Supercritical Fluid Chromatography System Research Articles

SFC Instrument Modification to Allow Greater Flexibility in Method Development by Generating Mixtures of Solvents and Modifiers On-Line for Mobile Phase B Optimization

A binary supercritical fluid chromatography (SFC) system was further modified by the addition of a quaternary HPLC pump. The flow outlet of this pump was connected to the flow inlet of the existing modifier pump (“pump B”) by way of a custom-designed reservoir in such a way that blends of methanol with up to three additional solvents and/or additives could be delivered to pump B on-the-fly. Small amounts of modifiers (0.1–5 %) are typically mixed with mobile phase B in SFC to improve either the peak shape or to optimize the selectivity of the separation. As the critical influence of these modifiers on the separation of complex mixtures cannot a priori be predicted, incremental changes in mobile phase B compositions typically have to be evaluated on a trial-and-error basis. The instrumental modifications described in this paper eliminate the need to prepare these combinations off-line and the concomitant time requirement to re-purge supply and de-gasser solvent lines with each change in composition. By eliminating these steps, a significantly decrease in the overall SFC method development time can be achieved. Furthermore, such an on-line arrangement allows for greater flexibility in method development, that is, in the “fine-tuning” of the method once the stationary phase and gradient conditions have been established.

Evaluation of mobile phase gradient supercritical fluid chromatography for impurity profiling of pharmaceutical compounds

The use of gradient supercritical fluid chromatography (SFC) for the impurity profiling of pharmaceutical products is not widely practiced. Historically, the limited advancement in SFC instrumentation and the lag in column development have resulted in marginal sensitivity, selectivity and reproducibility when compared with high performance liquid chromatography (HPLC). Using a recently developed commercial module, which allows an ordinary HPLC to be converted to a SFC system, a significant improvement in sensitivity (up to ~12-fold) has been obtained over previous studies. This has allowed for the first time a "real-world" head-to-head comparison of SFC to HPLC for impurity profiling of pharmaceutical products in a regulated environment. Retention time reproducibility and low level impurity detection were found to be comparable to reversed phase liquid chromatography (RPLC), that is, single digit %relative standard deviations (RSDs) were obtained for impurities present at less than 0.1 area%. Furthermore, these results were obtained with drug loading levels (≤2 mg/mL) that are not only comparable to those employed with HPLC, but are dictated by the limited solubility of many drug candidates. The elution of impurities was generally found to be orthogonal to that obtained with RPLC, but it was still challenging to find SFC conditions that would separate all of the components in the mixtures studied. In terms of enhancing selectivity, small amounts of mobile phase additives (0.1-1%) and temperature optimization were found to have a greater impact in SFC method development versus RPLC. However, unlike gradient RPLC, the relative changes in baseline noise and slope were found to be a complex function of the experimental conditions, with the largest differences in noise levels being generally observed for the widest and steepest gradients. It is likely that this gradient related noise is more apparent now because other sources of noise in SFC have been reduced significantly.

Evaluation of a New Preparative Supercritical Fluid Chromatography System for Compound Library Purification: The TharSFC SFC-MS Prep-100 System

Preparative HPLC-MS is often the method of choice for purification of small amounts (<100mg) of diverse new molecules, such as compound libraries for drug discovery. The method is robust, well proven, and widely applicable. In contrast, preparative supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) has seen only slow acceptance for the same application--despite some potential scientific and economic advantages. One of the reasons for slow adoption of SFC-MS is the lack of well-proven, robust, and commercially available instrumentation. In early 2009, TharSFC (a Waters Company, Pittsburgh, PA) introduced a new fully integrated system for preparative SFC-MS: The SFC-MS Prep-100. We report herein an objective evaluation of the SFC-MS Prep-100, including tests for pump and autosampler performance, sample recovery, sample carryover, fraction triggering, detector/fraction collector synchronization, and overall robustness. Our results suggest that the SFC-MS Prep-100 represents a significant advance over previous generation instrumentation.

Use of isopycnic plots in designing operations of supercritical fluid chromatography: I. The critical role of density in determining the characteristics of the mobile phase in supercritical fluid chromatography

In Supercritical Fluid Chromatography (SFC), the key chromatographic parameters of any compound, its retention and efficiency, are known to strongly depend on the density of the mobile phase. This indicates that iso-density, also called isopycnic, plots drawn on the pressure–temperature plane can provide an effective tool to analyze how SFC systems may operate under different combinations of inlet and outlet pressures and column temperature. To effectively use these isopycnic plots in designing the operations of SFC systems, however, a deeper understanding of the factors behind the dependence of the performance of these systems on the mobile phase density is required. The nature of this density dependence is explored with reference to the key physical properties of the mobile phase, its viscosity, diffusivity and solubility. This study is focused on the use of pure carbon dioxide as the mobile phase, but this method of investigation is applicable for other mobile phase combinations as well.

Applications of Supercritical Fluid Chromatography-Tandem Mass Spectrometry in Pharmaceuticals

Supercritical fluid chromatography (SFC) is a fully mature technique and can readily be coupled to mass spectrometers relatively easily. In the last years, supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) has had a great advance in productivity, due to increase in reliability and robustness of both SFC and MS systems, the need to push drug discovery faster, and as a potential solution to old problems unsolved and new problems yet to be encountered. This article will illustrate the role of SFC-MS analysis in drug discovery process.

Effect of pressure drop on solute retention and column efficiency in supercritical fluid chromatography Part 2: Modified carbon dioxide as mobile phase

The effect of pressure drop on the performance of supercritical fluid chromatography systems using a modified mobile phase (carbon dioxide + ethanol) was studied. Experiments were performed on a Lichrospher-RP-18 column with phenanthrene as a solute. A wide range of back pressures (130 to 210 bar) and modifier concentrations (2 to 7% w/w) have been explored. Experiments yielding both small and large pressure drops were performed. From these experiments, parameters to describe pressure drop, retention, and column efficiency were extracted, and were used to simulate the dynamics of the chromatographic column. A good match between the experimentally measured and calculated values of pressure drop, retention times, and column efficiency was observed. At low back pressure and modifier composition, significant loss of column efficiency was observed.

Characterisation of stationary phases in supercritical fluid chromatography with the solvation parameter model: V. Elaboration of a reduced set of test solutes for rapid evaluation

Characterisation of chromatographic systems with the solvation parameter model provides satisfactory information on the main non-ionic interactions developed in a chromatographic system. The procedure requires the analysis of a large number of compounds to warrant the relevance and the accuracy of the calculated models, and even if retention time is lower in supercritical fluid chromatography (SFC) than in HPLC (3–5 times lower), a decrease in the time required for that procedure would favour the use of this model in method development. Consequently, in order to establish a rapid testing procedure that would provide equivalent information, nine key solutes were carefully selected among the hundred we classically use. The separation factors calculated between these key solutes, taken two by two to establish new equations, allow the calculation of the model coefficients. The normal testing procedure is thus reduced from one or two days down to 2 h. Precision and accuracy of the models provided are assessed through back-calculation of the coefficients that served for the establishment of the procedure, then through calculation of the coefficients of 13 new SFC systems. The applicability of the rapid testing procedure in SFC is evidenced with three examples: the elaboration of a system map, by varying the modifier concentration in the mobile phase, and the comparison of six ODS phases bonded on the same silica base. The simplified procedure presented here does not pretend to characterize the chromatographic systems as precisely as the complete testing procedure does, but is only aimed at rapidly evaluating the chromatographic retention characteristics when operating parameters are varied.

Use of specially designed columns for antioxidants and antimicrobials enrichment by preparative supercritical fluid chromatography

A new, specially designed column has been developed for fractionation of supercritical fluid extract of rosemary by using a preparative supercritical fluid chromatography system (Prep-SFC). The column evaluated in this work was prepared using a new packing method consisting of a combination of slurry and supercritical CO2 with commercial silica particles coated with a stationary phase commonly used in gas chromatography, such as SE-54 (5% phenyl-, 95% methylsilicone). The new packing procedure provided columns with reasonable efficiencies, with high stability and useful at high-pressure range. A 25cm×10mm i.d. column packed with silica particles coated with 3% of SE-54 was prepared, and its separation power was tested for isolating fractions with high antioxidant and/or antimicrobial activity from a supercritical rosemary extract. The SFC conditions were selected based on a previous work done with a commercial LC-Diol packed column (130bar, 80°C), and different percentages of modifier in the mobile phase were tested (5 and 10%). Two cyclones were employed to collect the fractions which were then characterized by HPLC-diode array detection (DAD), GC, and in vitro antioxidant and antimicrobial assays. The use of coated packed columns allowed the fractionation of a complex mixture of rosemary supercritical extract with a minimum amount of modifier in the mobile phase (5% ethanol). At the optimum conditions it was possible to obtain two very active fractions in terms of antioxidant and antimicrobial activity, with no residual rosemary aroma and with improved activities compared to the original supercritical extract.

Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatography

The carbobenzyloxy (cbz) protecting group is evaluated for it's potential to enhance the resolution of chiral amine enantiomers using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A series of cbz derivatives of commercially available racemates was prepared and analyzed by enantioselective chromatography using a variety of mobile phases and polysaccharide and Pirkle-type chiral stationary phases (CSPs). The cbz-derivatized product consistently demonstrated enhanced chiral resolution under HPLC and SFC conditions. Improved selectivity and resolution combined with an automated preparative HPLC or SFC system can lead to the rapid generation of highly purified enantiomers of desirable starting materials, intermediates or final products.

Effect of pressure drop on solute retention and column efficiency in supercritical fluid chromatography

The effect of pressure drop on the performance of supercritical fluid chromatographic systems is studied. Experiments have been conducted at different pressures and at 55 and 65 ° C. Experiments at conditions leading to large and small pressure drops have been performed. Parameters to describe the pressure drop, retention time and efficiency have been extracted from these experiments. Using these parameters the dynamics of the chromatographic column have been modeled. Darcy’s law was used to describe the pressure drop. The efficiency was modeled by considering the contributions from axial dispersion, resistance to mass transfer from the fluid film, and to diffusion in the pores. Good description of the pressure drop, retention time and mass transfer characteristics under normal operating conditions was obtained. The parameters extracted were used to predict the elution profile by numerical simulations. Considerable loss in column efficiency was observed when operating the column at lower values of the back pressure.

Porous Electrodes Supported on Ion-Exchange Membranes as Electrochemical Detectors for Supercritical Fluid Chromatography

A conveniently assembled electrochemical cell, exploiting a porous electrode supported on a moist perfluorinated ion-exchange polymer, is proposed for profitable electrochemical detection in supercritical fluid chromatography. It consists of a porous Pt working electrode, contacted by the mobile phase from the chromatographic column, which is chemically deposited onto one side of a Nafion membrane. The rear uncoated side of this membrane, acting as a solid polymer electrolyte, is contacted by an electrolyte solution (1 M NaCl) contained in an internal compartment equipped with a Pt counter electrode and a Ag/AgCl, Cl(-) 1 M reference electrode. Ferrocene, eluted with supercritical carbon dioxide through a Spherisorb column installed in a supercritical fluid chromatographic system, was used as electroactive prototype analyte to test the performance of this detector, which turned out to be quite better than that provided by a conventional on-line UV absorbance detector. The recorded peaks were characterized by both a good reproducibility (4.5%) and a linear dependence of their height and area, which extended over a wide concentration range ( approximately 3 orders of magnitude). Moreover, they were not interfered by possible solvent front, unlike peaks recorded by the UV detector. The detection limit, estimated for a signal-to-noise ratio of 3 (4.2 x 10(-11) mol), was lower by approximately 1 order of magnitude than that found for the UV detector. Finally, the long-term stability of this detector was satisfactory in that only a approximately 6% decrease in the current responses was observed after a rather long period (2 months) of continuous use.

Fluidos supercríticos em química analítica. II. Cromatografia com fluido supercrítico: instrumentação

The first paper in this series discussed the basic theory involved in supercritical fluid chromatography (SFC) and how the technique progressed from gas and liquid chromatography. The first SFC instruments were simple adaptations of the commercially available liquid chromatographs with packed columns followed by modifications in gas chromatographs using open tubular capillary columns. In this paper, the most important aspects regarding instrumentation are covered, including practical, simple, and the most important, inexpensive solutions to build a home-made SFC system.

Development of comprehensive two‐dimensional packed column supercritical fluid chromatography

AbstractA comprehensive two‐dimensional supercritical fluid chromatographic system with conventional packed columns was developed. The system used a computer‐controlled ten‐port valve, a capillary trap, and a restrictor. The mobile phase was pure carbon dioxide. The first dimension column was operated under constant pressure in the stopped‐flow mode, while the second dimension column was operated in the constant flow mode. The effluent of the first column was depressurized through the restrictor and solute was trapped within the capillary trap. Then the trapped solute was transferred into the second column by switching the valve. Since the solute transfer process is very rapid, the valve can be switched back after a short injection period in order to collect next fraction. This allowed the sampling time to be changed without changing any of the other conditions, such as the second dimension run time. The system was applied to the separation of triglycerides in fats and oils. Separations were performed using the same ODS packing materials for both columns, and the first column was operated under subcritical conditions and the second column under supercritical conditions.

Purification of HTOS Libraries by Supercritical Fluid Chromatography

A commercially available preparative scale supercritical fluid chromatography (SFC) system has been customized and applied to the purification of high throughput organic synthesis (HTOS) libraries. Pilot reactions allow the triage between HPLC and SFC as the most appropriate purification technique, and when applicable, SFC has been proven to offer advantages in terms of decreased purification and sample dry‐down time and purification capabilities, somewhat complementary to HPLC. Standardized chemistry‐specific SFC methods have been determined and HTOS pilot reactions allow refinement of these standard methods to optimal conditions for a specific library core.

Coupling of a supercritical fluid chromatography system to a hybrid (Q-TOF 2) mass spectrometer: on-line accurate mass measurements.

This note describes a simple and economical method to couple a supercritical fluid chromatography (SFC) system (Berger Instruments, US) with a high-resolution hybrid mass spectrometer (Q-TOF 2; Micromass, UK). This experimental arrangement has three distinct advantages: (1) coupling between the two systems can be effected without the need for an interface or hardware modifications of either system, (ii) this experimental arrangement provides on-line accurate mass SFC/MS measurements which are indispensable for the characterisation of new chemical entities and unknown metabolites, and (iii) the characteristically fast spectral acquisition rate of the time-of-flight (TOF) analyser renders the present arrangement an important contribution to future semipreparative fraction collection setups which use mass spectrometry as a detector.

Open Tubular Supercritical Fluid Chromatography (ot-SFC) Applied to the Separation of Coal-Derived Products Obtained by Supercritical Fluid Extraction (SFE)

This work describes the construction and evaluation of a simple and versatile capillary supercritical fluid chromatographic (SFC) system and its application to the analysis of liquid fuels obtained by SFE. The modular supercritical fluid chromatography (SFC) system uses a syringe pump, a fused silica capillary column (100 μm i.d., coated with a 0.2-μm thick film of 5% phenylpolymethylsiloxane), a FID detector, and a high pressure injection valve with a 60-nL internal loop. Coal-derived samples were obtained by supercritical fluid extraction (SFE) of a Brazilian high-inertinite coal from Amando Simoes mine, located in Parana state (Brazil) using toluene as solvent and operating in the static mode. SFE was followed by preparative liquid chromatography fractionation into eight discrete fractions (PLC-8 method). Two of those fractions (F-2 and F-8) were used to evaluate the proposed set-up. The system described proved to be simple, easy-to-use, and versatile when used in the analysis of a range of samples from low molecular weight-low volatility (F-2) to higher molecular weight-high polarity asphaltols (F-8).

Purity testing of organometallic catalysts by packed capillary supercritical fluid chromatography

A packed capillary column supercritical fluid chromatography system with flame ionization detection has been used for purity testing of candidates for homogeneous catalysis such as methyl tricarbonyl pentamethylcyclopentadienyl tungsten [Cp*W(CO) 3Me], methyl tricarbonyl cyclopentadienyl tungsten [CpW(CO) 3Me], tetramethyl pentamethylcyclopentadienyl iridium (Cp*IrMe 4), trimethyl (1,4,7-trimethyl-1,4,7-triazocyclononane) rhodium (CnRhMe 3), trimethylphosphine hydride dicarbonyl cyclopentadienyl molybdenum [η 5-CpMoH(CO) 2PMe 3] and triphenylphosphine hydride dicarbonyl cyclopentadienyl molybdenum [η 5-CpMoH(CO) 2PPh 3]. A mass limit of detection of 240 pg was found for η 5-CpMoH(CO) 2PMe 3 when using a 60-nl injection volume and pure CO 2 as mobile phase on a 5 μm Kromasil C 18 column. The stability of the catalysts in solution has been examined. After 24 h more than 70% of η 5-CpMoH(CO) 2PMe 3 and 50% of η 5-CpMoH(CO) 2PPh 3 had decomposed. Due to the instability of the compounds the purity testing had to take place rapidly after sample dissolution.

Electrospray mass spectrometry and supercritical fluid chromatography of methylated β-cyclodextrins

Five commercial dimethylated β-cyclodextrin (DM-β-CD) samples were analysed by electrospray (or ionspray) mass spectrometry (ESI-MS) and supercritical fluid chromatography (SFC) with evaporative light scattering detection. A silica and a nitro-bonded silica were selected using CO 2–methanol–acetonitrile–water and CO 2–methanol as mobile phase, respectively. An extensive optimisation scheme was performed for mobile phase selection. Both SFC systems were used for analyses of complex DM-β-CD samples. Peak identifications were made using off-line ESI-MS. Commercial DM-β-CDs are impure mixtures of homologues and isomers and analysis reveals that every manufacturer produces a different mixture.

Feasibility of lovastatin analysis by packed column supercritical fluid chromatography with ultraviolet detection

A reliable supercritical fluid chromatography (SFC) method was developed for the analysis of lovastatin, a hypocholesterolaemic drug, from MEVACOR®. Methanol-modified carbon dioxide was shown to elute the drug, and its dehydrolovastatin and hydroxy acid lovastatin degradation products from a Hypersil® silica column. However, the hydroxy acid lovastatin was found to tail in this mobile phase. The phenomena was eliminated by the addition of trifluoroacetic acid [Haouck, S. Thomas, D. K. Ellison, Talanta 40 (1993) 491] to the mobile phase which permitted the drug and its two main degradation products to all elute from the Hypersil® silica column in under 6 min with symmetrical peak shape. Chromatographic limit of detection (LOD) and limit of quantification (LOQ), linear dynamic range (LDR), and injection precision were obtained in order to assess the chromatographic performance of the SFC system for the lovastatin separation.

Packed-column supercritical fluid chromatography coupled with solid-phase extraction for the determination of organic microcontaminants in water

A multiresidue method for the analysis of 35 common contaminants (including pesticides, polycyclic aromatic hydrocarbons and phenols) in lough and river waters from Castilla y León Spanish region, by using supercritical fluid chromatography (SFC) with five silica packed columns, is described. In order to decrease the detection limits, a preconcentration step by coupling solid-phase extraction to the SFC system was used. The different variables affecting the extraction procedure were studied and optimized, selecting the Isolute Env+ as the best sorbent. The detection limits achieved ranged from 0.4 to 2.6 μg l −1.