On-Line coupling of supercritical fluid extraction with high performance liquid chromatography

Supercritical fluid extraction was coupled directly with high performance liquid chromatograph. The system was evaluated for direct injection of supercritical CO₂ and modified supercritical CO₂ at high pressure and temperature onto a HPLC system with varying mobile phase compositions and flow rates. Injection of 9 μL supercritical CO₂ onto the HPLC using methanol/water mobile phases from 100% methanol to 80% with a flow of 1.0 mL/min did not adversely affect the baseline of UV detector. However at higher percentages of water, CO₂ solubility in the mobile phase decreased and caused baseline interferences on the UV detector. At higher HPLC mobile phase flow rates, supercritical CO₂ was injected to higher percentages of water without any effect on the UV baseline. Also, increasing the extraction pressure or modifier concentration did not change the results. Separations of polynuclear aromatic hydrocarbons and linear alkenebenzene sulfonate test mixtures were obtained using on-line SFE/HPLC interfaced system.     

Use of various filters for generating modified supercritical fluid mobile phases

Summary Adding various components to supercritical carbon dioxide in supercritical fluid chromatography can extend or significantly alter the solvating properties. Polar samples which are difficult to analyze with pure supercritical CO₂ because of their high polarity can be separated by addidng polar modifiers. In this paper, a new mixing method using an HPLC filter for adding polar modifier to CO₂ is described. Although several filters were tried, only one could keep the amount of modifier in the mobile phase constant for a long time. The amount of water or methanol dissolved in supercritical CO₂ was measured by an amperometric microsensor made of a thin film of perfluorosulfonate ionomer (PFSI).     

Retention behavior of polycyclic aromatic hydrocarbons in supercritical fluid chromatography on a chemically bonded stationary phases based upon liquid-crystalline polymer

Summary The retention behavior of a set of polycyclic aromatic hydrocarbons in supercritical fluid chromatography have been studied on a chemically bonded stationary phase based upon a side chain liquid crystalline polymer (LCP) with carbon dioxide-based mobile phase. The effects of the mobile phase pressure, column temperature and amount of mobile phase organic modifier have been investigated in order to detect a possible structural change in the liquid crystal polymer linked to the silica support. The influence of these factors on the selectivity coefficients has also been studied. Two distinctive behaviors with temperature are noted at low pressure on the one hand and at higher pressure on the other. This change in behavior is based on the density of the supercritical CO₂ and the PAH volatility rather than on any specific stationary phase structural change. Both lower mobile phase pressure and amount of mobile phase modifier are required to obtain better selectivities. Better planarity recognition is observed in SFC than in HPLC with these new bonded liquid crystal stationary phases. The bonded liquid crystal phase is only weakly affected by the addition of organic modifier in the supercritical CO₂.     

New device for controlling the amount of methanol in carbon dioxide mobile phase for supercritical fluid chromatography

A new device to accurately deliver a small amount of methanol into supercritical carbon dioxide fluid is described. Carbon dioxide, the most widely used mobile phase in supercritical fluid chromatography, is a relatively non-polar fluid, and hence the addition of a small amount of methanol could change the solvent strength of the mobile phase. In this work, supercritical CO₂ and methanol are delivered from the pump to a 100-μl mixing chamber in which a small magnetic bar is rotating. After passing through the mixing chamber, supercritical CO₂ is changed to a new mobile phase with different polarity. The modified mobile phase was successfully used for the separations of polar compounds and polyaromatic hydrocarbons (PAHs).     

Influence of temperature on FTIR spectra of various organic compounds analyzed by on-line carbon dioxide SFC/FTIR

The development of an on-line SFC-FTIR method with supercritical carbon dioxide as mobile phase requires information about the nature of the FTIR spectrum of a solute dissolved in supercritical or liquid CO₂. The wavenumber of maximum absorbance of the carbonyl stretching vibration was quantitatively studied versus temperature and CO₂ density. FTIR spectra recorded in supercritical or liquid CO₂ have been compared to their vapor-phase and condensed-phase spectra for various solutes.     

On-line SFC/FT-IR analysis of agriculturally-related compounds

Several agricultural compounds which are used as herbicide precursors have been analyzed by SFC and are detected on-line via a flow cell FT-IR interface. A mixture of ureas is separated on a microbore packed column; while a mixture of benzamides and anilides and a mixture of sulfonamides are separated on capillary columns. The use of 100% CO₂ as the mobile phase enables an infrared spectrum of each eluting analyte to be obtained on-line with good signal to noise ratio. The urea mixture is also separated with a conventional cyanopropyl bonded phase analytical scale packed column using a methanol-modified CO₂ mobile phase and UV detection. The separations achieved with and without modifier are compared.     

New Device for Adding Polar Modifiers to Carbon Dioxide Mobile Phase for Supercritical Fluid Chromatography

Abstract

Adding additional components to supercritical carbon dioxide in supercritical fluid chromatography can extend or significantly alter the fluid solvating properties. Polar samples which are difficult to be analyzed with pure supercritical CO₂ because of their high polarity can be separated by adding polar modifiers to supercritical CO₂. In this paper, a new mixing device using a teflon high capacity filter for adding polar modifiers to carbon dioxide mobile phase is introduced. This new mixing device could keep the amount of modifier in the mobile phase constant for a much longer time than a saturator column. The amount of water or methanol dissolved in supercritical CO₂ was measured by amperometric microsensor which is made of thin film of perfluorosulfonate ionomer(PFSI).     

Demonstration of a linear composition gradient during water saturation of CO2 in supercritical fluid chromatography

Summary Water was added to CO₂ by saturation to increase the solvation power of the mobile phase in supercritical fluid chromatography. The saturation was performed at a temperature above the boiling point of water (100°C) to increase the amount of water which could be loaded homogeneously into the CO₂ (2.5–3.0 mol% water as compared to about 0.25 mol% water at 25°C). A linear composition of water was produced by altering the density of the CO₂ during saturation. Modifications to the injector and CO₂ transfer lines prevented phase separation as a result of the instrumentation used in capillary supercritical fluid chromatography (SFC). After fitting vapor-liquid equilibria data to pressure, density, and temperature conditions, approximately 2.5–3.0 mol% of water was introduced in a linear gradient at 110°C. The effect of water on SFC performance was evaluated with standard steroid compounds. This paper provides further evidence for the need to examine vapor-liquid equilibria data prior to SFC.     

Evaluation of an ion mobility detector for supercritical fluid chromatography with solvent-modified carbon dioxide mobile phases

Summary An ion mobility detector (IMD) was evaluated for open tubular column supercritical fluid chromatography (SFC) when organic solvent-modified supercritical CO₂ was used as mobile phase. It was found that the SFC/IMD interface design in which the SFC capillaray restrictor was directly inserted into the ionization region of the IMD was not acceptable because of low sensitivity that resulted from the effect of the modifier on detector temperature and mechanism of detection. A new interface utilizing a heated nebulizer gas to provide heat to the restrictor and to minimize the formation of ion clusters, and a bent nozzle for enhancing the ionization efficiency of the solute in the IMD ion source are described. Using 5% acetonitrile in CO₂, the minimum detectable quantity (S/N=3) for pyrene was improved from 25.2 ng to 2.1 ng with the new detector design. This compares to a minimum detectable quantity of 0.1 ng when using neat CO₂ as mobile phase. The use of molecular connectivity calculations to predict the drift times of selected analytes is also successfully demonstrated.     

Characterization and classification of stationary phases in HPLC and SFC – a review

Packed column supercritical fluid chromatography (pSFC) is an attractive technique in drug discovery related analysis because it offers several advantages over the more commonly used high-performance liquid chromatography (HPLC) technique. The environmental-friendly CO₂ mobile phase, the high-throughput capacity, the increased efficiency and the lower operational costs give SFC additional benefits over HPLC in analysis related to drug development. The latter technique is well established and has been used for decades in the pharmaceutical industry. On the other hand, SFC is still in its infancy, even though the technique has been known for decades and researchers are still discovering the possibilities and limitations of this technique.     

Carbon Dioxide Supercritical Fluid Extraction with on-line Fluorescence Detection

Abstract

An HPLC detector was modified to operate as a low pressure on-line supercritical fluid extraction detector. Spectra of CO₂ and fluorene are presented of pressures ranging from 85 to 150 atm. Both the intensity and emission wavelength of fluorene were effected by CO₂ pressure. As pressure increased, the emission intensity also increased and the emission shifted to a shorter wavelength. The intensity of CO₂ emission decreased by increasing pressure. The emission wavelength of CO₂ was unaffected by pressure. A detection limit for fluorene was found to be 5 μg per extraction. The minimal detectable mass was 25 μg (± 8% RSD) per extraction.     

Evaluation of an amide‐based stationary phase for supercritical fluid chromatography

A relatively new stationary phase containing a polar group embedded in a hydrophobic backbone (i.e., ACE ® C18‐amide) was evaluated for use in supercritical fluid chromatography. The amide‐based column was compared with columns packed with bare silica, C₁₈ silica, and a terminal‐amide silica phase. The system was held at supercritical pressure and temperature with a mobile phase composition of CO₂ and methanol as cosolvent. The linear solvation energy relationship model was used to evaluate the behavior of these stationary phases, relating the retention factor of selected probes to specific chromatographic interactions. A five‐component test mixture, consisting of a group of drug‐like molecules was separated isocratically. The results show that the C₁₈‐amide stationary phase provided a combination of interactions contributing to the retention of the probe compounds. The hydrophobic interactions are favorable; however, the electron donating ability of the embedded amide group shows a large positive interaction. Under the chromatographic conditions used, the C₁₈‐amide column was able to provide baseline resolution of all the drug‐like probe compounds in a text mixture, while the other columns tested did not.     

Elution of Cationic Species with/without Ion Pair Reagents from Polar Stationary Phases via SFC

Traditionally, the application of supercritical fluid chromatography has been limited to compounds of relatively low polarity. Thus, we report here that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond Cyano-bonded silica column with the addition of sodium alkylsulfonate to the methanol-modified, carbon dioxide-based mobile phase. A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive has been proposed. In another set of experiments, the three amine salts readily eluted from both Ethyl-pyridine-bonded silica and Amino-bonded silica phases without the need of additive although the peak shapes were less than desirable. The addition of sulfonate salt to the mobile phase again sharpened the peaks. In the presence of a mixture of methanol and CO₂, we suggest that these stationary phases are positively charged. We describe here also for the first time the employment of a strong silica-based anion exchange (SAX) column for supercritical fluid separation of cationic species. Two elution mechanisms were proposed for the three amine salts depending upon whether the ionic additive was incorporated into the mobile phase. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged basic stationary phases was suggested to be operational without the ionic additive.     

Packed Column SFC of Gas Oils. Part II: Experimental Design Optimization of the Group-Type Analysis using CO2-SF6 as the Mobile Phase

An optimization procedure is presented for group-type analysis of diesel fuel by supercritical fluid chromatography using packed silica column and a mixed mobile phase. A set of five responses, four values of resolution of a performance mixture and analysis time, was modeled using a Doehlert matrix for experimental design. Optimized experimental conditions for the five responses were obtained from a response surfaces optimization, taking into account various constraints on SF₆ content in mobile phase and analysis time. The predicted and experimental resolutions were in good agreement for the different optimized conditions and one of them was selected to for application to a given diesel fuel for comparison with the results obtained by SFC using pure CO₂ and by HPLC. The conditions found in this study provide an alternative method for the determination of mono-, di-, and polyaromatic compounds in middle distillates.     

A brief study of the supercritical fluid chromatographic behavior of lanthanide β-diketonates

The chromatographic behaviors of lanthanide chelates of acetylacetone (ACAC), trifluoroacetylacetone (TFA), thenoyltrifluoroacetone (TTA), dipivaloylmethane (THD), 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedione (FOD), and the thenoyltrifluoroacetonepyridine (TTA·Py) adduct were investigated using packed column supercritical fluid chromatography. Mobile phases consisting of neat and alcohol modified CO₂ were used with a phenyl packed stationary phase. Lanthanide complexes of ACAC, THD, and FOD were shown to have better chromatographic performance compared to the corresponding chelates with TFA, TTA, and TTA·Py. In particular, TTA complexes such as Eu(TTA)₃ showed characteristic thermal decomposition in the mobile phase at elevated temperature. In addition, retention behavior was found to be a temperature dependent function of volatility and solubility for all chelates studied.     

Rhodium and palladium β-diketonate determination with on-line supercritical fluid extraction-high performance liquid chromatography via solid phase extraction

 An on-line system of supercritical fluid extraction (SFE) and high performance liquid chromatography (HPLC) via solid phase extraction (SPE) is described for the determination of palladium and rhodium 2,2,6,6-tetramethyl-3,5-heptanedione-(thd) as well as rhodium-acetylacetonate-(acac) and benzylacetonate-(bzac) chelates. The chelates were extracted with supercritical CO₂ from sand and humic acid, concentrated on SPE cartridges and analysed with HPLC. Two cartridge materials were tested and compared to off-line trapping. The percentage of the breakthrough and cartridge retained material were measured in liquid dichloromethane. The SFE conditions could be optimized to separate metal chelates during the extraction. The supercritical fluid (SF) behaviour of different ligands on rhodium were investigated. Received: 19 July 1996/Revised: 11 December 1996/Accepted: 14 December 1996     

Chromatographic resolution of closely related species: Drug metabolites and analogs

In this study, we investigate the separation of a variety of mixtures of drugs, metabolites, and related analogs including representatives of the carbamazepine, methylated xanthine, steroid hormone, nicotine, and morphine families using several automated chromatographic method development screening systems including ultra high performance liquid chromatography, core–shell HPLC, achiral supercritical fluid chromatography (SFC), and chiral SFC. Of the 138 column and mobile phase combinations examined for each mixture, a few chromatographic conditions afford the best overall performance, with a single achiral SFC method (4.6 × 250 mm, 3.0 μm GreenSep Ethyl Pyridine, 25 mM isobutylamine in methanol/CO₂) affording good separation for all samples. Four of these mixtures were also resolved by achiral SFC on the Luna HILIC and chiral SFC Chiralpak IB columns using methanol or ethanol with 25 mM isobutylamine as polar modifiers. Modifications of standard chromatography screening conditions afforded fast separation methods (from 1 to 5 min) for baseline resolution of all components of each of these challenging sets of closely related compounds.     

In situ FT-IR spectroscopic study on the conformational changes of isotactic polypropylene in the presence of supercritical CO2

The conformational changes of isotactic polypropylene (iPP) under supercritical CO₂ condition with different pressure and temperature have been carefully studied by in situ Fourier-transform infrared spectroscopy (FT-IR). Analysis of the corresponding spectra shows that the conformational ordering by supercritical CO₂ results in the intensity enhancement of the regularity bands of iPP. Due to the high CO₂ concentration and strong intermolecular interaction, iPP can reach an equilibrium state in a short time at high CO₂ pressure. The equilibrium time increases with soaking temperature. After supercritical CO₂ treatment, two mechanisms, the formation of short helix from amorphous phase and the extension of short helix into long one, happen simultaneously. The latter mechanism undergoes quickly at the beginning of induced conformational changes and then slows down, resulting in the slight increase of crystallinity. At the same time, the conformational ordering in amorphous phase happens continuously until a thermodynamic equilibrium. In summary, in the presence of supercritical CO₂, the conformational ordering of iPP chains occurs exclusively in the amorphous region, with no impact on the crystal part.     

Screening and confirmation of PAHs in vegetable oil samples by use of supercritical fluid extraction in conjunction with liquid chromatography and fluorimetric detection

A fast, simple, non-destructive method for the direct screening of polycyclic aromatic hydrocarbons (PAHs) in vegetable oil samples is proposed. The method uses a supercritical fluid extraction (SFE) system coupled on-line with a fluorimetric detector to determine PAHs. This special assembly avoids the main problems encountered in the determination of PAHs in complex matrices such as vegetable oils. PAHs are selectively extracted by using silica gel in the thimble and cleaned up by passage through a C18 column. Interferences are preferentially retained by the silica gel during the SFE process while PAHs are adsorbed in the C18 column and the remainder of the matrix is sent to waste. Finally, the C18 column is purged to remove residual CO₂ gas and adsorbed PAHs are recovered by desorption with a solvent. The extracts from positive samples are subsequently analyzed by liquid chromatography (LC) with fluorescence detection. The proposed method allows the confirmation of vegetable oil safety and hence provides a new tool for consumer protection.     

Packed capillary column supercritical fluid chromatography of fat-soluble vitamins using liquid crystal polysiloxane coated particles

Summary Liquid crystal polysiloxane stationary phases were prepared by coating two different polymers on deactivated porous silica particles (10 m diameter, 80 Å pores). Deactivation of the silica particles before coating was necessary to prepare highly efficient and inert stationary phases for supercritical fluid chromatography (SFC). Fat-soluble vitamins E, A, K₁, K₂, D₂, and D₃ were separated on these columns using neat supercritical CO₂ as mobile phase. The analyses were completed within 40 min at 70 °C. The results were compared to those obtained using a capillary column packed with less ordered liquid crystalm,m-cyanobiphenyl-substituted polysiloxane coated particles. Reduced shape selectivity was observed with this cyanobiphenyl phase. The response factors of vitamins A, E, K₁, K₂, D₂, and D₃ when using the flame ionization detector (FID) were determined to be very similar.