Optimization of resolution as a simultaneous function of solvent composition, pH and temperature in reversed-phase liquid chromatography.

BACKGROUND: The latest chromatographic retention models are capable of accurately describe the dependencies of retention over a wide range of experimental conditions. By using a suitable conversion, these models can be transformed into equations expressing the optimization criteria as function of multiples variables. Even though that theoretical models significantly reduce the experimental requirements for optimizations, these models have been barely used. Instead, most optimizations rely on empirical exploration of the relationships between criterions and variables. There is a need for a strategy to reduce the required number of experiments in multivariated optimization of separations, and Fundamental Models offer a clear opportunity for addressing it. RESULTS: A Fundamental Model is used to give the simultaneous dependence of chromatographic retention of seven ionizable pesticides on the three variables: solvent composition, temperature and pH (w, T, pH). Based on few experiments, the 10 parameters required to predict the chromatographic retention of those compounds, taken as model analytes, can be obtained. Two mathematical treatments to convert retentions into resolutions between pairs are used: one considering extracolumn dispersions and other neglecting these contributions. Using the Overlapped Resolutions Maps, extended to four dimensions, two optimal conditions can be found for the two different mathematical conversions. Chromatographic conditions were empirically evaluated obtaining the best results for the optimization considering extracolumn dispersions, proving that this condition is a true optimal. It was demonstrated that any small shift in any of the variables from this true optimal leads to a loss in resolution. SIGNIFICANCE: Fundamental Models describing chromatographic retention as a simultaneous function of multiple variables are nowadays very accurate. In this work is demonstrated that these models are useful not only to predict retentions, but also to optimize separations, even in the more challenging mode: isocratic, isothermal and iso-pH. However, the success in the optimization procedure depends also on the proper definition of the mathematical conversion of the Fundamental Models into optimization criteria.

Simultaneous analysis of cannabinoids and terpenes in Cannabis sativa inflorescence using full comprehensive two-dimensional liquid chromatography coupled to smart active modulation.

Nowadays, the higher peak capacity achievable by comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of vegetal samples is well-recognized. In addition, numerous compounds may be present in very different amounts. Cannabinoids and terpenes represent the main components of Cannabis sativa inflorescence samples, whose quantities are relevant for many application purposes. The analyses of both families are performed by different methods, at least two different separation methodologies, mainly according to their chemical characteristics and concentration levels. In this work, concentration differences and sample complexity issues were addressed using an LC×LC method that incorporates an optimized modulation strategy, namely smart active modulation, for the simultaneous analysis of cannabinoids and terpenes. The system was built by interposing an active flow splitter pump between both dimensions. This set up aimed to exploit the known advantages of LC×LC. In addition, here we proposed to use the splitter pump for online control over the splitting ratio to facilitate the selective dilution of different eluted fractions containing compounds with highly different concentrations. This work represents the first application and demonstration of smart active modulation (SAM) in LC×LC to simultaneously determine analytes with significant differences in concentration levels present in complex samples. The proposed method was tested with eight different strains, from which fingerprints were taken, and numerous cannabinoids and terpenes were identified in these samples. With this strategy, between 49 and 54 peaks were obtained in the LC×LC chromatograms corresponding to different strains. THCA-A was the main component in six strains, while CBDA was the main component in the other two strains. The main terpenes found were myrcene (in five strains), limonene (in two strains), and humulene (in one strain). Additionally, numerous other cannabinoids and terpenes were identified in these samples, providing valuable compositional information for growers, as well as medical and recreational users. The SAM strategy here proposed is simple and it can be extended to other complex matrices.

Enantioseparation of agrochemicals by gas chromatography. Exploring columns based on cyclodextrin derivatives dissolved into polysiloxanes.

The main goal of this work is to expand the availability of chiral columns for the analysis of agrochemicals by gas chromatography. A broader offer of chiral stationary phases would allow shifting toward enantioselective analytical techniques environmentally more friendly for those compounds. We prepared seven chiral capillary columns based on derivatives of either, ß-cyclodextrin or γ-cyclodextrins dissolved at high concentrations, in two typical polysiloxanes with different polarities, demonstrating not only the significance of the chiral selector but also of the polymer solvent for achieving adequate enantioseparation of some agrochemicals. The enantiorecognition ability of each column was evaluated with 20 volatile and semivolatile agrochemicals, possessing one or two chiral centers. Besides, to elute more polar agrochemicals, as well as to enhance enantioselectivity, three derivatization procedures targeting the carboxyl and/or amine group were evaluated. The results revealed that the prepared column consisting of octakis(2,3-di-O-acetyl-6-O-tertbutyldimethylsilyl)-γ-cyclodextrin dissolved in (14%-cyanopropyl-phenyl)-86%-methyl-polysiloxane provides the broadest enantiorecognition capacity. This column allowed the enantioseparation of seventeen chiral agrochemicals, including metalaxyl, furalaxyl, and four imidazolinones, which were not enantioseparated in the remaining columns. To the best of our knowledge, glufosinate, fluorochloridone, fenarimol, furalaxyl, and four imidazolinones were enantioseparated by gas chromatography for the first time.

Comprehensive two-dimensional liquid chromatographic method (Chiral × Achiral) for the simultaneous resolution of pesticides.

This work demonstrates the potential of two-dimensional liquid chromatography (2D-LC) to increase the resolution capacity of multiple pesticides in a single analysis of samples that contain both chiral and achiral compounds. The setup is based on the combination of a chiral column in the first dimension and an achiral column in the second dimension using the on-line full comprehensive mode (LC × LC). This method was optimized for the separation of 24 pesticides (17 chiral and 7 achiral). The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred to the second dimension and to select and optimize independently the flow rate in the first dimension. The first-dimension optimization involved the comparison of enantioresolution abilities of three different polysaccharides chiral stationary phases as well as different elution conditions, while in the second-dimension parameters like stationary phase and organic modifier were explored. Other experimental variables that influence the two-dimensional peak capacity (orthogonality, sampling frequency, shift gradients, etc.) are discussed.

Study of enantioseparation of ß-blockers using amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions.

In this study, we describe the experimental variables influencing enantioseparation of twelve ß-blockers when analyzed under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions on a column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase. Regarding polar-organic mode, two component mobile phases consisting of methanol, ethanol or acetonitrile with the addition of basic additives such as diethylamine, triethylamine, mono-ethanolamine, ethylendiamine or trifluoroacetic acid/diethylamine mixture were evaluated. Studies of retention at different temperatures were also performed. In reversed-phase mode, mixtures consisting of methanol or acetonitrile with either aqueous boric acid-borate buffer or sodium hydrogen carbonate-carbonate buffer solutions were compared aiming to study the influence of organic modifier as well as buffer type and pH on resolution. In addition, a systematic evaluation of the retention factors of ß-blockers enantiomers in hydro-organic eluents containing acetonitrile in presence of diethylamine as additive was carried out by increasing progressively the water content, in order to check for retention dependencies indicative of the interplay of both hydrophilic interaction liquid chromatography and reversed-phase modes.

Chiral separation of several pesticides on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column under polar-organic conditions. Influence of mobile phase and temperature on enantioselectivity.

In this work, the use of different solvents and temperatures was explored, aiming to evaluate their influence on the enantioseparation of pesticides by HPLC in polar-organic conditions, employing a column containing immobilized amylose tris(3-chloro-5-methylphenyl-carbamate). The chiral separation of seventeen different pesticides widely used as herbicides, fungicides, insecticides and precursors were studied. The mobile phases included methanol, ethanol, iso-propanol, n-propanol and acetonitrile; either pure or containing additives such as diethylamine, trifluoroacetic acid, formic acid, acetic acid or mixtures thereof. We studied the influence of these eluents on chiral separation of those pesticides in terms of retention factor, enantioselectivity, enantioresolution and peak symmetry. Regarding temperature influence, evaluated within the range 5 - 40 °C, nearly all the compounds decreased their retention and selectivity factors with the increase in temperature, although the effect was dependent on the mobile phase solvent. Moreover, estimation of thermodynamic parameters was performed based on linear van´t Hoff plots.

Efficiency of capillary GC columns based on phosphonium ionic liquids.

Gas chromatographic columns based on ionic liquids (ILs) are very promising since the selectivity of these columns can be tuned by both the cation and the anion chemical nature. In this paper, efficiencies of capillary columns based on four phosphonium ionic liquids were studied. The performance of seven columns containing the cation trihexyl(tetradecyl)phosphonium and the anions bromide, chloride, and bis(trifluoromethylsulfonyl)imide was evaluated by measuring the solute band broadening as a function of gas velocities at three temperatures. Hence, classical height equivalent to a theoretical plate (H) against gas velocity (u) plots corresponding to those columns were generated and the data were fitted to the Golay-Guiochon equation with the aim of seeking the optimum conditions to be operated each of them. Band broadening at practical gas velocities is mainly due to poor mass transfer properties of solutes in the (viscous) liquid phases, which limits the achieved efficiencies. These H/u plots proved to be necessary to characterize the column quality at a given temperature, to interpret the band broadening phenomena and thus, to establish the lower temperature limits and the expected plate counts at that temperature.

Determination of gas-liquid partition coefficients of several organic solutes in trihexyl (tetradecyl) phosphonium dicyanamide using capillary gas chromatography columns.

We report here gas-liquid partition constants and activity coefficients for thirty-seven volatile organic solutes in ionic liquid trihexyl(tetradecyl)phosphonium dicyanamide measured by gas-liquid chromatography using capillary columns. Four capillary columns with exactly known phase ratios were constructed and employed to measure the solute retention factors at four temperatures between 313.15 and 343.15 K. The partition coefficients were calculated from the slopes of the linear regression between solute retention factors and the reciprocal of phase ratio at a given temperature. The partition coefficients thus calculated are free from errors due to the contribution to retention from gas-liquid interphase adsorption. For these reason, reliable solute's infinite dilution activity coefficients can be obtained, as well as partition enthalpies and entropies. A thorough discussion of the uncertainties of the experimental measurements and the main advantages of the use of capillary columns to acquire the aforementioned relevant thermodynamic information was performed.

Determination of thermodynamic binding constants by affinity capillary electrophoresis.

A strategy to study thermodynamic binding constants by affinity capillary electrophoresis (ACE) is presented. In order to simplify mathematical treatment, analogy with acid-base dissociation equilibrium is proposed: instead of ligand concentration [X], negative logarithm of ligand concentration (or activity), pX = -log[X], is used. On this base, and taking into account ionic activities, a general procedure for obtaining thermodynamic binding constants is proposed. In addition, the method provides electrophoretic mobilities of the free analyte and analyte-ligand complex, even when binding constants are low and thus, the complexed analyte fraction is also low. This is useful as a base to rationally analyze a diversity of situations, i.e., different mathematical dependencies are obtained when analytes and ligands with different charges are combined. Practical considerations are given for carrying out a full experimental design. Enantiomeric ACE separation based on the use of chiral selectors is addressed. 2-hydroxypropyl-ß-cyclodextrin was chosen as a model ligand, and both enantiomeric forms of four pharmaceutical drugs (propranolol, pindolol, oxprenolol and homatropine methylbromide) were considered as model analytes. Practical aspects are detailed and thermodynamic binding constants as well as free and complexed analytes mobilities are determined.

Enantiomeric separations by capillary electrophoresis: Theoretical method to determine optimum chiral selector concentration.

A method to optimize the ligand concentration [S] in the background electrolyte of capillary electrophoresis separations is presented. It is based on the use of a model which predicts apparent electrophoretic mobilities as a function of ligand concentration (expressed as p[S] = -log[S]). This model is employed to compose the expression of a recently proposed criterion to qualify separations in electrophoresis. Two strategies to find the optimum p[S], leading to the best separation of all compounds, are explained: 1.- a graphical method using a windows map depicting the single separation criteria between all possible combination of compounds by pairs, and 2.- an analytical method where an extended multicriterion optimization function is composed and optimum p[S] is found by mathematical maximization. The procedure is applied to a hard-to-separate model system: enantiomeric separations of racemic mixtures. 2-Hydroxypropyl-ß-cyclodextrin was chosen as a model ligand, and four pharmaceutical drugs as model analytes. In order to demonstrate the performance of the procedure, results of electrophoretic separations obtained at p[S] found as optimum are compared with separations obtained at p[S] values slightly higher and lower than the optimum.

Determination of gas-liquid partition coefficients of several organic solutes in trihexyl(tetradecyl)phosphonium bromide using capillary gas chromatography columns.

In this paper, we report gas-liquid partition constants for thirty-five volatile organic solutes in the room temperature ionic liquid trihexyl(tetradecyl)phosphonium bromide measured by gas-liquid chromatography using capillary columns. The relative contribution of gas-liquid partition and interfacial adsorption to retention was evaluated through the use of columns with different the phase ratio. Four capillary columns with exactly known phase ratios were constructed and employed to measure the solute retention factors at four temperatures between 313.15 and 343.15K. The partition coefficients were calculated from the slopes of the linear regression between solute retention factors and the reciprocal of phase ratio at a given temperature according to the gas-liquid chromatographic theory. Gas-liquid interfacial adsorption was detected for a few solutes and it has been considered for the calculations of partition coefficient. Reliable solute's infinite dilution activity coefficients can be obtained when retention data are determined by a unique partitioning mechanism. The partial molar excess enthalpies at infinite dilution have been estimated from the dependence of experimental values of solute activity coefficients with the column temperature. A thorough discussion of the uncertainties of the experimental measurements and the main advantages of the use of capillary columns to acquire the aforementioned relevant thermodynamic information was performed.

Chiral separation of aryloxyphenoxy-propionate herbicides in a permethyl-ß-cyclodextrin based column. Influence of temperature and mobile phase composition on enantioselectivity.

We used a permethyl-ß-cyclodextrin chiral stationary phase under reversed-phase conditions for the chiral separation of four aryloxyphenoxy-propionate herbicides (fenoxaprop-p-ethyl, quizalofop-p-ethyl and tefuryl, and haloxyfop-p-methyl) with mixtures of methanol, ethanol, 2-propanol, n-propanol, tert-butanol, or acetonitrile and water as mobile phases and investigated the influence of mobile phase composition and column temperature (from 0 to 50°C) on the separation. The retention factors (k) and selectivity factors (α) of all the herbicides investigated decreased with increasing temperature. The lnα versus 1/T and lnk versus 1/T plots for the enantiomers of the chiral pesticides were linear within the range of 0-50°C with all alcohol/water mixtures constituting the mobile phase, but the lnk versus 1/T plots were nonlinear for all the enantiomers chromatographed in acetonitrile/water mixtures. The thermodynamic parameters based on linear van't Hoff plots were calculated. The influence of temperature and mobile phase composition on the enantioseparation of the solutes has rarely been considered simultaneously. The temperature and the solvents used in the mobile phase, however, were found to have a profound effect on the enantioseparation of these herbicides.

Quality criterion to optimize separations in capillary electrophoresis: Application to the analysis of harmala alkaloids.

In capillary electrophoresis (CE), resolution (Rs) and selectivity (α) are criteria often used in practice to optimize separations. Nevertheless, when these and other proposed parameters are considered as an elementary criterion for optimization by mathematical maximization, certain issues and inconsistencies appear. In the present work we analyzed the pros and cons of using these parameters as elementary criteria for mathematical optimization of capillary electrophoretic separations. We characterized the requirements of an ideal criterion to qualify separations within the framework of mathematical optimizations and, accordingly, propose: -1- a new elementary criterion (t') and -2- a method to extend this elementary criterion to compose a global function that simultaneously qualifies many different aspects, also called multicriteria optimization function (MCOF). In order to demonstrate this new concept, we employed a group of six alkaloids with closely related structures (harmine, harmaline, harmol, harmalol, harmane and norharmane). On the basis of this system, we present a critical comparison between the new optimization criterion t' and the former elementary criteria. Finally, aimed at validating the proposed methods, we composed an MCOF in which the capillary-electrophoretic separation of the six model compounds is mathematically optimized as a function of pH as the unique variable. Experimental results subsequently confirmed the accuracy of the model.

Enantioselective gas chromatography with functionalized cyclodextrins as chiral selectors. Fundamentals of the measurement of absolute association constants using capillary columns.

Chiral capillary GC columns containing different amounts of octakis(6-O-tert-butyldimethylsilyl-2,3-di-O-acetil)-γ-cyclodextrin as chiral selector dissolved in a polymeric matrix were constructed with the aim of determining enantiomeric association constants between a group of well resolved chiral N-trifluoroacetyl amino acid methyl esters and this specific selector at different temperatures. The most relevant sources of uncertainties in the experimental data (hold-up and retention times, and column phase ratios at each temperature) were assessed. These cyclodextrin-based columns are known to enantioseparate a wide variety of chemical compounds, thus, the measurement of the absolute enantioselective constants of a group of solutes with this selector can be useful for systematic studies aimed to a general understanding about how these selectors work. These absolute association constants were estimated from data collected from very simplified experimental systems, and by using the fundamental gas-liquid chromatography equations.

Effect of temperature on acid-base equilibria in separation techniques. A review.

Studies on the theoretical principles of acid-base equilibria are reviewed and the influence of temperature on secondary chemical equilibria within the context of separation techniques, in water and also in aqueous-organic solvent mixtures, is discussed. In order to define the relationships between the retention in liquid chromatography or the migration velocity in capillary electrophoresis and temperature, the main properties of acid-base equilibria have to be taken into account for both, the analytes and the conjugate pairs chosen to control the solution pH. The focus of this review is based on liquid-liquid extraction (LLE), liquid chromatography (LC) and capillary electrophoresis (CE), with emphasis on the use of temperature as a useful variable to modify selectivity on a predictable basis. Simplified models were evaluated to achieve practical optimizations involving pH and temperature (in LLE and CE) as well as solvent composition in reversed-phase LC.

Scope of partial least-squares regression applied to the enantiomeric composition determination of ketoprofen from strongly overlapped chromatographic profiles.

Valuable quantitative information could be obtained from strongly overlapped chromatographic profiles of two enantiomers by using proper chemometric methods. Complete separation profiles where the peaks are fully resolved are difficult to achieve in chiral separation methods, and this becomes a particularly severe problem in case that the analyst needs to measure the chiral purity, i.e., when one of the enantiomers is present in the sample in very low concentrations. In this report, we explore the scope of a multivariate chemometric technique based on unfolded partial least-squares regression, as a mathematical tool to solve this quite frequent difficulty. This technique was applied to obtain quantitative results from partially overlapped chromatographic profiles of R- and S-ketoprofen, with different values of enantioresolution factors (from 0.81 down to less than 0.2 resolution units), and also at several different S:R enantiomeric ratios. Enantiomeric purity below 1% was determined with excellent precision even from almost completely overlapped signals. All these assays were tested on the most demanding condition, i.e., when the minor peak elutes immediately after the main peak. The results were validated using univariate calibration of completely resolved profiles and the method applied to the determination of enantiomeric purity of commercial pharmaceuticals.

New method for sintering silica frits for capillary microcolumns.

One of the main steps in the manufacture of robust and efficient packed capillary microcolumns for electro- and capillary chromatography is the generation of porous devices to retain the packed beds. Frits based on sintered silica particles have been found to give the best results in terms of mechanical resistance and efficiency. The conventional procedure to produce these kinds of frits consists in a radial heating of the packed material with either a flame or an electrical resistance, but the frits thus obtained have many drawbacks as a result of the procedure rather than the silica per se as the base material. In the present work we investigated a new approach to produce silica-based retaining devices involving the frontal exposure of a short silica-particle bed packed at the end of a capillary tube. The capillary is radially insulated and frontally exposed to the heat of a muffle oven, generating a transfer of heat that is not radial but rather throughout the capillary axis. This procedure resulted in substantial advantages: an improved radial homogeneity, a protection of the external polyimide, and a generation of extremely short (400-600 µm) frits that were highly permeable and avoided bubble formation.

Measurements of association constants between enantiomers and chiral selectors by capillary gas chromatography. Theoretical and practical considerations.

The association constants of several volatile enantiomers with octakis(3-O-butanoyl-2,6-di-O-pentyl)-γ-cyclodextrin at temperatures between 50 and 100 °C were measured by gas-liquid chromatography using capillary columns coated with different amounts of chiral selector dissolved in polysiloxane OV-1701 and prepared with a precisely determined phase ratio. Simple expressions were deduced to estimate the apparent distribution constants from accurate hold-up and retention times along with that known phase ratio at each temperature. The enantiomer-chiral selector association constants were then calculated from the linear regression of the apparent constants as a function of the chiral selector concentration. One aim of this study consisted in discussing all the experimental uncertainties inherent in the determination of enantiomer/selector association constants with chiral analytes, and how these fundamental measurements can be performed precisely without resorting to the use of reference solutes.

Enantioseparation of α-amino acids by means of Cinchona alkaloids as selectors in chiral ligand-exchange chromatography.

A conventional nonchiral column was used for the enantioseparation of several racemic α-amino acids (native and derivatized) through the use of Cinchona alkaloids as chiral selectors along with Cu(II) ions in chiral ligand-exchange chromatography. The mobile phase composition (i.e., the organic modifier content and pH) was studied in order to modulate retention and enantioselectivity. Good enantioseparation of many amino acids was obtained using equimolar amounts of Cu(II) and either cinchonidine, quinine or quinidine as chiral selectors in the mobile phase. The molecular geometry of the diastereomeric complexes formed was modeled and energetic differences between both compounds were calculated by methods based on semi-empirical force-field. Good correlations were obtained between experimental enantioselectivity factors and calculated energetic differences.

A simple, robust orthogonal background correction method for two-dimensional liquid chromatography.

Background correction is a very important step that must be performed before peak detection or any quantification procedure. When successful, this step greatly simplifies such procedures and enhances the accuracy of quantification. In the past, much effort has been invested to correct drifting baseline in one-dimensional chromatography. In fast online comprehensive two-dimensional liquid chromatography (LC×LC) coupled with a diode array detector (DAD), the change in the refractive index (RI) of the mobile phase in very fast gradients causes extremely serious baseline disturbances. The method reported here is based on the use of various existing baseline correction methods of one-dimensional (1D) liquid chromatography to correct the two-dimensional (2D) background in LC×LC. When such methods are applied orthogonally to the second dimension ((2)D), background correction is dramatically improved. The method gives an almost zero mean background level and it provides better background correction than does simple subtraction of a blank. Indeed, the method proposed does not require running a blank sample.