Methodology for dyes purification by preparative bidimensional offline reversed-phase high-performance liquid chromatography separation based on mobile phase pH change: Case study of Justicia spicigera.

The extensive characterization of the natural dyes involves the purification of their colored compounds for their structural analysis and stability studies. As most natural compounds being ionizable, herein is presented the optimization of an easy and affordable preparative bidimensional offline reversed-phase high-performance liquid chromatography purification based on mobile phase pH change. At the analytical scale, several combinations of stationary phases and mobile phases at different pH values were investigated first. The orthogonality between the dimensions was quantitatively evaluated using the nearest-neighbor distance approach. The optimized separation method was transferred to the preparative scale for the successful isolation of two colored compounds from Justicia spicigera, a traditional dye plant from Central America. They were identified as perisbivalvine B (2-amino-8-hydroxy-7-methoxy-3H-phenoxazin-3-one) and one of its derivatives, also found in another tinctorial plant species, Peristrophe bivalvis growing in Asia.

Spectral characterisation of a traditional Mesoamerican dye: relationship between in situ identification on the 16th century Codex Borbonicus manuscript and composition of Justicia spicigera plant extract.

Characterisation of vegetal colourants in manuscripts is still a challenging task. Their identification using non-invasive techniques - since sampling is rarely possible - is valid only if there is evidence that the recorded signals are generated by compounds specific to the plant species. Otherwise, more extensive chemical characterisations are required to relate the non-invasive technique signals to the chemical composition of the dye extract and thus avoid misidentification. The case study of a traditional Mesoamerican dye found in the Codex Borbonicus is reported herein. The non-invasive identification of the colourant plant source and chemical characterisation of the coloured molecules were carried out through a multi-analytical technique approach. In situ Raman and UV-Vis emission fluorescence signals suggested the use of Justicia spicigera leaves to produce the brown paint layers of the manuscript. Analysis of the plant extract by liquid chromatography revealed that two compounds are mostly responsible for the colour, and these compounds were successfully isolated. Both contribute to the Raman spectra while only one of them is mainly responsible for the fluorescence recorded on Codex Borbonicus.

Towards a semiquantitative non invasive characterisation of Tyrian purple dye composition: Convergence of UV-Visible reflectance spectroscopy and fast-high temperature-high performance liquid chromatography with photodiode array detection.

In this paper, partial least square (PLS) regression is innovatively applied for a semi-quantitative non invasive study of the most precious dye of Antiquity: Tyrian purple. This original approach for the study of organic dyes in the cultural heritage field, is based on the correlation of spectrophotometric (UV-Visible) and chromatographic (Fast-HT-HPLC-PDA) data from an extensive set of textiles prepared with different snail species according to historical recipes. A cross-validated PLS model, based on the quantity of 6,6'-dibromoindigotin, displays an excellent correlation factor (R(2)Y = 0.987) between values determined by chromatography and those predicted from reflectance spectra. This indicates that the spectral features of Tyrian purple on textile fibre is strictly related to the amount of this indigoid component whose content may be non invasively predicted from reflectance spectrum. The studied correlation also highlights that, independently of the dyeing method and nature of the textile fibre used, the relative content of 6,6'-dibromindigotin may be used as a parameter to distinguish samples prepared with Hexaplex trunculus L. snails from those prepared with further mollusc species. To validate this model, archaeological textile fragments dating from the Roman period were successfully examined. The results achieved open an absolutely new way in Tyrian purple analysis in cultural heritage by non invasive spectroscopic techniques attesting their convergence with HPLC and giving them a semi-quantitative value.

Assessment of two-dimensional separative systems using the nearest neighbor distances approach. Part 2: separation quality aspects.

In this paper we propose a method for the evaluation of real separation quality in multidimensional separations based on the nearest neighbor distances (NND). This approach allows us to overcome the principal drawback of the orthogonality measurement which does not evaluate how good the real separation obtained with one system is, especially when compared to another one. Separation quality evaluation takes into account the distances (di(s)) between peaks in whole separation space. The distances between nearest neighbors were calculated in resolution scaled analysis space to overcome statistically different peak widths in each dimension. The obtained separation quality is ranked by harmonic mean (H̅(s)) of the distances di(s). The extent of peak spreading, described by arithmetic mean (A̅(s)), gives an appreciation of the effective analysis space of 2D separation. The classifications of systems obtained with the same retention data using separation quality and orthogonality approaches show important differences in ranking orders depending on two different targets of these evaluations: the separation potential of a 2D system and the divergence of selectivity between both separation directions. This study shows separation quality and orthogonality merit to be evaluated in parallel for the same systems. The other new threshold descriptor, minimal limit distance (dilim) derived from resolution dependent peak capacity scale, was used to predict the separation quality as a function of desired resolution. We also introduce here a composed descriptor for 2D systems: the optimality coefficient (Oc), which may be useful in the 2D separation optimization process. It takes into account the maximization of homogeneity of peak spreading (H̅(s)/A̅(s)) and the minimization of effective analysis space (or compactness, dilim/A̅(s)) terms.

Assessment of two-dimensional separative systems using nearest-neighbor distances approach. Part 1: orthogonality aspects.

We propose here a new approach to the evaluation of two-dimensional and, more generally, multidimensional separations based on topological methods. We consider the apex plot as a graph, which could further be treated using a topological tool: the measure of distances between the nearest neighbors (NND). Orthogonality can be thus defined as the quality of peak dispersion in normalized separation space, which is characterized by two factors describing the population of distances between nearest neighbors: the lengths (di(o)) of distances and the degree of similarity of all lengths. Orthogonality grows with the increase of both factors. The NND values were used to calculate a number of new descriptors. They inform about the extent of peak distribution, like the arithmetic mean (A̅(o)) of NNDs, as well as about the homogeneity of peak distribution, like the geometric mean (G̅(o)) and the harmonic mean (H̅(o)). Our new, NND-based approach was compared with another recently published method of orthogonality evaluation: the fractal dimensionality (DF). The comparison shows that the geometric mean (G̅(o)) is the descriptor behaving in the most similar way to dimensionality (DF) and the harmonic mean (H̅(o)) displays superior sensitivity to the shortest, critical distances between peaks. The latter descriptor (H̅(o)) can be considered as sufficient to describe the degree of orthogonality based on NND. The method developed is precise, simple, easy to implement, and possible to use for the description of separations in a true or virtual system of any number of dimensions.

Separation system suitability (3S): a new criterion of chromatogram classification in HPLC based on cross-evaluation of separation capacity/peak symmetry and its application to complex mixtures of anthraquinones.

A comparison of chromatograms obtained in a series of separation conditions for a given complex mixture may be done with a series of chromatographic descriptors. In this study, we used two descriptors: the number of critical pairs and symmetry of peaks, further rescaled and converted to the corresponding critical pairs' coefficient (CPc) and symmetry coefficient (Sc). Considering the difficulty of appreciating global separation quality using CPc and Sc criteria separately, as their respective values are usually uncorrelated, a double-criteria cross-evaluation system was required. For that purpose we tested the commonly used multi-criteria decision-making method - Derringer's desirability function (D) - as well as the recently introduced sum of ranking differences (SRD). To facilitate the graphical comparison of both approaches, the desirability function (D) was used in the inverse form (Dinv). The advantages and drawbacks of both evaluation methods, especially the respective under- or over-evaluation of outliers, caused us to introduce a new ranking approach, separation system suitability (3S). The obtained suitability rankings for the three tested approaches (Dinv, SRD and 3S) are different; nevertheless, 3S appears to be the most balanced and the easiest to interpret as well. The approach developed for selection of suitable systems was applied to the problem of separation of complex mixtures through the analysis of a series of standards of anthraquinone derivatives. To judge the pertinence of this evaluation, a sample containing a number of natural anthraquinones extracted from the bark of Indian mulberry (Morinda citrifolia) was analysed. In conclusion, the proposed methodology for the cross-evaluation of the series of chromatograms using single specific descriptors (CPc and Sc) through a global composite descriptor (3S) significantly simplifies the decision that separation systems are the most suitable for the separation of complex target mixtures of compounds.

Separation of 9,10-anthraquinone derivatives: evaluation of functionalised stationary phases in reversed phase mode.

A series of reversed phases bonded with several functional groups was investigated for separation of anthraquinone derivatives, following the previous work, dedicated to the selectivity of octadecyl silica bonded phases. Considering wide diversity of substitutions in hydrophobic anthraquinone skeleton, interactions like dipole-dipole, π-π or H-bond acceptor/donor, as well as inclusion complexes formation can be employed to improve separation. In this study, several phases with grafts like cyano, nitro, aromatic, PEG, diol, calixarene and cyclodextrin were used with water-acetonitrile gradient for separation of thirty anthraquinoids' standards. The evaluation of performances was measured using the symmetry parameter and the number of critical pairs of peaks formed. The results point out the aromatic and calixarene bonded silica as the most interesting in terms of symmetry and critical pairs number. Finally we tested the performance of Caltrex Resorcinaren, Pursuit XRs DP and Luna Phenyl-Hexyl on real samples of anthraquinone natural dye extracted from a red thread taken from a 15th C. tapestry. We observed and compared the retention behaviour of some new anthraquinoids additional to our standards set and showing behaviour particular to substituted anthraquinone carboxylic acids.

Separation of 9,10-anthraquinone derivatives: evaluation of C18 stationary phases.

Chromatographic study of natural products helps to determine their molecular composition and to identify their sources (biological, geographical, etc.). However, identifying anthraquinoids is still a challenge because this chemical family is composed of more than half a thousand molecules. In the present work, a series of C18 stationary phases were systematically studied to evaluate their separation potential for these compounds in high performance reversed phase liquid chromatography (RPLC). The stationary phases with various physicochemical properties were evaluated with respect to the separation of 30 standards. Selectivity of the studied stationary phases and symmetry of the obtained peaks were chosen as the evaluation criteria. Calculations of symmetry and separation capacity parameters have been proposed. These parameters are not dependent on the dimensions of the columns and were used for the classifications of phases. In conclusion, this study is intended to be a guide as to which stationary phases analysts can use for resolving separation of a complex mixture of anthraquinoids.

HPLC-PDA characterisation of Daphne gnidium L. (Thymemeacea) dyeing extracts using two different C-18 stationary phases.

Raw aqueous extracts and acid-hydrolysed extracts from leaves and stems of Daphne gnidium L. were investigated by HPLC-PDA under two different separation conditions. This approach provides improved characterisation of natural compounds, especially those with similar structures, thanks to better resolution of critical pairs which can be difficult to separate by modification of gradient parameters only. In the leaf extracts, the presence of luteolin-3',7-di-O-glucoside was found according to the results obtained for a standard sample. This compound is reported for the first time in this species. The presence of some other, previously reported flavonoids is confirmed. The stem extract is rich in coumarins and seems not to contain any significant amounts of flavonoids. Further investigation of hydrolysed extracts is a useful additional step in the indirect identification of flavonoid and coumarin glycosides.

Identification of "insoluble" red dyewoods by high performance liquid chromatography-photodiode array detection (HPLC-PDA) fingerprinting.

The paper describes a high performance liquid chromatography-UV/Vis spectrometry detection analytical approach to the identification of some redwood species of historical importance in textile dyeing. The group of extracted dyestuffs considered as "insoluble" because of their non-aqueous or alkaline extraction conditions is present in the wood of the Pterocarpus family and Baphia nitida species. First, the crude extracts of tinctorial and related species and their chromatographic fingerprints were studied. This part of work shows that some species not yet mentioned in the literature have potential dyeing properties. Subsequent experiments performed on the redwood cargo of a 200-year-old archaeological shipwreck allowed identification of the water-logged wood species. Furthermore, the different methods of dyestuff extraction used for dyeing according to traditional recipes and their impact on analytical results were studied. They show that standard recovery obtained by acid hydrolysis of dyestuff from dyed yarns is inadequate. Hence, alternative solvent-based procedures were proposed. The identification of species in textile threads then becomes possible. The applied approach was validated by analysis of dyed reference yarns with some indications of crude material extraction mode. The employed method of analysis seems to be useful for "insoluble" wood species identification in cultural heritage artifacts as well as for phytochemical purposes, despite the fact that very few detected color compounds were chemically identified.

Composition of the black crusts from the Saint Denis Basilica, France, as revealed by gas chromatography-mass spectrometry.

The organic fraction of black crusts from Saint Denis Basilica, France, is composed of a complex mixture of aliphatic and aromatic compounds. These compounds were studied by two different analytical approaches: tetramethyl ammonium hydroxide (TMAH) thermochemolysis in combination with gas chromatography-mass spectrometry (GC-MS), and solvent extraction, fractionation by silica column, and identification of the fraction components by GC-MS. The first approach, feasible at the microscale level, is able to supply fairly general information on a wide range of compounds. Using the second approach, we were able to separate the complex mixture of compounds into four fractions, enabling a better identification of the extractable compounds. These compounds belong to different classes: aliphatic hydrocarbons (nalkanes, n-alkenes), aliphatic and aromatic carboxylic acids (n-fatty acids, alpha,omega-dicarboxylic acids, and benzenecarboxylic acids), polycyclic aromatic hydrocarbons (PAH), and molecular biomarkers (isoprenoid hydrocarbons, diterpenoids, and triterpenoids). With each approach, similar classes of compounds were identified, although TMAH thermochemolysis failed to identify compounds present at low concentrations in black crusts. The two proposed methodological approaches are complementary, particularly in the study of polar fractions.