Received on 28.04.2015 Modified on 06.06.2015

Research J. Pharm. and Tech. 8(6): June, 2015; Page 775-786

DOI: 10.5958/0974-360X.2015.00125.0

INTRODUCTION:

The supercritical fluid extraction (SFE) technology has advanced tremendously since its inception and is a method of choice in many food processing industries. Over the last two decades, SFE has been well received as a clean and environmentally friendly “green” processing technique and in some cases, an alternative to organic solvent-based extraction of natural products. The most recent advances of SFE applications in food science, natural products, by-product recovery, pharmaceutical and environmental sciences have been published in extensive reviews Solvent extraction (SFE) is one of the old methods of separation known and certainly dates back to Paleolithic age.

The science of solvent extraction has evolved 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. Hannay and Hogarth’s (1879) early observations of the dissolution of medium. However, it is only quite recently (around1960) that commercial process applications of supercritical fluid extraction have been extensively examined. Since the end of the 1970s, supercritical fluids have been used to isolate natural products; industrial applications of SFE have experienced a strong development since the early 1990s in terms of patents As will be seen throughout this paper, the main supercritical solvent used is carbon dioxide. Carbon dioxide (critical conditions tc=31.3C and pc=72.8 bar, dc=0.467gm/ml) is cheap, environmentally friendly and generally recognized as safe by FDA and EFSA^[^1]. In many countries, health and safety regulations are getting stricter in addressing environmental problems created by the use of organic solvents and these issues are forcing the industries to search for alternative processing methods. The solvent is unsafe to handle and unacceptable as it is harmful to human health and the environment, restricting its use in the food, cosmetic and pharmaceutical industries. Furthermore, the major drawback of the solvent extracted products is the high level of residues left in the final products that must be desolventized before consumption. Therefore, SC-CO₂ is seen as a more favourable alternative to organic solvents in the extraction of fats and oils, and meets the growing consumer demand for safe natural fats and oils of excellent quality ²⁵. Pressure, temperature, particle size and sample pre-treatment are most important factors in oils as well as high value bioactive desired compounds extraction from the natural sources using supercritical fluid, because of the influence they have on the quality of the extracts.

What is Supercritical Fluid Extraction?:

Supercritical fluids have been investigated since last century, with the strongest commercial interest initially focusing on the use of supercritical toluene in petroleum and shale oil refining during the 1970s. Supercritical water is also being investigated as a means of destroying toxic wastes, and as an unusual synthesis medium. The biggest interest for the last decade has been the applications of supercritical carbon dioxide, because it has a near ambient critical temperature (310⁰C), thus biological materials can be processed at temperatures around 350⁰C. The density of the supercritical CO2 at around 200 bar pressure is close to that of hexane, and the salvation characteristics are also similar to hexane; thus, it acts as a non-polar solvent. Around the supercritical region, CO₂ can dissolve triglycerides at concentrations up to 1% mass. The major advantage is that a small reduction in temperature, or a slightly larger reduction in pressure, will result in almost the entire solute precipitating out as the supercritical conditions are changed or made sub critical. Supercritical fluids can produce a product with no solvent residues. Examples of pilot and production scale products include decaffeinated coffee, cholesterol-free butter, low-fat meat, evening primrose oil, squalling from shark liver oil, etc. The salvation characteristics of supercritical CO₂ can be modified by the addition of an entrained, such as ethanol, however some entrained remains as a solvent residue in the product, negating some of the advantages of the "residue-free” extraction. Supercritical fluid extraction (SFE) is the process of separating one component (the extract ant) from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is usually from a solid matrix, but it can also be from liquids. SFE can be used as a sample preparation step for analytical purposes, or on a larger scale to either strip unwanted material from a product (e.g. decaffeination) or collect a desired product (e.g. essential oils). Carbon dioxide (CO₂) is the most used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol. Extraction conditions for supercritical CO₂ are above the critical temperature of 31°C and critical pressure of 74 bar. Addition of modifiers may slightly alter this. Supercritical extraction mostly uses carbon dioxide at high pressure to extract the high value products from natural materials. Unlike other processes, the extraction process leaves no solvent residue behind. Moreover the CO2 is non-toxic, non-flammable, odourless, tasteless, inert, and inexpensive. Due to its low critical temperature 31°C, carbon dioxide is known to be perfectly adapted in food, aromas, essential oils and nutraceutical industries.

Definition:

Supercritical Fluid Extraction:

Supercritical fluid extraction (SFE) may be defined as separation of chemicals, flavours from the products such as coffee, tea, hops, herbs, and spices which are mixed with supercritical fluid to form a mobile phase. In this process, the mobile phase is subjected to pressures and temperatures near or above the critical point for the purpose of enhancing the mobile phase solvating power. The process begins with CO2 in vapour form. It is then compressed into a liquid before becoming supercritical. While supercritical, the extraction takes place ^[^2].

Critical conditions:

· Temperature (tc)= 30.9 ⁰C

· Pressure (pc)=73.8 bar

· Density (dc)=0.467gm/ml [4,5]

Principle:

The first guiding principle is the optimization of the solubility of materials to be extracted (lipids, heavy metals, natural products) in supercritical CO₂ and the improvement of the fractionation with respect to a particular lipid species, natural products Supercritical fluid extraction facilitates the detachment of the extract from the supercritical fluid solvent by simple expansion. An added benefit is derived from the liquid like densities of the supercritical fluids with superior mass transfer distinctiveness that enables the easy release of solutes, compared to other liquid solvents. This uniqueness is owed to the high diffusion and very low surface tension of the supercritical fluid that enables easy infiltration into the permeable make-up of the solid matrix to reach the solute . Since the early 1980s, the use of SC-CO2 in the extraction of oil or lipid from various sources, both plants and animals [19,20] has been studied extensively. In addition, the application of SC-CO₂ in the extraction of minor constituents from various plant sources has also been widely studied Recently, Pourmortazavi et al. reported that carbon dioxide is used in more than 90% of all analytical supercritical fluid extractions. [3]

HISTORY:

The first reported observation of the occurrence of a supercritical phase was made by Baron Cagniard de la Tour in 1822.

· He noted visually that the gas-liquid boundary disappeared when the temperature of certain materials was increased by heating each of them in a closed glass container.

· From these early experiments, the critical point of a substance was first discovered.

· The first workers have been done to demonstrate the solvating power of supercritical fluids for solids in 1879.

· In 1970 a significant development in supercritical fluid extraction (SFE), provided incentive for extensive future work, which involved decaffeination of green coffee with CO₂.

Supercritical Fluid:

The supercritical fluid extraction (SFE) has been applied only recently to sample preparation on an analytical scale. This technique resembles” Soxhlet extraction” except that the solvent used is a supercritical fluid, substance above its critical temperature and pressure. This fluid provides a broad range of useful properties. One main “advantage” of using SFE is the elimination of organic solvents, thus reducing the problems of their storage and disposal in the iridologist laboratory. Furthermore, several legislative protocols (such as the EPA Pollution Prevention Act in the USA) have focused on advocating a reduction in the use of organic solvents which could be harmful to the environment. Besides ecological benefits, one of the most interesting properties of SFE is the high diffusion coefficients of lipids in supercritical fluids, far greater than in conventional liquid solvents. Thus, the extraction rates are enhanced and less degradation of solutes occurs. Several studies have shown that SFE is a replacement method for traditional gravimetric techniques. In addition, carbon dioxide, which is the most adopted supercritical fluid has low cost, is a non-flammable compound and devoid of oxygen, thus protecting lipid samples against any oxidative degradation[4].

The definition of a supercritical fluid is best described by using a typical pressure-temperature phase diagram as shown in Figure No: 1.

Figure 1: Show pressure temperature phase diagram demonstrating the SFE region and its relation to liquid and gas phase region.

The range of solvating power of practical supercritical fluids for SFC is of primary importance, and ultimately defines the limits of application. The solubility of analyses typically increases with density and a maximum rate of increase in solubility with pressures generally observed near the critical pressure, where the rate of increase of density with pressure is greatest. There is often a linear relationship at constant temperature between log [solubility and fluid density for dilute solutions of non-volatile compounds (up to concentrations where solute-solute interactions become important).

At constant pressure, when solute volatility is extremely low, and at densities less than or near the critical density, increasing temperature will typically decrease solubility. However, solute entrainment in the fluid may increase at sufficiently high temperatures, where solute vapour pressure also becomes significant .Under conditions of constant density, solubility generally increases with temperature. Thus, while the highest supercritical fluid densities (at constant temperature) are obtained near the critical temperature, the greatest solubility’s and lowest chromatographic retention will often be obtained at somewhat lower densities, but at higher temperatures. As with liquids, polar solutes are most soluble in polar supercritical fluids, although nominally non polar fluids can be remarkably good solvents for many moderately polar compounds. Carbon dioxide, for example, can exhibit solvating properties at higher pressures, intermediate between liquid n-pentane and dichloromethane. A comparison of the effective solvent polarity of seven fluids as a function of reduced density is shown in Figure No: 2

Solvent polarity is defined in terms of solvent polarizability (x*) which was developed by Kamlet et aL75 to correlate different solvent solute interactions based on the solvatochromic effect of the solvent on the x-x* electronic transition of probe solutes. In this plot, x* contains terms to account for solvent polarity (i.e., dipolarity) and polarizability, but does not include effects from potential hydrogen bonding interactions.

Figure 2: Shows Solvent polarizability/polarity parameter (x*) for various supercritical fluids as a function of reduced density at a reduced temperature of 1.03. Supercritical fluids: (a)NH,, (b) CO,, (c) N,O, (d) Xe, (e) CCl,(F) C,H, (g) SF.

At equal reduced densities, the various fluids have quite different x* values, indicating that there are large differences in their effective polarities/polarizabilities. Ammonia has the largest x* value, which supports the fact that it is the most polar solvent. The solvatochromic method also demonstrates the variable solvent properties of a supercritical fluid as a function of density. Many polar solvents would offer highly specific solvating power but have excessively high critical temperatures, precluding practical operation with current stationary phases. The thermo stability limits of the analyses themselves can also be exceeded. This has generated interest in mixed or binary fluid mobile phases that can have enhanced solvating power at lower critical temperatures. Solvatochromic studies suggest that such fluid mixtures have a net enrichment of polar modifiers in the hypotactic region (nearsneighboursalvation sphere) of the analyse[5].

Characteristics of SF:

It is both the liquid-like and gas-like characteristics of supercritical fluids that make them unique for chemical separation. In particular, supercritical fluid densities, diffusivities, and viscosities fall into ranges between those of liquids and gases. Under practical analytical operating conditions, pressures from 50-500 atm and temperatures from ambient to 300⁰C, densities of supercritical fluids range from one to eight-tenths of their liquid densities. Diffusivities of analyses in supercritical fluids throughout this operating range vary between10-3 and 10-4cm’/s compared to values of less than 10-5cm²/s for liquids. Viscosities of supercritical fluids are typically 10-100 times less than those of liquids. On the other hand, viscosities of supercritical fluids are considerably higher and diffusivities considerably lower than in gases. Moreover, densities of supercritical fluids can be 100-1000 times greater than those of gases. Advantages of supercritical fluids over liquid phases rest with improved mass transfer processes due to lower fluid viscosities and higher analyse diffusivities, while advantages over gas phases rest with increased molecular interactions due to higher densities.

Other Characteristics of Supercritical fluids:

That is important to consider include the operational temperature and pressure range. Table.1 provides a list of nine of the most common supercritical fluids used in extraction and chromatography along with temperature, pressure, density, and dipole moment information. These nine are chosen primarily because of the convenience of their critical temperatures and critical pressures. These temperatures and pressures are low enough for use with commercial instrumentation. The polarity of the supercritical fluid, as reflected in its dipole moment and polarizability. The density at 400 atm (p and I; = 1.03 was calculated from compressibility data. ‘Measurements were made under saturated conditions if no pressure is specified or were performed at 25°C if no temperature is specified. [6]

Properties of Supercritical Fluids:-

· A supercritical fluid is any substance above its critical temperature and critical pressure. In the supercritical area there is only one state-of-the-fluid and it possesses both gas- and liquid-like properties.

· A supercritical fluid exhibits physicochemical properties intermediate between those of liquids and gases Supercritical fluids have highly compressed gases, which combine properties of gases and liquids in an intriguing manner.

Supercritical fluids can lead to reactions, which are difficult or even impossible to achieve in conventional solvents.

Supercritical fluids have solvent power similar to light hydrocarbons for most of the solutes. However, fluorinated compounds are often more soluble in supercritical CO₂ than in hydrocarbons; this increased solubility is important for polymerization.

Solubility increases with increasing density (that is with increasing pressure).

Rapid expansion of supercritical solutions leads to precipitation of a finely divided solid. This is a key feature of flow reactors.

The fluids are commonly miscible with permanent gases (e.g. N₂ or H₂) and this leads to much higher concentrations of dissolved gases than can be achieved in conventional solvents.

7. Characteristics of a supercritical fluid:

· Dense gas

· Solubility’s approaching liquid phase

· Diffusivities approaching gas phase. [7]

Figure No: 3 Shows Phase diagram (P---T):

Critical Temperature (TCU):

The highest temperature at which a gas can be converted to a liquid by an increase in pressure.

Critical pressure (Pc):

The highest pressure at which a liquid can be converted to a traditional gas by an increase in temperature.

Triple point (Top):

A point at which the gas, liquid and solider phases all exist in equilibrium. Therefore, the properties of gas-like diffusivity, gas-like viscosity, and liquid-like density combined with pressure-dependent solvating power have provided the impetus for applying supercritical fluid technology to various problems. [8] All the above terms are mentioned in Figure No: 3.

Density considerations:

For a material at temperatures just above the critical temperature of the substance, liquid-like densities are rapidly approached with modest increases in pressure. Higher pressures are required to attain liquid-like densities for temperatures further above the critical temperature Lists the densities at the critical point and at 400 atm and Tc for various fluids employed for SFE.

Characteristics of Super-Critical Fluids Relevant to Separation Science

· In the absence of actual phase equilibrium data, simple mole fraction additively methods used to obtain mixture critical parameters can result in considerable error and lead to inadvertent operation in the vapour liquid region. More complex predictive methods utilizing equations of state [9] or surface fraction functions (Chueh and Prausnitz method)[10] generally provide more accurate estimates of the true critical parameters. These considerations are important when pressure programming methods are used, but are of lesser importance when relatively high isobaric pressures are used.

Extraction Method:

Often the analysis of complex materials requires as a preliminary step separation of the analyse or analyses form a sample matrix. Ideally, an analytical separation method should be rapid, simple and inexpensive; should give quantitative recovery of analyses without loss or degradation; should yield a solution of the analyte this is sufficiently concentrated to permit the final measurement to be made without the need for concentration; and should generate little or no laboratory wastes that have to be disposed of

Figure 4: Shows Relation between the Extraction time (min.) and Extracted amount(%)

It must be noticed that the fast back-diffusion of analyses in the supercritical fluid reduces the extraction time since the complete extraction step is performed in about 20 min instead of several hours, shown in Figure No: 4. A common practice in SFE, which must be mentioned in connection -with the physicochemical properties of supercritical fluids, is the use of modifiers (co-solvents). [11]

The system must contain a pump for the CO₂, a pressure cell to contain the sample, a means of maintaining pressure in the system and a collecting vessel. The liquid is pumped to a heating zone, where it is heated to supercritical conditions. It then passes into the extraction vessel, where it rapidly diffuses into the solid matrix and dissolves the material to be extracted. The dissolved material is swept from the extraction cell into a separator at lower pressure, and the extracted material settles out. The CO₂ can then be cooled, recompressed and recycled, or discharged to atmosphere.

Pumps:

Carbon dioxide is usually pumped as a liquid, usually below 5°C and a pressure of about 50 bars. The solvent is pumped as a liquid as it is then almost incompressible. As a supercritical fluid, much of the pump stroke will be "used up" in compressing the fluid, rather than pumping it. For small-scale extractions (up to a few grams/minute), reciprocating CO₂ pumps or syringe pumps are often used. For larger scale extractions, diaphragm pumps are most common. The pump heads will usually require cooling, and the CO₂ will also be cooled before entering the pump.

Pressure Vessels:

Pressure vessels can range from simple tubing to more sophisticated purpose built vessels with quick release fittings. The pressure requirement is at least 74 bars, and most extractions are conducted at less than 350 bar. However, sometimes, higher pressures will be needed, such as extraction of vegetable oils, where pressures of 800 bars are sometimes required for complete miscibility of the two phases. The vessel must be equipped with a means of heating. It can be placed inside an oven for small vessels, or oil or electrically heated jacket for larger vessels. Care must be taken if rubber seals are used on the vessel, as the CO₂ may dissolve in the rubber, causing swelling, and the rubber will rupture on depressurization.

Pressure Maintenance:

The pressure in the system must be maintained from the pump right through the pressure vessel. In smaller systems (up to about 10 mL/min), a simple restrictor can be used. This can be either a capillary tube cut to length, or a needle valve, which can be adjusted to maintain pressure at different flow rates. In larger systems a backpressure regulator will be used, which maintains pressure upstream of the regulator by means of a spring, compressed air, or electronically driven valve. Whichever is used, heating must be supplied, as the adiabatic expansion of the CO₂ results in significant cooling. This is problematic, if water or other extracted material is present in the sample, as this may freeze in the restrictor or valve and cause blockages.

Collection:

The supercritical solvent is passed into a vessel at lower pressure than the extraction vessel. The density, and thus, dissolving power, of supercritical fluids varies sharply with pressure, and hence, the solubility in the lower density CO₂ is much lower, and the material precipitates for collection. It is possible to fractionate the dissolved material using a series of vessels at reducing pressure. The CO₂ can be recycled or depressurized to atmospheric pressure and vented. For analytical SFE, the pressure is usually dropped to atmospheric, and now the gaseous carbon dioxide is bubbled through the solvent to trap the precipitated components, heat must be provided to prevent excessive cooling. For small-scale extractions, such as for analytical purposes, it is usually sufficient to pre-heat the fluid in a length of tubing inside the oven containing the extraction cell. The restrictor can be electrically heated, or even heated with a hair dryer. For larger systems, the energy required during each stage of the process can be calculated using the thermodynamic properties of the supercritical fluid.

Heating and cooling:

This is an important aspect. The fluid is cooled before pumping to maintain liquid conditions, and then heated after pressurization. As the fluid is expanded into the separator,

Schematic diagram of SFE apparatus

Modifiers (co-solvents):

These are compounds that are added to the primary fluid to enhance extraction efficiency. Thus, addition of 1 to 10% of methanol or ethanol to CO₂ expands its extraction range to include more polar lipids. When the extraction was performed with supercritical carbon dioxide and 20% of ethanol, more than 80% of the phospholipids were recovered from salmon roe.[18]

Instrumentation:

Instrument components include a fluid source, commonly a tank of carbon dioxide followed by a syringe pump having a pressure rating of at least 400 atm a valve to control the flow of the critical fluid into a heated extraction cell having a capacity of a few ml, and lastly an exit valve leading to a flow restrictor that depressurizes the fluid and transfers it into a collection device. Figure No: 5 shows the flow diagram of SFE apparatus.

Figure No: 5 Shows SFE Flow diagram.

1. Mobile phase:

Mobile phase:

The most widely used mobile phase for SFE is carbon dioxide. It is an excellent solvent for a variety of organic molecules. In addition, it transmits in the ultraviolet and is odorless, nontoxic, readily available, and remarkably inexpensive when compared with other chromatographic mobile phases which has been shown in Table No: 1.

Table No: 1 Shows Comparison of the physical properties of supercritical CO2 and those of ordinary gases and liquids [19]

Phase	Density (g/cm²)	Viscosity (g/cms)	Diffusion coefficient (cm²/s)
Gases	0.0001-0.002	0.0001-0.0003	0.1-0.4
Supercritical CO₂	0.47	0.003	0.0007
Liquids	0.6-1.6	0.002-0.03	0.000002-0.00002

Sample Matrix Parameters that influence Supercritical Fluid Extraction:

· Particle size and shape

· Surface area and porosity

· Moisture content

· Changes in morphology

· Sample size

· Extractable level

The parameters effect on solubility:

· The vapour pressure of the component

· Interaction with the supercritical fluid

· Temperature, pressure, density and additives.

A generalized solubility isotherm for a solute-supercritical fluid system as a function of pressure and at two different temperatures, r, and T2, is shown in Figure No: 6. Upon initial pressurization of the system, there is a decrease in solute solubility in going from the respective pressures designated by points A and A’ to B and B’. At a certain pressure beyond B and B’, the solute’s solubility begins to increase with pressure. Frequently, this pressure regime is called the “threshold pressure” [12], since there is a large measurable solubility -enhancement of the solute in the dense fluid solvent. However, it has been noted [13] that the above-reported solubility trends and threshold pressures are very dependent on the technique that is utilized to measure the solute’s solubility in the supercritical fluid media. However, the differential extraction behaviour-

Figure 6: Shows Generalized solubility isotherms as a function of pressure

-exhibited between points A and A’ or B and B’ can obviously be used as a basis for the selective extraction of target analyses. Similarly, fractionation of solute mixtures can be performed in the pressure interval between B or B’ and C and C’, although the relative separation factor between individual solutes is not always large. Note that the solubility isotherms may cross at a particular pressure called the “cross-over pressure” [14], at which the solubility of one solute can- be enhanced in the fluid phase relative to the other. Solute fractionation at the solubility maxima, C and c’. As shown in Figure-14, is also possible, but the resultant a values may be low, since many solutes will extract into the supercritical fluid at these high pressures. For this reason, some analysts avoid conducting extractions in the solubility maxima region. However, as shown by King and co-workers [15], this pressure region is to be preferred for exhaustively extracting bulk phases, such as lipid materials from insoluble sample matrix components. Also, extractions conducted in this region generally can be completed much more rapidly, since the solutes have considerably higher solubility in the supercritical fluid under these conditions of equal importance in the above solubility criteria are the mass transfer properties of the extracted solutes in the supercritical fluids. Solute extraction fluxes from a sample matrix are directly proportional to the product of the solute’s solubility in the supercritical fluid times its diffusivity in the fluid. Therefore, as a solute’s solubility increases with pressure, its corresponding diffusivity in the super-critical fluid can decrease over two orders of magnitude. The net effect of the above two trends can best be measured in terms of mass transfer coefficients or dimensionless transport numbers. For example, the ratio of the Reynolds number (Re) for CO, at 200 atm and 55°C to those for the liquid solvents cited in Table No: 2, at an equivalent fluid velocity, is 6.5, 5.0, and 1.74 for methanol, n-hexane and methylene chloride, respectively.

Table No: 2 Shows Comparison of physical properties of supercritical CO₂ with liquid solvents at 250°C (T 1.9)

Parameter	CO2	n- Hexane	Methylene chloride	Methanol
Density (g/ml)	0.746	0.660	1.326	0.791
Kinematic viscosity (m²/s x 10⁷	1.00	4.45	3.09	6.91
Diffusivity of Benzoic acid (m²/s x 10⁹)	6.0	4.0	2.9	1.8

In this case, the larger fluid turbulence that occurs in the CO2 should greatly enhance the rate of solute extraction. The kinetics for solute extraction into a supercritical fluid follow a similar pattern to that observed for liquid extraction. As we know initial stage of the extraction is governed by the distribution coefficient of the solute between the dense fluid -phase and the sample matrix, giving way to a. diffusion-controlled process in the latter stages of the extraction. The implications of the curve shown in Figure No: 7 on the extent and time of SFE has been treated theoretically by Bartle and co-workers [16] in terms of the “hot ball” model, where the mass of extractable material remaining in the sample matrix m to the mass of original extractable material mo is given by

Where, n is an integer; D is the diffusion coefficient of the Solute in the hypothetical spherical matrix of radius r; and t is the extraction time. This Expression can be rewritten in terms of reduced time tr =2Dt/r2 , to yield an expression for m/mo in terms of an exponential decay series expansion. The final expression, given in (2) is

Figure 7: Shows Generalized extraction curve of percent solute extracted as a function of volume of extraction fluid or time of extraction

The latter equation has been found to describe analytical

SFE kinetics from such diverse sample matrices as railroad bed soil, crushed rosemary, and comminutes polypropylene pellets. In many cases, slow solute extraction kinetics or limiting analyse solubility in the fluid phase, can be overcome by the addition of modifiers or co solvents to the supercritical fluid phase. Examples of solubility enhancements for selected solutes that have been realized by adding modifiers into supercritical CO2 are shown in Table No: 3.

Table.3 Shows Solubility enhancement with supercritical co2 with various modifiers

Solute	Modifier	Enhancement
Acridine	3.5% MeOH	2.3
2-amino benzoic acid	3.5% MeOH	7.2
Cholesterol	9% MeOH	100
Hydroquinone	2% Tributyl phosphate	300
Tryptophan	AOT, Octanol	>>100

The addition of methanol to CO2 not only enhances the solubilisation of polar solutes, such as alcidine and 2-amino benzoic acid, but increases the solubility of highly soluble lipophilic solutes, like cholesterol, over lOO-fold. Certain specific modifiers, such as tributyl phosphate, act as completing agents [17], thereby enhancing the extraction of a donor molecule, hydroquinone, over 300-fold.

Fluid reservoir:

A gas cylinder provides a source of SF (e.g., CO2).Both syringe and reciprocating pumps can be used as solvent delivery systems

Pumps:

a) Reciprocating pump, [18] b) Syringe pump, [19] c) Other pump modules (like supplementary modifier pump)

For the instrumentation used in some analysis, a syringe pump was employed. Although syringe pumps are relatively expensive, they deliver pulse-free flow over a large range of flow rates.

Example:

Quantitative Analysis of Additives in Low Density Polyethylene Using On-line Supercritical Fluid Extraction. A supplementary modifier pump is used if the analyse/ matrix to be extracted requires a polar modifier. Stainless steel or fused silica tubing is used to connect the various parts of the extraction apparatus.

9. Extraction cell (or) Columns (stationary phase):

The extraction chamber or vessel is the compartment where the sample is placed for subjection to the action of the SF. It must be capable of withstanding high pressure (300-600 atm). The extraction vessel is usually a stainless steel cylinder of varying length and inner diameter shown in Figure No: 8. The high pressure rating and the absence of leaks are characteristic of SFE vessels 1. The vessel is in turn placed in a temperature-controlled zone, which is required, since the critical temperature of most SFs is above room temperature.

Figure 8: Shows Types of extraction cells

Open tubular capillary columns:-

Open tubular columns for SFE must possess the usual qualities of high efficiency, inertness, and lasting stability, which .are characteristic of open tubular columns for GC. The main differences in the preparation of the columns are related to the smaller internal diameters characteristic of SFE columns. Immobilization (generally cross-linking of the polymeric phase) is an essential ingredient in the preparation of open tubular columns. It must be performed to resist dissolution, but without lowering solute diffusion within the phase. [20]

9.2 Packed Columns:-

In the packed column, the stationary phase is normally near monomolecular thickness and is polymerized and chemically bonded to the support. Both open-tubular and packed columns are used for SFC although currently the former are favoured. Open-tubular columns are similar to the fused-silica columns with internal coatings of bonded and crossed-linked siloxanes of various types.

For example In the on-line SFE-SFC system used in the additive analysis, a linear fused silica capillary was employed as a vessel outlet restrictor. [21]

Restrictors:

The pressure change from supercritical conditions in the extraction vessel to the prevailing atmospheric conditions is effected via an interface known as restrictor. Commercially available restrictors are of two types: fixed restrictors, shown in Figure No: 9 which are manufactured in various designs (e.g., linear, tapered, integral, pinhole, and frit), and variable restrictors 1. Heating of the restrictor is usually required to avoid plugging through freezing. [22]

a) Fixed restrictors:

i) Linear restrictor (fused-silica)

ii) Tapered desire

iii) Integral restrictor

iv) Ceramic frit restrictor

v) Metal restrictor (platinum, platinum-iridium or steel)

b) Variable restrictors:

i) Variable nozzle (HP)

ii) Backpressure regulator (BPR) (Jasco)

Collector (trapping system):

Following the restrictor is a trapping device. There are three basic types of SFE systems characterized by the way in which the solutes are isolated from the SFE media used

· In the first type, solutes are separated from the extraction media based on pressure reduction, which causes a solubility decrease.

· In the second type, a temperature change issued to bring about a decrease in solubility from the extraction media,

· And in the third type solutes are absorbed onto an appropriate absorb ate. Often a combination of the first and second types is used, where after extraction the SF is simply evaporated to leave the solutes of interest. The simplest way of collection is when the restrictor outlet is inserted through the septum of a collection vial containing a few millilitres of solvent. The most common way of collection is solid phase trapping.

The materials used for this purpose are column pickings or inert surfaces. The solid phase trapping system is often heated or cooled depending on the volatility of the target analyses. In any case, this collection mode involves an additional step which is desorption of the analyses from the adsorbent by elution with a small amount of solvent for subsequent analysis or, alternatively, thermal desorption and sweeping of the trap by the fluent if an on-line coupled system is used. The trapping temperature depends on whether the analyses are to be isolated from the fluid. The collection chamber should be sealed in order to avoid losses of the analyses. In this research, a cryogenic trap served as the interface between SFE and SFC. Thermal desorption and sweeping the trap with SF CO2 was employed to flush analyses onto the SFC column

Detectors:

A major advantage of SFC /SFE over HPLC is that the flame ionization detector of gas chromatography can be employed. Mass spectrometers are also more easily adapted as detectors for SFE than HPLC. [23]

i) UV detector

ii) Fluorescence detector

iii) Flame ionization detector

iv) Electron capture detector

v) Mass spectrometric detector

12.1 Different modes of Supercritical fluid extraction:

(i) Static extraction mode (steady state extraction),

(ii) Dynamic extraction mode (non-steady state extraction),

(iii) Recalculating mode

Contact between the SF and sample from which extraction takes place can be established in a static or dynamic modern a static extraction, the sample matrix is soaked in a fixed amount of SF. This type of extraction is often compared to a teabag in a cup of water. In a dynamic extraction, SF continuously passes through the sample matrix. This is analogous to a coffee maker 1. Typically a dynamic extraction can be more exhaustive than a static extraction. SFE can be performed in the dynamic mode, static mode or a combination of the two. In order to develop an efficient and quantitative extraction method, many experimental parameters must be optimized. The extraction pressure is an important variable because the density, and hence the solvating power of SF is directly related to the pressure. The effect of temperature is more complicated than that of pressure. Increasing the temperature increases the diffusion coefficients of the solutes, whereas at the same time it also decreases the density. In addition, the considerations of fluid flow rate, addition of a modifier, and extraction time should be explored to achieve highest recoveries.

12.2 Types of SFEs:

SFE is generally not selective enough to isolate specific solutes from the matrix without further clean-up or resolution from co-extracted species prior to qualitative and quantitative analysis. Consequently, for analytical applications, SFE is usually used in conjunction with chromatographic techniques, to improve the overall selectivity of the process in isolating specific solutes. SFE combined with chromatography can be either “off-line” or “on-line.[24]

i) Off-line:- In the off-line process, SFE takes place as a separate and isolated process to the chromatography. A block diagram is shown in Figure No: 10.

ii) On-line:- In the on-line process, SFE and chromatography are coupled to form an integrated process.

In other words, the extracted species are passed directly to the chromatograph, usually via a trap or sample loop and a valves witching device shown in Figure No: 11. Among all these coupling techniques, on-line SFE/SFC is the most feasible combination.

a) SFE-GC

b) SFE-MS

c) SFE-LC

d) SFE-SFC

Fig no 9 ; Shows Off-l

Advantages of on-line SFE:

1. Direct coupling of the analyse-containing supercritical fluid to a chromatographic separation system with appropriate detection.

2. Eliminating sample handling after loading in the extraction

Disadvantages of on-line SFE:

1. Long periods of time

2. Understand the nature of analyses

Scaling-up SFE and preparation of the crude extract:

After the SFE conditions were optimized, an ideal optimization conditions are shown in Table No: 6 the extraction was scaled up by 100 times using a preparative system. 5 kilograms amount of sample (40–60 mesh) was placed into an extraction vessel with a 1.0×104 ml capacity, and extracted statically for 1 h followed by another 5 h dynamically under the optimized conditions at 45⁰C, 25MPa. The flow-rate of carbon dioxide supercritical fluid was set at 40 kg/h, and the extract in supercritical fluid was depressed directly into a separate vessel. The SFE extract before methanol washing (crude extract I) was light yellow semi-solid and then re-dissolved in methanol, and the methanol soluble fraction (crude extract II)was obtained and evaporated to dryness under[25]r reduced pressure at 60⁰C, which was subjected to subsequent HSCCC isolation and separation.

Optimizing flow rate:

The flow rate of CO2 should be measured in terms of mass flow rather than by volume because the density of the CO2 changes according to the temperature both; before entering the pump heads and during compression. Carioles flow meters are best used to achieve such flow confirmation. To maximize the rate of extraction, the flow rate should be high enough for the extraction to be completely diffusion limited (but this will be very wasteful of solvent). However, to minimize the amount of solvent used, the extraction should be completely solubility limited (which will take a very long time). Flow rate must therefore be determined depending on the competing factors of time and solvent costs, and also capital costs of pumps, heaters and heat exchangers. The optimum flow rate will probably be somewhere in the region where both solubility and diffusion are significant factors.

Applications SFE:

1. Application of SFE to Enantiomeric Separations:

Choral separation is a very important issue for the pharmaceutical industry. The applicability of SFE as an effective and green technique for enantioseparations is known since the late 1990s. In these processes, diastereomeric salts or complexes of the racemic compounds and resolving agents are formed before the extraction step. The selected resolving agent is added in less than stoichiometric ratio to the racemic compound. The unreached enantiomers are extracted with the supercritical solvent, and are collected as a powder after depressurization of the solution[34]

2. Metals Recovery using Supercritical Fluids:

Removal of heavy metals from solid matrices and liquid remain a big challenge and, although various methods have been described for this purpose, SFE seems to be one of the most promising. Completing agents used in conventional solvent extraction can also be used in SFE complication of metal ions

Test no.	Factors
Matrix	A: Pressure (MPa)		B: Temp. (°C)		C: Particle size (mesh)		D: Modifier (methanol %)^a
1	A1	15	B1	45	C1	10-20	D1	0
2	A2	15	B2	55	C2	20-40	D2	10
3	A3	15	B3	65	C3	40-60	D3	20
4	A1	25	B1	45	C1	20-40	D1	20
5	A2	25	B2	55	C2	40-60	D2	0
6	A3	25	B3	65	C3	10-20	D3	10
7	A1	35	B1	45	C1	40-60	D1	10
8	A2	35	B2	55	C2	10-20	D2	20
9	A3	35	B3	65	C3	20-40	D3	0

3. SFE in Food Toxicology and Ecotoxicology:

There are several compounds with serious health implications which determination can be done using SFE, the main areas of application include food toxicology and ecotoxicology

4. Solvent Removal and new Drug Delivery Formulations:

In order to enhance the bioavailability of poorly water soluble drugs, an increasing number of pharmaceutical formulation technologies are being developed; these include micronization, complex formation and solid dispersions. In the case of polar compounds which are not soluble in supercritical fluids (particularly CO2), SCFs could be used as ant solvent; in this process, a solution consisting of an organic solvent, completely miscible with the SCF, and a solid material dissolved in this solvent, is sprayed into a high-pressure vessel filled with SCF [197]. In these processes the supercritical fluid is used to extract the solvent instead of the analyse the spectroscopic and chromatographic characterization of triflusal (2-acetoxy-4- (trifluoromethyl) benzoic acid) delivery systems prepared by using supercritical

3. SFE in food Toxicology and Ecotoxicology:

There are several compounds with serious health implications which determination can be done using SFE, the main areas of application include food toxicology and ecotoxicology

4. Solvent Removal and new Drug Delivery Formulations:

CONCLUSION:

The supercritical fluid extraction is a separation method in which co₂ solvent are use for separation of chemical from plant are other species. The most recent advances of SFE applications in food science, natural products, by-product recovery, pharmaceutical and environmental sciences have been published in extensive reviews Solvent extraction (SFE) is one of the old methods of separation known and certainly dates back to Paleolithic age. The supercritical fluid extraction (SFE) has been applied only recently to sample preparation on an analytical scale. This technique resembles” Soxhlet extraction” except that the solvent used is a supercritical fluid, substance above its critical temperature and pressure.

ACKNOWLEDGMENT:

The author wants to acknowledge the library of Rungta College of Pharmaceutical Sciences and Research, Kohka-Kurud road Bhilai, for providing necessary literature for the compilation of the work. The authors also want to thanks Shri Santosh Rungta, Chairman, Santosh Rungta Group of Institution for providing necessary facility and infrastructure for the completion of the work.

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