Usual, unusual and unbelievable retention behavior in achiral supercritical fluid chromatography: Review and discussion.

Retention rules are well known in liquid chromatography. For the mobile phase composition, retention decreases when adding organic solvent to water for reversed-phase or increasing water proportion for hydrophilic interaction liquid chromatography, and a decrease in temperature usually increases retention. For supercritical fluids, the fluid density, which is related to temperature and column back-pressure, is significant for neat CO2 and with low percentages of organic modifiers, i.e. with compressible mobile phases. The increase in the modifier percentage reduces the fluid compressibility, leading to retention behaviors close to those observed with liquid mobile phases, for instance for temperature changes. Moreover, adsorption of carbon dioxide or modifiers modify the solutes/stationary phase interactions, further complicating the understanding of the observed retention changes, either with low amount of modifier, or with specific modifiers. Besides, the polar and nonpolar stationary phases (SPs) do not always behave identically, depending on physico-chemical properties. Silica, amino or ethyl-pyridine polar phases display mostly identical behavior for classical differences of compounds of different polarity, but can provide different retention order for more subtle differences, such as the position of polar groups. Moreover, the nature of the silica, inorganic or hybrid, or the additional charges onto the silica surface can also lead to different results. Even if the C18-bonded phases are not as popular as polar SPs, the non-polar SPs provide very high separation performances for suited compounds, i.e. for non-polar compounds, which are perfectly solubilized by supercritical fluids. Recently, unusual retention behaviors were observed with some specific C18-bonded phases, which display polar interactions in addition to dispersion interactions. Whatever the SPs used, supercritical fluids appear to favor specific effects that are not observed with liquid mobile phases that are more uniform in terms of physico-chemical properties. The objective of this paper is to describe different separation behaviors observed in SFC, to improve the general understanding of the specificities of the association of supercritical fluids and varied SPs.

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.

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.

Combined supercritical fluid chromatographic tests to improve the classification of numerous stationary phases used in reversed-phase liquid chromatography.

In this paper, we present a combination of a key-solute test based on retention and separation factors of large probe solutes (carotenoid pigments) and a quantitative structure-retention relationship analysis based on the retention factors of small probe solutes (aromatic compounds), both performed in supercritical fluid chromatography, to investigate the different chromatographic behaviour of octadecylsiloxane-bonded stationary phases of all sorts: classical, protected against silanophilic interactions or not, containing polar groups (endcapping groups or embedded groups). The results indicate that the two approaches chosen (carotenoid test and solvation parameter model) are complementary and provide precise information on the chromatographic behaviour of ODS phases. The applicability of the classification to the selection of stationary phases is evidenced with some examples of separations.

Σpider diagram: A universal and versatile approach for system comparison and classification. Part 2: Stationary phase properties.

Many different sorts of bonded phase chemistries may be used in high-performance liquid chromatography (HPLC) in the reversed-phase mode: C8, C18 (type A or B), polar-embedded C18, phenyl, pentafluorophenyl, or cyanopropyl. To assess their retention and selectivity properties, chromatographic tests exist. The data obtained from these tests may be presented in three ways. First, simple classification diagrams may be plotted, when only two or three parameters are studied. Secondly, chemometric treatments such as principal component analyses (PCA) or hierarchical cluster analyses (HCA) may be computed, when at least 4 parameters are studied. These are sometimes uneasy to interpret. Thirdly, the "distance" between one column and a reference column may be estimated, through calculated ranking (F or CDF) or selectivity (s) factors. In this paper, another treatment type is applied to the data of Euerby (Tanaka test) and Snyder (Hydrophobic Subtraction Model), each of these tests having 6 parameters. This treatment produces a visual classification, called spider diagram. In the first part of this series, this type of classification was applied to the classification of solvents. A logical and easily comprehensible classification is obtained for the varied types of bonded phases, with a clear location, which can be related to the chromatographic properties. The comparison of these diagrams shows that the classification based on Snyder's hydrophobic subtraction model discriminates the stationary phases more effectively than the one based on the Tanaka test. Finally, on the basis of the parameter relevance and in order to favour comparison between these two tests and a third one called the carotenoid test, simplified classification maps are proposed. For Tanaka test, the selected parameters are the pentylbenzene retention factor (hydrophobicity), the benzylamine/phenol separation factor at pH 7.6 (polar surface activity) and the triphenylene/ortho-terphenyl (shape selectivity) separation factor. For Snyder test, the parameters selected are the ethylbenzene retention factor (hydrophobicity) the C term at pH 7.0 (polar surface activity) and the S* term (steric electivity). The location of some stationary phases onto the maps and their rankings are compared and shown to agree well between the three tests.

Combined supercritical fluid chromatographic methods for the characterization of octadecylsiloxane-bonded stationary phases.

In this paper, we present a combination of a key-solute test based on retention and separation factors of large probe solutes (carotenoid pigments) and a quantitative structure-retention relationship analysis based on the retention factors of small probe solutes (aromatic compounds) to investigate the different chromatographic behavior of octadecylsiloxane-bonded stationary phases of all sorts: classical, protected against silanophilic interactions or not, containing polar groups (endcapping groups or embedded groups). Varied chemometric methods are used to enlighten the differences between the 27 phases tested. The results indicate that the two approaches chosen (carotenoid test and solvation parameter model) are complementary and provide precise information on the chromatographic behavior of ODS phases.

Description and comparison of chromatographic tests and chemometric methods for packed column classification.

The main tests developed in last 20 years to investigate the chromatographic behaviour and the stationary phase properties are described in this paper. These properties are the hydrophobicity, depending on the surface area and the bonding density, the number of accessible residual silanol groups having sometimes different acidity, which can interact with neutral solutes by hydrogen bonds or with the ionic form of basic compounds and the shape or steric selectivity, depending on both the functionality of the silanising agent and the bonding density. Two types of tests are performed, either based on key solutes having well defined properties such as phenol, caffeine, amitriptyline, benzylamine, acenaphtene, o-terphenyl, triphenylene, p-ethylaniline, carotenoid pigments, or on retention models (solvation parameter, hydrophobic subtraction) obtained from the analyses of numerous and varied compounds. Thus, the chromatographic properties are either related to selectivities or retention factors calculated from key solutes, or they are described by interaction coefficients provided by multilinear regression from retention models. Three types of comparison methods are used based on these data. First, simple plots allow the study of differences between the columns as regards to one or two properties. Columns located in the same area of the plot display close properties. Second, chemometric methods such as principal component analysis (PCA) or hierarchical cluster analysis (HCA) can be performed to compare columns. In this case, all the studied properties are included in the comparison, done either by data projection to reduce the space in which the information is located (PCA) or by distance calculation and comparison for drawing a classification (HCA). Neighbouring columns are expected to provide identical chromatographic performances. These two chemometric methods can be used together, PCA before HCA. The third way is to calculate a discrimination factor from a reference column, through calculation methods based on the Pythagorean Theorem: the lower this factor, the closer the column properties. Following the presentation of the analytical conditions, the compounds and the data treatments used by the teams working in this field, the pertinence of the different selectivities, i.e. of the different probe solute couples or of the different interaction coefficients, are discussed as regards their discrimination capacity. The accuracy of chemometric treatments in the discrimination of stationary phases having different functionalities (octadecylsiloxane (ODS), cyano, fluorinated, phenyl, polar embedded group or "aqua" type) will be discussed, as well as their performances in the finer ODS discrimination. New two-dimensional plots, from data gained by different studies will be suggested, to improve the classification of stationary phases having different nature of bonded chains.

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 2h. 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.

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model IV. Aromatic stationary phases.

The purpose of the present work was to systematically study the chromatographic behaviour of different aromatic stationary phases in a subcritical fluid mobile phase. We attempted to assess the chemical origin of the differences in retention characteristics between the different columns. Various types of aromatic stationary phases, all commercially available, were investigated. The effect of the nature of the aromatic bonding on interactions between solute and stationary phases and between solute and carbon dioxide-methanol mobile phase was studied by the use of a linear solvation energy relationship (LSER): the solvation parameter model. This study was performed to provide a greater knowledge of the properties of these phases in subcritical fluid chromatography, and to allow a more rapid and efficient choice of aromatic stationary phase in regard of the chemical nature of the solutes to be separated. Charge transfer interactions naturally contribute to the retention on all these stationary phases but are completed by various other types of interactions, depending on the nature of the aromatic group. The solvation vectors were used to compare the different phase properties. In particular, the similarities in the chromatographic behaviour of porous graphitic carbon (PGC), polystyrene-divinylbenzene (PS-DVB) and aromatic-bonded silica stationary phases are evidenced.

Characterization of stationary phases in subcritical fluid chromatography by the solvation parameter model. I. Alkylsiloxane-bonded stationary phases.

Varied types of alkylsiloxane-bonded and fluoroalkylsiloxane-bonded stationary phases, all commercially available, were investigated with subcritical fluid mobile phase. The effect of the alkyl chain length (from C4 to C18) and of the nature of the bonding (fluorodecylsiloxane, phenyl-C18 and polar-embedded-C18) on the chromatographic behaviour was investigated by the use of a linear solvation energy relationship (LSER), the solvation parameter model. A large set of test compounds provides precise and reliable information on the intermolecular interactions responsible for retention on these stationary phases used with a subcritical mobile phase. First of all, the results underline the close properties between subcritical fluid and organic liquid. The use of non aqueous mobile phases reduces the cavity energy and the mobile phase acidity generally encountered with aqueous liquid phases, allowing other interactions to take a part in retention. As expected, an increase in the alkyl chain length favours the dispersive interactions between the solutes and the stationary phases. Changes in basicity and acidity of the stationary phases are also related to the chain length, but, in this case, mobile phase adsorption onto the stationary phase is supposed to explain these behaviours. The addition of a phenyl group at the bottom of the C18 chain, near the silica, does not induce great modifications in the retentive properties. The fluorodecylsiloxane and the polar-embedded alkylsiloxane phases display very different properties, and can be complementary to the classical alkylsiloxane-bonded phases. In particular, the fluorinated phase does not favour the dispersive interactions, in comparison to hydrogenated stationary phases, when the basicity of the polar-embedded phase is obviously greater than the one of classical alkylsiloxane-bonded phases, due to the amide function. Finally, logk-logk curves plotted between the different phases illustrate the effect of the interaction properties on the retention of different classes of compounds.

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases.

In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.

Characterisation of stationary phases in subcritical fluid chromatography by the solvation parameter model. II. Comparison tools.

This study is an investigation of numerical and graphical tools for the comparison of the chromatographic stationary phases previously characterized with the solvation parameter model [C. West, E. Lesellier, J. Chromatogr. A, in press]. Numerous methods are presented and discussed: the coefficient ratio comparison, principal component analysis (PCA) and calculation of the distance. The coefficient ratio comparison allows to estimate the weight of a particular type of interaction relative to dispersive interactions, but is not practical when a lot of chromatographic systems need to be compared. The principal component analysis is mainly used to reduce the set of correlated variables, but is not useful for non correlated variables such as the solvation parameters. The distance calculation is an interesting tool to measure the differences between two systems but suffers from a confusion between two notions: the difference in the type of interactions involved in the chromatographic system and the intensity of these interactions. Finally, we chose to associate each stationary phase to a vector in a five-dimensional space, according to a method proposed by Ishihama and Asakawa [Y. Ishihama, N. Asakawa, J. Pharm. Sci., 88(12) (1999) 1305] for the comparison of lipophilicity scales. Thus, the angles between the different vectors are used to compare the selectivities of the stationary phases, and the lengths of the vectors are used to compare the relative intensities of the interactions. This method was applied to different alkyl phases characterized in subcritical fluid chromatography (SubFC). The angle values are well suited to the description of the differences in chromatographic behaviour. As expected, a small angle value is obtained between C8, C12 and C18, when a greater one is noticed between C4 and the longer chain length alkyl bonded phases, showing the different acidity and basicity of the C4 phase. Moreover, a satisfactory correlation is obtained between the length of the vectors and the carbon number of the alkyl chain. The differences between classical alkyl-bonded silica phases and polar-embedded alkyl-bonded phases or fluorinated phases are also conveniently evidenced. Finally, graphical tools are investigated and a new type of representation, based in part on a radar plot, is proposed. This plot allows the comparison of stationary phases without reducing the number of studied variables. The comparisons based on this method are consistent with the observed chromatographic behaviour of the phases compared.

Classification of special octadecyl-bonded phases by the carotenoid test.

Amongst the numerous base-deactivated ODS phases obtained by increasing the bonding density or/and by efficient endcapping treatments, some particular stationary phases have been developed, to limit the additional interactions of basic compounds with residual silanols, to work at extreme pH or with rich water mobile phases. Horizontal polymeric phases, sterically protected ones, hybrid silicas, propylene bridge, are particularly used for this purpose. Octadecyl chains with embedded polar groups and hydrophilic endcapping are also used in this goal. The properties of these phases were studied with a simple test consisting in the injection of carotenoid pigments in Subcritical Fluid Chromatography. The molecules used and the nature of the mobile phase allow the determination of hydrophobicity, polar site accessibility and type or/and bonding density of the stationary phases. Whatever the type of the phases, the particular stationary phases do not show any remarkable property, in comparison to other base-deactivated C18-bonded phases. On the other hand, embedded and polar-endcapped phases display a specific behaviour in regard of hydrophilic interactions, which are highlighted by the absence of water in the subcritical fluid. Additional properties of these phases are described, such as steric recognition and retention performances. As expected, polar-embedded phases are less retentive than classical ODS ones, but are sometimes able to provide greater steric recognition. On the other hand, the polar-endcapped phases display greater hydrophobicity than polar-embedded ones. From a simple classification diagram based on chromatographic properties, differences can be noticed between the polar-embedded groups (amide, carbamate, ether, sulfonamide) and between embedded and endcapped phases. Surprising behaviours are also noticed for some on the tested phases.

Effects of modifiers in subcritical fluid chromatography on retention with porous graphitic carbon.

The effect of different modifiers in subcritical fluid chromatography (SubFC) on interactions between solute and porous graphitic carbon (PGC) and between solute and carbon dioxide-modifier mobile phases was studied by the use of linear solvation energy relationships (LSERs). This study was performed to allow efficient optimization of the composition of the carbon dioxide-modifier mobile phase in regard of the chemical nature of the solutes to be separated. With all modifiers tested (methanol, ethanol, n-propanol, isopropanol, acetonitrile, tetrahydrofuran and hexane), the solute/stationary phase interactions are greater than the solute/mobile phase ones. Dispersion interactions and charge transfer between electron donor solute and electron acceptor PGC mainly explain the retention on this surface, whatever the modifier. These interactions are quite constant over the range of modifier percentage studied (5-40%). For acidic compounds, the retention variation is mainly related to the change in the basic character of mobile and stationary phase due to the variation of modifier percentage. Changes in eluting strength are mostly related to adsorption of mobile phase onto the PGC with methanol and acetonitrile, and to the increase of dispersion interactions between the solute and the mobile phase for other modifiers. Relationships between varied selectivities and solvation parameter values have been studied and are discussed in this paper.

Separation of substituted aromatic isomers with porous graphitic carbon in subcritical fluid chromatography.

The ability of porous graphitic carbon (PGC) to separate structural isomers has been reported in high-performance liquid chromatography (HPLC). This paper presents studies carried out in subcritical fluid chromatography (SubFC). Various polar and nonpolar modifiers were added to the carbon dioxide mobile phase, in proportions ranging from 5 to 40%. The effects of both the nature and the percentage of the modifier on aromatic isomer separations were studied. Two types of selectivity behaviour appear. The first one, related to steric recognition, is due to the number of contact points between the compounds and the flat surface of PGC. In this case, retention orders are often identical to that reported in HPLC. The second is related to the favourable interaction between the polar moieties of the solutes and the stationary phase. In this case, the retention and selectivity strongly depend on the mobile phase composition. Thus, the separations obtained are greatly enhanced, compared to those obtained in HPLC. The retention and selectivity variations observed when the composition of the mobile phase is changed are discussed based on linear solvation energy relationships (LSERs). Practical applications are presented, namely benzene, toluene, ethylbenzene and xylenes (BTEX) and flavour molecules separations.

Modelling of ceramide interactions with porous graphite carbon in non-aqueous liquid chromatography.

Interactions of solutes on porous graphitic carbon (PGC) with non-aqueous mobile phases are studied by the linear solvation energy relationship (LSER). Studies have been carried out with eight binary mixtures composed of a weak solvent (acetonitrile or methanol) and a strong solvent (tetrahydrofuran, n-butanol, CH2Cl2, 1,1,2-trichloro-2,2,1-trifluoroethane). The systematic analysis of a set of test compounds was performed for each solvent mixture in isocratic mode (50:50). The results were compared to those obtained on PGC with hydro-organic liquids and supercritical fluids. They were then correlated with the observed retention behaviour of lipid compounds, more particularly ceramides.

Σpider diagram: a universal and versatile approach for system comparison and classification: application to solvent properties.

Classification methods based on physico-chemical properties are very useful in analytical chemistry, both for extraction and separation processes. Depending on the number of parameters, several classification approaches can be used: by plotting two- or three-dimensional maps (triangles, cubes, spheres); by calculating comparison values for one system with reference to another one, i.e. the ranking factor F, or the Neue selectivity difference s(2); or with chemometric methods (principal component analysis-PCA or hierarchical cluster analysis-HCA). All these methods display advantages and drawbacks: some of them are limited by the number of studied parameters (e.g. three for triangle or sphere plots); others require a new calculation when changing the reference point (F; s(2)), while for chemometric methods (PCA, HCA), the relationships between the clusters and the physico-chemical properties are not always easily understandable. From previous studies performed in supercritical fluid chromatography for stationary phase classification on the basis of linear solvation energy relationships (LSER) including five parameters, we developed a classification map called the Σpider diagram. This diagram allows plotting in a two-dimensional map the location of varied systems, having as many parameters as the ones required getting a satisfactory classification. It can be three, five, eight, or any number. In the present paper, we apply this diagram, and the calculation mode to obtain this diagram, to different solvent classifications: Snyder triangle, solvatochromic solvent selectivity, Hansen parameters, and also to LSER Abraham descriptors and COSMO-RS parameters. The new figure based on Snyder data does not change the global view of groups, except by the use of corrected data from literature, and allows adding the polarity value onto the map. For the solvatochromic solvent selectivity, it leads to achieve a better view of solvents having no acidic character. For Hansen parameters, the "flattening" of the spherical view down to a single plane could be found easier to use. For COSMO-RS and with Abraham descriptor, a more subtle classification is achieved, mainly due to the use of five parameters instead of three. A strong reversed correlation is established between the Rohrschneider polarity P' and the normalized V (molecular volume) parameter. The study of the location of solvents used for reversed-phase liquid chromatography and the Arizona system for counter-current chromatography is discussed, as well as the replacement of unsafe solvents by greener ones, or the use of these classifications for the study of compound solubility. Besides, this paper also shows the ability to the spider diagram to plot on a single plane three axes from principal component analyses.

Method developments approaches in supercritical fluid chromatography applied to the analysis of cosmetics.

Analyses of complex samples of cosmetics, such as creams or lotions, are generally achieved by HPLC. These analyses are often multistep gradients, due to the presence of compounds with a large range of polarity. For instance, the bioactive compounds may be polar, while the matrix contains lipid components that are rather non-polar, thus cosmetic formulations are usually oil-water emulsions. Supercritical fluid chromatography (SFC) uses mobile phases composed of carbon dioxide and organic co-solvents, allowing for good solubility of both the active compounds and the matrix excipients. Moreover, the classical and well-known properties of these mobile phases yield fast analyses and ensure rapid method development. However, due to the large number of stationary phases available for SFC and to the varied additional parameters acting both on retention and separation factors (co-solvent nature and percentage, temperature, backpressure, flow rate, column dimensions and particle size), a simplified approach can be followed to ensure a fast method development. First, suited stationary phases should be carefully selected for an initial screening, and then the other operating parameters can be limited to the co-solvent nature and percentage, maintaining the oven temperature and back-pressure constant. To describe simple method development guidelines in SFC, three sample applications are discussed in this paper: UV-filters (sunscreens) in sunscreen cream, glyceryl caprylate in eye liner and caffeine in eye serum. Firstly, five stationary phases (ACQUITY UPC(2)) are screened with isocratic elution conditions (10% methanol in carbon dioxide). Complementary of the stationary phases is assessed based on our spider diagram classification which compares a large number of stationary phases based on five molecular interactions. Secondly, the one or two best stationary phases are retained for further optimization of mobile phase composition, with isocratic elution conditions or, when necessary, two-step gradient elution. The developed methods were then applied to real cosmetic samples to assess the method specificity, with regards to matrix interferences, and calibration curves were plotted to evaluate quantification. Besides, depending on the matrix and on the studied compounds, the importance of the detector type, UV or ELSD (evaporative light-scattering detection), and of the particle size of the stationary phase is discussed.

Analysis of non-saponifiable lipids by super-/subcritical-fluid chromatography.

Because of the particular properties of carbon dioxide or carbon dioxide/modifier mobile phases, super- or subcritical-fluid chromatography (SFC) can be an alternative to more classical chromatographic methods such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) for the separation of unsaponifiable lipids. These fluids can also be helpful in the extraction and/or the concentration steps of sterols, tocopherols or carotenoids from complex samples. Supercritical extraction, off-line prefractionation or semi-preparative supercritical fluid chromatography, carried out before the analysis are described. The effects on separation of analytical parameters such as pressure, nature of and modifier percentage or stationary phase nature are also reported. The performance of capillary, packed or capillary packed columns is discussed, as well as the consequences of their use (choice of stationary phases, type of coupled detector). Numerous examples of fine separations are reported.

Study of dead volume measurement in packed subcritical fluid chromatography with ODS columns and carbon dioxide-modifier mobile phases.

Studies were done for providing a simple, rapid and reliable procedure of void volume measurement in packed subcritical fluid chromatography (pSubFC), with CO2-modifier mobile phases containing high modifier amounts. Methods used in RPLC with ODS columns were applied in pSubFC: gravimetric, homologous series linearisation and unretained marker injection. Results lead us to propose the method of marker injection to determine the void volume in pSubFC. Acetonitrile was chosen as the void volume marker among six tested markers. Furthermore, void volume variations vs. the modifier volume (from 5 to 45%) were studied for nine organic modifiers. The void volume variations were related both to adsorption-desorption phenomena between the mobile phase and the stationary phase and to mobile phase density changes. These variations allowed the classification of the modifiers into four groups on the basis of the molecular interactions.