Fundamental conditions in pressure-programmed supercritical fluid chromatography-mass spectrometry and some applications to vitamin analysis

Summary A combined system of pressure-programmed packed capillary supercritical fluid chromatography-mass spectrometry (SFC/MS) was constructed using a self-soupting interface assisted by vacuum nebulizing. For the optimum operation of the SFC/MS system, fundamental analytical conditions such as the flow rate of the mobile phase, the pump pressure and the composition of the mobile phase were examined. The use of large packing materials indicated that the capacity factor for a sample solute is almost constant under a given pump pressure regardless of the flow rate of the supercritical fluid. The SFC/MS system was applied to the analysis of both water- and fat-soluble vitamins. Both types vitamins were clearly separated under basically the same SFC conditions. High quality mass spectra of the vitamins were obtained; selected ion monitoring (SIM) traces of the vitamins are also reported as well as their UV traces.     

Capillary supercritical fluid chromatography of limonoids

Capillary supercritical fluid chromatography (SFC) with carbon dioxide as the mobile phase has been used to separate a mixture of limonoid standards. Chromatographic conditions were optimized to enable analysis of plant extracts. The seed of Aphanamixis polystacha (Meliaceae) and the stem bark of Harpephyllum caffrum (Anacardiaceae) were extracted with hexane and the extracts were analyzed by SFC to demonstrate the potential of the technique as a screening tool. SFC analysis indicated the presence of limonoids in the hexane extract of the bark of Entandrophragma delevoyii (Meliaceae). One limonoid and two protolimonoids isolated from the extract were separated by SFC.     

Use of isopycnic plots in designing operations of supercritical fluid chromatography: II. The isopycnic plots and the selection of the operating pressure-temperature zone in supercritical fluid chromatography

In SFC, the key chromatographic parameters, the retention factors and the column efficiency, strongly depend on the density of the mobile phase. This indicates that the isodensity or isopycnic plots, drawn on the pressure-temperature plane, can provide an effective tool to help analyzing how the chromatograms obtained in SFC evolve, when the experimental conditions, the inlet and outlet pressures and the column temperature, are changed. In a companion paper, we analyzed the role of density in controlling the physical properties of the mobile phase, which in turn controls solute retentions and column efficiencies. In this report, we analyze the operating conditions in SFC with reference to the isopycnic plots of carbon dioxide. This analysis clarifies the differences and similarities between the operating conditions selected in the subcritical zone and those located in the supercritical zone. It also sets out an operational map illustrating how retention factors vary with respect to the operating temperatures and pressures. This study is focused on the use of pure carbon dioxide as the mobile phase, but the same method of investigation is also applicable when the mobile phase contains a modifier.     

Supercritical fluid chromatography tandem-column method development in pharmaceutical sciences for a mixture of four stereoisomers

A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns was achieved for baseline separation of a mixture of chiral pharmaceutical compounds (i.e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobile phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50:50 v/v). On the contrary, this mixture (mixture A) could not be baseline separated by SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns. The effects of various mobile phases on elution order, capacity factor, selectivity, and resolution were determined with mixture A on the individual aforementioned columns to develop the tandem-column method.     

Synthetic ceramic clay material as stationary phase in packed column supercritical fluid chromatography

Summary A novel inorganic synthetic clay material (SC) has been evaluated as the stationary phase in packed-column, supercritical fluid chromatography (SFC). The molecular recognition capability of the SC stationary phase in SFC for polycyclic aromatic hydrocarbons has been evaluated using carbon dioxide and carbon dioxide modified with methanol as the mobile phase. This recognition derives from the layer structure of the SC material which acts as a slit to distinguish non-planar solutes from the molecular-molecular interaction between solute and stationary phase and leads to smaller retention for non-planar solutes. The recognition capability is also dependent on the SFC conditions such as column pressure and column temperature.     

Reviews on recent trends in chromatography/mass spectrometry coupling. Part IV. Reasons why supercritical fluid chromatography is not so easily coupled with mass spectrometry as originally assessed

Summary When SFC was rediscovered in the early 1980s, it was frequently estimated that a strong driving force to its development would be the ease of devising a simple SFC/MS interface. This was believed to be easily achieved if analytical conditions were limited to capillary SFC columns as a general separation tool, and to the choice of neat CO₂ as the unique supercritical fluid. The low flow rate of mobile phase delivered by capillary columns was easy to accommodate by the vacuum equipment of standard mass spectrometers, and the specific physical properties of CO₂ made possible solute ionization by different ion-molecule reactions, especially charge exchange ionization. This approach has lived up to all of its promises. The major causes of the observed mismatch are the large variations of the MS source pressure as a result of the CO₂ pressure gradient at the SFC column inlet, the low sensitivity of charge exchange ionization at these high MS source pressures, and the inability to handle polar and nonvolatile molecules. Adaptation of LC/MS interfaces, such as the thermospray interface or the particle beam interface, to SFC/MS conditions was a step forward, but these devices have their own limitations. Alternative methods to direct SFC/MS coupling have been investigated recently. They are based on the use of packed columns rather than capillaries, and on solute ionization at atmospheric pressure rather than under a vacuum, by means of either gas-phase corona discharge ionization or liquidphase electrospray ionization. These new developments may revive research into the design of reproducible and sensitive SFC/MS systems where the number of recent studies is still low compared with other chromatography/mass spectrometry coupling studies.See [1] for part III     

Development of capillary supercritical fluid chromatography/Fourier transform infrared spectrometry

Summary The feasibility of a new technique, supercritical fluid chromatography/Fourier transform infrared spectrometry (SFC/FTIR), for separation and identification of nonvolatile and labile compounds in complex mixtures is discussed in this report. The design of a high pressure (flow cell) interface is described. The results of a continuing study of possible mobile phases for SFC/FTIR are presented. Finally, SFC/FTIR was applied to a standard test mixture. The current detection limits of one component of the mixture, 2,6-di-t-butylphenol, are presented.     

Efficiency for unretained solutes in packed column supercritical fluid chromatography. I. Theory for isothermal conditions and correction factors for carbon dioxide

A general theory for efficiency of nonuniform columns with compressible mobile phase fluids is applied to the elution of an unretained solute in packed-column supercritical fluid chromatography (pSFC). The theoretical apparent plate height under isothermal conditions is given by the Knox equation multiplied by a compressibility correction factor f1, which is equal to the ratio of the temporal-to-spatial average densities of the mobile phase. If isothermal conditions are maintained, large pressure drops in pSFC should not result in excessive efficiency losses for elution of unretained solutes.     

超临界流体色谱中溶质的保留行为

超临界流体色谱中热力学参数最佳化和动力学最佳化同等重要。溶质的保留行为和物质对的分离受柱固定相和流动相各种参数的影响。在本研究中,使用纯CO_2作为流动相,饱和烃、芳烃、以及它们的硝基取代物作为溶质,对上述影响进行了系统地考察。结果发现影响物质对分离的(以分离系数,α,表示)最主要因素是流体的密度。而温度的影响并不明显。柱固定相的性质也有一定程度的影响。在柱固定相相同的情况下,改变操作参数,溶质峰的洗馏顺序可能出现倒置。表明在超临界流体色谱中流动相溶解能力的重要作用。实验结果为了解和优化超临界流体色谱热力学参数提供了依据。     

Supercritical fluid solid chromatography using novel porous glassy carbon. Evaluation of retention mechanisms

The use of a porous glassy carbon (PGC) material as a packed-column SFC stationary phase has been previously demonstrated [1]. The material is further characterized in terms of its retention characteristics. The effects of variations in mobile phase composition, pressure, and temperature conditions are evaluated. Variation of temperature and pressure yielded expected results, specifically, decreased solute capacity factors with increased mobile phase density. The choice of supercritical fluid mobile phase allows the most notable control of solute retention; this was evaluated by adding low percentages of organic modifiers of varying molecular weights to the supercritical carbon dioxide mobile phase. PGC-SFC provides reversed phase characteristics similar to those found for PGC-HPLC. Porous glassy carbon has selectivity characteristics previously unavailable in supercritical fluid chromatography. Use of porous glassy carbon in supercritical fluid chromatography may provide distinct advantages in difficult analytical separations, allowing separations of molecules with only slight structural differences.     

Retention behaviour of carboxylic acid methyl esters in supercritical fluid chromatography

The study on retention behavior in supercritical fluid chromatography (SFC) is necessary to understand the mechanism of the various interactions in SFC. The retention of SFC in carboxylic acid methyl ester/polymethylsiloxane/CO2 system was studied systematically and the retention behavior of this kind of compounds under various typical operation conditions was described using the method of an alternative unified theory of chromatographic retention. The results illustrated that expression: Ink.= a + b/T + cp + dp/T + ep2/T can be used to describe quantitatively the retention behavior of carboxylic acid methyl ester/polymethylsiloxane/CO2 system in the ranges of reduced density from 0.549 to 1.411. It was also found that the entropy of solute in stationary phase is dependent on the density of supercritical fluid (SF) under typical operating conditions of SFC.     

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.

     

Isolation and purification of osthole and imperatorin from Fructus Cnidii by semi-preparative supercritical fluid chromatography

Fructus Cnidii, the dried ripe fruit of Cnidium monnieri (L.) Cusson., has been widely used in traditional Chinese medicine. Osthole and imperatorin are the main active ingredients of Fructus Cnidii and had been found of antispasmodic, anti-HIV, anti-fungal, anti-viral, anti-tumor, anti-mutagenic, anti-arrhythmic, hypotensive, and broad-spectrum antimicrobial effects. A supercritical fluid chromatography (SFC) method for isolation and purification of osthole and imperatorin from Fructus Cnidii was established in this work. The separation conditions, including the stationary phase, the organic modifier, the composition and the flow rate of the mobile phase, column backpressure and column temperature, were optimized on analytical scale at first. And then a semi-preparative SFC (SP-SFC) method was developed based on the conditions of analytical scale SFC. SP-SFC was accomplished on YMC-Pack NH₂ column. Ethanol was used as the modifier and its percentage in the mobile phase was 3%. The flow rate of the mobile phase was 20?mL/min, column backpressure was 13?MPa, column temperature was 318?K, detection wavelength was 310?nm, and injection volume was 0.2?mL. Under the optimum conditions, osthole and imperatorin were obtained with high purities as determined by high performance liquid chromatography. The chemical structures of the obtained compounds were identified by nuclear magnetic resonance and mass spectrum analysis.     

Retention in SFC at high density

The elucidation of the detailed mechanism of retention in supercritical fluid chromatography (SFC) is a complex problem, especially for high-density supercritical fluid mobile phases. Martire and Boehm developed a theory of SFC retention based on a statistical thermodynamic treatment, and introduced a density-squared equation for retention in SFC: In k = a + b/T + cρ + dρ/T + eρ²/T. In this paper the systems n-alkane-polydimethylsiloxane-CO₂ and n-carboxylic acid methyl ester-polydimethylsiloxane-CO₂ have been treated using the method introduced by Martire and Boehm. In the ranges of reduced temperature from 1.079 to 1.293 and reduced SF mobile phase density from 0.705 to 1.632, two problems were discovered in the treatment of SFC retention behavior by the method of Martire and Boehm: (1) the experimental value of the site of CO₂ in the lattice field, r_a, of the two systems was ca 0.9, which is obviously different from the theoretical values of 3.6 or 5.4; and (2) the difference between experimental and calculated retention values increased with increasing SF mobile phase density. The minimum in SFC retention predicted by Martire and Boehm was not observed. On the basis of some assumptions and the experimental data, the following retention equation was derived: In k = a + b/T + cρ + dρ/T + eρ²/T + fρ³/T + gρ⁴/T. This equation was consistent with the experimental data and can conveniently be used to explain other SFC retention behavior.     

Enantioselective separation of biologically active basic compounds in ultra-performance supercritical fluid chromatography

The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC.     

Fundamental challenges and opportunities for preparative supercritical fluid chromatography

The use of supercritical fluids as mobile phases in chromatography was suggested nearly fifty years ago. In spite of some major potential advantages, this mode of chromatography, generally known as SFC, is only now beginning to be considered by the mainstream community but it still does not yet enjoy a popularity comparable to those of gas or liquid chromatography. This seems to be largely due to a combination of (1) the serious instrumental difficulties that took many years to solve; (2) the complexity of the behavior of supercritical fluids in chromatographic systems when their temperature, pressure, or composition changes; (3) the long-lasting absence of any substantial incentive to use more complex systems, when the simpler and more robust approaches provided by HPLC are available. This situation, however, has begun to significantly change during recent years. The incentive of employing green, sustainable technologies in industrial processes as well as in analyses is increasing. Because mobile phases generally used in SFC tend to be less environmentally harmful and less expensive than those used in HPLC, SFC presents strong economical and regulatory advantages over the latter technique. Added to that, steady advancements in LC techniques in the last three decades has solved many instrumental difficulties related to SFC, which is now taking full advantages of many of these advances. One factor, however, has remained mostly unresolved. A clearer understanding of the physico-chemical behavior of supercritical fluids in preparative chromatographic columns under nonlinear conditions is still needed. This seems to be the main obstacle to the establishment of SFC as a sustainable separation tool. One aim of this review is to highlight these issues in more detail through a survey of the state-of-the-art techniques available for the design and operation of SFC. Another aim is to outline a possible series of investigations, which are necessary to develop a better physical understanding of SFC.     

Fast Separation Method Development for Supercritical Fluid Chromatography Using an Autoblending Protocol

Supercritical fluid chromatography (SFC) is a powerful separation technique particularly in the area of enantioseparations. With rapid analysis speed, wide polarity compatibility, higher column efficiency and lower cost of the mobile phase, SFC is regarded as a better choice than high-performance liquid chromatography for drug discovery. In the development of separation method, the choice of modifier and/or additive is the key point of optimum separation. However, such screening of SFC is typically time-consuming. In this study, an autoblending protocol was introduced to speed up the modifier and/or additive screening process, which was performed on a separate programmable gradient proportioning system. The protocol prepares mobile phases on the fly to speed up the screening of modifiers and/or additives and reduces the waste of solutions. Furthermore, by switching mobile phase in the same run, separation of different types of compounds could also be achieved. This system was successfully applied to screen modifier–additive combinations of three alkaloids and three polyphenols by switching to two mobile phase conditions, as well as by a ternary additive mobile phase on an SFC system. The proposed protocol allows fast separation method development of SFC, which was proved to be rapid, simple, and reproducible.     

Zur analytischen und präparativen Trennung von funktionellen Carbosilanen und Phosphanen mit Hilfe der SFC (Supercritical-Fluid-Chromatography)

Analytical and Preparative Separation of Functional Carbosilanes and Phosphanes by Means of SFC (Supercritical Fluid Chromatography) Analytical and preparative separations of functional silanes, carbosilanes and phosphanes by means of SFC (supercritical fluid chromatography) using CO₂ or CF₃Cl as mobile phases are reported. The detecting system (an IR high pressure cell) of the apparatus which was developed by us is depicted. The separation of mixtures containing silanes with strongly polarizing groups proceeds successfully with Nucleosil-C₁₈ and CO₂ according to the molecular weights. Large differences in the polarity of the compounds give rise to a separation according to molecular weights, smaller ones to the types of compounds. The advantages of a separation by SFC are demonstrated using a mixture of 1,3,5-trisilacyclohexanes with SiH, CCl₂ and SiF groups. The preparative separation is demonstrated using a mixture of 4 different groups of a toral 22 carbosilanes. Eight fractions were obtained each of which containing the compounds with the same number of Si atoms. The final separation was achieved by means of a pressure program (Nucleosil-C₁₈, CO₂). The preparative isolation of a cyclic phosphinoborane from a mixture of three components of the same type as well as the isolation of (Me₃Si)₃P from a mixture of P-rich silylphosphanes is reported.     

Separation of geometrical isomers of sesquiterpene and diterpene alcohols on bare silica by carbon dioxide supercritical fluid chromatography

Summary The application of supercritical fluid chromatography (SFC) with modified carbon dioxide to the separation of geometrical isomers of several sesquiterpene or diterpene alcohols is reported. The chromatographic system consists of bare silica (250×4.6 mm i. d.) with carbon dioxide-methanol mobile phase and UV detection. A mixture of sesquiterpene alcohols, (2E-6E)-farnesol, -bisabolol, (E)-nerolidol and (Z)-nerolidol, and also of two diterpene alcohols, (R)- and (S)-geranyllinalol, was separated by SFC using a mixture of carbon dioxide modified with 0.5–1.0% methanol as mobile phase.