Separation Technique Has Relevant Insight Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Separation technique has relevant insight in drug development. Solvent extraction is one of the oldest methods of separation. Among the different extraction techniques used at analytical scale, Supercritical Fluid Extraction provides unique perspective as far as drug development is concerned. Combining supercritical Fluid extraction (SFE) with Supercritical Fluid Chromatography (SFC) technique is remarkably advantageous since it couples both solvating property of liquid and transport property of gas.

SFE is a technique in the field of analytical chemistry, having evolved as an alternative method of sample preparation for analysis. SFE offers many advantages such as distillation, extraction with liquid solvents or low resolution liquid chromatography. In practice SFE can provide an appreciable saving in time and cost associated with sample preparation.

SFC is the separation technique in which mobile phase is a supercritical fluid, is more important because it permits the separation and determination of a group of compounds not conveniently handled by either GC or LC. SFE/SFC uses supercritical fluid as a mobile phase for separation and extraction. Supercritical fluid Carbon dioxide has been used almost exclusively as the extraction media due to the inertness and non-toxic properties of the gas. Hence the use of hazardous organic solvents can be kept to a minimum in SFE/SFC procedure.

Combined SFE-SFC technique has gained importance in separation in diverse fields such as pharmaceuticals, forensics, food industry, environmental etc

Considering the potential of SFE-SFC in separation of complicated mixtures, the present review focused on different aspects of SFE-SFC. The basic theoretical consideration in Supercritical fluid Extraction has also been discussed. It also encompasses the basics of Supercritical fluid Chromatography while elaborating various instrumentation aspects. Coupling of SFE with SFC has proved its significance. The utility of SFE-SFC technique has now widened with different advanced technique like SFC- FTIR, SFC-MS, SFC-LC-MS, SFC-GC-MS, SFC-MS-MS, etc. The review covers all these hyphenated techniques and their applications.

The review also discussed wide applications SFE-SFC in diverse fields like pharmaceutical applications like chiral separation, drug delivery system, cosmetics, natural product etc. and other applications such as in food industry, environmental etc.

In nutshell the present review emphasizes on the basics of SFE-SFC, its advancements and its applications in diverse fields of science covering most recent literature.

1. Introduction

Solvent extraction is one of the known methods of separation. However, around 1960 commercial process applications of supercritical fluid extraction (SFE) have been extensively examined. Hannay and Hogarth's in 1879 by observed the dissolution of solutes in supercritical fluid media introduced the possibility of a new solvent medium. In recent years, the supercritical fluid extraction has received special consideration in the fields of solid material extraction . SFE method is very advantageous and environmentally friendly over other conventional methods either solvent or enzyme extraction methods for recovering, for example natural oil. Use of SFE technology offers suitable extraction and fractionation appears to be promising for the food and pharmaceutical industries.( Lili Xu et al.,2011)

Supercritical fluid extraction evolved as an alternative method of sample preparation for analysis. SFE offers many advantages such as distillation, extraction with liquid solvents, or low resolution liquid chromatography. The most unique property of supercritical fluids is "solubilising power" via mechanical compression and additionally via temperature. In practice, SFE can provide an significant savings in time and cost associated with sample preparation.

( Milton L. Lee et al., 1999)

Supercritical fluid extraction is a technique that employs a fluid phase having intermediate properties between a gas and liquid, to effect the solubilisation of solutes. The advantages that are gained by employing SFE can be traced to the distinctive physical properties that these fluids possess. The properties of Supercritical Fluid compared to liquid solvents, supercritical fluids have lower viscosities and higher diffusivities, thus allowing more efficient mass transfer of solutes from sample matrices .Another advantage of supercritical fluids is that the solvent power can be adjusted through mechanical compression of the extraction fluid. This attribute not only permits selective extraction to be accomplished, but allows the concentration of analytes after extraction, free from any contaminating solvent. Appropriate choice of the extraction fluid will also allow the analyst to conduct the extraction at low temperatures, a feature which makes SFE particularly amenable to the treatment of thermally-labile substances.( Jerry W. King And John E. France,1992)

Supercritical Fluid

Supercritical fluid (SF) is defined as a fluid at a temperature and a pressure exceeding its critical values. Most of the SF's become gases and dissipate automatically under ambient conditions. Therefore, the lengthy cost and the potential loss of volatile analytes usually associated with conventional liquid solvent extraction are minimized.( Sheng-Meng Wang, et al.,2003)

The characteristics associated with 'supercritical' fluids were first observed by Baron Cagniard de la Tour in 1822, when he acknowledged that a liquid could be converted into a gas without a phase transition, if the liquid is heated above a specific temperature. Nearly 50 years later (1869), Andrews further developed the idea of a critical point (a 'critical temperature' Tc and a 'critical pressure' Pc) above which only a single phase existed. In this context, the word 'super' is only intended to indicate 'above. At the 'triple point,' solid, liquid, and gas are all present. One line continues nearly vertically and separates the liquid and solid forms of the compound. (Berger T.A., 2007.)

A supercritical fluid is a substance remain where distinct liquid and gas phases do not exist at critical temperature & critical pressure. It can diffuse through solids like a gas, and dissolve materials like a liquid. In addition, close to the critical point, little changes in pressure or temperature result in large changes in density, allowing many properties of a supercritical fluid to be "fine-tuned". Supercritical fluids are suitable as a substitute for organic solvents in a range of industrial and laboratory processes. All supercritical fluids are completely miscible with each other so for a mixture a single phase can be assured if the critical point of the mixture is exceeded. The critical point of a binary mixture can be estimated as the arithmetic mean of the critical temperatures and pressures of the two components, (Wikipedia, the free encyclopedia.)

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

Increasing use of SFC in separation also stems from the valuable properties of supercritical fluid. Compared to the carrier gas of a GC operated at somewhat elevated temperatures, the cold SF of an SFC can more simply and safely solvate thermally labile and/or non-volatile compounds. The analytes eluting from the column outlet are in vapour phase and ready for detection by common detectors such as flame ionization detector and mass spectrometer. The main supercritical solvent used is carbon dioxide. Carbon dioxide (critical conditions = 30.9 â-¦C and 73.8 bar) is cheap, environmental friendly and generally recognized as safe by FDA and EFSA. Supercritical CO2 is also attractive because of its high diffusivity combined with its easily tuneable solvent strength. Another advantage is that CO2 is gaseous at room temperature and pressure, which makes analyte recovery very simple and provides solvent-free analytes. Also, important for food and natural products sample preparation, as the ability of SFE using CO2 to be operated at low temperatures using a non-oxidant medium, which allows the extraction of thermally labile or easily oxidized compounds.( Miguel Herrero et al.,2009 )

At temperatures and pressures lower than 374 0C and 218 atm, subcritical water has widely tunable properties such as dielectric constant, surface tension, viscosity, and dissociation constant achieved by simply adjusting the temperature with a moderate pressure to keep water in the liquid state. At elevated temperatures, water acts like a weak polar organic solvent. Thus, subcritical water has been used as a green eluent to replace hazardous solvents commonly used as organic modifiers in RPLC.( Yang Y., 2007)

Table 1 Critical temperature and pressure and acentric factor for common SCF solvents.( Dana E. Knox, 2005)

Table 2 Critical temperature and pressure and acentric factor for common cosolvents for carbon dioxide.( Dana E. Knox, 2005)

1.2. Properties and cycles of Supercritical Fluid

1.2.1. Heat transfer

Supercritical fluids have large compressibility and density values compared with those of a gas which tends to abnormal behaviour in their hydrodynamic and heat transfer properties. Among the mechanisms for relaxation of a supercritical fluid there is the piston effect, which is a thermal wave that moves through a compressible fluid causing expansion and compression of the hydrodynamic boundary layer. The small variations in piston effect affects the temperature ,convection diffusion effects affect the density; while acoustical effects affect strongly on pressure . (Hiroshi Machida et al., 2011)

1.2.3. Transcritical cycles

Practically much concerns are given over Transcritical cycles, since there is no boiling or condensation during the main heat transfer that occurs.Thermodynamic cycles that use fluids for heat transport in their supercritical state are known as Transcritical cycles. The presence of only a single-phase during heating or cooling means that there is a continuous heat transfer command which gives improved control and efficiency over heat transfer that occurs in two-phase regions.( Hiroshi Machida et al., 2011)

Selection of the Operating Parameters

Selection of the operating parameters depend on the compound to be extraced. Molecular weight and polarity have to be taken into account as per case; but some general rules can be applied. Temperature for thermolabile compounds has to be fixed between 35 and 60 °C in SFE e.g. closed to the critical point and can be prevented from degradation. The raise of temperature reduces the density of Supercritical CO2 (for a fixed pressure) thus reducing the solvent power of the supercritical solvent; but it increases the vapour pressure of the compounds to be extracted. Therefore, the propensity of these compounds to pass in the fluid phase is increased. However, the most relevant process parameter is the extraction pressure that can be used to tune the selectivity of the SF. The common rule is the higher is the pressure, the larger is the solvent power and the smaller is the extraction selectivity. Frequently, the solvent power is described in terms of the Supercritical CO2 density at the given operating conditions. CO2 density can vary from about 0.15 to 1.0 g/cm3 and is connected to both pressure and temperature.( Ernesto Reverchon and Iolanda De Marco, 2006)

Other important parameters to be control in SFE are CO2 flow rate, particle size of the matrix and duration of the process.The proper selection of these parameters has the capacity of producing the complete extraction of the desired compounds in the shorter time. As the parameters are connected to the thermodynamics (solubility) and the kinetics of the extraction process in the specific raw matter.The suitable selection depends on the mechanism that controls the process: the slowest one determines the overall process velocity. CO2 flow rate is a relevant parameter if the process is controlled by an external mass transfer resistance or by equilibrium: the amount of supercritical solvent supply to the extraction vessel, in this case, determines the extraction rate. Particle size plays a determining role in extraction processes controlled by internal mass transfer resistances, since a smaller mean particle size reduces the length of diffusion of the solvent. However, if particles are too small, they can give problems of channelling inside the extraction bed. Part of the solvent flows through channels formed inside the extraction bed and does not get in touch with the material to be extracted thus causing a loss of efficiency and yield of the process. Particles with mean diameters ranging approximately between 0.25 and 2.0 mm are used. The optimum dimension can be chosen case by case considering water content in the matrix and the quantity of extractable liquid compounds that can produce phenomena of coalescence among the particles thus favouring the asymmetrical extraction along the extraction bed. Moreover, the production of very small particles by grinding could produce the loss of volatile compounds. Process duration is interconnected with CO2 flow rate and particle size and has to be properly selected to maximize the yield of the extraction process.( Ernesto Reverchon and Iolanda De Marco, 2006)

2.Supercritical Fluid Chromatography

Supercritical Fluid Chromatography may be defined as a technique that separate the components of a compound or mixture by using supercritical fluid as a mobile phase, which is above and relatively close to its critical temperature and pressure. In this type of chromatography, the use of a supercritical fluid as the mobile phase makes it different from other chromatographic techniques like gas chromatography (GC) and high performance liquid chromatography (HPLC). It is a normal phase chromatography. It can be considered as hybrid of gas and liquid chromatography because when the mobile phase is below its critical temperature and above its critical pressure, it acts as a liquid, and when the mobile phase is above its critical temperature and below its critical pressure, it acts as a gas.( Sethi N. et al., 2010)

Most of the separations are performed using liquid chromatography. One important advantage of using supercritical fluids instead of liquids as solvents is the reduction of use of organic solvents, hence it is significantly less expensive. A high productivity of the supercritical chromatographic process is possible because the low viscosity enables high flow rates at a moderate pressure drop and a high number of theoretical plates can be reached as a consequence of the high diffusion coefficients of supercritical fluids.

The regeneration of SFC is recognized in 1981-1982 with Hewlett-Packard's introduction of SFC instrumentation for packed column SFC.( Larry T. Taylor, 2009)

2.1. Instrumentation Supercritical Fluid Chromatography

The basic components of an analytical-scale SFE-SFC apparatus includes:-

2.1.1. Supercritical Fluid source

High-purity, i.e., SFE grade, Supercritical fluid (SF) contained in aluminium or stainless steel cylinders. Cylinders are usually equipped with a dip tube, a pressurized headspace and a cooling device to warrant stable equilibrium and regular SF delivery.

2.1.2. Pumping system

A high-pressure pump delivers the SF at a regular controlled pressure and flow rate. An additional (optional) pump is used to introduce the modifier. An supreme system would provide a wide range of pressure between 1000 and 10,000 psi along with reproducible and non-pulsating flow-rates between 1.0 (l m/min and 90 ml/min). Most pumps are either syringe or dual piston pumps.

2.1.3. Extraction chamber

Extraction chamber is also called sample vessel or cell and is used to hold the sample while extraction is in progress. The typical volume is from 0.1 to 50 mL. Following figure shows a typical cell consisting of the extractor body and a couple of frit lids along with threaded seal for tight fit. The frit lids prevent the SF from sweeping the solid sample. The cell will withstand high pressure and is fitted with a safety valve to protect the operator in the event of system malfunction. The temperature of the cell is controlled either by placing the cell in a GC oven or a heating tube.




Fluid reservoir




Fig. A typical SFE-SFC cell.

2.1.4. Controller system

It controls the flow rate of the SF and the pressure and temperature of the extraction chamber, ensuring the fluid is entering in the extraction chamber in SF state. More functions, such as the valve switching and extraction time setting etc., are available in commercial systems, providing automatic operation

2.1.5. Collector system

A restrictor is connected between the cell outlet and the collector inlet, maintaining the required pressure in the extraction chamber. The extracts are usually collected using an appropriate solvent.

2.1.6. Supercritical Chromatograph

The construction of an SFC is similar to that of an HPLC, excluding that the past needs additional components to control and maintain the column temperature and pressure. Most of the instruments are currently use packed columns and LC based detectors. This meets the requirements of higher flow rate and the regular use of modifiers with packed columns. The foregoing restrictor or a backpressure device also helps convert the eluting SF into vapor phase and makes the eluents ready for detection. The SF source, pumps and controller are similar to those employed in a SFE. (Sheng-Meng Wang et al., 2003)

2.2. Stationary Phases

Supercritical fluid chromatography has benefited considerably from innovations in column design for liquid chromatography even if the separation conditions employed are generally quite different. The mobile phase composition and column operating conditions play an interactive role in modifying selectivity in supercritical fluid chromatography by altering analyte solubility in the mobile phase and through selective solvation of the stationary phase resulting in a wider range and intensity of intermolecular interactions with the analyte.( Poole C.F, 2012)

The analytical column contains a highly viscous liquid i.e. a stationary phase into which the analytes are adsorbed and then released according to their chemical nature. Due to this some analytes remain longer in the column and thus it allows the separation of the mixture. There are different types of stationary phases available with varying compositions and polarities like absorbents such as alumina, silica or polystyrene or stationary phases insoluble in Supercritical CO2. The recent packed columns consist of bonded non-extractable stationary phases such as octadecylsilyl (C 18) or aminopropyl bonded silica. (Sethi N., et al., 2010)

Craig White et al worked on, Stationary phase used for investigation and evaluation of supercritical fluid chromatography for achiral batch purification, is the 2 ethyl pyridine stationary phase was selected for the achiral SFC optimisation, purchased pre-packed column from Princeton Chromatography with dimensions; 25 cm Ã- 4.6 mm (6 μm particle size), 10 cm Ã- 4.6 mm (5 μm particle size), 10 cm Ã- 3.0 mm (5 μm particle size), 5 cm Ã- 4.6 mm (5 μm particle size), 5 cm Ã- 3.0 mm (5 μm particle size), 5 cm Ã- 4.6 mm (3 μm particle size) and 5 cm Ã- 3.0 mm (3 μm particle size). The CN, premier, silica, diol, diol-HL and 2CN:diol columns with a 5 cm Ã- 4.6 mm diameter (5 μm particle size) were also evaluated for acidic and zwitterionic compounds, also supplied by Princeton Chromatography. The preparative column of choice has dimensions 15 cm Ã- 21.2 mm (5 μm particle size). (Craig White, et al., 2005)

West C et al present the characterization of eleven Hydrophilic Interaction Liquid Chromatography (HILC)-type stationary phases used with carbon dioxide-methanol mobile phases in the isocratic mode. The columns are compared in terms of their retention and separation characteristics assessed by the solvation parameter model, and based on peak shapes. For this purpose, hundred and forty-six low molecular weight molecules, comprising neutral, basic and acidic compounds, were eluted on each column. Data analysis is carried out with hierarchical cluster analysis and principal component analysis in order to define three clusters of columns with similar selectivity: the first cluster comprises neutral stationary phases like amide and diol phases; the second one comprises basic stationary phases like aminopropyl-bonded silica; the last cluster comprises bare silica stationary phases. ( West C. et al., 2012)

Chiral Stationary Phases(CPSs)

2.2.1. Macrocyclic selectors CSP's

2.2.2. Low-molecular weight selectors CSPs

2.2.3. Macromolecular selectors CSPs

2.2.1. Macrocyclic Selectors CSPs

Cyclodextrine and Macrocyclic antibiotics are the examples of Macrocyclic CSPs. Cyclodextrine have the shape of a truncated cone with a relatively hydrophobic interior chiral cavity and a hydrophilic exterior surface surrounded by hydroxyl groups. The presence of hydrophobic cavity enables the entrapment of hydrophobic part of molecules. The secondary hydroxyl groups on the outside allow the selector to interact with analytes via hydrogen-bonding and/or dipole-dipole interaction, leading enantioselective separation.( Larry T. Taylor, 2009)

Ren-Qi Wang et al worked on chemically bonded cationic β- Cyclodextrine derivatives and their applications in SFC. Cationic β-Cyclodextrine (CD) perphenylcarbamoylated derivatives were chemically bonded onto vinylized silica using a radical co-polymerization reaction. The derivative materials were used as chiral stationary phases in SFC. Enantioseparations were successfully demonstrated on 14 racemates encompassing flavanones, thiazides and amino acid derivatives. The electrostatic force between the analytes and the cationic moiety on β-Cyclodextrine derivative was found to be important for retention and enantioseparation of the racemates. Aromatic cation moiety on β-Cyclodextrine enabled better enantioseparation than aliphatic cation moiety. The presence of acid additives would result in lower retention of the analyte but often assist the chiral separation. (Ren-Qi Wang et al., 2012)

Macrocyclic Antibiotic:- the examples of this class stationary phases are vancomycin , teikoplanin, teikoplanin without saccharide component, avoparcin, ristocetin A etc. Due to the numerous active chiral interaction sites within their macromolecular structure, these stationary phases show a wide range-enantioselectivity. They consist of a similar aglycone part containing fused Macrocyclic glycopeptide rings and linked carbohydrates moieties. These stationary phases show good stability because this type of selectors is always borided on the silica, but long equilibration times have been reported as a disadvantage. (Dungelová J. Et al.,2003 ; Michel Perrut, 2004)

2.2.2. Low-Molecular Weight Selectors CSPs

The example of the Low-Molecular Weight Selectors CSPs is Pirkle type. Pirkle type also called as Brush type CSPs. Brush-type or Pirkle-type stationary phases were the first chiral stationary phases reported in the literature . These stationary phases are practically designed to target specific chiral interactions with an analyte. Structurally they consist of single strands with either Π-donor or Π-acceptor aromatic fragments, as well as hydrogen bonding agents and dipole-stacking inducing functional groups that are covalently bonded onto a silica surface. Enantioselectivity on these CSPs arises from a three-point interaction between the analyte and chiral selector which forms a labile diastereomeric complex with one enantiomer, while the other enantiomer forms a diastereomeric complex through a two-point interaction. This will result in different complex stabilities, different retentions and, hence enantioseparation. This type of stationary phases shows the advantage of having an excellent physical stability under relatively extreme pH condition.( Michel Perrut, 2004)

2.2.3. Macromolecular Selectors CSPs

Polysaccharide and synthetic polymers are the examples of macromolecular selector CSPs. Polysaccharide based CSPs are most successful CSPs and have taken a dominant position in chiral SFC because of their easy accessibility and broad enantioselectivity. Underivatized polysaccharides, such as cellulose and amylose, only show limited enantioselectivity, since their helical structure is too dense to allow inclusion and enantiorecognition of many molecules. (Katrijn De Klerck et al,2012)

Chiral bigotry can happen from inclusion interaction inside the polysaccharide helical structure, aromatic functional groups may undergo π-π interaction with the selector , and hydrogen binding may add to enantioselectivity. Synthetic polymer-based CSPs are developed by the polymerization of single chiral monomers to a three dimensional polymer network. The limited stability of these CSPs under high pressure makes them more suitable for SFC than the HPLC applications. (Patrick J. Arpinoa et al., 1995; Reverchon E. Et al., 2007)

2.3. Column

2.3.1. Packed Column

Many researchers have expected a new chromatographic technique that overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development, chromatography plays an central role in the evaluation of safety and efficacy of a new compound.

Koji Yaku et al provides an overview of the separation of drugs by Packed-column supercritical fluid chromatography. The effects of the chromatographic parameters were studied for the separation of steroids. In chiral separation, the successful results were shown and compared with HPLC.( Koji Yaku et al., 2000)

It uses the identical injector and packed column configurations as in HPLC. It is more robust and more adaptable to a broader spectrum of compound classes than just low molecular weight polymeric compounds and non-ionic surfactants. Teething troubles with back-pressure regulation, consistent flow rates, modifier addition, sample injection, automation, stationary phases, etc. have been determined. Furthermore, the hyphenation of packed column SFC to mass spectrometry and ultraviolet detectors is experimentally more straightforward than for open tubular column SFC. Chromatographers readily agree that SFC is normal phase chromatography without most of the problems usually associated with normal phase HPLC. Composition, pressure, and temperature can all be programmed with rapid recovery to initial conditions between analyses. Traces of water do not cause the variations in retention often encountered in normal phase HPLC. The advantages of packed column SFC relative to more seemingly mature HPLC methodologies are clear: (a) lower viscosity and higher diffusivity of supercritical mobile phases relative to liquids which lead to both faster, more efficient separations per unit time and shorter turn-around time between injections, (b) an inert, environmentally "green" and more volatile carbon dioxide-based mobile phase for large scale separations and energy efficient isolation of the desired product, (c) longer, stacked columns with the same or multiple phases with total theoretical plates in excess of 100,000, (d) selectivity that matches reversed phase HPLC, but it is more easily adjustable , and (e) HPLC applications can be run on SFC instrumentation. When working with water-soluble compounds, however, reversed phase HPLC remains the first choice. But for certain applications such as chiral separations and high-throughput screening, SFC has clear advantages.( Larry T. Taylor, 2009)

Phinney KW shows the Enantioselective separations by packed column subcritical and supercritical fluid chromatography. Enantioselective separations have been one of the most flourishing applications of supercritical fluid chromatography (SFC). Although analytical scale separations have dominated the literature, the use of SFC for preparative chiral separations is rising. Both analytical and preparative scale SFC separations seek to take advantage of the high efficiency, high throughput, and rapid method development associated with the technique. (Phinney K.W., 2005)

2.3.2. Capillary Column

Capillary column is the novel combination of SF mobile phases and open tubular column SFC. Early greater emphasis was placed on open tubular columns during that time than on packed columns. Capillary SFC experienced an explosive growth mainly due to the novel combination of supercritical mobile phases and open tubular fused silica column technology. The wide acceptance of open tubular column SFC at this point, unfortunately, did not reduce the heated exchanges between open tubular SFC users and packed column SFC users. Column efficiency was noted to markedly decrease with carbon dioxide density programming because flow varied with pressure and temperature using fixed restrictors which continues to be the norm even today. Nevertheless, many fantastic separations were reported employing capillary columns. Many such applications were unique, with no other practicable solution. ( Larry T. Taylor, 2009)

3. Advancements In SFE-SFC Technique

Following its rediscovery during the early 1980s, most of the work initially done in the field of supercritical fluid chromatography was concentrated on the use of capillary columns. The expectation of high efficiency combined with low volumetric flow-rates which should allow for an easy interfacing to most of the common gas chromatography (GC) detectors and, in particular, also to mass spectrometry (MS) rapidly led to the development of commercially available capillary SFC equipment. The most common approach to interface SFC to MS consisted in the direct insertion of a restrictor, fixed to the end of a capillary column, into the ion source of a mass spectrometer operating in electron impact (EI) or chemical ionisation (CI) mode. (Michael T. Combs et al., 1997)

The packed column supercritical fluid chromatography (SFC) coupled directly with mass spectrometry (MS) using either analytical scale or microbore columns that have typically been used for liquid chromatography analysis. Techniques and applications of analytical instruments combining a chromatographic technique, including liquid chromatography and supercritical fluid chromatography, with mass spectrometry (LC/MS and SFC/MS). It is shown that still many different methods co-exist and have both definite advantages and limitations. SFC/MS appears easier to run for many compounds so far analyzed by conventional LC/MS methods. (Andreas Seubert and Ralf Meinke, 1994)

In the previous years, supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) has had a great advance in productivity, due to increase in reliability and robustness of both SFC and MS systems, the need to push drug discovery faster, and as a potential solution to old problems unsolved and new problems yet to be encountered.( Maria Espinosa Bosch et al., 2009)

Christine Aurigemma et al studied the Fast Track to supercritical fluid chromatographic purification: Implementation of a walk-up analytical supercritical fluid chromatography/mass spectrometry screening system in the medicinal chemistry laboratory. To obtain pure compounds for biological assays, the removal of side products and final compounds through purification is often necessary. Prior to purification, chemists often utilize open-access analytical LC/MS instruments because mass confirmation is fast and reliable, and the chromatographic separation of most sample constituents is satisfactory. SFC is often used as an orthogonal technique to HPLC or when isolation of the free base of a compound is desired. In laboratories where SFC is the predominant technique for analysis and purification of compounds, a reasonable approach for speedily determining suitable purification conditions is to screen the sample against different columns. This can be a bottleneck to the purification process. To commission SFC for open-access use, a walk-up analytical SFC/MS screening system was implemented in the medicinal chemistry laboratory.( Christine Aurigemma et al., 2010)

Takeshi Bamba et al worked on high throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics. They developed an analytical system that enables the simultaneous rapid analysis of lipids with varied structures and polarities through the use of supercritical fluid chromatography-mass spectrometry (SFC-MS). The separation conditions for SFC (column, modifier, back pressure, etc.) and the detection conditions for mass spectrometry (ionization method, parameters, etc.) were investigated to develop a simultaneous analytical method for lipid mixtures that included phospholipids, glycolipids, neutral lipids, and sphingolipids.( Takeshi Bamba et al., 2008)

Wen-Lin Liua et al worked on Headspace solid phase microextraction in-situ supercritical fluid extraction coupled to gas chromatography-tandem mass spectrometry for simultaneous determination of perfluorocarboxylic acids in sediments. Headspace solid phase microextraction (HS-SPME) in-situ supercritical fluid extraction (SFE) was investigated for the determination of