Caroline West

The many faces of packed column supercritical fluid chromatography--a critical review.

Packed column sub- and supercritical fluid chromatography (SFC) is a versatile separation method: on the one hand the number of parameters acting on the quality of a separation is very large, and the effects of these parameters can be complex (and not always fully understood). But on another hand, due to numerous advantageous properties of CO2-based mobile phases, method development is a fast task. This paper is a review of the main features of SFC, focusing essentially on achiral separations. However, several fundamental aspects discussed here are also relevant to chiral SFC separations. This is not intended to be an extensive review, as the way to practice SFC has somewhat evolved with time. We rather wished to provide an expert opinion on the characteristics of the method, pointing at the sources of difficulty and displaying the wide possibilities that it offers. A large number of selected applications concerning several different areas are also presented.

Porous graphitic carbon: A versatile stationary phase for liquid chromatography

Chromatographic stationary phases based on porous graphitic carbon were invented 30 years ago, while columns have been commercially available for 20 years. This special occasion deserved a complete review on this material. In this paper, we describe current knowledge on graphitic carbon stationary phases, based on over 400 fundamental studies and applications.

Modern analytical supercritical fluid chromatography using columns packed with sub-2 μm particles: a tutorial.

This tutorial provides an overview of the possibilities, limitations and analytical conditions of modern analytical supercritical fluid chromatography (SFC) using columns packed with sub-2 μm particles. In particular, it gives a detailed overview of commercially available modern SFC instrumentation and the detectors that can be employed (UV, MS, ELSD, FID, etc.). Some advice on the choice of the stationary phase dimensions and chemistries, the nature of the mobile phase (choice of organic modifier and additives) and its flow rate as well as the backpressure and temperature are also provided. Finally, several groups of potentially problematic compounds, including lipophilic compounds, hydrophilic substances and basic drugs, are discussed in detail. All these families of analytes can be resolved with SFC but require specific analytical conditions.

A unified classification of stationary phases for packed column supercritical fluid chromatography.

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.

Investigations on the chromatographic behaviour of zwitterionic stationary phases used in hydrophilic interaction chromatography

Two commercial stationary phases possessing a sulfobetaine zwitterionic bonded ligand (ZIC-HILIC and Nucleodur HILIC) were compared under hydrophilic interaction chromatographic (HILIC) conditions. First of all, the separation of 12 model compounds chosen among neurotransmitters and presenting a diversity of ionization states (anionic, cationic and zwitterionic) was studied under varied operating conditions. The effects of the percentage of acetonitrile, ammonium acetate concentration and temperature of the mobile phase were compared on the two columns. Secondly, a generally applicable retention model was established, based on chromatographic retention data (logk) acquired for 76 model compounds. The chosen compounds are small molecules presenting a wide diversity of molecular structures and are relevant to biomedical and pharmaceutical studies. To account for their retention behaviour, a modified version of the solvation parameter model was designed: two additional molecular descriptors were introduced, to account for ionic interactions with anionic and cationic species. The retention equations obtained allow a rationalization of the interactions contributing to retention and separation in the HILIC systems considered.

Approach to hydrophilic interaction chromatography column selection: application to neurotransmitters analysis.

This paper presents the comparison between 12 hydrophilic interaction liquid chromatography columns that are commercially available. The main factors influencing the retention and selectivity toward 12 neurotransmitters for these different HILIC systems have been studied. For additional information, the retention data have been analyzed statistically by factor analysis. Principal component analyses (PCA) were calculated to evidence different separation behaviour between the stationary phases, based on the retention data measured for three compound classes: anionic acidic compounds, cationic basic compounds and zwitterionic amino acids. Finally, a generic procedure is suggested for optimization of HILIC analyses, depending on the ionization state of the analytes.

Orthogonal screening system of columns for supercritical fluid chromatography

The application of supercritical fluid chromatography is expanding nowadays, particularly in the pharmaceutical industry and for natural extracts. In order to select appropriate columns from the continuously increasing number of potentially suitable ones, a test that evaluates stationary phases properties, based on the solvation parameter model, was performed earlier. In this study, it is investigated whether the number of stationary phases can be reduced to an optimized set with only highly orthogonal systems. Such a set of orthogonal chromatographic systems having different selectivities may provide the initial separation for method development. Moreover, it was considered important to include systems with good overall separation performance in our final set. Thus the columns are also selected based on their ability to provide sharp and symmetric peaks. An example application based on the analysis of seven sunscreen molecules is presented as a validation of the suggested column set.

Characterization and use of hydrophilic interaction liquid chromatography type stationary phases in supercritical fluid chromatography

All stationary phase chemistries are useful to achiral packed-column supercritical fluid chromatography (pSFC), but the majority of recent applications are based on polar stationary phases of the type used in normal-phase HPLC. Some manufacturers have recently introduced pSFC-tailored stationary phases, while others simply advocate the use of selected HPLC columns among their brands for SFC use. Indeed, any column developed for HPLC use can be used with supercritical fluids. Besides, hydrophilic interaction liquid chromatography (HILIC) is facing an increasing interest. Consequently, a number of HILIC-devoted stationary phases are now commercialized by most column manufacturers. As HILIC stationary phases are polar, their possible use in pSFC seems obvious, although literature examples are not numerous. However, whether they provide acceptable peak shapes in SFC conditions is a serious concern for them to find applicability. Indeed, columns optimized for HILIC mobile phases may not be optimum for SFC mobile phases. We present the characterization of eleven HILIC-type stationary phases used with carbon dioxide-methanol mobile phases in the isocratic mode. The columns are compared in terms of their retention and separation characteristics assessed by the solvation parameter model, and based on peak shapes. For this purpose, hundred and forty-six low molecular weight molecules, comprising neutral, basic and acidic compounds, were eluted on each column. Data analysis is carried out with hierarchical cluster analysis and principal component analysis in order to define three clusters of columns with similar selectivity: the first cluster comprises neutral stationary phases like amide and diol phases; the second one comprises basic stationary phases like aminopropyl-bonded silica; the last cluster comprises bare silica stationary phases. Sample applications with three different test mixtures relevant to pharmaceutical applications (barbiturates, benzodiazepines and propionic acids) are presented to assess the complementarity of some selected columns.

Improved separation of furocoumarins of essential oils by supercritical fluid chromatography.

Separation of furocoumarins has become of a great interest for cosmetic industry and human health, since the recent directive of the European Union. Furocoumarins are a class of compounds presenting varied substituents linked mainly in two positions to an identical skeleton made by a furan ring bonded to a coumarin nucleus (Psoralen). The substituents are mainly methoxy, or alkyl chains, which can contain double bonds, hydroxyl or epoxy groups. Due to the variety of compounds, and their subtle structure differences, their separation requires high-performance methods. Multi-gradient high-performance liquid chromatography (HPLC) and two-dimensional chromatography are usually applied. This paper describes a new approach, by using super/subcritical fluid chromatography (SFC), with a green mobile phase: CO(2)-ethanol. The choice of the stationary phase from varied types of phases, and the effects of numerous analytical parameters (flow rate, modifier percentage, temperature and outlet pressure) are studied, described and discussed, on the basis of the separation of a complex sample: lemon residue. From these studies, isocratic conditions are determined to obtain a satisfactory separation in 10 min. A two-dimensional analysis was also investigated, by performing first a class fractionation of compounds on an ethylpyridine (EP) phase, then by separating each class on a pentafluorophenyl phase (Discovery HS F5) with the selected isocratic mobile phase. A gradient elution is also studied to improve separation of some minor compounds. Structure of the eluted compounds was determined by comparison with standards, HPLC-DAD, HPLC-MS analysis, and NMR analysis of collected fractions. All these approaches allow relating structure of compounds to retention behaviour, which is unusual due to the selected pentafluorophenyl stationary phase.

Effects of mobile phase composition on retention and selectivity in achiral supercritical fluid chromatography

In supercritical fluid chromatography (SFC), the effects of adding a co-solvent to carbon dioxide are numerous. In this work, the changes in retention and selectivity occurring when the nature of the co-solvent (methanol, ethanol, isopropanol, acetonitrile) in the SFC mobile phase is varied, are studied on seven columns with different stationary phase chemistry (cyanopropyl-, pyridine-ethyl-, phenyl-oxypropyl-, phenyl-propyl-, phenyl-hexyl-, pentafluorophenyl-propyl- and octadecyl-bonded silica). Hierarchical cluster analysis and quantitative structure-retention relationships with the solvation parameter model are employed to evaluate these effects, based on the observed retention for over a hundred probe compounds. It appears that some columns are somewhat more affected than others by the change of solvent nature. Acetonitrile induces the strongest differences, essentially due to its poor capabilities as an eluent for bases and possibly because it does not adequately cover residual silanol groups. Ethanol appears to be a possible green alternative to the most-used methanol. Some hints on a possible partition-adsorption mixed mechanism are offered. Finally, sample applications with three different test mixtures relevant to cosmetic and pharmaceutical applications (flavanones, non-steroidal anti-inflammatory drugs and barbiturates) are presented.