Facilitated on-line supercritical fluid extraction - supercritical fluid chromatography for nonpolar and polar compounds from milk thistle seeds.

Plant seeds, as those from milk thistle (Silybum marianum), are a valuable source of nonpolar and polar compounds with potentially interesting biological activity. The main nonpolar compounds are triglycerides, which are also the main components of all vegetable oils. In addition, specific polar compounds - flavonolignans, called silymarin, have been found in large amounts in milk thistle seeds extract. These flavonoids derivatives have different biological activity, for instance hepatoprotective effects. In order to extract and analyze both nonpolar (triglycerides) and polar compounds (flavonolignans) from milk thistle seeds through a sequential methodology, an on-line supercritical fluid extraction - supercritical fluid chromatography (SFE-SFC) method was developed. Different ways of transferring the extracts from SFE to SFC (i.e. direct on-column transfer and loop transfer) were compared, and particularly for their effect on chromatographic quality. In this respect, nonpolar and polar compounds caused different issues, especially as polar compounds required a significant portion of co-solvent in the extraction step, favoring early elution in the chromatographic column. First, on-line SFE-SFC was used for triglycerides analysis and allowed the comparison of transfer modes. Then, on-line kinetics were performed to measure defatting time before polar molecules extraction. Finally, the eventual benefit of loop transfer was also investigated for the analysis of flavonolignans, polar molecules whose analysis can be difficult by on-line SFE-SFC. The aim of this paper is to discuss the versatility of on-line SFE-SFC and how challenging the coupling can be, especially when both non-polar and polar molecules must be analyzed independently in a single sample.

Defining a generic column set for achiral supercritical fluid chromatography applied to pharmaceuticals or natural products.

When starting a method development in supercritical fluid chromatography (SFC), the first step is usually to screen several stationary phases based on previous experience or simply based on what is available in the laboratory. However, as there are now a large number of stationary phases available for SFC, the choice of an adequate set of columns to rapidly achieve a satisfying result can be difficult. In this project, 16 columns comprising a wide diversity of stationary phases and polarities ranging from the most polar (like bare silica gel) to the least polar (like octadecylbonded-silica) were compared, based on the gradient analysis of 129 probe compounds. The set mostly comprised active pharmaceutical ingredients, natural products and a few metabolites. The columns were ranked with the help of Derringer desirability functions taking account of (i) the number of compounds eluted from the column, (ii) the elution time in a suitable time frame, (iii) the average peak width, (iv) the average peak symmetry and (v) the spreading of retention along the gradient time. The five criteria selected showed no correlation. Overall, it appeared that those columns that had a high overall score were good for several reasons, like bare silica gel, propanediol-bonded silica or pentabromobenzyloxy-bonded silica. Initially, the columns had been screened with a gradient elution starting from 5% co-solvent and ending with 50% co-solvent in CO2. However, for some most retentive columns like amide-bonded silica, too many compounds remained non-eluted from the column. To examine this column more fairly, a second elution gradient was applied that ended with 100% co-solvent. This proved effective in restoring good overall performance through the elution of the most polar compounds.

Characterization of stationary phases in supercritical fluid chromatography including exploration of shape selectivity.

Achiral packed column supercritical fluid chromatography (SFC) has shown an important regain of interest in academic and industrial laboratories in the recent years. In relation to this increased concern, major instrument manufacturers have designed some stationary phases specifically for SFC use. SFC stationary phases have been widely examined over the last two decades, based on the use of linear solvation energy relationships (LSER), which relate analyte retention to its properties and to the interaction capabilities of the chromatographic system. The method provides some understanding on retention mechanisms (normal phase, reversed phase or mixed-mode) and the possibility to compare stationary phases on a rational basis, especially through a spider diagram providing a visual classification. The latter can be used as a primary tool to select complementary stationary phases to be screened for any separation at early stages of method development, before optimization steps. In this context, the characterization of the 14 columns from the Shim-pack UC series (Shimadzu Corporation, Kyoto, Japan), which are dedicated to SFC and more broadly to unified chromatography (UC), was performed, using the LSER methodology. As in previous works, seven descriptors, including five Abraham descriptors (E, S, A, B, V) and two descriptors describing positive and negative charges (D- and D+) were first employed to describe interactions with neutral and charged analytes. Secondly, two more descriptors were introduced, which were previously employed solely for the characterization of enantioselective systems and expressing shape features of the analytes (flexibility F and globularity G). They brought additional insight into the retention mechanisms, showing how spatial insertion of the analytes in some stationary phases is contributing to shape separation capabilities and how folding possibilities in flexible molecules is unfavorable to retention in other stationary phases.

Hyphenation of ultra-high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 2. Study of chromatographic and mass spectrometry parameters for the analysis of natural non-polar compounds.

An analytical method based on Ultra-High-Performance Sub/Supercritical Fluid Chromatography (UHPSFC) coupled with High-Resolution Mass Spectrometry (HR-MS) equipped with an Atmospheric Pressure Chemical Ionisation source (APCI-Q-TOF-HRMS) was developed for the screening of various compounds in oily samples. The hyphenation was achieved using a hybrid UHPSFC system for the vegetable oil analysis, mainly composed of fatty acids, diacylglycerols, triacylglycerols but containing also some minor bioactive compounds. No split was used with this ionisation source in this optimized configuration, allowing the introduction of all compounds in the mass spectrometer, ensuring a better sensitivity. This configuration is preserving peak shapes and efficiency because the dead volume of this interface is optimized by the supplier for the hyphenation with open cell detectors such as Evaporative Light Scattering Detector (ELSD) or MS detectors. The influence of analytical conditions (UHPSFC and APCI parameters) on MS response was studied to understand the behaviour of analytes in UHPSFC-APCI-MS. The tested parameters were: the corona discharge value, the nebulising gas pressure, the drying gas flow rate, MS source temperature and the mobile phase flow rate (at constant modifier percentage). A factorial experimental design was carried out for this study, which displayed the major and negative role of increased nebulising gas pressure. The factorial design was renewed with the four remaining parameters, allowing to enlighten and explain their different effects on MS responses. Finally, some vegetable oils were analysed by UHPSFC-APCI-HRMS with these optimal conditions to determine the chemical structures of unknown compounds from oily samples.

Hyphenation of ultra high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 1. Study of the coupling parameters for the analysis of natural non-polar compounds.

An analytical method based on Ultra-High-Performance Supercritical Fluid Chromatography (UHPSFC) coupled with Atmospheric Pressure Chemical Ionization - High-resolution mass spectrometry (APCI-Q-TOF-HRMS) was developed for compounds screening from oily samples. The hyphenation was made using a commercial UHPLC device coupled to a CO2 pump in order to perform the chromatographic analysis. An adaptation of the injection system for compressible fluids was accomplished for this coupling: this modification of the injection sequence was achieved to prevent unusual variations of the injected volume related to the use of a compressible fluid. UHPSFC-HRMS hyphenation was optimized to enhance the response of the varied compounds from a seed extract (anthraquinones, free fatty acids, diacylglycerols, hydroxylated triacylglycerols and triacylglycerols). No split was used prior to the APCI ionization source, allowing introducing all the compounds in the spectrometer, ensuring a better sensitivity for minor compounds. The effects of a mechanical make-up (T-piece) added before this ionization source was discussed in terms of standard deviation of response, response intensity and fragmentation percentage. The location of the T-piece with regards to the backpressure regulator (BPR), the flow rate and the nature of the make-up solvent were studied. Results show that the effects of the studied parameters depend on the nature of the compounds, whereas the make-up addition favours the robustness of the mass response (quantitative aspect).

Selective enrichment in bioactive compound from Kniphofia uvaria by super/subcritical fluid extraction and centrifugal partition chromatography.

Nowadays, a large portion of synthetic products (active cosmetic and therapeutic ingredients) have their origin in natural products. Kniphofia uvaria is a plant from Africa which has proved in the past by in-vivo tests an antioxidant activity due to compounds present in roots. Recently, we have observed anthraquinones in K. uvaria seeds extracts. These derivatives are natural colorants which could have interesting bioactive potential. The aim of this study was to obtain an extract enriched in anthraquinones from K. uvaria seeds which mainly contains glycerides. First, the separation of the seed compounds was studied by using supercritical fluid chromatography (SFC) in the goal to provide a rapid quantification method of these bioactive compounds. A screening of numerous polar stationary phases was achieved for selecting the most suited phase to the separation of the four anthraquinones founded in the seeds. A gradient elution was optimized for improving the separation of the bioactive compounds from the numerous other families of major compounds of the extracts (fatty acids, di- and triglycerides). Besides, a non-selective and green Supercritical Fluid Extraction (SFE) with pure CO2 was applied to seeds followed by a Centrifugal Partition Chromatography (CPC). The CPC system was optimized by using the Arizona phase system, to enrich the extract in anthraquinones. Two systems were selected to isolate the bioactive compounds from the oily extract with varied purity target. The effect of the injection mode for these very viscous samples was also studied. Finally, in order to directly apply a selective process of extraction to the seeds, the super/subcritical fluid extraction was optimized to increase the anthraquinone yield in the final extract, by studying varied modifier compositions and nature, as well as different temperatures and backpressures. Conditions suited to favour an enrichment factor bases on the ratio of anthraquinone and trilycerides extracted are described.