A promising "metastable" liquid crystal stationary phase for gas chromatography.

The liquid crystal state is an ordered physical state between a solid and a liquid. Previous research, in gas chromatography, proved that it provides a geometric selectivity, which allows the separation of geometric position isomers and cis-trans isomers that are difficult to separate on conventional gas chromatography stationary phases (polydimethyl siloxane derived and polyethylene glycol stationary phases). However, their use was generally very limited by the rather high temperature at which they must be operated, normally above the solid-liquid crystal transition temperature. In the present study we are interested in a new synthesized material, 1,4- bis (4-bromohexyloxy benzoate) phenyl (BHOBP). The first characterizations of BHOBP were carried out by thermogravimetric analysis, hot-stage optical microscopy and differential scanning calorimetry to control the thermal stability of the BHOBP as well as the nematic texture of the mesophase highlighted in a well-defined temperature range (120 °C-200 °C). When heated, the solid compound led to a stable liquid crystal state. Its cooling has revealed "a new metastable physical state, which is the supercooled liquid crystal phase". After these first characterizations, the new material was used as a stationary phase for gas chromatography. The BHOBP was deposited in a capillary column by the dynamic method. The inverse gas chromatography study of the column revealed a solid-stable nematic phase transition temperature, in agreement with the first characterization methods. The stable liquid crystal phase showed good resolutions in the analysis of some geometric isomers of low volatility as PAHs. The presence of the supercooled liquid crystal state in the chromatographic column has also been confirmed. This new metastable state is particularly interesting because it enlarged the scope of this material by improving the resolution of several mixtures. Thus, the separation of highly volatile mixtures of geometric isomers (e.g. cis and trans-decalin) was achieved only through this metastable mesophase confirming its unique selectivity. The metastable liquid crystal, used at 80 °C, has also exhibited an original behavior by its stability after several weeks of use at the same temperature, maintaining constant retention factors and selectivity.

Active modulation in neat carbon dioxide packed column comprehensive two-dimensional supercritical fluid chromatography.

After demonstrating in a first paper the feasibility of SFCxSFC without decompression of the mobile phase, a modified interface has been developed in order to perform active modulation between the two SFC dimensions. In this paper, it is shown that the new interface enabled independent control of modulation parameters in SFCxSFC and performed a band compression effect of solutes between the two SFC dimensions. The effectiveness of this new modulation process was studied using a Design of Experiments. The SFCxSFC prototype was applied to the analysis of a real oil sample to demonstrate the benefits of the active modulator; in comparison to our previous results obtained without active modulation, better separation was obtained with the new interface owing to the peak compression occurring in the modulator.

Synergetic Use of Principal Component Analysis Applied to Normed Physicochemical Measurements and GC × GC-MS to Reveal the Stabilization Effect of Selected Essential Oils on Heated Rapeseed Oil.

Lipid oxidation leads to the formation of volatile compounds and very often to off-flavors. In the case of the heating of rapeseed oil, unpleasant odors, characterized as a fishy odor, are emitted. In this study, 2 different essential oils (coriander and nutmeg essential oils) were added to refined rapeseed oil as odor masking agents. The aim of this work was to determine a potential antioxidant effect of these essential oils on the thermal stability of rapeseed oil subject to heating cycles between room temperature and 180 °C. For this purpose, normed determinations of different parameters (peroxide value, anisidine value, and the content of total polar compounds, free fatty acids and tocopherols) were carried out to examine the differences between pure and degraded oil. No significant difference was observed between pure rapeseed oil and rapeseed oil with essential oils for each parameter separately. However, a stabilizing effect of the essential oils, with a higher effect for the nutmeg essential oil was highlighted by principal component analysis applied on physicochemical dataset. Moreover, the analysis of the volatile compounds performed by GC × GC showed a substantial loss of the volatile compounds of the essential oils from the first heating cycle.

Combination of partial least squares regression and design of experiments to model the retention of pharmaceutical compounds in supercritical fluid chromatography.

This work presents a first attempt to establish a model of the retention behaviour for pharmaceutical compounds in gradient mode SFC. For this purpose, multivariate statistics were applied on the basis of data gathered with the Design of Experiment (DoE) methodology. It permitted to build optimally the experiments needed, and served as a basis for providing relevant physicochemical interpretation of the effects observed. Data gathered over a broad experimental domain enabled the establishment of well-fit linear models of the retention of the individual compounds in presence of methanol as co-solvent. These models also allowed the appreciation of the impact of each experimental parameter and their factorial combinations. This approach was carried out with two organic modifiers (i.e. methanol and ethanol) and provided comparable results. Therefore, it demonstrates the feasibility to model retention in gradient mode SFC for individual compounds as a function of the experimental conditions. This approach also permitted to highlight the predominant effect of some parameters (e.g. gradient slope and pressure) on the retention of compounds. Because building of individual models of retention was possible, the next step considered the establishment of a global model of the retention to predict the behaviour of given compounds on the basis of, on the one side, the physicochemical descriptors of the compounds (e.g. Linear Solvation Energy Relationship (LSER) descriptors) and, on the other side, of the experimental conditions. This global model was established by means of partial least squares regression for the selected compounds, in an experimental domain defined by the Design of Experiment (DoE) methodology. Assessment of the model's predictive capabilities revealed satisfactory agreement between predicted and actual retention (i.e. R2=0.942, slope=1.004) of the assessed compounds, which is unprecedented in the field.

Alternative solvents for improving the greenness of normal phase liquid chromatography of lipid classes.

An evaluation of solvents alternative to n-heptane (d-limonene and hexamethyldisiloxane) and chloroform (cyclopentyl methyl ether, 2-methyltetrahydrofuran and isopentyl acetate) was developed for lipid classes separation of non-polar cholesteryl ester to highly polar phospholipids by high-performance liquid chromatography on bare silica stationary phase and evaporative light-scattering detection. Screening of alternative solvents was used to estimate their compatibility with liquid chromatography and evaporative light-scattering detection and to evaluate their chromatographic selectivity. This work shows that n-heptane can be advantageously replaced by hexamethyldisiloxane. An increase of non-polar lipids retention is observed with hexamethyldisiloxane as weak solvent. Chloroform, which is largely used for lipid analysis, might be replaced efficaciously by cyclopentyl methyl ether, 2-methyltetrahydrofuran or isopentyl acetate. Aside from offering a different selectivity, the gradients composed by one or both alternative solvents gave efficient and comparable or even better separations than those obtained with conventional solvents.

Validation of a headspace trap gas chromatography and mass spectrometry method for the quantitative analysis of volatile compounds from degraded rapeseed oil.

Due to lipid oxidation, off-flavors, characterized by a fishy odor, are emitted during the heating of rapeseed oil in a fryer and affect the flavor of rapeseed oil even at low concentrations. Thus, there is a need for analytical methods to identify and quantify these products. To study the headspace composition of degraded rapeseed oil, and more specifically the compounds responsible for the fishy odor, a headspace trap gas chromatography with mass spectrometry method was developed and validated. Six volatile compounds formed during the degradation of rapeseed oil were quantified: 1-penten-3-one, (Z)-4-heptenal, hexanal, nonanal, (E,E)-heptadienal, and (E)-2-heptenal. Validation using accuracy profiles allowed us to determine the valid ranges of concentrations for each compound, with acceptance limits of 40% and tolerance limits of 80%. This method was then successfully applied to real samples of degraded oils.

Innovative green supercritical fluid chromatography development for the determination of polar compounds.

In the context of green analytical chemistry, a supercritical fluid chromatography method was developed. In order to prove the potential of this technology, a worst case was selected, i.e. the separation of very polar compounds. For that purpose, an innovative methodology based on design of experiments (DoE) and design space (DS) was previously developed and successfully tested on liquid chromatography. For the first time, this methodology was applied to a supercritical fluid chromatography (SFC) separation. First, a screening design was used to select the stationary phase and the nature of the mobile phase based on a maximization of the number of peaks eluted and a minimization of the number of co-eluted peaks. Then, a central composite design with orthogonal blocks defined a set of experiments used to model the retention times of each peak at the beginning, the apex, and the end. The gradient slope, the isocratic plateau before the gradient, the temperature, and the concentration of trifluoroacetic acid (TFA) in the mobile phase were the potentially influential factors. The critical quality attributes (CQAs), i.e. the separation (S) between peaks of the most critical pair, and the analysis time were the criteria considered to assess the quality of the separation. The DS was computed as the multidimensional subspace where the probability for the separation and analysis time criteria to be within acceptance limits was higher than a defined quality level. The DS was computed propagating the prediction error from the modeled responses to the quality criterion using Monte Carlo simulations. The optimal condition was predicted at a gradient slope of 3.8% min(-1) to linearly modify the modifier proportion between 5 and 40%, an isocratic time of 3min, a concentration of TFA of 25mM, and a temperature of 60.5°C. This optimal condition was experimentally tested to confirm the prediction. Furthermore, chromatographic conditions included in the DS and on the limits of the DS were experimentally tested to assess the robustness of the developed SFC method.

Feasability of neat carbon dioxide packed column comprehensive two dimensional supercritical fluid chromatography.

The design and implementation of comprehensive two dimensional supercritical fluid chromatography (SFC) using neat carbon dioxide as the mobile phase is described. Two conventional supercritical fluid chromatographs were hyphenated via an on line comprehensive 2D liquid chromatography like interface; it consisted of a two loop switching valve allowing the collection of the first dimension column effluent, the second dimension separation of a fraction being performed during the time allowed for the collection of the subsequent fraction of the first dimension eluent. Both dimension separations were monitored via UV detection; for the second dimension, the main flow was diverted to implement flame ionisation detection for the detection of hydrocarbons and the construction of the corresponding colour plots. Some key parameters related to the interfacing of the two dimensions and the chromatographic conditions used in both dimensions are discussed. In this preliminary report, the feasibility of comprehensive 2D SFC is demonstrated on synthetic mixtures of hydrocarbons and its potential on real sample analysis is illustrated by the separation of coal derived vacuum distillate.

Impact of the probe solutes set on orthogonality evaluation in reverse phase chromatographic systems.

Two-dimensional liquid chromatography (2DLC) is a very attractive technique for the characterization of complex samples due to its separation power obtained via the coupling of two separation modes exhibiting different mechanisms, i.e. orthogonality. In reverse phase, orthogonality is mainly governed by three factors: the pH of the mobile phase, the structure of the stationary phase and the nature of the organic modifier. In the present paper, we studied the impact of the nature of the probe solutes on orthogonality evaluation. A set of 63 compounds with various physicochemical properties was used to evaluate 32 reverse phase chromatographic systems (2 pH × 8 stationary phases × 2 organic modifiers). Principle component analysis revealed that the solutes could be split into three subsets according to their charge in the experimental conditions. The factors affecting orthogonality and the magnitude of their effect were shown to depend on the charge of the compounds. For positively charged (basic) compounds, the pH was the most important factor, followed by the nature of the stationary phase. For negatively charged (acidic) compounds, the nature of the stationary phase had the highest impact. For neutral compounds, only the nature of the stationary phase and, to a smaller extent, the organic modifier had an influence. The present study also showed that a reduced set of only 9 test compounds instead of whole set of the 63 could enable an appropriate orthogonality evaluation.

Feasibility of ultra high performance supercritical neat carbon dioxide chromatography at conventional pressures.

The implementation of columns packed with sub-2 µm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 µm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.

Comparison of different statistical approaches to evaluate the orthogonality of chromatographic separations: application to reverse phase systems.

Selectivity of phase system is of primary concern when designing a bidimensional chromatographic system and looking for the highest degree of orthogonality between the two separations. Several statistical or geometrical criteria can potentially be used to measure the degree of orthogonality. A comparison of eight candidate criteria has been carried out in this study. Analysis of variance (ANOVA) was used to evaluate the relevance of each criterion and its ability to reveal the significance of the influence of factors like pH, stationary phase, and organic modifier. Experimentally, a set of 32 chromatographic systems was evaluated by the same generic gradient with 63 probe solutes, likely to be present in biological and/or environmental samples and covering a wide range of physico-chemical properties: acidic, basic and neutral compounds with different pKa, molecular mass and hydrophobicity (logP). Each chromatographic system was defined by the nature of the stationary phase (8 different silica or grafting chemistries), the pH of the aqueous fraction of the mobile phase (2.5 or 7.0) and the nature of the organic modifier (acetonitrile or methanol). The orthogonality of the 496 couples of chromatographic systems was evaluated and ranked using the eight different approaches: the three correlation coefficients (Pearson, Spearman and Kendall), two geometric criteria characterizing the coverage of the 2D separation space, Slonecker's information similarity and two chi-square statistics of independence between normalized retention times. In fact, there were only seven distinct criteria, since we established the analytical equivalence between the rankings with the likelihood ratio statistics and Slonecker's information similarity. Kendall's correlation coefficient appeared to be the best measure of orthogonality since, according to ANOVA, it exhibited the highest sensitivity to all experimental factors. The chi-square measures, and hence Slonecker's information similarity, performed equally well provided the discretization of the separation space was carried out appropriately. Finally, from the compared study of the factors acting upon orthogonality carried out by ANOVA, it is possible to draw the conclusion that the pH of the mobile phases has the highest impact on the selectivity followed by the type of stationary phase and finally by the organic modifier.

The discriminant pixel approach: a new tool for the rational interpretation of GCxGC-MS chromatograms.

GCxGC is now recognized as the most suited analytical technique for the characterization of complex mixtures of volatile compounds; it is implemented worldwide in academic and industrial laboratories. However, in the frame of comprehensive analysis of non-target analytes, going beyond the visual examination of the color plots remains challenging for most users. We propose a strategy that aims at classifying chromatograms according to the chemical composition of the samples while determining the origin of the discrimination between different classes of samples: the discriminant pixel approach. After data pre-processing and time-alignment, the discriminatory power of each chromatogram pixel for a given class was defined as its correlation with the membership to this class. Using a peak finding algorithm, the most discriminant pixels were then linked to chromatographic peaks. Finally, crosschecking with mass spectrometry data enabled to establish relationships with compounds that could consequently be considered as candidate class markers. This strategy was applied to a large experimental data set of 145 GCxGC-MS chromatograms of tobacco extracts corresponding to three distinct classes of tobacco.

Comprehensive two-dimensional GC of supercritical fluid and pressurized liquid extracts of tobaccos.

The developments of an analytical strategy based on rapid extraction techniques coupled to comprehensive bi-dimensional gas chromatography (GCxGC) for the characterization of the volatile fraction of tobaccos are presented. The high-peak capacity of GCxGC allows considering global extraction techniques that do not focus on restricted chemical families of products. Thus, potential of pressurized fluid extraction and supercritical fluid extraction (SFE) to provide informative extracts is evaluated and compared. As expected, both techniques enable rapid extraction of compounds of interest. However, because of the easy removal of the extracting fluid, SFE provides more concentrated extracts, allowing a GCxGC direct injection without any concentration step. Moreover, the use of pure carbon dioxide with tuneable extraction power favors the extraction of the compounds of interest while reducing the extraction of interfering heavy compounds. GCxGC conditions, especially concerning the set of columns used, are selected thanks to the comparison of separations obtained on model compounds. The developed methodology is applied not only to compare the three main types of tobaccos but also to discriminate between different tobaccos of the same type.

Combination of dynamic time warping and multivariate analysis for the comparison of comprehensive two-dimensional gas chromatograms: application to plant extracts.

Comprehensive two-dimensional gas chromatography (GC x GC) is now recognized as the preferred technique for the detailed analysis and characterization of complex mixtures of volatile compounds. However, for comparison purposes, taking into account all the information contained in the chromatogram is far from trivial. In this paper, it is shown that the combination of peak alignment by dynamic time warping and multivariate analysis facilitated the comparison of complex chromatograms of tobacco extracts. The comparison is shown to be efficient enough to provide a clear discrimination among three types of tobacco. A tentative interpretation of loadings is presented in order to give access to the compounds which differ from one sample to another. Once located, mass spectrometry was used to identify markers of tobacco type.

Pharmaceutical quality of docetaxel generics versus originator drug product: a comparative analysis.

INTRODUCTION: The aim of this study was to evaluate the quality of 31 commercially available generic formulations of docetaxel purchased in 14 countries by comparing their docetaxel content, impurity levels and pH versus those of the proprietary product Taxotere (Tx). RESEARCH DESIGN AND METHODS: Generic formulations were purchased in 14 countries in Asia, Africa, the Middle East and Latin America. Levels of docetaxel and impurities (chromatographic peaks above 0.05%) were obtained for each sample using reverse-phase liquid chromatography with ultraviolet detection. The pH of aqueous solutions of generic docetaxel formulations and Tx was also measured. A global evaluation of quality was conducted on each product using a multicriteria desirability analysis based on standards defined by the International Conference on Harmonisation guidelines and the US Pharmacopeia paclitaxel injection monograph. RESULTS: Most generic formulations contained a lower than expected amount of docetaxel and/or a high level of impurities: 21 generic docetaxel formulations had an average mass of docetaxel that was <90% of the expected mass and 23 generic docetaxel formulations had a total impurity content of >3.0%, almost twice the level of impurities in Tx 20 mg. In total, 33 impurities not present in Tx were detected in the generic samples. Desirability analysis demonstrated that none of the generic docetaxel formulations had composition characteristics similar to those of Tx. CONCLUSIONS: This study demonstrated that from an analytical point of view, 90% of the generic docetaxel formulations evaluated contained insufficient active drug, high levels of impurities or both. This has the potential to affect both efficacy and safety of the drug.