction is an expensive process and should not be used simply because it is a ‘novel’ technique. Unless use is being made of its unique features there is no rationale for its implementation if a cheaper separation process can meet the requirements of the separation as effectively. The one clear advantage that Co 2 does offer for food applications is its lack of toxicity. In the current climate of growing consumer concern regarding food safety this feature will undoubtedly promote its use in the food industry. 3.4.1. Pepper extraction Supercritical fluid extraction is the most versatile separation technology now being employed. It has high extraction selectivity from a mixture of components because of the pressure-temperature dependent solubility in the solvents. The pepper raw material is loaded into the extractor and brought into contact with the supercritical solvent at relatively high pressures of 80– 350 bar, at temperatures of 35–70ºC. The solute mixes into the supercritical solvent and both are passed through a pressure-reducing valve. The pressure on the separator side is about 40–60 bar, while the temperature is lower due to sudden expansion of the supercritical solvent. These conditions lower the solubility of the pepper raw materials in the solvent. When the material 22


starts to separate, the gas is again compressed back to extract the material. Solvent recycling is achieved by means of a compressor. Supercritical CO2 is an ideal solvent for extraction of pepper, because it is cheap, abundant, inert, non-toxic, non-corrosive, non-inflammable and does not pollute the environment. Separation can be carried out at low temperature, residual solvent content can be reduced to near zero, solubility variation of active constituents can be easily manipulated, fractions can be extracted easily, the process consumes little energy, transfer rates are high and there are no fire hazards. Pepper extraction has been very successful with about 98% extraction of piperine and 81% of essential oil. The quality of the product is high compared to conventional extraction process. The extracted oleoresin is also used for the separation of piperine by centrifuging the oleoresin in a basket centrifuge. From the oleoresin numerous secondary products have been developed having specified flavour strength and other properties. Such products include seasonings, emulsions, solubilised spices, dry soluble spices, encapsulated spices, heat resistant spices, fat based spices, etc. 3.4.2. Seed oil extraction Extraction and processing of seed oils is a large-scale commercial operation with high throughput. The type of oilseeds processed depends almost entirely on regional agricultural policies; in the USA soya oil is by far the largest commodity, whereas in Canada rapeseed oil is more common. However, seed oils all contain the same basic triglyceride units, though the distribution of individual carboxylic acids in the triglycerides is a unique feature of each oil which imparts individual characteristics. The overall solubility of seed oils in SEF Co 2 does not appear to vary much and basic principles and conditions established for the extraction of one oil translate reasonably well to another. In the conventional process for oilseed extraction the pretreated seeds are extracted using hexane. This also removes phospholipids (lecithin) which, although beneficial to health, present physical problems when the oil is used for cooking. In the refining process a degumming stage is therefore required to remove the phospholipids from the oil which is then bleached (to reduce colour) and deodorised. Most of the pioneering work on the SEF Co2 extraction of seed oils has been carried out in the USA and West Germany. The solubility of soya oil in SEF Co 2 has been measured at high pressure (Friedrich et al., 1982). The most important feature of Friedrich’s measurements was that at high pressures and temperatures (800 bar, 70°C) the oil became completely miscible with Co 2. This suggested the possibility of an efficient high-pressure SEF extraction process. Under these extreme conditions the rate of extraction from soya flake was found to be rapid, and nearly complete extraction could be achieved in 20 min. Moreover, since the solubility falls markedly at lower pressures, most of the oil could be separated from the Co 2 stream without having to undergo complete decompression. Probably the most significant feature of the SEF extraction of oilseeds is that phospholipids are not co-extracted. This eliminates the need for chemical degumming of the oil. If the phospholipids are required as a separate commodity they can be extracted from the seeds in a secondary extraction with a solvent such as hexane. Oil extracted with SEF Co 2 is also often reported as having a lighter colour. Although showing many advantages over conventional processing techniques SEF extraction is not currently economically viable for large-scale oilseed extraction. This is due to the low bulk value of seed oils, high plant cost and inconvenience of batch processing large quantities of solid materials under high-pressure conditions. There has been much interest and 23


speculation concerning the development of systems for continuously feeding solids into highpressure vessels. Implementation of SEF extraction of bulk commodity seed oils will probably have to await the development of continuous processing plant or the tightening of legislation governing the use of petrochemical solvents. SEF extraction is, however, appropriate for smallerscale processes involving high-value oils such as evening primrose and wheatgerm for which there is a growing market in the health sector. 3.4.3. Extraction of antioxidants with supercritical fluid carbon dioxide A modern method is extraction with gases, usually carbon dioxide, under supercritical conditions. The method and its application for fats and oils were reviewed by King and List. Propane/butane, methanol, ethanol and other substances may be used as co-solvents, improving yield or selectivity. Extraction with carbon dioxide is relatively selective, generally better than that of organic solvents. This treatment was proposed for removal of volatiles, preceding extraction with organic solvents (both polar and non-polar). The safety aspect should be considered because of the high pressure used although carbon dioxide, being a gas at atmospheric pressure, is easily removed so that solvent residues present no risk factor. A big disadvantage of supercritical extraction is the high operation pressure, which requires expensive equipment. Several suitable devices have been proposed and critical reviews are available. The cost of the process is high making it unsuitable for the extraction of main food components, such as lipids. Antioxidants are, of course, a more expensive group of food preparations so that price would not play a crucial role if it is compensated by other advantages, such as high purity of extracts and great efficiency of the process. The application of supercritical solvent extraction to the preparation of natural antioxidants has, until now, been limited. A few examples of application of supercritical carbon dioxide to lipid and oilseed extraction are reviewed. The procedure was used for the extraction of rosemary and sage leaves. Phenolic substances can be removed from sunflower extracted meals using supercritical fluid extraction with carbon dioxide. Phenolics may be used as natural antioxidants and the residual protein possesses a higher nutritional value. It can be hoped that applications will be more frequent in the near future, when the procedure becomes better investigated and the equipment becomes cheaper. 3.4.4. Lowering cholesterol levels in foods Although the correlation between dietary intake and levels of cholesterol in the blood is not universally accepted, initial suspicions have led to strong consumer aversion to high cholesterol foods. This has been compounded by advertising campaigns claiming low levels of cholesterol, even in products that would not be expected to contain it. On solubility grounds there would appear to be a good chance of selectively extracting cholesterol from oils and fats using SFE. It is clear that the solubility of cholesterol is significantly greater than that of triglyceride oils.

24


Figure 9. Process for supercritical CO2 extraction of cholesterol from butter oil. (Tiwari. 1995) Krukonis (1988) has tested the feasibility of removing cholesterol from butter, egg yolk and beef tallow by measuring the partition coefficients of the individual components in SFE. Selectivity at 60°C and 150 bar was found to be greatest for egg yolk and least for butter. This trend is in accord with the relative solubilities of the component oils in SFE. In these trials 90% removal of the cholesterol from butter was reported with an overall yield of 70% of cholesterolreduced product. Studies on the effect of extraction conditions upon the composition of SEF CO 2 extracted egg yolk powder are in broad agreement with available solubility data. The process for cholesterol-free dairy products involves separation of fats from the milk in a centrifugal separator. The cholesterol is then removed from the fats by extraction with supercritical carbon dioxide. Use of methanol as entrainer increases the solubility of cholesterol in fluid phase by an order of magnitude. The cholesterol-free fats are reblended into the milk by conventional methods. A typical plant for supercritical fluid extraction of cholesterol from butter oil is shown in Figure 9. Butter oil is fed into the extraction column between two packed sections. The supercritical carbon dioxide at 40°C and 175 bar is fed at the bottom of the column for countercurrent extraction. The cholesterol-laden carbon dioxide flowing up through the top packed section is contacted with the cholesterol-rich extract which is refluxed at the top. The cholesterol-rich extract is obtained as the top product and low-cholesterol butter oil is obtained as the bottom product. The carbon dioxide is recycled from the separator to the bottom of the column. Conditions in the separator are subcritical for carbon dioxide and require the carbon dioxide to be recompressed with make up carbon dioxide for reuse in the process. In case entrainer is used for the separation, it is fed with the butter oil. Two columns may be used in place of two packed sections in one column. Some plants use sieve plate columns for the extraction process.

25


4. AQUEOUS TWO-PHASE EXTRACTION Biomaterials derived from or produced by the plants, animals or the micro-organism mainly consist of proteins, fats and carbohydrates. Production of biomaterials on a commercial scale involves cell disruption followed by separation of the desired products. The cost of the separation step may be as high as 90% of the total cost of the production. For the separation of enzymes and proteins, aqueous two-phase extraction process has become a versatile and efficient method. The method can be used for the removal of cell debris as well as further purification of the biomolecules. Although, not of current interest, the developments in the field of biotechnology for separation using aqueous two-phase extraction will be of importance for macromolecules of interest to the food industry. The advantages of the aqueous two-phase extraction process (ATPE) are: (1) biocompatibility, (2) easy processing, (3) high capacity, (4) easy and precise scale-up, (5) high product yields, (6) high potential for continuous processing, and (7) low investment cost. The aqueous two-phase systems have extreme physical properties compared to the conventional Hquid-hquid extraction systems. The interfacial tension is in the range of 10 -3-10 mN/m; phase viscosities are usually 1-103 mPa.s; and density differences between phases are low, ranging from 20-100 kg/m3. The performance of extraction equipment for the aqueous two-phase systems is markedly different from that of conventional equipment. Aqueous Two-Phase Extraction Also called aqueous biphasic extraction, this technique generally involves use of two incompatible water-miscible polymers [normally polyethylene glycol (PEG) and dextran, a starch-based polymer], or a water-miscible polymer and a salt (such as PEG and Na2SO4), to form two immiscible aqueous phases each containing 75+% water. This technology provides mild conditions for recovery of proteins and other biomolecules from broth or other aqueous feeds with minimal loss of activity .The effect of salts on the liquid-liquid phase equilibrium of polyethylene glycol + water mixtures has been extensively studied .

Figure 10 Equilibrium phase diagram for PEG 6000 + Na2SO4 + water at 25°C. (Timothy C. Frank, et.al. 2008 ) 26


A typical phase diagram, for PEG 6000 + Na2SO4 + water, is shown in Figure 10. The hydraulic characteristics of the aqueous two-phase system PEG 4000 + Na2SO4 + water in a countercurrent sieve plate column have been reported. Two immiscible aqueous phases also may be formed by using two incompatible salts. An example is the system formed by using the hydrophilic organic salt 1-butyl-3-methylimidazolium chloride and a water-structuring (kosmotropic) salt such as K3PO4. 4.1. Protein partitioning Protein partitioning in ATPE depends on many factors such as phase polymers, the ionic composition and the partitioned substance. The type of polymer as well as their molecular weights and the presence of certain chemical groups influence the partitioning of proteins. The ionic composition is of vital importance as the sign and magnitude of the interfacial electric potential are determined by the ions present. The properties such as size, charge and biospecific surface properties, presence of receptors and biospecific ligands and chirality govern partitioning. The partitioning of a protein between the top and bottom phases is defined by the partition coefficient (m) and is the ratio of concentrations in the top and bottom phases. Increased polymer concentration shifts the phase system away from the critical point and the physical properties of the coexisting phases become more different. The partitioning of the protein becomes more favourable. However, cell organelles adsorb more strongly and selectively to the interface when polymer concentration increases. Molecular weight of the polymer affects the partitioning of high molecular weight proteins. The higher the molecular weight of the polymer, the lower will be the partition coefficient. Salts have a paramount effect in the partitioning of all kinds of molecules and cell particles. Salts with different ions have different affinities for the two phases. An electric potential is created between the phases. A salt with two ions that have different affinities for the two phases will generate larger potential difference. The isoelectric point of proteins can be determined by a cross-partition and partition coefficient is determined as a function of pH with two different salts. Presence of a charged polymer in one of the phases has a stronger effect on the partitioning of charged macromolecules than that of the salts. Hydrophobic groups such as palmitate bound to polyethylene glycol (PEG) show increased affinity for proteins with hydrophobic binding and cause protein to be partitioned in the PEG-rich phase. Affinity ligands attached to one of the polymers can be used to extract ligand-binding proteins and nucleic acids into the corresponding phase. The composition of the two phases changes with temperature. Proteins partition more equally between phases of a two-polymer system when temperature is increased. This effect may be counteracted by using higher concentrations of polymer. 4.2. Aqueous two-phase extraction for purification and concentration of betalains Partitioning behavior of the betalains in ATPE is an important criterion for the purification. The partitioning of the betalains is influenced by large number of factors such as tie line length, phase volume ratio and concentration of neutral salt (NaCl). Polyethylene glycol (PEG) (MW 6000) / ammonium sulphate was found to be the most suitable system for the purification of betalains. Differential partitioning of betalains and sugars was achieved in aqueous two phase extraction at a higher tie line (34%), wherein 70– 75% of betalains partitions to the top phase and 80–90% of sugars present in the beet partitions to the 27


bottom phase, thus purifying the betalains. The polymer (PEG) was separated from betalains, by organic aqueous extraction and the polymer obtained can be recycled for subsequent extraction. Beet extract was prepared as shown in the flow diagram (Figure 11). Predetermined quantities of PEG 6000 and ammonium sulphate were weighed and added to crude beet extract to make the total weight of the system 100% (w/w). The contents were mixed thoroughly using a magnetic stirrer for equilibration and were allowed for phase separation for 4–5 h. After the separation of two phases, the volumes of top and bottom phases of the system were noted and analyzed for betalain as well as sugar content. All the experiments were carried out at 25 ± 1 °C and the pH of the system was in the range of 5–5.5. In this pH range, the betalains are more stable 5. LIQUID MEMBRANE EXTRACTION One way of achieving both a large distribution coefficient and a reduction in the solvent duty was proposed and patented by Norman Li in 1968. The main feature of this new separation technique, called the liquid membrane process, was that it was a three-phase system consisting of two phases of a similar nature but different composition (aqueous–aqueous, organic–organic, gas–gas) separated by a third phase of a different nature and as insoluble as possible into the other two. The middle phase is the liquid membrane. Figure 12.a shows the first configuration presented by Li, the single-drop liquid membrane, in which the membrane is formed by coating liquid drops or bubbles with a liquid film layer and by subsequently dispersing the resulting particles into a continuous liquid phase containing a solute. This configuration is now only of historical importance. There are two other configurations that have been more widely investigated due to their potential industrial application: (1) the emulsion liquid membrane, also called the surfactant liquid membrane; and (2) the supported liquid membrane. In the emulsion liquid membrane configuration, the liquid membrane is formed by dispersing into the feed (phase 1) an emulsion of the stripping phase (phase 3) in an organic phase (phase 2) containing an emulsifying agent. This configuration is shown in Figure 12.b. Here the liquid membrane is the continuous phase of the emulsion and the viability of the process depends primarily on the stability of the emulsion. In the supported liquid membrane process, the liquid membrane phase impregnates a microporous solid support placed between the two bulk phases (Figure 12.c). The liquid membrane is stabilized by capillary forces making unnecessary the addition of stabilizers to the membrane phase. Two types of support configurations are used: hollow fiber or flat sheet membrane modules. The formulation of the three phases must be such that the liquid membrane extracts the solute from one of the phases and the third phase strips it from the membrane. Thus extraction and stripping take place in the same contactor, and the stripping phase is where the solute is accumulated, instead of the organic phase as in the case of conventional solvent extraction. This allows for a middle phase of small volume that, being thin, behaves like a membrane. The main advantage of this process becomes clear when applying Eq. (5.1) to the threephase system. (5-1) D M = [M]T,org / [M]T,aq where DM is the solute distribution ratio and [M]T is the sum of the concentrations of all Mspecies in a given phase, and the second subscript indicates the organic and the aqueous phase.

28


Fresh Beetroots

Washing, peeling, slicing, and grinding

Aqueous Extraction

Centrifugation

Beet extract stored at 4°C

ATPE

Top phase rich in Betalains

Bottom phase containing sugars

Organic-Aqueous phase extraction

Betalains

BEG

As the liquid membrane phase does not accumulate the solute, the distribution ratio that is Lyophilization

Betalains powder

Figure11. Flow chart for the differential partitioning of betalains. 29


relevant to the efficiency of this process is that between phases 1 and 3. At equilibrium, this equation applies to both pairs of phases: 2–1 and 3–2: (5.2) D M 2-1 = [M]2 / [M]1 (5.3) D M 3-2 = [M]3 / [M]2 The distribution ratio between phases 3 and 1 is then given by: (5.4) D M 3-1 = [M]3 / [M]1 = DM 2-1 * DM 3-2 As the distribution ratio between phases 1 and 3 is the product of those in the two pairs of fluids, the potential effectiveness of the liquid membrane process is considerably greater than that of conventional solvent extraction. Thus the liquid membrane process is particularly suitable for the treatment of dilute feeds. In addition, if the liquid membrane is an organic phase, its small volume reduces the solvent duty considerably.

Figure 12 Liquid membrane configurations: (a) Single-drop liquid membrane; (b) emulsion globule; (c) supported liquid membrane. (Susana and David. 2004) 5.1. Mechanisms of solute transfer in liquid membranes As in most membranes, the liquid membrane must have selective permeability to specific solutes. The overall mechanism of solute transfer consists of three steps: (1) extraction of the solute into the liquid membrane; (2) diffusion of the extracted species through the membrane; and (3) stripping into the third phase. As in conventional solvent extraction, solute transfer into the membrane can be achieved by selective solubility or by chemical reaction with a component in the liquid membrane. Stripping at the interface of the liquid membrane with the third phase can be equally achieved by either physical or chemical mechanisms. The mechanism of solute separation requires analysis as it affects the conditions that control the overall rate of transfer between the first and the third phase. Application of Eq. (5.1) to the liquid membrane process highlights one of the main advantages of the process, i.e., the high solute distribution coefficient that can be obtained between phases 3 and 1. However, another factor that must be considered when evaluating a separation process performance is the kinetics of transfer. The two main mechanisms of solute separation by liquid membranes involve chemical reactions. In the case 30


illustrated in Figure 13.a, the solute first dissolves in the liquid membrane, then diffuses toward phase 3 due to the buildup of a concentration gradient, and finally transfers to phase 3 at the second interface.

A

AB

B A

(a)

B C A A AB C (b) Figure 13 Basic mechanisms of liquid membrane extraction: (a) type I facilitated transport (A +B→AB); (b) type II facilitated transport (A + B ↔ B+C). Phase 3 contains a reagent that reacts irreversibly with the solute to form products that are insoluble in the membrane, and therefore incapable of diffusing back through the membrane, thus maintaining the solute gradient in the membrane. Ammonia, sulfidric acid, and phenol are among the various compounds that have been successfully removed using H 2SO4 and NaOH as reactants in phase 3. A major disadvantage associated with this mechanism is the difficulty of achieving selective separations of solutes of similar size and chemical properties, as membranes are usually permeable to all of them. The other type of chemical mechanism is more selective and is used when the solute is not soluble in the membrane phase, therefore requiring the addition of a selective reactant into the membrane to form a complex or an ion pair with the solute. The reaction product then diffuses across the membrane and at the second interface it reacts with a 31


species added to phase 3 so that stripping also takes place by chemical reaction (Figure 13.b). This mechanism is called carrier-mediated membrane transfer. The reagent recovered from the reversed reaction then transfers back to the extraction interface. This is usually called the reagent shuttle mechanism. A typical application of carrier-mediated transfer is the recovery of metal cations from aqueous phases. The overall reactions involved in the extraction and stripping stages can be represented by the following reversible reaction: (5.5) Mn+(aq) + n.RH(org) ↔ RnM(org)+ n.H+(aq) where Mn+ is a metal cation of valence n, RH is an oil-soluble liquid ion-exchange reagent, and RnM is the metal complex. In this example, the forward reaction takes place at the interface between phase 1 and the membrane, and the reverse reaction at the other membrane interface. Equation (5.5) provides the guidance for the formulation of both the liquid membrane and the stripping phase, as for a given concentration of metal ion in the feed a high concentration of reactant favors the forward reaction, whereas a high complex concentration and a low pH facilitate the reverse reaction. The latter indicates that one of the conditions required in order to improve the flux through the membrane is that the pH of phase 3 must be substantially lower than that of phase 1. 5.2. Emulsion liquid membrane process The emulsion liquid membrane (Figure 12b) is a modification of the single drop membrane configuration presented by Li in order to improve the stability of the membrane and to increase the interfacial area. The membrane phase contains surfactants or other additives that stabilize the emulsion. Depending on the mechanism of extraction, the liquid membrane may also contain a carrier that reacts with the solute; the internal phase of the emulsion is the stripping phase and must be formulated accordingly. In this configuration, the liquid membrane is the continuous phase of the emulsion, and the extent of the interface between the feed and the liquid membrane depends on the size of the emulsion globules dispersed into the feed, which in turn depends on the physical properties of the phases and the mode and intensity of the mixing. The interfacial area on both sides of the liquid membrane is relevant to the rate of extraction. However, both the size of the emulsion globules dispersed in a stirred contactor, which provides the extraction interfacial area, and the size of the emulsion droplets that leads to the calculation of the extent of the stripping interface are difficult to measure. Reported emulsion globule sizes measured in stirred tanks are of the order of 0.2–0.4mm, whereas emulsion droplet sizes are in the range of 1–10 mm. Figure 14 illustrates schematically the different stages of a continuous separation process using the emulsion liquid membrane. There are four main stages in the flow sheet: (1) emulsification of the stripping phase with the liquid membrane phase; (2) dispersion of the emulsion into the feed; (3) separation of the emulsion from the raffinate phase; and (4) demulsification. This final stage separates the stripping solution that contains the species extracted from the feed, from the liquid membrane phase, which is recycled to the emulsification stage. In common with the supported liquid membrane, the emulsion liquid membrane yields a solute partition coefficient of a higher order of magnitude than that obtained with the conventional solvent extraction process, thus allowing a high separation percentage from dilute feeds, and concentrated stripping solutions in just one contact stage. However, the process has its disadvantages; one is that the

32


Organic phase + surfactant

Feed

(Stripping Agent) Emulsification

Demulsification Figure 14 Emulsion liquid membrane process. (Susana and David. 2004). need to produce a stable emulsion requires the use of additives that slow down the rate of extraction and, even if their solubility is negligible, they may contaminate the raffinate. Another disadvantage is the problem posed by emulsion rupture in the contactor. Emulsion rupture is usually due to emulsion swelling caused by the transport of the external phase into the emulsion. Three different mechanisms have been identified as causes of emulsion swelling: (1) occlusion due to entrainment of the external phase; (2) secondary emulsification of the external phase caused by an excess of surfactant in the liquid membrane phase; and (3) external phase permeation through the liquid membrane. The latter includes osmosis and, in the case of external aqueous feed, the transport of hydration water attached to complexes and water transport due to the presence of reverse micelles in the organic membrane. Swelling and emulsion rupture can be greatly decreased by including additives and especially designed components to the membrane phase. 5.3 Supported liquid membrane As shown in Figure 12.c, the supported liquid membrane consists of a microporous solid support impregnated with the membrane phase and placed between the two bulk phases. In this case, the interfacial area and the thickness of the liquid membrane can be selected by choosing the solid membrane porosity, size, and thickness. The main advantages of supported liquid membranes over emulsion liquid membranes are their well defined and easily measurable interfacial mass transfer area and membrane thickness, and the absence of surfactant additives that in general reduce the membrane flux and may contaminate the bulk phases. The main disadvantages are the difficulty in controlling the pressure on both sides of the membrane in order to avoid blowing the membrane out of the support, and the washing of the membrane from the support caused by shear forces. The former leads to contamination of the separated phases, and the latter to the need to reimpregnate the membrane. 33


5.4. Extraction equipment The extraction equipment for contact of emulsion phase and the feed phase can be a continuous contactor or a battery of mixer-settlers. A recycle arrangement for the oil phase is necessary to conserve the oil and the carrier after the oil phase is separated in a demulsification unit. 6. NONDISPERSIVE SOLVENT EXTRACTION Nondispersive solvent extraction is a novel configuration of the conventional solvent extraction process. The term nondispersive solvent extraction arises from the fact that instead of producing a drop dispersion of one phase in the other, the phases are contacted using porous membrane modules. The module membrane separates two of the immiscible phases, one of which impregnates the membrane, thus bringing the liquid–liquid interface to one side of the membrane. This process differs from the supported liquid membrane in that the liquid impregnating the membrane is also the bulk phase at one side of the porous membrane, thus reducing the number of liquid–liquid interfaces between the bulk phases to just one. There are two different arrangements for the process. One uses two modules: one for extraction and the other for stripping, making it formally closer to conventional solvent extraction. The other configuration is closer to the liquid membrane process, as the three phases flow through the same module: the liquid membrane phase in the shell, and the feed and the stripping phase through the lumen of different fibers in the module. Therefore, this is a three-liquid phase system and although the liquid membrane may not be as thin as in the emulsion or supported liquid membrane configurations, extraction and stripping take place simultaneously in the same contactor, thus keeping the thermodynamic advantages of the three-phase system. The main benefits of nondispersive solvent extraction over the conventional process are: (1) it avoids the need of a settling stage for phase disengagement and the consequent risk of dispersed phase carryover; (2) the value of the interfacial area per unit volume can be much higher than in a liquid–liquid dispersion as there is no risk of phase inversion; and (3) the interfacial area is easily calculated and scale-up of the process is straightforward. 7. REVERSE MICELLE EXTRACTION This scheme involves use of microscopic water-in-oil micelles formed by surfactants and suspended within a hydrophobic organic solvent to isolate proteins from an aqueous feed. The micelles essentially are microdroplets of water having dimensions on the order of the protein to be isolated. These stabilized water droplets provide a compatible environment for the protein, allowing its recovery from a crude aqueous feed without significant loss of protein activity. The main reason for the interest in W/O is that the presence of the aqueous microphase in the extracting phase may enhance the extraction of hydrophilic solutes by solubilizing them in the reverse micellar cores. However, this is not always the case and it seems to vary with the characteristics of the system and the type of solute. Furthermore, in many instances the mechanism of extraction enhancement is not simply solubilization into the reverse micellar cores. Four solubilization sites are possible in a reverse micelle, as illustrated in Figure 15. An important point is that the term solubilization does not apply only to solute transfer into the reverse micelle cores, but also to insertion into the micellar boundary region called the palisade. 34


1

Surfactant

1 4 Solubilzate 3 4

3

1. Surface 2. Pallisade 3. Deep palisade

2 4. Core 2 Figure 15 Possible solubilizate locations in a micelle. (Susana and David. 2004). 7.1. Micellar extraction potential applications 7.1.1. Extraction from synthetic mixtures The ease that certain protein mixtures can be separated using reverse micelle extraction was clearly demonstrated by Jarudilokkul et al., who investigated a series of binary and ternary protein mixtures. In two cases, they were able to quantitatively extract cytochrome c and lysozyme from a ternary mixture of these proteins with ribonuclease A. The separation of a mixture of ribonuclease A and concanavalin A showed that the system behaved ideally and that there was no interaction between the proteins. 7.1.2. Extraction of extracellular enzymes Rahaman et al. demonstrated the use of reverse micelles (AOT/ isooctane) for the recovery of an extracellular alkaline protease (MW, 33k Dalton; pI, 10) from a whole fermentation broth. Purification factors as high as 6 and yields of 56% were achieved in a threestage cascade. The combination of a cascade with a higher aqueous/organic ratio, and the use of true cross-flow designs, shows promise for purification without dilution. The application of the reverse micelle technique by extracting an α-amylase broth of Bacillus licheniformis using a CTAB/isooctane/5% octanol reverse micelle system has also demonstrated. Jarudilokkul extracted lysozyme from egg white, and while extractions as high as 98% were achievable, a variety of demulsifiers added to the mixture could actually enhance yields substantially. 7.1.3. Extraction of intracellular enzymes Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water 35


pool of surfactant aggregates. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 8. A PRESPECTIVE ON THE FUTURE Liquid-liquid extraction as a separation process is less energy intensive compared to other processes. By selecting a suitable solvent having high selectivity and distribution coefficient for the solute, the energy requirement in the separation step of the extraction process may be significantly reduced. This is of paramount importance in view of the ever increasing energy cost. There is an increasing public awareness of the health, environment and safety hazards associated with the use of organic solvents in food processing and the possible solvent contamination of the final products. The high cost of organic solvents and the increasingly stringent environmental regulations together with the new requirements of the medical and food industries for ultra-pure and high added value products have pointed out the need for the development of new and clean technologies for the processing of food products. In view of the above, the extractors have to be designed on the basis of experimental data for specific systems. Multistage extractors with reduced backmixing will be of interest. The mixed solvents and specially designed solvents will find more use in future. Supercritical fluids are known to provide good reaction media due to their capacity to homogenize a reaction mixture, high diffusivity and controlled phase separations and distribution of products. For the past three decades, the commercial application of supercritical fluid technology remained restricted to few products due to high investment costs and for being new and unfamiliar operation. Future technology should permit reduced cost of equipment and lower pressure operations. Aqueous two-phase extraction (ATPE) has been recognized as a superior and versatile technique for the downstream processing of biomolecules. ATPE has potential to achieve the desired purification and concentration of the product in a single step. Flavouring substances, dipeptides and nucleotides from acid hydrolysis in food industry can be easily handled with ATPE. The use of ATPE in isolation/purification of such compounds will be of interest in future. Liquid membrane extraction as a novel separation techniques discussed in this chapter offer some advantages over conventional solvent extraction for particular types of feed, such as dilute solutions and the separation of biomolecules. Some of them, such as the emulsion liquid membrane and reverse micellar extraction, have been investigated at pilot plant scale and have shown good potential for industrial application. However, despite their advantages, many industries are slow to take up novel approaches to solvent extraction unless substantial economic advantages can be gained. Nevertheless, in the future it is probable that some of these techniques will be taken up at full scale in industry. Areas for which new technology could be beneficial include, among others, development of extractants that can be readily incinerated; detailed information concerning the kinetics of extraction of various solutes; and perhaps, development of contactors with very short residence times. Extraction kinetics must be more carefully investigated in the future to be able to take advantage of kinetic differences.

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