Chromatography supercritical-fluid

Chromatography with a supercritical fluid as mobile phase was first reported more than twenty years ago [1], although only in recent years have its advantages been realized. These are especially relevant to oligomers, polymers, and polymer additives, and supercritical fluid chromatography (SFC) is finding increasing use in analyses in polymer chemistry. 

The range of molecular masses (MM) which may be eluted in SEC is virtually unequalled, but the efficiency of separation is inevitably low because of the proportionality between retention volume and the logarithm of MM. The separating power of gas chromatography (GC) with capillary columns is unparalleled, but the limited volatility and thermal stability of polymers severely limits its application. SFC allows the high-resolution separation of high-MM solutes at temperatures well below those of thermal decomposition. 

Above the critical point (3 PC and 73 bar for carbon dioxide) a substance has remarkable properties it has a high dissolving power, which can be varied by changing the density the viscosity is similar to that of a gas and solute diffusion coefficients are intermediate between those in a gas and those in a liquid. These properties are, of course, particularly relevant to chromatography, and the properties of some of the fluids which have been used as mobile phases in chromatography are listed in Table 9.1. 

The basic principle of SFC is partition between the supercritical mobile phase and the stationary phase, which is either a packing of silica particles on the surface of which are bonded a variety of functional groups (cf. high- 

For both SFC and SEC, the solute distribution coefficient between stationary and mobile phases is K, which is related to the standard free energy difference for a solute in the two phases. SFC is an enthalpy-controlled process, so that the retention parameter k is positive, and retention volumes, Fr, for the members of a homologous series in SFC are related to molecular mass, M  

Supercritical fluids can dissolve a variety of solutes such as polymers with high molecular weight and low volatility. The solubility may be easily varied by changing the density via the applied pressure. The low viscosity means that the pressure drop across the column is small, and the consequent permeability allows capillary columns with high efficiencies to 

The narrow columns necessary in SFC make small injection and detector volumes necessary, but SFC is compatible with both GC and HPLC detectors. Coupling to spectrometric detectors such as those of mass, Fourier transform infrared (FT-IR), and even NMR spectrometers can also be carried out. 

The technology for the preparation of capillary columns for SFC is now well advanced. Column diameters below 100 pm are necessary because the solute diffusion coefficients are smaller than in GC. Practical efficiencies of up to 5000 plates per metre are possible for 50 pm id columns. 

For very high molecular weight polymers, hydrocarbon (e.g., pentane) mobile phases modified with polar additives such as alcohols and ethers are required. Many such separations have been reported by SFC using packed columns, often with gradient elution, but such an approach poses special problems in capillary SFC because of the low flow rates. Capillary SFC with solvent programming is a likely future growth area. 

SFC has been used to determine oligomers in PET [124] and low molecular weight, high-density polyethylene wax [125]. Carbon dioxide, propane, and propane-modified carbon dioxide have been studied as eluents. 

Supercritical fluid chromatography (SEC) was first reported in 1962, and applications of the technique rapidly increased following the introduction of commercially available instrumentation in the early 1980s due to the ability to determine thermally labile compounds using detection systems more commonly employed with GC. However, few applications of SEC have been published with regard to the determination of triazines. Recently, a chemiluminescence nitrogen detector was used with packed-column SEC and a methanol-modified CO2 mobile phase for the determination of atrazine, simazine, and propazine. Pressure and mobile phase gradients were used to demonstrate the efficacy of fhe fechnique. 

Supercritical fluid chromatography (SFC) is a relatively recently developed chromatographic technique. Because of its ability to deal with compounds that are either polar or of high molecular weight, much attention has recently focused on applications of SFC to the analysis of different analytes using a variety of fluids or fluid mixtures to provide differing solvent capabilities and select vities. As a result there is a large amount of research currently underway both in SFC method development and in hardware development. 

Packed columns are conventional stainless steel columns that contain small deactivated particles to which the stationary phases adheres. Capillary columns are open tubular columns of narrow internal diameter made of fused silica, with 

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 and into the detector by a pressure restrictor placed either after the detector or at the end of the column. The restrictor is a vital component it keeps the mobile phase supercritical throughout the separation and often must be heated to prevent clogging both variable- and fixed-restrictors are available. 

Capillary SFC is particularly useful for compounds that are difficult to detect using LC and too unstable for GC. Nevertheless, at present there have not been enough applications to justify an investment (74, 75). In contrast, packed-column SFC has undergone a renaissance thanks to evaporative light-scattering detection (ELSD). Packed-column SFC-ELSD is a suitable method for analyzing various compounds, with or without chromophores, and with diverse polarities, as found in drug, steroid, and ionophore complex mixtures (76-79). 

In addition, SFC permits the use of a flame ionization detector instead of the classic UV detector. Because the response factor of the former detector is much less dependent on the compounds to be analyzed, this is a very important advantage when analyzing compounds, which, for calibration purposes, are not available as pure substances (80). 

Supercritical-fluid chromatography (SFC) was extensively evaluated in the late 1980s, in both the capillary and the packed-column formats, for a variety of compounds, including pharmaceuticals—primarily with negative results. Subsequent evaluation of SFC has shown that the primary source of this disillusionment is that supercritical carbon dioxide is substantially less polar than initially predicted. In its simplest form, supercritical carbon dioxide can be thought of as providing approximately the same polarity as hexane. As a 

If the polarity is considered equivalent to hexane and polar modifiers are added to the supercritical fluid, then the separation may be considered similar to normal-phase HPLC. However, the viscosity and mass transfer properties of supercritical fluids are more favorable and can lead to increased separation efficiencies and decreased analysis times. Berger and Wilson,for example, have demonstrated that separations with up to 260,000 theoretical plates can be achieved by serially coupling 10 HPLC columns without the deleterious pressure effects that would be encountered in separations using a liquid mobile phase. For applications that are not limited by polar matrices, SFC is, therefore, a viable option. 

Systematic method development guidelines akin to those available for HPLC have not been developed. However, details beyond the scope of this chapter are available.Separation conditions should be evaluated based on the polarity of the solute and the polarity of the stationary phase. Stationary-phase polarity increases in the order C18 C8 phenyl cyano silica amine diol. For nonpolar solutes on nonpolar stationary phases, separation may be achieved using pure carbon dioxide. As solute or stationary-phase polarity increases, carbon dioxide modified with methanol (or isopropanol, ethanol, or acetonitrile) or carbon dioxide modified with solvent and an additive such as TFA, acetic acid, triethylamine, or isopropylamine (0.5% or less) is required. 

The capillary format of SFC is attractive because of the potential of interfacing with a wide array of detectors available when carbon dioxide is used as the mobile phase. Several advances, beyond the issue of mobile-phase polarity, are, however, required prior to the technique becoming viable for pharmaceutical analyses. Capillary SFC instrumentation has lacked the requisite analytical performance for pharmaceutical analyses, and difficulties are encountered due to the acidic nature of fused silica and the problem of measuring impurities while, at the same time, not overloading the stationary phase with the main component.  

Results of the study indicate that it is possible to simultaneously detect the active drug substance and most related substances at 0.1% (w/w). Furthermore, the method provides different selectivity than reversed-phase HPLC. As a broader conclusion, this indicates orthogonality to reversed-phase HPLC and suggests the viability of SFC in support of early-phase method development. 

Supercritical fluid chromatography (SFC) is a hybrid technique of GC and HPLC that combines some of the best features of both methods. SFC is a relatively recent separation method, having only been commercially available since the 1980s. 

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 the supercritical temperature (T ) and supercritical pressure (Pe) respectively and are the defining boundaries on a phase diagram for a pure substance. Beyond these boundaries, the substance has properties that are intermediate between a liquid and a gas and is called a supercritical fluid. 

What differentiates SFC from other separation techniques such as GC and HPLC is the use of this supercritical fluid as the mobile phase. Analytes that cannot be vaporised for analysis by GC and have no functional groups for sensitive detection with HPLC, can often be separated and detected using SFC. In SFC, the sample is carried by a supercritical fluid (typically carbon dioxide) through a separating column where the mixture is divided into unique bands based on the amount of interaction between the analytes and the stationary phase in the column. As these bands leave the column their identities and quantities are determined by a detector. 

A supercritical fluid chromatograph consists of a gas supply, usually carbon dioxide, a pump, the column in an oven, a restrictor to maintain the high pressure in the column and a detector. In general there are two possible hardware setups  

An HPLC-like setup with two reciprocating pumps designed to provide amixed mobile phase with a packed analytical column placed in an oven followed by a detector, where the pressure and flow rates can be independently controlled. 

Gradual reduction in pressure from the supercritical state leads into a gas phase, without any noticeable physical changes. Gradual reduction in temperature from the supercritical state leads into the liquid state, without any changes in physical appearance. 

A supercritical fluid has lower density and lower viscosity than a liquid, but higher than a gas (Table 5.1). 

This means that a supercritical fluid mobile phase results in lower column backpressure than a liquid. Due to the higher diffusion rates in a supercritical fluid than in a liquid, the column effldency should, in principle, be higher in supercritical fluid chromatography (SFC) than in liquid chromatography (LC), but lower than that in gas chromatography (GC). However, for long columns with a density gradient over the column, improved efficiency compared to HPLC is not necessarily the case. 

Chromatography Basic Principles, Sample Preparations and Related Methods, First Edition. 

Elsa Limdanes, Leon Reubsaet and Tyge Greibroldc. 

In supercritical fluid chromatography (SFC) the mobile phase is a supercritical fluid, such as carbon dioxide [15]. A supercritical fluid can be created either by heating a gas above its critical temperature or compressing a liquid above its critical pressure. Generally, an SFC system typically has chromatographic equipment similar to a HPLC, but uses GC columns. Both GC and LC detectors are used, thus allowing analysis of samples that cannot be vaporized for analysis by GC, yet cannot be detected with the usual LC detectors, to be both separated and detected using SFC. SFC is also in other 

The first application of a supercritical fluid as a chromatographic solvent was reported in the separation of thermally labile porphyrins using supercritical 

Temperature programming and particularly pressure programming provide an added dimension, along with the relative strengths of interactions of solute molecules with both the mobile fluid phase and the stationary phase, which contribute to the separation of compounds by SFC. Thus, SFC may prove to be more selective than HPLC in certain situations as the relative dominance of partition or adsorption interaction can be altered by pressure programming. Pressure is unquestionably the single most important parameter in SFC, and without pressure programming, some solutes would not be eluted. 

Supercritical fluid chromatography employing carbon dioxide as an eluent is frequently operated in a temperature range up to 100°C (110-115, 316). Pressures that conform to an eluent density of 200 to 500 times greater than that of the gas are usually employed. Other eluents frequently utilized besides carbon dioxide include ammonia (T = 132.4 °C), nitrous oxide (Tc = 36.5°C), ethane (Tc = 32.2 °C), ethylene (T = 9.21 °C), propane (T = 96.7 °C), pentane (Tc= 196.5 °C), and chlorotrifluoromethane (Tc = 28.9°C) (129). Low concentrations of a moderator, usually a low molecular-weight alcohol such as methanol, ethanol, or isopropanol can be added to enhance resolution (314). 

The increased solubilizing powers of a supercritical fluid cannot be entirely explained on the basis of an increased density of the fluid phase. Thus, supercritical carbon dioxide is a suitable solvent for SFC in terms of mobilizing a solute, but supercritical argon and nitrogen are not (116). 

A solvation type interaction also contributes. Lewis acid-base interactions and co-complexation undoubtedly enhance the solvent qualities of carbon dioxide while hydrogen-bonding also contributes to those of ammonia (116). 

A separation technique in which the mobile phase is a supercritical fluid. 

Colorplate 11 shows the phase transition of liquid CO2 to supercritical CO2. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all types of samples. Gas chromatography, particularly when using capillary columns, provides for rapid separations with excellent resolution. Its application, however, is limited to volatile analytes or those analytes that can be made volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally as the flame ionization detector commonly used in gas chromatography. 

The most common mobile phase for supercritical fluid chromatography is CO2. Its low critical temperature, 31 °C, and critical pressure, 72.9 atm, are relatively easy to achieve and maintain. Although supercritical CO2 is a good solvent for nonpolar organics, it is less useful for polar solutes. The addition of an organic modifier, such as methanol, improves the mobile phase s elution strength. Other common mobile phases and their critical temperatures and pressures are listed in Table 12.7. 

In addition to gases, supercritical fluids can be used to carry samples into an IMS after chromatography or directly after extraction. Supercritical fluids have two primary advantages for sample introduction. First, unlike gases, supercritical fluids 

FIGURE 3.10 Experimental setup of GC-DMS system. Chemical analysis is performed using gas chromatography differential mobility spectrometry (GC/DMS). Several user-defined parameters were selected factorial experiments (a) the RF voltage of the DMS sensor, (b) nitrogen carrier gas flow rate through the DMS, (c) solid phase microextraction (SPME) filter type, and (d) GC cooling profile. (From Molina et al.. Anal. Chim. Acta 368(2), 2008.) 

IMS has been compared with the UV detector after separation with SFC of benzoates and esters, demonstrating the ability of IMS for the detection of compounds that do not have sensitive chromophores for UV detection Often, polymers do not have sufficient UV-visible (Vis) absorbance for detection after LC or SFC, while SFC-IMS can be used for both efficient separation and detection of a variety of polymeric materials. IMS detection of a variety of drugs, such as various steroids, opiates, and benzodiazepines, after SFC separation demonstrated the ease and utility of acquiring ion mobility spectra at ambient pressure. While SFC has only captured a small portion of the separation maiket, the potential of IMS for detecting compounds that cannot be easily seen with a standard UV-Vis approach has led to its use as a stand-alone detector for LC. 

A compound such as COj is a gas at normal temperature and pressure (NTP) and, like all gases below a certain critical temperature, further increasing the pressure results in the formation of a liquid. Above this critical temperature, increasing the pressure increases the density of the fluid but a distinct transition to and boundary with a liquid phase never forms. At the critical temperature and pressure, the density of the gas phase and the liquid phase are the same. This state is neither a true 

SFC is rarely applied to ionic surfactants. Ionic materials have poor solubility in CO2, which is the usual SFC mobile phase. If another supercritical fluid is substituted for CO2, then the main advantage of SFC is lost, i.e., the ability to use the flame ionization detector. Applications of SFC to ionic surfactant characterization are listed in Table 1. 

Analysis of anionics by SFC is only of academic interest, since other techniques are more useful. Soap may be analyzed as the free fatty acids, with elution on a nonpolar packing in order of boiling point (1). Some anionic surfactants, such as phosphate esters, may be analyzed by SFC of the methyl esters (2). As is also true in gas chromatography. 

Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 

TABLE 1 Analysis of Ionic and Amphoteric Surfactants by Supercritical Fluid Chromatography 

C8-C24 Fatty acids separation by alkyl chain length Ethylvinylbenzene/divinyl-benzene copolymer, 5 pm particles, 750 pm x 15 cm steel CO2, pressure programmed, 150-C FID 1 

Unlike paper and thin-layer chromatography (TLC Chapters 5 and 6) techniques in which very little new work has been published since the 1970 s, supercritical fluid chromatography (SFC) is a rapidly expanding area. 

Beyond its critical point, a substance can no longer be condensed to a liquid, no matter how great the pressure. As the pressure increases, however, the fluid density approaches that of a liquid. Because solubility is closely related to density, the solvating strength of the fluid assumes liquid-like characteristics. Its diffusivity and viscosity, however, remain. SFC can use the widest range of detectors available to any chromatographic technique. As a result, CSFC has already demonstrated a great potential in application to polymer additives. 

SFC is now one of the fastest growing analjnical techniques. The first paper on the technique was by Klesper and co-workers [2], but SFC did not catch the analyst s attention until Novotny and co-workers [3] published the first paper on CSFC. 

Most supercritical fluid chromatographs use carbon dioxide as the supercritical eluent, as it has a convenient critical point of 31.3 °C and 7.3 MPa. Nitrous oxide, ammonia and -pentane have also been used. This allows easy control of density between 0.2 ml/g and 0.8 ml/g and the utilisation of almost any detector from liquid chromatography (LC) or GC. 

SFC uses detectors from both LC and GC. A summary of detection systems used in SFC has been documented (Later and co-workers [4]). 

Typical properties of a gas (helium) under gas chromatographic conditions, a liquid (water) under liquid chromatographic conditions, and carbon dioxide under low and high density conditions, as might be used in supercritical fluid chromatography, are 

Change in density of supercritical fluid carbon dioxide with pressure and temperature 

Representative properties of typical chromatographic mobile phases 

Mobile phase Temperature (°C) Pressure (atm) Density (g/ml) Diffusivity (cm /s) Viscosity (cP) 

A variety of modern instrumental analytical techniques have attracted considerable attention in the last decades as alternative separation and analysis methods with respect to HPLC. This includes, in particular, supercritical fluid chromatography (SFC), which utilizes condensed carbon dioxide (above or near its critical temperature of 

SFC has been undergoing a renaissance in its use in modem analytical laboratories over the last decade, particularly in chiral and achiral analysis of pharmaceutical compounds. Many of the problems associated with implementing SFC have come from the teclmical and operational difficulties in earlier instruments [134, 135]. New developments in SFC include improved backpressure-regulation, more consistent SFC flow rates, more reliable sample injection systems and improved flow cell designs [136,137]. 

Keith D. Bartle, Anthony A. Clifford and Naila Malak 

Edited by K. Blau and /. M. Halket 1993 John Wiley Sons Ltd 

Since part of the ethos of SFC is to extend high resolution analysis to high molecular weight, reactive and thermolabile compounds not accessible to GC without derivatization, and to use universal detection, applications of derivatization in SFC have been sparse. They fall into two main groups derivatization to increase solubility in the supercritical mobile phase, and hence to extend further the molecular weight range of SFC and derivatization to provide selectivity and increase sensitivity in detection. 

Myoinositol is a naturally occurring vitamin with a hexahydroxycyclohexane structure its phosphorylated forms are readily converted to trimethylsilyl derivatives which, as has been observed for alkyl ethoxy phosphates, can be separated by SFC at temperatures some hundreds of degrees below those necessary for GC of such compounds [14]. Both silylated inositol triphosphate and phytic acid (inositol hexaphosphate) were eluted 

A number of groups have shown how enantiomeric resolution of amino acids derivatized with non-chiral reagents is possible in SFC with chiral stationary phases. N-Acetylamino acid t-butyl ester racemates were rapidly resolved [17] on (N-formyl-L-valylamino)propyl silica with CO2 modified with methanol, acetonitrile and diethyl ether. A similar stationary phase allowed (18 rapid ( 5 min) separation of racemic N-4-nitrobenzoyl-amino acid isopropyl esto-s with methanol-modified CO2 the enantioselectivity in SFC was comparable with that in HPLC with isopropanol/n-hexane as mobile phase. Capillary column SFC on polysiloxane stationary phases containing chiral side chains has been employed 

Capillary SFC using carbon dioxide as mobile phase and a FID as detector has been applied to the analysis of several essential oils and seemed to give more reliable quanti cation than GC, especially for oxygenated compounds. However, the separation ef ciency of GC for monoterpene hydrocarbons was, as expected, better than that of SFC. Manninen et al. (1990) published a comparison of a capillary GC versus a chromatogram obtained by capillary SFC from a linalool-methyl chavicol basil oil chemotype exhibiting a fairly good separation by SFC. 

1 Applications, Analysis of aikylbenzene sulphonates and alkyl sulphonates on a fused silica open tubular column (10 m x 0.53 or 0.25 mm i.d.) coated with 0.1 or 0.2 micron SE54 with carbon dioxide as mobile phase and FID is described in [8]. The anionics were derivatised before analysis. Among the many examples of analysis of nonionic surfactants are the following  

Capillary SFC offers unprecedented versatility in obtaining high-resolution separations of difficult compounds. 

SFC uses detectors from both LC and GC. A summary of detection systems used in SFC has been documented [115]. One of the most commonly used detection systems is electron capture detector. A sensitivity to about 50 pg (minimum detection limit) on a column is obtainable [116]. 

Beyond its critical point, a substance can no longer be condensed to a liquid, no matter how great the pressure. As pressure increases, however. 

Supercritical fluid chromatography finds its applications in compounds that are either difficult or impossible to analyse by liquid chromatography or gas chromatography. Supercritical fluid chromatography is ideal for analysing either thermally labile or non volatile non chromatophoric compormds. The technique will be of interest to water chemists as a means of identifying and determining the non volatile components of water. 

Wall [17] has discussed recent developments including timed-split injection, extraction and detection systems in supercritical fluid chromatography. 

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Supercritical fluid chromatography has found many applications in the analysis of polymers, fossil fuels, waxes, drugs, and food products. Its application in the analysis of triglycerides is shown in Figure 12.38. 

Example of the application of supercritical fluid chromatography to the analysis of triglycerides. (Chromatogram courtesy of Alltech Associates, Inc. Deerfield, IL). 

SFC/MS. supercritical fluid chromatography and mass spectrometry used as a combined technique SID. surface-induced dissociation (or decomposition)... 

Jinno, K., Hyphenated Techniques in Supercritical Fluid Chromatography and Extraction, Elsevier, Amsterdam, 1992. 

INFRARED TECHNOLOGY AND RAMAN SPECTHOSCOPY - INFRARED TECHNOLOGY] (Vol 14) Sfc. See Supercritical fluid chromatography. 

ANALYTICALTffiTHODS - HYPHENATED INSTRUTffiNTS] (Vol 2) Supercritical fluid chromatography... 

The use of separation techniques, such as gel permeation and high pressure Hquid chromatography interfaced with sensitive, silicon-specific aas or ICP detectors, has been particularly advantageous for the analysis of siUcones in environmental extracts (469,483—486). Supercritical fluid chromatography coupled with various detection devices is effective for the separation of siUcone oligomers that have molecular weights less than 3000 Da. Time-of-flight secondary ion mass spectrometry (TOF-sims) is appHcable up to 10,000 Da (487). 

Mixtures can be identified with the help of computer software that subtracts the spectra of pure compounds from that of the sample. For complex mixtures, fractionation may be needed as part of the analysis. Commercial instmments are available that combine ftir, as a detector, with a separation technique such as gas chromatography (gc), high performance Hquid chromatography (hplc), or supercritical fluid chromatography (96,97). Instmments such as gc/ftir are often termed hyphenated instmments (98). Pyrolyzer (99) and thermogravimetric analysis (tga) instmmentation can also be combined with ftir for monitoring pyrolysis and oxidation processes (100) (see Analytical methods, hyphenated instruments). 

Supercritical Fluid Chromatography. Supercritical fluid chromatography (sfc) combines the advantages of gc and hplc in that it allows the use of gc-type detectors when supercritical fluids are used instead of the solvents normally used in hplc. Carbon dioxide, -petane, and ammonia are common supercritical fluids (qv). For example, carbon dioxide (qv) employed at 7.38 MPa (72.9 atm) and 31.3°C has a density of 448 g/mL. 

Cyclopentadiene oligomers up to octamers can be effectively analy2ed and quantified by supercritical fluid chromatography using a chemically bonded methyl siUcone capillary column. 

M. E. Eee and K. E. Mai kides, Analytical Supercritical Fluid Chromatography and Extraction, Qiromatography Conferences , Provo, USA (1990). 

H. Daimon and Y. Hirata, Direct coupling of capillary supercritical fluid chromatography with superaitical fluid extraction using modified carbon dioxide , J. High Resolut. Chromatogr. 17 809-813 (1994). 

T. A. Berger, Practical advantages of packed column supercritical fluid chromatography in supporting combinations chemistiy , in Unified Chromatography, J. P. Parcher and T. L. Chester (Eds), ACS Symposium Series 748, American Chemical Society, Washington, DC, pp. 203-233 (2000). 

T. L. Chester and J. D. Pinkston, Pressure-regulating fluid interface and phase behavior considerations in the coupling of packed-column supercritical fluid chromatography with low-pressure detectors , ]. Chromatogr. 807 265-273 (1998). 

Supercritical fluid extraction (SFE) has been extensively used for the extraction of volatile components such as essential oils, flavours and aromas from plant materials on an industrial as well as an analytical scale (61). The extract thus obtained is usually analysed by GC. Off-line SFE-GC is frequently employed, but on-line SEE-GC has also been used. The direct coupling of SEE with supercritical fluid chromatography (SEC) has also been successfully caried out. Coupling SEE with SEC provides several advantages for the separation and detection of organic substances low temperatures can be used for both SEE and SEC, so they are well suited for the analysis of natural materials that contain compounds which are temperature-sensitive, such as flavours and fragrances. 

A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

SUPERCRITICAL FLUID EXTRACTION COUPLED WITH SUPERCRITICAL FLUID CHROMATOGRAPHY... 

An on-line supercritical fluid chromatography-capillary gas chromatography (SFC-GC) technique has been demonstrated for the direct transfer of SFC fractions from a packed column SFC system to a GC system. This technique has been applied in the analysis of industrial samples such as aviation fuel (24). This type of coupled technique is sometimes more advantageous than the traditional LC-GC coupled technique since SFC is compatible with GC, because most supercritical fluids decompress into gases at GC conditions and are not detected by flame-ionization detection. The use of solvent evaporation techniques are not necessary. SFC, in the same way as LC, can be used to preseparate a sample into classes of compounds where the individual components can then be analyzed and quantified by GC. The supercritical fluid sample effluent is decompressed through a restrictor directly into a capillary GC injection port. In addition, this technique allows selective or multi-step heart-cutting of various sample peaks as they elute from the supercritical fluid... 

Figure 12.20 SFC-GC analysis of a sample of aviation fuel (a) SFC separation into two peaks (b and c) coixesponding GC ttaces of the respective peaks (flame-ionization detection used throughout). Reprinted from Journal of High Resolution Chromatography, 10, J. M. Levy et ah, On-line multidimensional supercritical fluid chromatography/capillary gas chromatography , pp. 337-341, 1987, with permission from Wiley-VCH.

Figure 12.22 SFC-GC analysis of aromatic fraction of a gasoline fuel, (a) SFC trace (b) GC ttace of the aromatic cut. SFC conditions four columns (4.6 mm i.d.) in series (silica, silver-loaded silica, cation-exchange silica, amino-silica) 50 °C 2850 psi CO2 mobile phase at 2.5 niL/min FID detection. GC conditions methyl silicone column (50 m X 0.2 mm i.d.) injector split ratio, 80 1 injector temperature, 250 °C earner gas helium temperature programmed, — 50 °C (8 min) to 320 °C at a rate of 5 °C/min FID detection. Reprinted from Journal of Liquid Chromatography, 5, P. A. Peaden and M. L. Lee, Supercritical fluid chromatography methods and principles , pp. 179-221, 1987, by courtesy of Marcel Dekker Inc.

Figure 12.23 SFC-SFC analysis, involving a rotaiy valve interface, of a standard coal tar sample (SRM 1597). Two fractions were collected from the first SFC separation (a) and then analyzed simultaneously in the second SFC system (h) cuts a and h are taken between 20.2 and 21.2 min, and 38.7 and 40.2 min, respectively. Peak identification is as follows 1, tii-phenylene 2, chrysene 3, henzo[g/ i]perylene 4, antliracene. Reprinted from Analytical Chemistry, 62, Z. Juvancz et al, Multidimensional packed capillary coupled to open tubular column supercritical fluid chromatography using a valve-switcliing interface , pp. 1384-1388, copyright 1990, with permission from the American Chemical Society.

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