Scale Supercritical Fluid Chromatography Research Articles

Analytical scale supercritical fluid chromatography for the analysis of nine tocochromanols in 24 different cold-pressed plant oils: Method development, validation, and isolation of tocotrienols and plastochromanol-8

In the present study, four tocotrienol (T3) homologues and plastochromanol-8 (PC-8) were isolated from plant oils; Linum usitatissimum (PC-8), Bixa orellana (γ-T3 and δ-T3), and Triticum spelta bran (α-T3 and β-T3) by analytical scale supercritical fluid chromatography (SFC). The optimized method of tocopherols, tocotrienols, and PC-8 separation via SFC with UV detection was validated on a biphenyl core-shell column. The sample preparation conditions of the plant oils involved a simple dilution in 2-propanol (1:9, v/v), followed by direct injection into the SFC. For the analysis of 24 different plant oils we recommended the following final analytical scale SFC method conditions: 5 µL for the injection volume and sample oil:2-propanol dilution ratio of 1:9. The final assay developed facilitated the rapid (<15 min) and sensitive (limit of detection within the range of 2.5–7.4 μg/mL and the limit of quantitation within the range of 7.7–22.6 μg/mL) analyses of tocochromanols in obtained cold-pressed oils from twenty-four different plant species. Furthermore, the method was repeatable and reproducible with % RSD values in the context of standard retention times which ranged within 0.10–0.31 intraday and 0.59–0.79 interday.

RPLC-HILIC and SFC with Mass Spectrometry: Polarity-Extended Organic Molecule Screening in Environmental (Water) Samples.

Trace organic compounds are important in environmental analysis because they impact water quality and introduce potential (eco)toxicological effects. Current analytical methods mostly rely on gas chromatography (GC) or reversed-phase liquid chromatography (RPLC) coupled with (tandem) mass spectrometry. However, neither method can easily separate very polar molecules. This study presents two chromatographic separation strategies, a serial RPLC-hydrophilic interaction liquid chromatography (RPLC-HILIC) coupling and an analytical scale supercritical fluid chromatography (SFC) system, and validates their separation effectiveness as polarity-extended chromatographic methods for 274 environmentally relevant compounds. Compounds tested were grouped into three polarity classes, "very polar" {log D (pH 7) below -2.5}, "polar" {log D (pH 7) -2.5 to +2}, and "non-polar" {log D (pH 7) higher than +2}). Nearly all compounds could be retained in both systems with relative standard deviations of retention times (RT; n = 6) typically between 2 and 5%. Both techniques have considerable benefits when combined with accurate mass spectrometric detection. Molecules RT and accurate mass were recorded in a database for each set up. This information was used for compound screening measurements like "hidden-target screening" in complex environmental matrices (such as wastewater treatment plant effluents). Results of both techniques are complementary and useful for all types of molecules polarity. In this study, more than 80% of the compounds found in wastewater treatment plant effluent samples possessed a negative log D (pH 7) value. This result highlights the basic necessity to include "very polar" compounds in water monitoring techniques and protocols.

Isolation of Non‐methylene Interrupted or Acetylenic Fatty Acids from Seed Oils Using Semi‐preparative Supercritical Chromatography

AbstractPreparative scale supercritical fluid chromatography was used for isolating and purifying uncommon non‐methylene interrupted or acetylenic polyunsaturated fatty acids ethyl esters from seed oils. Fractionation of Biota orientalis seed oil ethyl esters was performed by supercritical fluid chromatography to obtain juniperonic acid [(5Z,11Z,14Z,17Z)‐eicosa‐5,11,14,17‐tetraenoic acid], a non‐methylene interrupted polyunsaturated fatty acid. Fractionation of sandalwood seed oil ethyl esters yielded ximenynic acid [(E)‐octadec‐11‐en‐9‐ynoic acid], an acetylenic polyunsaturated fatty acid. The effects of CO2 flow rate, column stationary phase and particle size were explored to optimize ximenynic and juniperonic ethyl ester recovery and purity from ethyl ester mixtures using online UV/Vis detection. Particle size, followed by the stationary phase, were found to be the most important parameters to achieving good separation. Under optimized conditions, ximenynic and juniperonic ethyl ester purities greater than 99 and 95%, respectively, were achieved in a one step process without co‐solvent. The isolation and recovery of juniperonic acid from biota seed oil free fatty acids was also attempted. Using free fatty acids as the feed material, the non‐methylene interrupted polyunsaturated sciadonic acid was also able to be separated from other compounds including juniperonic acid under some conditions, and gave an increase in concentration of more than 17 times.

Evaluation of scale-up from analytical to preparative supercritical fluid chromatography

An approach for reliable transfer from analytical to preparative scale supercritical fluid chromatography was evaluated. Here, we accounted for the conditions inside the columns as well as to the fact that most analytical instruments are volume-controlled while most preparative scale units are mass-controlled. The latter is a particular problem when performing pilot scale experiments and optimizations prior to scaling up to production scale. This was solved by measuring the mass flow, the pressure and the temperature on the analytical unit using external sensors. Thereafter, it was revealed with a design of experiments approach that the methanol fraction and the pressure are the two most important parameters to control for preserved retention throughout the scale-up; for preserved selectivity the temperature was most important in this particular system. Using this approach, the resulting chromatograms from the preparative unit agreed well with those from the analytical unit while keeping the same column length and particles size. A brief investigation on how the solute elution volume varies with the volumetric flow rate revealed a complex dependency on pressure, density and apparent methanol content. Since the methanol content is a parameter of great importance to control during the scale up, we must be careful when changing operational and column design conditions which generates deviations in pressure, density and methanol content between different columns.

Separation of derivatized glucoside anomers using supercritical fluid chromatography

Preparative scale supercritical fluid chromatography was used to separate derivatized anomeric monosaccharides. Methyl 2,3,4,6-tetra-O-benzoyl-α-d-glucopyranoside and methyl 2,3,4,6-tetra-O-benzoyl-β-d-glucopyranoside were synthesized by benzoyl derivatization of the parent anomerically pure monosaccharides. The elution profiles of each anomer were studied, suggesting that separation was possible. Mixtures of the compounds were then prepared at different ratios and separation was achieved using carbon dioxide/ethanol mixtures as the mobile phase and GreenSep™ 5micron particle size silica as the stationary phase. Ethanol co-solvent in the mobile phase was required to give a successful separation of the anomers however over the range 6.5–10.4wt% ethanol concentration studied here there was little variation in the degree of separation. In fractionation trials, separation of mixtures into fractions with >98% purity was achieved, with recoveries greater than 96%.

Instrumentation for analytical scale supercritical fluid chromatography

Analytical scale supercritical fluid chromatography (SFC) is largely a sub-discipline of high performance liquid chromatography (HPLC), in that most of the hardware and software can be used for either technique. The aspects that separate the 2 techniques stem from the use of carbon dioxide (CO2) as the main component of the mobile phase in SFC. The high compressibility and low viscosity of CO2 mean that pumps, and autosamplers designed for HPLC either need to be modified or an alternate means of dealing with compressibility needs to be found. The inclusion of a back pressure regulator and a high pressure flow cell for any UV–Vis detector are also necessary. Details of the various approaches, problems and solutions are described. Characteristics, such as adiabatic vs. isothermal compressibility, thermal gradients, and refractive index issues are dealt with in detail.

Enantioselective separations by packed column subcritical and supercritical fluid chromatography

Enantioselective separations have been one of the most successful applications of supercritical fluid chromatography (SFC). Although analytical scale separations have dominated the literature, the use of SFC for preparative chiral separations is growing. Both analytical and preparative scale SFC separations seek to take advantage of the high efficiency, high throughput, and rapid method development associated with the technique. This review will cover recent developments in the application of SFC to enantioseparations.

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.

Customization of a Commercially Available Prep Scale SFC System to Provide Enhanced Capabilities

Preparative Scale Supercritical Fluid Chromatography is emerging as a powerful alternative to HPLC for the purification and separation of complex chemical reaction mixtures. Advantages include greatly reduced solvent usage (and thus lower cost and environmental impact), higher throughput, and in some cases higher resolution. While there are commercially available prep SFC instruments, none currently offer all the features desired by many medicinal chemists engaged in the drug discovery process. These include: the ability to collect an unlimited number of fractions per sample with high recovery and negligible carryover, fully automated capacity to collect several hundred fractions, and the ability to collect fractions into the same disposable test tubes and racks which are already employed in HPLC. This article describes the customization of a preparatory scale SFC system purchased from Berger Instruments, Inc., Newark, DE. (a subsidiary Mettler-Toledo International, Inc., of Greifensee, Switzerland) in order to provide these capabilities.

Customization of a Commercially Available Prep Scale SFC System to Provide Enhanced Capabilities

Preparative Scale Supercritical Fluid Chromatography is emerging as a powerful alternative to HPLC for the purification and separation of complex chemical reaction mixtures. Advantages include greatly reduced solvent usage (and thus lower cost and environmental impact), higher throughput, and in some cases higher resolution. While there are commercially available prep SFC instruments, none currently offer all the features desired by many medicinal chemists engaged in the drug discovery process. These include: the ability to collect an unlimited number of fractions per sample with high recovery and negligible carryover, fully automated capacity to collect several hundred fractions, and the ability to collect fractions into the same disposable test tubes and racks which are already employed in HPLC. This article describes the customization of a preparatory scale SFC system purchased from Berger Instruments, Inc., Newark, DE. (a subsidiary Mettler-Toledo International, Inc., of Greifensee, Switzerland) in or...

Peak compression in semi-preparative supercritical fluid chromatography

Peak compression of a dihydropyridine drug, clevidipine, is obtained in both analytical and semi-preparative scale supercritical fluid chromatography, resulting in extremely high apparent efficiencies. The observed effect, when utilising a carbon dioxide/2-propanol mobile phase with a bare silica stationary phase, is achieved when the retention of the clevidipine peak is controlled to coalesce with a system peak, generated as a result of having water in the sample. Apparent efficiencies of 350,000 and 170,000 plates meter−1 were obtained when 0.25 and 0.5 mg, respectively, are directly injected to a 200×4.6 mm ID 5 μm Hypersil silica packed column. The effect was extended to a semi-preparative system where apparent efficiencies in the region of 2,000,000 plates meter−1 were observed when 0.3 mg of a clevidipine sample containing 80% water was injected to a 250×10 mm I.D. column containing 5-μm Hypersil silica particles.