Separation Strategies for Isoprenoids from Aqueous Solutions - Term Paper Example

?Separation Strategies for Isoprenoids from Aqueous Solutions Executive summary Isoprenoids are a of biomolecules of great interest given theirbiological activity. Thus several separation techniques have been developed to isolate them from their natural sources, which in most case are aqueous medium. These techniques include conventional ones such as the distillation techniques, chromatographic techniques, crystallization and solvent extraction, and an emerging separation technique that is based on bioaccumulation of isoprenoids by metabolically engineered microorganisms. The simplicity, cost and energy efficiencies as well as the separation capability of solvent extraction, make it the best among these techniques. Introduction Found in all classes of living organisms, isoprenoids are the largest and a diverse group of biomolecules. Also known as terpenoid, isoprenoids are derived from five-carbon isoprene units (2-methyl-1,3-butadiene) assembled and modified in thousands of ways (Encyclop?dia Britannica). In isoprenoids, two to thousands of the isoprene units, through one or neither of its double bonds, are linked into larger molecules to form linear or ring structures. As biomolecules, isoprenoids play a wide variety of roles in plant and animal physiological processes and as intermediates in the biological synthesis of other important biomolecules. The flavors, fragrances of essential oils and other plant-derived substances are due to these molecules. Geraniol, an isoprenoid, is a contributor to the fragrance of rose perfume. These molecules are also extracted from plants or chemically synthesized to be used as pharmaceuticals (e.g. taxol, bisabolol, lycopene, artemisinin), animal feed supplements and food colorants (various carotenoids) For instance, lycopene is the red pigment in tomatoes while carotene, an isoprenoid and precursor of vitamin A, is responsible for the pigment in carrots. Given the biological importance and applications of these molecules, numerous chemical techniques have been developed for their isolation from their natural sources, which inevitably contains some amount of water. Conventional separation techniques such as distillation, fractional distillation, stream distillation, crystallization, solvent extraction, enfleurage, and chromatography are used. The chemical and physical properties of the compound as well as its abundance and distribution in nature, influenced the choice of technique. For instance, while volatile and plentiful isoprenoids such as turpentine are isolated by distillation of oleoresins, extremely rare compounds such as insect’s hormones are separated from the substrate by chromatography. Currently, fundamental research has been directed towards extraction of these molecules from their natural source by bioaccumulation in microorganism, from which these isoprenoids can be extracted (Clark, Maury and Asadollahi 29). This article seeks to discuss the various conventional and emerging separation techniques used for the separation of isoprenoids from aqueous substrate. This discussion will include overview of the underlying principle involved in the process, design considerations with respect to the technique, fundamental challenges associated with the technique and suggestion of the best technique with respect to performance, safety, cost, and energy efficiency. In addition, specific applications of the best technique will be given. Conventional separation techniques for the isolation of isoprenoids Conventional technologies employed include, simple distillation, fractional distillation, stream distillation, vacuum distillation, solvent extraction, crystallization, and chromatographic techniques. Simple distillation Distillation involves the conversion of a liquid into vapor and the subsequent condensation of the vapor to back to liquid form. Distillation, as performed in the industry or laboratory is based differences in their volatilities (boiling point) of the mixture. Thus distillation is a physical separation process, and not a chemical reaction. Basically, the operation, as performed in the laboratory, involved the use of a heat source, a distilling flask, a condenser, and a receiver to collect the distillate. This simple apparatus is adequate for the separation of liquids of widely divergent boiling points such as isoprenoids from aqueous solutions (Encyclop?dia Britannica). It is usually expected that in heating a mixture of substances, the most volatile or the lowest boiling component distills first and the others subsequently or not at all. However, this is far from the reality; even an ideal system do not follows this expectation. Assuming that vapor-liquid equilibriums are attained, idealized models of distillation are governed by Raoult’s law and Dalton’s law. Raoult's law assumes that the percentage of a component in a mixture and its vapor pressure when pure determine its contribution to the total vapor pressure of the mixture while Dalton’s law state that the total vapor pressure is the sum of the vapor pressures of each individual components in the mixture. Under the ideal condition governed by Raoult’s law and Dalton’s law, the components are not “cleanly” separated. The more volatile components have higher partial pressure and thus are more concentrated in the vapor, but the less volatile components also evaporate, though they are less concentrated in the vapor. The idealized model works for chemically similar liquids in which the interactions between the components are the same as those between the molecules of each component by itself; for instance benzene and toluene. Thus for an aqueous mixture containing isoprenoids, Raoult’s law fails. However, given that the vapor pressures of the components – isoprenoids and water – are sufficiently different, Raoult’s law may be neglected due to the insignificant contribution of the less volatile component and; thus good to excellent separation is achieved. Indeed, simple distillation has been used to isolate volatile and plentiful substances such as turpentine from a mixture of oleoresins and water and rose oil from rose water (Eikani, Golmohammad and Rowshanzamir 555). Fractional distillation When the difference in boiling point of the components is less than 25 °C, fractional distillation is the distillation of choice provided the isoprenoid is not heat-sensitive. This has been applied to the separation of rosin acids and fatty acids from crude mixture of tall oil and water, a by-product obtained in the manufacture of paper pulp from pine wood (Setsuo, Sales and Jorge 7). Basically, a simple laboratory fractional distillation set-up consist of heat source, distilling flask, receiving flask, fractionating column, distillation head, thermometer and adapter if needed, condenser, such as a Liebig condenser, Graham condenser or Allihn condenser, vacuum adapter. The mixture is placed in the distilling flask and the fractionating column fitted into the top. As the mixture boils, vapor rises up the fractionating column and condenses on the glass platforms, known as trays, inside the column. The condensed vapor runs back into the liquid in the flask, where it is re-distilled. The process is known as refluxing. Usually, the column is heated from the bottom, with the hottest tray at the bottom and the coolest at the top. Under steady state conditions, the vapor and the liquid on each tray are in equilibrium. The most volatile of the vapors remains in the gaseous form up to the top, where it escapes from the column into the condenser and liquefies. The efficiency of separation is improved by increasing the number of trays in the column. A simple laboratory set-up as described above has been use to separate isoprenoids from a mixture of water and crude oil (Vyskrebentsev 614). The efficiency of separation can also be enhanced by working at reduced pressure (Setsuo, Sales and Jorge 7).The same principle applies when fractional distillation is used in the industry. Steam distillation Steam distillation is an alternative distillation technique for separating isoprenoids from their substrates at temperatures lower than the normal boiling point of the isoprenoid. It is the most popular of all the separation techniques used for isolation isoprenoids from the natural sources (Encyclop?dia Britannica). It is applicable when the material to be distilled is immiscible and chemically nonreactive with water. This is a method of choice for most aqueous mixtures of isoprenoids. Indeed, steam distillation was used to isolate isoprenoids and other volatile compounds from the leaves of glanded and glandless Artemisia annua L (Tellez, Canel and Rimando 1035). The underlying principle of steam distillation is that when a mixture of two practically immiscible liquids such as sesquiterpenes, an isoprenoid, and water, is heated while being agitated to expose the surfaces of both liquids to the vapor phase, each component independently exerts its own vapor pressure as a function of temperature as if the other constituent were not present. This will results in an increase in the vapor pressure of the whole system. Since boiling commences when the sum of the partial pressures of the two immiscible liquids just exceeds the atmospheric pressure , many organic compounds, such as isoprenoids, that are insoluble in water can be purified at a temperature well below the point at which decomposition occurs. Because of their lower volatility and temperature sensitivity, sesquiterpenes, are isolated from their natural sources by steam distillation (Encyclop?dia Britannica). Vacuum distillation Like steam distillation, heat-sensitive isoprenoids are separation by a variation of distillation whereby the pressure above the liquid mixture to be distilled is reduced to less than its vapor pressure (usually less than atmospheric pressure) causing evaporation of the most volatile liquid(s) (those with the lowest boiling points). This distillation technique, known as vacuum distillation, can be carried out with or without heat. Beta-carotene, an isoprenoid that is heat-sensitive, is usually isolated from aqueous mixture by this technique (Encyclop?dia Britannica). Compare with steam distillation, vacuum distillation is greener than the former since it leaves lower amounts of residues. Apart from isolation, vacuum distillation is very effective in the purification of isoprenoids (Davisson, Woodside and Neal 4768). Its purification efficiency can be enhanced by combining it with fractional distillation (Encyclop?dia Britannica). Safety is very important during vacuum distillation given that an implosion can occur. Chromatography Chromatography is a collective term for a set of separation and purification techniques that separates components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase (Encyclop?dia Britannica). The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid. The sample is dissolved in the mobile phase and due to interaction between the mobile phase and the stationary phase, separation is achieved. As a separation technique, chromatography has a number of advantages over older techniques such as distillation, crystallization, and solvent extraction. It has the capability to separate all components of a chemical compound without an extensive prior knowledge of the nature and concentration of the components. In addition, its resolving power is unequal among separation techniques. In chromatography, kinetic molecular motion continuously exchange solute molecules between the mobile phase and the stationary phase. If, for a particular solute, the distribution (exchange) favors the mobile phase, the molecule will spend most of the time in this phase and is eluted (transported) faster than the other components. In this way, separation takes place since the different components are differently distributed in the phases and, thus travel at different speed. By manipulating the driving force for solute migration in the mobile phase and the solute affinity for the stationary phase, separation is achieved. This principle applies for all types of chromatographic separation. The most important chromatographic methods generally used for separation of isoprenoids are high-performance liquid chromatography (Fraser, Elisabete and Pinto 551), gas chromatography (Berthou and Friocourt 393) and thin layer chromatography (Ikan 44). High-performance liquid chromatography In liquid chromatography, the mobile phase is a liquid. The separation is carried out in either a column or a plane. In high-performance liquid chromatography, relatively high pressure and very small packing particles are used. The sample is forced through a column that is packed with a stationary phase composed of irregular or spherically shaped particles, or a porous membrane, by a the liquid mobile phase at high pressure. Generally, in liquid chromatography separation can be achieved by adsorption, distribution, ion exchange, or exclusion mechanisms. The mechanism of distribution is based on dispersion forces that occur between molecules that have no permanent dipoles or can have no dipole induced on them. Adsorption is based on polar interactions, which arise from electrical forces between localized charges such as permanent or induced dipoles. In ion exchange, ionic interactions involving permanent negative and positive charges on the molecules control the separation process. The mechanism of exclusion is based on a molecular sieving effect. With respect to separation of isopronoids from an aqueous mixture, all mechanisms have been used with good results obtained (Lankes, Muller and Weber 915; Hooff, Volmer and Wood 673). Gas chromatography Also known as gas-liquid chromatography, (GLC), this technique is similar to fractional distillation since the separation is based on differences in boiling point (volatility) though the component’s affinity for the stationary phase is very important. Typically, gas chromatography is used to separate compounds that can be vaporized with decomposition. Though this technique has not been used to separate isoprenoids from aqueous mixtures, it has been used in the qualitative identification of isoprenoids. In fact, a two dimensional gas chromatography (coupling two gas chromatographs together) was used in fingerprinting of isoprenoids (Ventura, Raghuraman and Nelson 1026). In gas chromatography, the mobile phase is a carrier gas, usually an inert gas such as helium or nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support called a column. The mixture/sample is introduced into the column with a syringe. By temperature programming of the oven in the gas chromatograph, the sample is vaporized. The vaporized compound interacts with the walls of the stationary phases coated on the column. Given that different components in the mixtures have different affinity for the stationary and mobile phases, they travel at different rates. This results in a separation of the components, which are eluted at different times known as retention time. Crystallization Crystallization is solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. This consists of two steps, first nucleation and crystal growth. Nucleation normally occurs at nucleation sites on surfaces contacting the liquid medium. During nucleation, the solute molecules form clusters on the nanometer scale that, if are thermodynamically stable under the operating conditions, become nucleation sites. The nucleation sites constitute sites for crystal growth or formation. The crystal growth is the transfer of solute from the liquid phase to the nucleation site. Crystal growth is a necessary step for the aggregated clusters to reach a critical size to prevent dissolution. Thus nucleation and crystal growth occur simultaneously when the solution is supersaturated. Supersaturation is the driving force of crystallization; hence the rate of nucleation and crystal growth is dependent on the degree of supersaturation. As crystals are formed, the degree of supersaturation reduces until the solution is diluted and crystallization stops. Many isoprenoids can be separated from aqueous medium by crystallization (Morimoto, Cantrell and Bailey 69). Simply seeding the mixture (adding a little crystal of the isoprenoid of interest) initiates crystallization and the isoprenoid is separated from the aqueous medium (Encyclop?dia Britannica; Morimoto, Cantrell and Bailey 69). Solvent extraction Solvent extraction, also known as liquid-liquid extraction, is a technique that separate components of a mixture based on their relative solubility in two different immiscible liquids, usually and polar (aqueous) and a non-polar phase (organic). Precisely, it is use to extract a component of a mixture from one liquid phase into another liquid phase. It is the simplest and cheapest of all the separation techniques if the appropriate extracting solvent is selected. A simple separatory funnel is sufficient to carry out solvent extraction. Given that the polarity of isoprenoids and that of water differs considerably, solvent extraction is the best and cheapest method of separating isoprenoids from aqueous mixtures. For instance, by mean of sequential extraction with a biphasic mixture of petroleum ether (b.p. 60–80°C) and methanolic saline, the isoprenoid, quinines, was isolated from an aqueous medium (Minnikin, O'Donnell and Goodfellow 233). The distribution ratio (D), which is the concentration of a solute in the organic phase divided by its concentration in the aqueous phase, is a measure of extractability of a component in a mixture. The distribution ratio is a function of the temperature, the concentration of components in mixture and other parameters such as free energy, ?G, of the extraction process. Emerging technologies for separating isoprenoids from aqueous mixture In living organisms, isoprenoids are synthesized and bioaccumulated for diverse biological purposes. This has form the background for the biotechnological processes that employed microorganisms for the synthesis and accumulation of isoprenoids. Indeed, there is a lot of interest in this field due to its numerous advantages. For instance, employing microorganisms in the synthesis of isoprenoids offer the advantages of large scale synthesis of isoprenoids from cheap, bio-renewable sources. Microorganisms can be metabolically engineered to accumulate these isoprenoids in their system thereby offering the opportunity of separating them from their aqueous mixture (Clark, Maury and Asadollahi 29). Thereafter, the isoprenoids are extracted from the microorganisms. This approach, though requires a lot of technical expertise, is claimed to be greener and cheaper than other conventional approaches. It also presented as a single step (one pot) approach since the microorganisms synthesize and separate the isoprenoids in a single step. As promising and attractive this technology may look, a critical look reveals that is will be expensive and tedious given that metabolically engineered microorganisms will be expensive and that the bioaccumulated isoprenoids must be extraction from the microorganism by solvent extraction. Conclusion Among the separation techniques discussed, the solvent extraction remains the best approach for the large scale, industrial separation and even simple laboratory separation of isoprenoids from aqueous mixtures. The set-up is cheap; a simple separatory funnel is adequate when implemented in the laboratory. There is no need for technical expertise, as a first year science student can carried out extraction process once the appropriate solvent is identified. The safety concerns are the lesser than any of the separatory techniques discussed above. The energy requirement is lesser than any of the other separation techniques; heating is not required in most cases. In addition to these merits, it is an efficient approach of isolating and purifying isoprenoids from the aqueous mixtures. Works Cited Berthou, F and M P Friocourt. "Gas chromatographic separation of diastereomeric isoprenoids as molecular markers of oil pollution." Journal of Chromatography A (1987): 393-402 . 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