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.

Rapid assessment of fatty acyls chains of phospholipids and plasmalogens by atmospheric pressure chemical ionization in positive mode and high-resolution mass spectrometry using in-source generated monoacylglycerol like fragments intensities.

We recently published a new concept using monoacylglycerol-like fragments [MG+H-H2O]+ (ions B) produced in-source by atmospheric pressure photoionization in positive mode and high-resolution mass spectrometry for the determination of the fatty acyl (FA) composition of triacylglycerols (TGs) from plant oils. This study extends the concept to the phospholipids (PLs) category and shows that the APCI+ source can also be used. Moreover, the coupling with NP-LC allows to simultaneously analyze different PLs classes in the same sample. We compared the relative intensities of the ions B produced in-source to the % composition of FAs determined by GC-FID. In the case of PLs from natural extracts composed exclusively of diacyl-PLs, the relative intensities of ions B are close to the % of the FAs obtained by GC-FID. This approach is not directly useable for extracts containing plasmalogens (P-PLs). For these PLs, acidic hydrolysis by HCl fumes allows hydrolyzing selectively vinyl ether functions to form lyso-PLs. The analysis of hydrolyzed extracts makes it possible to obtain the composition of P-PLs FAs thanks to the lyso-PLs thus formed, while the diacyl-PLs composition remains unchanged. Unlike GC-FID FAs determination, this approach allows a distinction between the diacyl-PLs and P-PLs FAs composition. We also found that the ion B intensities were consistent among the PL classes (PG, PE, PA, PI, CL, PS and PC) and lyso- forms (LPE and LPC). In the case of the diacyl-PLs extracts analyzed, no statistically significant differences were found between the PLs FAs compositions calculated from ion B intensities and the corresponding GC-FID data. A weighting coefficient was applied to correct ion B intensities issued from polyunsaturated FAs with three or more double bonds. The fatty alkenyls composition of P-PLs could also be calculated from the % intensities of specific ions.

Rapid assessment of triacylglycerol fatty acyls composition by LC-APPI+-HRMS using monoacylglycerol like fragments intensities.

We present a new analytical approach for the analysis of triacylglycerol fatty acyls distribution by normal phase liquid chromatography (NPLC) coupled with APPI+-HRMS. The NPLC method used allows the separation of more than 30 classes of lipids. The energy of the APPI+ source enables the formation of low-intensity ions B fragments ([RC = O+74]+ <3%), characteristic of lipids with a glycerol esterified by one or more fatty acyls. We found the relative intensities of ions B were close to the fatty acyl distribution. To establish the proof of concept, we decided to focus on the triacylglycerols (TGs) class, the major component of plant oils. By either NPLC or FIA, the TGs class appeared as a single peak. In our experimental conditions, ions B are always present in the mass spectra of TGs and each ion B is specific to a fatty acyl group. The Orbitrap mass spectrometer featured high enough resolution and accuracy to identify ions B and distinguish them from other TG fragment ions. A further adjustment of the fatty acyls relative quantities calculation from ions B intensities was computed using weighting coefficients of ions B response. The methodology was developed and validated using plant oils characterized by a GC-FID reference method. NPLC-APPI+-HRMS method offers the advantage of analyzing the fatty acyl composition of complex lipid extracts without the need for sample preparation.

New anthracenyl polar embedded stationary phases with enhanced aromatic selectivity, a combined experimental and theoretical study: Part 1-experimental study.

New polar embedded aromatic stationary phases of different functionalities (mono- and trifunctional) and on different silica supports (Fully Porous Particle (FPP) and Superficially Porous Particle (SPP)) were synthesized to determine the impact of the functionality on the retention process and the selectivity towards aromatic compounds. A full experimental characterization was performed using a combination of techniques (elemental analysis, thermogravimetric measurements, infrared spectroscopy and solid-state NMR) to differentiate unambiguously the mono- and trifunctional Stationary Phases (SP). Commercially available columns with either an aromatic group or a polar embedded group were compared to the new stationary phases. The latter presented enhanced affinity for polycyclic aromatic hydrocarbons (PAH) structures compared to alkylbenzenes, especially when using methanol instead of acetonitrile as the organic modifier.

Fast non-aqueous reversed-phase liquid chromatography separation of triacylglycerol regioisomers with isocratic mobile phase. Application to different oils and fats.

The distribution of fatty acid species at the sn-1/3 position or the sn-2 position of triacylglycerols (TAGs) in natural fats and oils affects their physical and nutritional properties. In fats and oils, determining the presence of one or two regioisomers and the identification of structure, where they do have one, as well as their separation, became a problem of fundamental importance to solve. A variety of instrumental technics has been proposed, such as MS, chromatography-MS or pure chromatography. A number of studies deal with the optimization of the separation, but very often, they are expensive in time. In the present study, in order to decrease the analysis time while maintaining good chromatographic separation, we tested different monomeric and polymeric stationary phases and different chromatographic conditions (mobile phase composition and analysis temperature) using Non-Aqueous Reversed Phase Liquid Chromatography (NARP-LC). It was demonstrated that mixed polymeric stationary bonded silica with accessible terminal hydroxyl groups leads to very good separation for the pairs of TAGs regioisomers constituted by two saturated and one unsaturated fatty acid (with double bond number: from 1 to 6). A Nucleodur C18 ISIS percolated by isocratic mobile phase (acetonitrile/2-propanol) at 18°C leads to their separations in less than 15min. The difference of retention times between two regioisomers XYX and XXY are large enough to confirm, as application, the presence of POP, SOP, SOS and PLP and no PPO, SPO, SSO and PPL in Theobroma cacao butter. In the same way, this study respectively shows the presence of SOS, SOP and no SSO, PSO in Butyrospermum parkii butter, POP, SOP, SOS and no PPO, PSO and SSO in Carapa oil and finally POP and no PPO in Pistacia Lentiscus oil.

Thermal pretreatments of superficially porous silica particles for high-performance liquid chromatography: Surface control, structural characterization and chromatographic evaluation.

This study reports the impact of thermal pretreatment between 400 and 1100°C on superficially porous silica particles (e.g. core-shell, fused-core; here abbreviated as SPP silica). The different thermally pretreated SPP silica (400°C, 900°C and 1100°C) were chemically bonded with an octadecyl chain under microwave irradiation. The bare SPP silica, thermally untreated and pretreated, as well as the chemically bonded phases (CBPs) were fully characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), and solid state cross polarization magic angle spinning (CP-MAS) (29)Si NMR. The chromatographic properties of the overall set of C18-thermally pretreated SPP silica stationary phases were determined using the Tanaka test. Complementary, the simplified Veuthey test was used to deeply study the silanol activity, considering a set of 7 basic solutes with various physicochemical properties. Both tests were also performed on different commercial SPP silica columns and different types of bonding chemistry (C18, Phenyl-hexyl, RP-amide, C30, aQ). Multivariate data analyses (hierarchical cluster analysis and principal component analysis) were carried out to define groups of stationary phases with similar chromatographic properties and situate them in relation to those commercially available. These different C18-thermally pretreated SPP silicas represented a wide range of stationary phases as they were spread out along the score plot. Moreover, this study highlighted that the thermal pretreatment improved the chemical stability of the SPP silica compare to untreated SPP silica and untreated porous silica. Consequently, higher thermal pretreatment can be applied (up to 900°C) before functionalization without destruction of the silica matrix. Indeed, a significantly lower dissolution of the thermally pretreated SPP silica under aggressive conditions could allow the use of the corresponding functionalized stationary phases at high temperature (60°C) with good lifetime of the columns.

A new methodology to determine the isoeluotropic conditions on ultra-performance flash purification stationary phases from analytical reversed liquid chromatography stationary phase.

Nowadays, the determination of the experimental chromatographic conditions to be used in Reversed Phase Liquid Ultra-Performance Flash Purification is still challenging. This is due to four different items. In most cases, flash purification stationary phases are not available with geometry of column used in analytical chromatography. The flash purification columns are single-use only. From the point of view of selectivity and retention, few RPLC phases exist with properties of separation identical for analytical and flash purification supports. Characterization methods and databases used for comparing analytical RPLC columns do not include stationary phases for RP flash purification columns. The goal of this work is to develop a new method development strategy which permits the determination of the experimental chromatographic conditions on RP ultra-performance flash purification columns. It relies on the knowledge of any isocratic conditions obtained on any given initial reversed stationary phase. The final conditions to implement on the RP ultra-performance flash purification phase enable either to keep the retention range of a selected solute constant, or to set it around a previously chosen value. The rules of transfer in linear gradient mode are also described. The methodology was valid, whatever the initial RP stationary and mobile phases, for different chemical classes, whatever the bonding, particle diameter, porous or core shell particle, towards different RP alkyl and analogues stationary and mobile phases.

Comparison of iso-eluotropic mobile phases at different temperatures for the separation of triacylglycerols in Non-Aqueous Reversed Phase Liquid Chromatography.

Triacylglycerols (TAGs) are a large class of neutral lipids that naturally occur in both plant and animal oils and fats. Their analyses in Non-Aqueous Reversed Phase Liquid Chromatography (NARP) require a mixture of weak solvent (mostly acetonitrile) and strong solvent. In the present work, we have established eluotropic solvent strength scale of several binary mobile phases on C18 bonded silica at different temperatures (acetonitrile/methylene chloride, acetonitrile/acetone, acetonitrile/ethyl acetate, acetonitrile/propan-2-ol, and acetonitrile/butan-1-ol at 25°C, 43°C, 63°C and 85°C); it is based on the methylene selectivity and the use of homologous series. We show that this scale is well suited to the TAGs analysis. The analysis of nine seed oils (Aleurites fordii, Calophyllum inophyllum, Glycina max, Olea europea, Orbignya olifeira, Pinus koraiensis, Pistacia lentiscus, Punica granatum and Ribes nigrum) in iso-eluotropic conditions leads to propose unambiguously the couple MeCN/BuOH at 25°C as the best system to separate TAGs. The use of butanol, as strong solvent, provides very good TAGs congeners separations and avoids the use of chlorinated solvents which gave to this day the best separations.

High-resolution magic angle spinning description of the interaction states and their kinetics among basic solutes and functionalized silica materials.

Modeling of the interaction is crucial to understanding and predicting chromatography. However, the complexity and variety of the grafted motifs render the creation of an accurate model overwhelmingly challenging, so that most often the classification of column separation properties is described by monitoring the retention times of carefully selected control molecules. We analyzed here the characteristics of the interplay of compounds of basic nature by (1)H HRMAS NMR, which provide relevant descriptors for products with pharmaceutical properties, with chromatographic phases for Reversed Phase Liquid Chromatography. Eight grafted silica phases were selected, differing to enhance specific structural properties (monomeric and polymeric grafts, endcapping or not, carbon content, alkyl with polar embedded group or alkyl bonded chain, chemical nature of end capping, native silica). These materials were put in interaction with five basic molecules, previously chosen as probes for the evaluation of efficient base deactivated liquid stationary phases using five theoretical molecular descriptors to cover a large scale of molecular volume, polar surface area, LogP, hydrogen-bond donor capacity and finally hydrogen-bond acceptor capacity. (1)H HRMAS NMR was capable of describing qualitatively a wealth of interaction states, characterized both thermodynamically and kinetically. In one case (penbutolol) up to five interaction states could be differentiated. Variable temperature experiments revealed the complexity of the retention process on grafted silica as in some cases the kinetics of the interaction is shown to slow down on increasing the temperature.

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.

Determination of triacylglycerol regioisomers using electrospray ionization-quadrupole ion trap mass spectrometry with a kinetic method.

The kinetic method was applied to differentiate and quantify mixtures of regioisomeric triacylglycerols (TAGs) by generating and mass selecting alkali ion bound metal dimeric clusters with a TAG chosen as reference (ref) and examining their competitive dissociations in a quadrupole ion trap mass spectrometer. This methodology readily distinguished pairs of regioisomers (AAB/ABA) such as LLO/LOL, OOP/OPO and SSP/SPS and consequently distinguished sn-1/sn-3, sn-2 substituents on the glycerol backbone. The dimeric complex ions [ref, Li, TAG((AAB and/or ABA))](+) generated by electrospray ionization mass spectrometry were subjected to collision induced dissociation causing competitive loss of either the neutral TAG reference (ref) leading to [Li(AAB and/or ABA)](+) or the neutral TAG molecule (TAG((AAB and/or ABA))) leading to [ref, Li](+). The ratio of the two competitive dissociation rates, defined by the product ion branching ratio (R(iso)), was related via the kinetic method to the regioisomeric composition of the investigated TAG mixture. In this work, a linear correlation was established between composition of the mixture of each TAG regioisomer and the logarithm of the branching ratio for competitive fragmentation. Depending on the availability of at least one TAG regioisomer as standard, the kinetic method and the standard additions method led to the quantitative analysis of natural TAG mixtures.

Simple complementary liquid chromatography and mass spectrometry approaches for the characterization of triacylglycerols in Pinus koraiensis seed oil.

A new simple strategy to identify triacylglycerols (TAGs) in oils and fats was performed using on line coupling of non aqueous reversed phase chromatography-electrospray ionization-mass spectrometry (NARP-LC-ESI-MS(2)) with silver nitrate (AgNO(3)) as post-column additive, and chromatographic data (partition number information and both the graphs of log k vs. number of double bond (DBN) and carbon number (CN)). NARP liquid chromatography permitted to separate TAGs composed of Δ5 and Δ9 but not from Δ11 double bond location on alkyl chain of fatty acid residues. Silver cationization improved the sensitivity by a factor one hundred. MS(2) information gave unambiguously the nature of three fatty acid residues bonded to glyceryl backbone of TAGs while log k against DBN and CN curves discriminated between the same molecular mass TAG isomers (whose constitutive fatty acid residues are double bond position and configuration isomers). Combination of structural information given by MS with chromatographic retention laws led to the development of a general methodology for determination of the structure of TAGs in lipids. This methodology was applied to Pinus koraiensis seed oil for which some uncommon TAGs are present. It permitted the identification of 58 TAGs in this oil. The experimental proof of 29 uncommon TAGs as component of this oil is demonstrated. Among them 26 were minor constituents.

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.

The celebrated écorchés of honoré Fragonard, part 2: The details of the technique used by Fragonard.

It is remarkable that the famous écorchés of Honoré Fragonard have survived the centuries to reach us today. Studies carried out by several teams have established details of the technique used by Fragonard that help to explain their longevity. The injection of the vessels was achieved by means of a mixture of mutton tallow and pine resin diluted in essence of turpentine and essential oils. This gave Fragonard a very high success rate. Above all, he did not add pigments to his mixture while injecting the veins, and this facilitated the procedure. The vessels were painted after preservation to give them the vivid colors that we can still see today. Another detail that explains their exceptional conservation is that the varnish used by Fragonard was composed of Venice turpentine, made from larch resin and known to repel insects.

Regioisomer characterization of triacylglycerols by non-aqueous reversed-phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent.

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag(+) after their separation by non-aqueous reversed-phase liquid chromatography (NARP-LC) before MS to improve MS sensitivity. Electrospray ionization-MS (ESI-MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO(3)](+) ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS(5) spectra. The observation of ions produced by LC-MS(5) of on-line Ag(+)-cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS(5) experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils.

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates.

The natural (13)C/(12)C isotope composition (delta(13)C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the (12)C/(13)C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific delta(13)C of individual carbohydrates. Here, we examined two complementary methods for measuring the delta(13)C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro (12)C/(13)C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC approximately EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the (12)C/(13)C isotope effect is rather small and it is not affected by the use of heavy water (D(2)O).

Method development for a quantitative analysis performed without any standard using an evaporative light-scattering detector.

A new method for quantitative analyses using an evaporative light-scattering detector (ELSD) is proposed. It is based on the preliminary determination of the calibration curve of an ELSD which correlates coefficient b and loga, the two coefficients from the equation: A=am(b), that characterize the law of the quantitative response for an ELSD. Dilutions of the mixture to be analyzed allow the determination of coefficient b for each component of the mixture. The knowledge of the b value and the experimental relationship correlating b with loga allows to determine the loga value and consequently to quantify each compound of the mixture. This method is an alternative to the quantitative method which uses an internal normalization without any response coefficient. This internal normalization method used with an ELSD provides inaccurate results and this inaccuracy increases when the analytes are in very different proportions. The relevance of the new method proposed in this work lies in the quantification of all the components present in a complex mixture when some of them are not available as standards.