Supercritical fluid chromatography

The following is an article obtained from the website http://www.pharmainfo.netreviews/super-critical-fluid-chromatography-fundamentals-and-applications.html and is a fairly simple and easy-to understand explanation of supercritical fluid chromatography. The references have not been included here and the interested students can go to the above web page to view the references ( labeled as 1,2 etc. throughout the article)

When supercritical fluid chromatography was first developed in 1960, it was considered as a science fiction chromatography and a revolutionary separation technique. But its reputation has slowly ebbed over the years and it is now moving forward by leaps and bounds as a stable analytical method with many advantages over the existing chromatographic methods.

Supercritical fluid chromatography is more versatile than high performance liquid chromatography, more cost-efficient, user friendly, with higher throughput, better resolution and faster analysis times than general liquid chromatographic methods. The instrumentation that is required for supercritical fluid chromatography is versatile because of its multi-detector compatibility. Due to this, supercritical fluid chromatography has formed a niche in the pharmaceutical industry. The present article reviews the fundamentals, instrumentation and varied applications of supercritical fluid chromatography in the analytical arena.

Introduction

The phenomenon and behavior of supercritical fluid (SCF) has been the subject of research right from 1800’s. Hanny and Hogarth in 1879 first demonstrated solubility in (SCF) but first suggestion of supercritical fluid chromatography (SFC) was put forward in 1958 by Lovelock. In 1962 Klesper Corvin and Turner used SFC for separation of porphyrins. Giddings in 1966 and sie rijender in 1967 were responsible for further developments of SFC. jentoft and gouw in 1972 successfully carried out analysis of petroleum derived mixture by SFC. Novotny and Lee et al. demonstrated the first experiments on capillary SFC in 1982. The first commercial packed column of SFC was made available in 1981 and the first commercial capillary column SFC instrument was introduced in 1985 ¹ .

Super Critical fluids: Fundamentals and Properties

Supercritical fluid may be defined from a phase diagram for a pure substance (Fig.1), in which the regions corresponding to solid, liquid and gaseous state are clear. A substance such as CO ₂ can exist in solid, liquid and gaseous phases under various combinations of temperature and pressure. For every substance there is a temperature above which it can no longer exist as a liquid, no matter how much pressure is applied. Likewise, there is a pressure above which the substance can no longer exist as a gas no matter how high the temperature is raised. These points are called critical temperature and critical pressure respectively ² and are the defining boundaries on a phase diagram for a pure substance. At this point, the liquid and vapour have the same density and the fluid cannot be liquefied by increasing the pressure. Above this point, where no phase change occurs, the substance acts as a supercritical fluid. So SCF can be described as a fluid obtained by heating above the critical temperature and compressing above the critical pressure ³ . There is a continuous transition from liquid to SCF by increasing temperature at constant pressure or from gas to SCF by increasing pressure at constant temperature. The term, compressed liquid is used frequently to describe a supercritical fluid, a near critical fluid, an expanded liquid or a highly compressed gas ⁴ .

Fig. 1: Phase Diagram for Pure Substance ⁴

Important Properties of Super CRITICAL fluids ⁵

SCFs have high densities (0.2-0.5gm/cm ³ ) due to which they have a remarkable ability to dissolve large, non-volatile molecules, for example, SC - CO ₂ readily dissolves n-alkanes containing 5 to 30 carbon atoms, di-n-alkyl phthalates with dialkyl group containing 4-16 carbon atoms and several polycyclic and aromatic compounds with many rings. Solvation strength of SCF is directly related to the fluid density. Thus solubility of solid can be manipulated by making slight changes in temperatures and pressures. Certain important processes are based upon the high solubility of organic species in SC -CO ₂ , for example; it has been employed for extracting caffeine from coffee beans to get decaffeinated coffee and for extracting nicotine from cigarette tobacco.

A second important property of SCFs is that dissolved analytes can be easily recovered by simply allowing the solutions to equilibrate with the atmosphere at low temperatures, for example an analyte dissolved in the SC- CO ₂ can be recovered by simply reducing the pressure and allowing to evaporate under ambient laboratory conditions. This property is particularly useful with thermally unstable analytes.

Another advantage of many SCFs is that they are inexpensive, innocuous, ecofriendly and non-toxic. With SCFs at hand, there is no need of any organic solvents. Finally SCFs have the advantage of higher diffusion constants and lower viscosities relative to liquid solvents. The low viscosity means that pressure drop across the column for a given flow rate is greatly reduced. The greater diffusibility means longer column length can be used. Higher diffusion coefficient means higher analysis speed that increases in the order HPLC, SFC and GC. These advantages are important in both, chromatography and extractions with SCFs.

SCFs are finding applications in fractionation of low vapour pressure oils, in several reactions in different areas of biochemistry, polymer chemistry, environmental sciences as well as food, polymer and material industries ⁵ .

Table I: SCFs have densities, viscosities and other properties that are intermediate between those of a substance in gaseous and liquid state ⁶

Property	Gas (STP)	SCF	Liquid
Density (g/cm 3 )	(0.6-2) x 10 -3	0.2-0.5	0.6-2
Diffusion coefficient (cm 2 /s)	(1-4) x 10 -1	10 -3 x 10 - 4	(0.2-2) x 10 -5
Viscosity (G Cm -1 s -1 )	(1-4) x 10 - 4	(1-3) x 10 - 4	(0.2-3) x 10 -2

Table II: Critical properties of some commonly used SCFs ^{7, 8}

Fluid	Critical Temperature (K)	Critical Pressure (bar)
Carbon dioxide	304.1	73.8
Ethane	305.4	48.8
Ethylene	282.4	50.4
Propane	369.8	42.5
Propylene	364.9	46.0
Trimethoflurane	299.3	48.6
Chlorotrifluoromethane	302.0	38.7
Trichloromethane	471.2	44.1
Ammonia	405.5	113.5
Water	647.3	221.2
Cyclohexane	553.5	40.7
n-Pentane	469.7	33.7
Toluene	591.8	41.0

The two supercritical fluids of particular interest are , carbon dioxide and water.

Carbon dioxide ⁹

It is a non-flammable, nontoxic and ecofriendly solvent with low critical temperature of 304K and moderate critical pressure of 73bar.It is miscible with variety of organic solvents and is readily recovered after processing. As it’s a small and linear molecule, it diffuses faster than conventional liquid solvents. It is often used to replace freons and certain organic solvents.

Water ⁹

It has a critical temperature of 647K and critical pressure of 220bar due to its high polarity. The character of water at supercritical conditions changes from one that supports only ionic species at ambient conditions to one that dissolves paraffins, aromatics, gases and salts. Due to this unique property, research has been carried out on supercritical water for reaction and separation processes to treat toxic wastewater. Control of reactions that depend on the dielectric constant of a medium is also possible in supercritical water as its dielectric constant changes from about 78 at room temperature and atmospheric pressure to roughly 6 at critical conditions.

The final choice of SCF depends on the specific application as well as other factors like safety, flammability, phase behavior, solubility at operating conditions and cost of fluid.

Supercritical Fluid Chromatography

Chromatography is an analytical technique used for the separation of complex chemical mixtures into individual components. In SFC, the sample is carried through a separating column by a supercritical fluid where the mixture is divided into unique bands based on the amount of interaction between the individual analytes and the stationary phase in the column. As these bands leave the column, their identities and quantities are determined by a detector ¹⁰ .

SFC is a 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, so the technique is liquid chromatography (LC) and when the mobile phase is above its critical temperature and below its critical pressure, it acts as a gas so the technique is gas chromatography (GC) ¹¹ . Thus SFC combines some of the best features of each, LC as well as GC. SFC is important because it permits separation and determination of group of compounds that are not conveniently handled by either GC or LC. For example, GC is inapplicable for nonvolatile or thermally unstable compounds. Similarly, LC cannot be employed for compounds with those functional groups that cannot be detected by either spectroscopic or electrochemical detectors used in LC.SFC is a relatively recent chromatographic technique and there is a large amount of research currently underway both in SFC method development and in hardware development.

SFC instrumentation ^{10, 12, 13}

The instrumentation of SFC is similar in most regards to instrumentation for HPLC because the pressure and temperature required for creating supercritical fluid from several gases or liquids lie well within the operating limits of HPLC equipment However, there are two main differences between the two. First, a thermostated oven similar to that of GC, is required to provide precise temperature control of the mobile phase and second, a restrictor or a back pressure device to maintain the pressure in the column at a desired level and to convert the eluent from SCF to a gas for transfer to detector ⁷ .

Fig. II Flow Diagram of Construction of SFC Instrument ¹⁰

In SFC, the mobile phase is initially pumped as a liquid and is brought into the supercritical region by heating it above its supercritical temperature before it enters the analytical column. It passes through an injection valve where the sample is introduced into the supercritical stream and then into the analytical column. It is maintained supercritical as it passes through the column into the detector by a pressure restrictor placed either after the detector or at the end of the column.

Pumps

In contract to HPLC pumping system, pressure rather than flow control is necessary and pulseless operation is more critical. In general, the type of high-pressure pump used in SFC is determined by the column type. For packed columns, reciprocating pumps are generally used while for capillary SFC, syringe pumps are most commonly employed. Reciprocating pumps allow easier mixing of the mobile phase or introduction of modifier fluids. Syringe pumps provide consistent pressure for a neat mobile phase.

Injector

Injection in SFC is usually achieved by switching of the content of a sample loop into the carrier fluid at the column entrance by means of a suitable valve. For packed column SFC, a conventional HPLC injection system is adequate, but for the capillary column SFC, the sample volume depends on column diameters and small sample volumes must be quickly injected into the column, therefore pneumatically driven valves are used.

Oven

A thermostated column oven is required for precise temperature control of the mobile phase. Conventional GC or LC ovens are generally used.

Columns

The strong solvating abilities of mobile phase in SFC makes the careful selection of stationary phases imperative. Basically two types of analytical columns are used in SFC, packed and capillary. Earlier work employed absorbents such as alumna, silica or polystyrene or stationary phases insoluble in SC -CO ₂ . More recent packed column work has involved bonded non-extractable stationary phases such as octadecylsilyl (C ₁₈ ) or aminopropyl bonded silica.

Restrictor or Back-Pressure Device

This is a device, which is used to maintain desired pressure in the column by a pressure-adjustable diaphragm or controlled nozzle so that the same column-outlet pressure is maintained irrespective of the mobile phase pump flow rate. It keeps the mobile phase supercritical throughout the separation and often must be heated to prevent clogging. The pressure restrictor is placed either after the detector or at the end of the column.

A typical restrictor for a 50 or 100 µm open tubular column consist of a 2-10 cm length of 5-19 capillary tubing attached to the column. Alternately the restriction may be integral part of the column formed by drawing down the end of the column in the flame.

Microprocessor

The commercial instruments for SFC are ordinarily equipped with one or more microprocessors to control such variables as pumping pressures, oven temperature and detector performance.

Detector

SFC utilizes mobile phases, which can either be liquid like or gas like. Therefore it is compatible with both HPLC and GC detectors. Conventional gas-phase detectors such as flame ionization detectors and flame photometric detectors, liquid-phase detectors like refractive index detectors, ultraviolet-visible spectrophotometric detectors and light scattering detectors have been employed for SFC. Mass spectrometry and fourier transform infrared spectrometry can also be used effectively with SFC.The choice of detectors will depend upon the mobile phase composition, column type, flow rate and ability to withstand the high pressures of SFC.

Effect of Pressure

Part of the theory of separation in SFC is based on the density of the supercritical fluid which corresponds to solvating power. As the pressure in the system is increased, the density of the supercritical fluid increases and correspondingly its solvating power increases. This in turn shortens the elution time for the eluent as pressure changes in SFC have a pronounced effect on the retention of analytes. This effect is general and similar to programmed temperature in GC or gradient elution in HPLC.

Mobile Phase

There are a number of possible fluids, which may be used in SFC as a mobile phase. However, based on its low cost, low interference with chromatographic detectors and good physical properties (nontoxic, nonflammable, low critical values) CO ₂ is the most used mobile phase for SFC. It is an excellent solvent for a variety of nonpolar organic molecules. In addition, it transmits in the UV. It permits a wide selection of temperatures and pressures without exceeding the operating limits of modern HPLC equipments.

Modifiers ^{6, 9, 14}

CO ₂ is not a very good solvent for high molecular weight, ionic and polar analytes. This can be overcome by adding a small portion of a second fluid called modifier fluid. This is generally an organic solvent, which is completely miscible with carbon dioxide (alcohols, cyclic ethers) but can almost be any liquid including water. Therefore in some applications methanol is introduced in small concentrations (1-20 mol%) to modify solvation power of CO ₂ . Including chemical additives like acids and bases in the modifier can further enhance the solubility. Modifiers can also enhance selectivity of separation and improve separation efficiency by blocking some of the highly active sites on the stationary phase. Small amount (3.5%)of methanol to CO ₂ increases solubility of cholesterol. If an analyte is only soluble in an aqueous solution, it is probably a poor candidate for SFC. Apart from methanol other solvents are also used as modifiers like acetonitrile, ethanol and1-propanol. For highly retained nonpolar solutes, modifiers increase the column efficiency. For polar solutes, they improve retention and efficiency, both.

Comparison of SFC with Other Types of Chromatography

SFC combines some of the characteristics of gas and liquid chromatography, as several physical properties of SCF are intermediate between gases and liquids. Like GC, SFC is inherently faster than LC because the lower viscosity makes use of higher flow rates. Diffusion rates in SCFs are intermediate between gases and liquids. As a consequence, band broadening is greater in SCFs but less, than in gases. Thus, the intermediate diffusivities and viscosities of SCFs result in faster separation than is achieved in LC, accompanied by lower zone broadening than is encountered in GC ¹⁰ .

The mobile phases play different role in GC, LC and SCF. In GC, the mobile phase causes the zone movement. In LC, the mobile phase transports the solute molecule and also interacts with them thus influencing the selectivity. When a molecule dissolves in supercritical medium, the process resembles volatilization but at much lower temperature than that of GC. Thus, at a given temperature the vapor pressure for a large molecule in SCF may be 10 ¹⁰ greater than in the absence of that fluid. As a consequence, high molecular weight compounds, thermally unstable spieces, polymers and large biological molecules can be eluted from a column at a reasonably low temperature. The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds. This is appreciated in the pharmaceutical fields since roughly 20% of all drugs candidates fall in this category. Unlike GC, by changing the mobile phase the selectivity can be varied in SFC ¹⁵ .

Due to the thermally unstable or non- –volatile nature of many nitrogen and / or sulfur containing compounds, they cannot be analyzed by GC. Even if HPLC is applicable to analyze these compounds, it generates a large number of organic solvents, which need to be ultimately disposed. The disposal cost of organic solvents typically ranges from $5 to $10 per gallon and is constantly rising due to the strict environmental regulations. With the desire for environmentally conscious technology, the use of organic chemicals as used in HPLC could be reduced with the use of SFC. Because SFC generally uses carbon dioxide, collected as a byproduct of other chemical reactions or is collected directly from the atmosphere, it contributes no new chemicals to the environment ¹⁶ .

Like GC, SFC is inherently faster than HPLC, because of its lower viscosity and higher diffusion rates. It is well documented that SFC provides a combination of 3-5 times increase in the speed of analysis and a decrease in the analysis cost through saving in organic solvent ¹⁷ .

Unlike GC or HPLC where the mobile phase dominates the type of detector to be used, SFC utilizes mobile phase, which can be either liquid like or gas like. Therefore both GC and HPLC detectors are applicable to SFC. This multidetector compatibility makes SFC a technique of unparallel success in the analysis of thermally liable species and/or relatively high molecular weight compounds.

Supercritical fluid chromatography has several main advantages over conventional chromatographic techniques (GC and HPLC). The biggest advantage that SFC has over HPLC lies within the differences in the mobile phases. Supercritical fluids are less viscous, possess a higher diffusivity than liquids under HPLC conditions and allow lower pressure drops along an analytical column. This provides not only the ability to increase column lengths, but also allows for faster flow rates. These factors in turn affect capacity ratios, selectivities and theoretical plate heights. It has been reported that 200,000 theoretical plates have been achieved by using eleven analytical (4.6mm i.d.) columns in series. Additionally, SFC can be set up for sub ambient temperatures, which has been key in many chiral separations ¹⁷ .

Applications

By now SFC has been applied to wide variety of materials ¹⁸ including natural products, drugs, foods, pesticides, herbicides, surfactants, polymers and polymer additives, fossils fuels, petroleum, explosives and propellants. Some of the important applications are as follows.

Natural Products

Lipophilic – amphiphilic compounds with properties between volatiles and hydrophilic compounds often create problems in connection with their isolation and analytical determination resulting in an analytical gray area, But SFC has been found to give relatively fast and simple procedures for determination of oil constituents such as chlorophyll and its derivatives ¹⁹ , carotenoids, tocopherols ^{20, 21} vitamins ^22,23 and phenolics ²⁴ which may be important for the oil quality. Thereby it gives a tool to determine the origin of oil and improved possibilities of determination of relations between oil constituents and physical as well as biochemical properties of oil.

Separation of bile salts ²⁵ and common free bile acids like ursodeoxycholic acid and chenodeoxycholic acid in pharmaceutical preparations has been reported using phenylbonded silica column and SFC-CO ₂ modified with methanol ²⁶ .

SFC has been successfully utilized for the separation of underivatized triterpene acids ²⁷ , estimation of caffeine from tea ²⁸ and conjugated bile acids ²⁹ . Capillary-SFC has been used for analysis of panaxadiol / panaxatriol in ginseng and its preparations ³⁰ , vegetable carotenoids ³¹ and pyrrolizidine alkaliods ³² .

Pesticides

Supercritical fluid extraction and chromatography has been used for the analysis of pesticide residues in canned foods, fruits and vegetables wherein pyrethroids, herbicides, fungicides and carbamates have been tested ³³ .

Surfactants

Separation of the oligomers in a sample of the nonionic surfactant Triton X100 has been reported where the detection was by measuring the total ion current produced by the chemical ionization mass spectrometer ³⁴ .

Lipids

As SFC operates at low to moderate temperature, it is most suited for the analysis of high molecular weight lipids like triacylglycerols. Even though HPLC methods give excellent resolutions, the elution times are relatively long and quantitative detection becomes a problem. With GC there is a possibility if thermal cracking of stationary phase or of the sample. Separation of paraffin wax, free fatty acid, mono-di-and tri acyl glycerol detergents like Triton X-100 has been achieved using a capillary column coated with a nonpolar stationary phase at a temperature between 60°-120° at which no thermal damage to lipids is observed ^3.

SFC has also been applied to analyze phospholipids after conversion to diacylglycerol derivatives ³⁵ . Separation of fatty acid methyl esters ³⁶ , biosynthetic polyunsaturated fatty acids (PUFA) ³⁷ , nonsaponifiable lipids ³⁸ , cholesterol and its esters in human serum ^39,40 and food samples ⁴¹ , mono-, di- and triglycerides in pharmaceutical excipients ⁴² has been carried out by SFC successfully. SFC has also been applied to analysis of archaebacterial lipids and glycosphingolipids ⁴³ . A number of review articles have appeared in recent years on this topic ^{44, 45, 46} and should be consulted for more detailed information.

Polymers

SFC provides elution of high molecular weight compounds, polymers and large biological molecules from a column at a reasonably low temperature. Separation of the series of dimethyl polysiloxane oligomers ⁴⁷ and polycyclics aromatic hydrocarbon extracted from carbon black using fluorescence detection ⁴⁸ has been reported. SFC has also been applied to analysis of polyethoxylated alkylphenols ⁴⁹ , polyolefinic antioxidants /light stabilizers ⁵⁰ and polynuclear aromatic hydrocarbons in automobile exhaust ⁵¹ .

Drugs

Modern drug substances are commonly nonvolatile and thermally or chemically labile therefore analysis by HPLC is common over GC. In SFC the conditions are mild and no volatilization is required so it is possible to handle such drug substances by SFC. Separation of various categories of drugs like antidepressants ⁵² , phenothiazine antipscychotics ⁵³ , beta blockers ⁵⁴ , felodipine ⁵⁵ , a new dihydropyridine drug-clevidipine ⁵⁶ , methylated betacyclodextrins ⁵⁷ , vasodialators ⁵⁸ like isosorbide mononitrate, isosorbide dinitrate, cyclandelate, nimodipine, amlodipine, pentifylline,pentoxifylline, lovastatin ⁵⁹ ,pyrethrins ⁶⁰ , isosorbide –5- mononitrate and related compounds in bulk substances and tablets ⁶¹ , atropine ⁶² , indol-3-yl methyl oligomers and ascorbigens ⁶³ , tolnaftate and related impurities ⁶⁴ have been carried out by SFC. Separation and trace estimation of benzidine and its macromolecular adducts ⁶⁵ , fusarium mycotoxins ⁶⁶ , oestrogens ⁶⁷ , combinations of various nonsteroidal antiinflammatory drugs ⁶⁸ like flufenamic acid, mefenamic acid, fenbufen, indomethacin mixtures, flufenamic acid, mefenamic acid, acetyl salicylic acid, ketoprofen and fenbufen mixtures and mixtures of ibuprofen,fenoprofen, naproxen, ketoprofen and fenbufen by SFC has been reported. SFC has also been applied for estimation of prostaglandins ⁶⁹ , determination of mefloquine in blood ⁷⁰ , anticancer drugs like cyclophosphamide, diaziquone, mitomycin C, thiotepa ⁷¹ , sorbitan trioleate in metered-dose inhalers ⁷² , steroids ⁷³ , [14C] propranolol ⁷⁴ , underivatized 2,4-dichlorophenoxy acetic acid ⁷⁵ , determination of urinary metabolites of styrene ⁷⁶ , high speed screening of combinatorial libraries ⁷⁷ , determination of phenylbutazone and its major metabolite oxyphenbutazone in serum, phenylbutazone in dosage forms ⁷⁸ and sulphadoxin in blood plasma ⁷⁹ , mesoprostol from tablets ⁸⁰ , simultaneous SFC of ibuprofen and methocarbomal in solid dosage form. ⁸¹

Chiral compounds

Chiral separation by SFC was first documented in 1985 ⁸² . Due to the high efficiency, fast separation, low temperature analysis and applicability to wide variety of detectors, SFC has now become an attractive alternative for chiral drug separation ⁸³ . The success of SFC in the field of bioanalytical chemistry is well documented. A real break through for SFC in the bioanalytical field has been its contribution to chiral separations and in the near future it may surpass HPLC in the ability to provide appreciable selectivity of molecular stereoisomers ⁴ . SFC has been applied to separation of a large number of enantiomers, diasterioisomers and geometrical isomers like achiral and chiral analysis of camazepam and its metabolites ⁸⁴ , diasterioisomers of Du P105- a novel oxazolidinone antibacterial agent ⁸⁵ , chiral separation of 1,3 dioxolane derivatives ^{86, 87} , diasterioisomers of 2-bromomethyl-2- [(2,4-dichlorophenyl)-1,3-dioxolan-4-yl] methyl benzoate ⁸⁸ , enantiomers of ibuprofen ⁸⁹ , chiral antifungal agents ⁹⁰ , enantiomeric separation of aminoalcohols ⁹¹ , triadimefon and triadimenol enantiomers anddiasterioisomers ⁹² , albendazole sulfoxide enantiomers ^{93, 94} , chiral separation of drugs based on macrocyclic antibiotics ⁹⁵ , separation of cis and trans beta carotene enantiomers ⁹⁶ and resolution of D- and L- alpha amino acid derivatives ⁹⁷ , enetiomeric seperation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole, and tetraconazole ⁹⁸ , enetiomeric seperation of racemic mixtures of five acidic drugs namely dichlorprop , ketoprofen ,warfarin, coumachlor and thalidomide using macrocyclic antibiotic chiral stationary phases (CSPs) ⁹⁹ .

Organometallics

Separation of metal chelates and organometals of thermally labile category, chelates of transition metals, heavy metals, lanthenides and actinides as well as organometallic compounds of lead, mercury and tin has been carried out by SFC. Determination of solubility of organometallic compounds by SFC is also reported. ¹⁰⁰ .

SFC-MS in Pharmaceuticals

In pharmaceutical industry, analyte concentrations in the picogram -per-milliliter or lower range are a commonplace. In order to detect the realistic concentration levels, a detector with highest sensitivity, broadest selectivity and best resolution must be used. Currently, the detector that fits all of these criteria is the mass spectrometer.

MS vs. Other Available Detectors for SFC ¹⁰¹

The pressure requirements for packed column supercritical fluid chromatography (pSFC) significantly limit the type of detector that can be used successfully. LC detectors need to have pressure tolerances of upto 400 bar. This upper limit is possible with special pressure-resistant absorbance detector cells, but not with the orthogonal window cell designs found in fluorimeters. GC detectors that have been used with both capillary supercritical fluid chromatography (cSFC) and pSFC include, mass spectrometers, flame ionization (FID), electron capture, nitrogen-phosphorus and phosphorus sensitive detectors, just to name a few. In all, these detectors give a wide variety of sensitive selectivity, but each in itself can be restricted in application. The only truly universal detectors available to SFC are FID and MS. FID will produce high background noise in the presence of SFC polar modifiers, concluding that the practical universal detector of choice should be MS.

Some of the applications of SFC-MS include -

Separation of avermectines, a group of potent broad spectrum antiparasitic agents by chemical ionization SFC-MS, determination of impurity profile of macrolid antibiotics using SFC–EIMS and of erythromycin-A from fermentation broth using SFC–CIMS, separation of distereoisomers of cephalosporins using SFC– EIMS, Separation of mixture of 6 pesticides namely atrazine, simazine, dimethirimol, terbacil, manazon, and ethirimol by packed column SFC-EIMS ^{102 ,} Separation of the mixture of 7 triglycerides namely trilaurin, trimyristin, tripalmitin, tristearin, triolein, trilinolien and trilinolenin, using cSFC-EIMS ¹⁰³ , identification of milk fat triacyl glycerols ¹⁰⁴ , Separation of gamma- and alpha – lenolenic acid containing triacyl glycerols in berry oils ¹⁰⁵ , analysis of beta agonists ¹⁰⁶ , characterization of triglycerides in vegetable oils ¹⁰⁷ , analysis of long chain

polyprenols ¹⁰⁸ , analysis of artemisinin ¹⁰⁹ ,determination of allicin in garlic extracts ¹¹⁰ , estimation of trichothecenes ¹¹¹ , characterization of N-linked glycans ¹¹² , characterization of glycosphingolipids by SFC-MS ^113, analysis of organic metallic containing iron, silicon, tin, chromium, arsenic, lead, mercury and antimony using plasma spectrometric detection and inductively coupled plasma mass spectrometry (ICPMS) interfacing SFC ¹¹⁴ , separation of mixture of sulphonamides ^115. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interference from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than in UV and much shorter analysis times compared to normal phase/reversed phase liquid chromatography ¹¹⁶ .

Conclusion

Lately SFC has found a niche in the field of pharmaceutical chemistry and has gained much support in the field of bioanalytical applications. In the overall ranking of chromatographic techniques, it has been judged that SFC falls somewhere between HPLC and GC as the chromatographic method of choice. There are too many examples in the literature testifying to the practicality of using SFC to separate specific compounds. The list of compounds separated by SFC is increasing day by day. SFC enjoys many advantages over the existing chromatographic techniques. But the most important contribution that SFC has made is towards separation of chiral compounds. SFC is enjoying great success in meeting the challenges of stereoisomer separation and may have already surpassed HPLC in the ability to provide appreciable selectivity of molecular stereoisomers. The biggest advantage that SFC holds over GC is the ability to separate thermally labile compounds, which is a very significant application in the pharmaceutical field as 20% of all drug candidates fall in this category. With the advent of SFC-MS, even picogram per milliliter concentrations can be detected easily which is not possible with other techniques. The enforcement of strict quality standards has produced a need for fast, complete and sensitive analysis of drug candidate. SFC can provide the fast and complete analysis and MS can provide universal, sensitive detection. SFC-MS shows great potential in the field of bioanalytical chemistry, but especially in chiral separation and detection. As the science advances, it would be reasonable to foresee the practicality of this analytical technique reach into mainstream of analytical chemistry.