Use of Database of Plots of Pesticide Retention (R F ) Against Mobile-Phase Compositions for Fractionation of a Mixture of Pesticides by Micropreparative Thin-Layer Chromatography

Relationships between R _F values and mobile-phase composition have been determined for urea herbicides and fungicides in normal-phase systems (NP) of the type silica-nonpolar or weakly polar diluent (heptane, toluene, diisopropyl ether) – polar modifier (ethyl acetate, tetrahydrofuran, dioxane, ethyl-methyl ketone and 2-propanol). These relationships constitute a retention database which has enabled to choose optimum systems for preliminary fractionation of a multicomponent mixture of pesticides by zonal micropreparative TLC. The mixture was applied from the edge of the layer in the frontal + elution mode which increased the separation efficiency because or displacement effects. The separated simpler fractions were applied to a silica plate and rechromatographed. The plate was videoscanned, furnishing a real picture of the plate showing preliminary separation of the simpler pesticide fractions. Complete separation of the fractions was carried out by two-dimensional thin-layer chromatography on plates with chemically bonded-cyanopropyl silica stationary phase using non-aqueous eluent in the first direction and aqueous reversed-phase eluent in the second direction.     

Use of a database of plots of pesticide retention (R F) against mobile-phase composition.Part I. Correlation of pesticide retention data in normal- and reversed-phase systems and their use to separate a mixture of ten pesticides by 2D TLC

Summary Ten pesticides have been completely separated by two-dimensional (2D) development on TLC plates coated with coupled layers of octadecyl silica (reversed-phase, RP) and plain silica (normal-phase, NP). The binary mobile phases, aqueous-organic for RP chromatography and nonaqueous for NP chromatography, were chosen from plots ofR _F against mobile-phase composition and graphicalR _F(RP)-R _F(NP) correlations. The different selectivity of the RP and NP systems enabled dispersion of spots over the plate area and good separation.     

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.     

Use of a Graphical Method to Predict the Retention Times of Selected Flavonoids in HPLC from Thin-Layer Chromatographic Data

Similarities and differences between the retention characteristics of octadecylsilica wettable with water used in TLC and RP-18 used in HPLC have been elucidated by use of the linear relationships between log k and R_M. The stationary phases compared were investigated with the same mobile phases—binary mixtures of methanol and water, acetonitrile and water, and tetrahydrofuran and water. For these adsorbents of the same type but differing in specific surface area the correlation line was shifted by log (_systemI/_systemII). High values of the correlation coefficients obtained over the whole range of mobile phase organic modifier concentration examined indicated that the TLC systems could be used to predict HPLC conditions for flavonoid separation.     

Comparison of chromatographic properties of cyanopropyl-, diol- and aminopropyl- polar-bonded stationary phases by the retention of model compounds in normal-phase liquid chromatography systems.

Polar-bonded stationary phases, such as CN-, diol- and NH2-silica, have been characterised by the retention of model solutes (phenols, aromatic amines and quinoline bases) in normal-phase systems using n-heptane--polar modifier (2-propanol, tetrahydrofuran or dioxane) mixtures as eluents. The selectivity of separation for the particular groups of substances has been analysed by the log kI versus log kII relationships for CN- and diol, CN- and NH2- and NH2- and diol phases in examined eluent systems by the plotting of correlation lines. The values of regression coefficient r indicate either the similarity of the retention mechanisms of model solutes in some examined systems where r>0.9, or differences among various systems where r<0.9. The values of slopes of correlation lines show the selectivity of separation for particular group of compounds. The selectivity of separation has also been characterised by deltalog k values. The effect of modifier (2-propanol, tetrahydrofuran and dioxane) on selectivity of model solutes on these phases has also been discussed.     

A refined physico-chemical model of solute retention in reversed-phase high-performance liquid chromatography with ternary mobile phases

Summary In our previous publication we have introduced a new model of solute retention in RP-HPLC systems with ternary mobile phases of the B+AB₁+AB₂ type (B: acetonitrile or tetrahydrofuran; AB₁: methanol; AB₂: water). That model proposed no stoichiometric differentiation between acetonitrile and tetrahydrofuran, alternatively present in the solvent system; moreover, it made some very rough assumptions only as to the intermolecular interactions among the mobile phase constituents.This paper introduces a significant refinement to the already established retention model, which is based on the simple quantitative relationships between acetonitrile and tetrahydrofuran, and the remaining components of the ternary liquid system. The refined model is tested with same experimental data.     

Enantiomeric separation of six chiral pesticides that contain chiral sulfur/phosphorus atoms by supercritical fluid chromatography

Six chiral pesticides containing chiral sulfur/phosphorus atoms were separated by supercritical fluid chromatography with supercritical CO₂ as the main mobile phase component. The effect of the chiral stationary phase, different type and concentration of modifiers, column temperature, and backpressure on the separation efficiency was investigated to obtain the appropriate separation condition. Five chiral pesticides (isofenphos‐methyl, isocarbophos, flufiprole, fipronil, and ethiprole) were baseline separated under experimental conditions, while isofenphos only obtained partial separation. The Chiralpak AD‐3 column showed a better chiral separation ability than others for chiral pesticides containing chiral sulfur/phosphorus atoms. When different modifiers at the same concentration were used, the retention factor of pesticides except flufiprole decreased in the order of isopropanol, ethanol, methanol; meanwhile, the retention factor of flufiprole increased in the order of isopropanol, ethanol, methanol. For a given modifier, the retention factor and resolution decreased on the whole with the increase of its concentration. The enantiomer separation of five chiral pesticides was an “enthalpy‐driven” process, and the separation factor decreased as the temperature increased. The backpressure of the mobile phase had little effect on the separation factor and resolution.     

Reversed-Phase Liquid Chromatographic Separation, on Cellulose Chiral Stationary Phases, of the Stereoisomers of Methoxytetra-hydronaphthalene Derivatives, New Agonist and Antagonist Ligands for Melatonin Receptors

A stereoselective high-performance liquid chromatographic method has been established for chiral separation of melatoninergic derivatives with one or two chiral centers, new agonist and antagonist ligands for melatonin receptors. Reversed-phase separations were performed on cellulose-based chiral stationary phases—tris-3,5-dimethylphenylcarbamate (Chiralcel OD-RH) or tris-methylbenzoate (Chiralcel OJ-R). Water–modifier (methanol or acetonitrile) mixtures in different proportions were used as mobile phases. The effects of organic mobile-phase modifier concentration, temperature, and compound structure were examined. Baseline separation (R_S > 1.5) was readily obtained for many of the compounds.     

Enantioselective separation of thalidomide on an immobilized α1-acid glycoprotein chiral stationary phaseglycoprotein chiral stationary phase

Summary An easy and rapid enantioselective separation for assay of racemic thalidomide on an immobilized α₁-acid glycoprotein chiral stationary phase (GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modifier, buffer concentration to control the ionic strenth, and mobile phase pH were studied. These variations have consequences in terms of chromatographic retention (k), resolution (R _s), selectivity (α), and peak asymmetry (USP tailing factor). The main condition affecting chromatographic retention was mobile phase pH. At pH 4.5, no separation of thalidomide enantiomers was achieved whereas at pH 7.9 chiral separation was optimum. Peak tailing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF concentration-dependent, and that the separation factor (α) is the best parameter for evaluating enantioselectivity. The best mobile phase was pH 7.0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions validation including linearity, recovery, and precision was performed. The suitability of this method has been successfully proved in a limited in-vivo study after intravenous administration of thalidomide to a New Zealand male rabbit.     

Addition of organic modifiers to control retention order of enantiomers of dihydropyridines on chiral-AGP

Summary The retention order of the enantiomers of clevedipine (solute no 1), a shortacting blood pressure reducer, was controlled by type of organic modifier. With 1-propanol as, mobile phase modifier the (R)-form eluted first and by using methanol, the (S)-form was first to elute. These effects could also be seen for the hydrolysed analogue, to clevedipine, an acid (solute no 2). The reversal of retention order was then obtained when 1-propanol was replaced by acetonitrile. An oxidized analogue to clevedipine, a pyridine (solute no 3), was also tested regarding reversal of retention order of its enantiomers. In this case the retention order could not be controlled by type of organic modifier. The influence of the mobile phase buffer pH on enantioselective retention was also studied for the three substances. For clevidipine and the oxidized analogue, a high mobile phase pH favored enantioselective resolution while the opposite result was obtained for the hydrolysed analogue. Temperature studies were also performed, and enthalpies and entropies at different mobile phase pH:s using different organic modifiers were calculated, in order to promote an understanding of the thermodynamic driving forces for retention in the systems. Optimized chromatographic systems were used to determine less than 0.1% of an enantiomeric impurity in (R)- and (S)-clevidipine.     

Roles of Organic Modifiers in Micellar Electrokinetic Capillary Chromatography

In the present work, four organic modifiers, viz. urea, dioxane, methanol, and tetrahydrofuran, were comparatively and systematically studied in terms of their effects on electrokinetic migration behavior and the retention mechanism of homologous solutes in MECC. The results showed that the electroosmotic mobility, μ_eo, and the electrophoretic mobility of a micelle, μ_ep,mc, decrease linearly with increasing organic modifier concentration. The ability of organic modifiers to lower μ_eo is greater than their ability to lower μ_ep,mc. The negative values of the slopes of these linear relationships, D_eo and D_ep,mc, increase along the series the order urea < methanol < dioxane < tetrahydrofuran. The logarithm of the capacity factor (ln k′) of uncharged homologous solute, which is mainly determined by the hydrophobic interaction, decreases linearly with increasing organic modifier concentration, due not only to the decrease in the partition coefficient but also to the decrease in the phase ratio. A linear relationship was observed between the slope of the plot of ln k′ vs. organic modifier concentration and the carbon number of homologous compound. The slope of such a relationship can characterize the hydrophobicity of the organic modifier. The hydrophobicity of such organic modifiers is also found to increase along the series urea < methanol < dioxane < tetrahydrofuran.     

Retention and selectivity of aromatic hydrocarbons with mono-substituted polar groups in ternary RP-HPLC systems

Summary The variation in selectivity for aromatic hydrocarbons with mono-substituted polar groups is investigated in ternary mobile-phase systems on C₁₈ stationary phases. The dependence of log k′ on the proportions of two modifiers was determined for the solutes within the concentration range of ternary systems obtained by mixing two binary eluents: methanol + water and tetrahydrofuran + water or acetonitrile + water and tetrahydrofuran + water. The nature of the relationships is explained in terms of molecular shape, molecular interactions between solute and extracted modifier and the ordering of solvated ligands on the stationary phase.     

Optimization of chromatographic systems for the high-performance thin-layer chromatography of flavonoids

Summary To characterize the retention and selectivity of separations of 23 flavonoids (aglycones and glycosides) relationships betweenR _F and modifier concentration were determined for silica and diol adsorbents (with mixtures of ethyl acetate and methanol as mobile phases), for cyanopropyl silica (with mixtures of ethyl acetate and dichloromethane as mobile phases), for aminopropyl silica (with mixtures of ethyl acetate, methanol and water as mobile phases) and for octadecyl silica (with mixtures of methanol and water as mobile phases). Owing to large polarity differences between aglycones and glycosides, these groups of compounds cannot be separated other than by use of reversed-phase systems, for which the selectivity is lower. It follows from correlation plots ofR _F1 againstR _F2 that for some pairs of adsorbents (e. g. silica and diol) selectivity differences are small; for others the points in the plot are widely dispersed, indicating selectivity differences. The chemometric database obtained can be used to choose optimum chromatographic systems for the separation of given sets of flavonoids and for planning gradient elution programs for separation of flavonoid aglycones and glycosides in a single TLC experiment.     

Modeling the retention mechanism for high‐performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu²⁺(l ‐phenylalanine)₂ using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu²⁺(l ‐phenylalanine)₂ complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed‐phase column with an optimized mobile phase composed of 6 mmol/L of l ‐phenylalanine or N,N‐dimethyl‐l ‐phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.     

Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

Orthogonal array design experiments for optimizing the separation of nine pesticides by micellar electrokinetic chromatography

Separation in micellar electrokinetic chromatography is influenced by many factors. The separation of 9 pesticides was optimized using orthogonal array designs (OADs), five relevant factors were investigated: type and concentration of surfactant, concentration of buffer, pH and concentration of urea modifier. In the first experiment, five factors were examined at two levels using an OA₁₆(2¹⁵) matrix, by which the effect of each factor was estimated using individual contributions as response function, based on the results of the first experiment, three more important factors were chosen for further optimization using OA₉(3⁴) matrix to locate the exact levels for each variable. Finally, 9 pesticides achieved baseline-separation within 15 min.     

An empirical retention model for the optimization of on-line normal-phase LC-GC for the multi-analyte determination of hydrophobic compounds in fatty samples

Summary Normal-phase LC (NPLC) is a powerful method for the clean-up of fatty samples in the determination of organochlorine pesticides (OCPs). The injected sample deactivates the stationary phase and the triglyceride matrix therefore serves as a polarity modifier in the NPLC separation. Thus, the amount of sample injected is the key to both selectivity and sensitivity in matrixmodified LC coupled to capillary GC. In coupled LC-GC the NPLC separation becomes particularly critical because only a limited amount of the LC eluent can be transferred to the GC and the triglyceride matrix must be prevented from entering the GC, because it degrades the performance of the injector and the column. In previous applications method development was seriously hampered by these boundary conditions and tedious and lengthy trial-and-error experiments were required to determine suitable experimental conditions. In this study an empirical model was developed that describes the NPLC separation process in terms of column dimensions and fat loadability. The output is given as the probability of achieving successful LC-GC analysis of a particular set of analytes, thus furnishing a useful tool for the development of new applications in the field of exposure assessment and analysis of residues of apolar compounds in fatty samples. The limitations of current procedures—maximum transfer volumes and minimal separation—are also discussed.     

A new gas chromatographic retention index for pesticides and related compounds

A new gas chromatographic (GC) retention index based on a homologous series of tri-n-alkylamines is proposed for use in the detection of pesticides and related compounds because the standard n-paraffin hydrocarbons used for the Kovats index do not show up well on the nitrogen-phosphorus detectors commonly used in pesticide analysis. Using fused silica bonded phase capillary columns (DB-1 or DB-5), the trialkylamine indices of 106 selected pesticides and related compounds were measured and their relationship to the Kovats index determined.     

Chemometric characterization of TLC systems of the type silica-binary non-aqueous mobile phase in the analysis of flavonoids

Summary Retention-mobile phase composition relationships have been determined for eighteen flavonoids chromatographed with non-aqueous systems of the type silicadiluent+polar modifier (S). The slopes of the plots depended on the molecular structures of the solutes, primarily on the number of hydroxyl groups. The selectivities and sequences depended on the diluent and modifier applied; plots ofR _M against log [S] (where [S] is the percentage concentration of S) were approximately linear, although deviations from linearity were observed occasionally. The plots enable selection of mobile phases of optimum composition for qualitative and quantitative analysis of given sets of flavonoids.     

Combined column–mobile phase mixture statistical design optimization of high-performance liquid chromatographic analysis of multicomponent systems

A statistical approach for the simultaneous optimization of the mobile and stationary phases used in reversed-phase liquid chromatography is presented. Mixture designs using aqueous mixtures of acetonitrile (ACN), methanol (MeOH) and tetrahydrofuran (THF) organic modifiers were performed simultaneously with column type optimization, according to a split-plot design, to achieve the best separation of compounds in two sample sets: one containing 10 neutral compounds with similar retention factors and another containing 11 pesticides. Combined models were obtained by multiplying a linear model for column type, C8 or C18, by quadratic or special cubic mixture models. Instead of using an objective response function, combined models were built for elementary chromatographic criteria (retention factors, resolution and relative retention) of each solute or pair of solutes and, after their validation, the global separation was accomplished by means of Derringer's desirability functions. For neutral compounds a 37:12:8:43 (v/v/v/v) percentage mixture of ACN:MeOH:THF:H₂O with the C18 column and for pesticides a 15:15:70 (v/v/v) ACN:THF:H₂O mixture with the C8 column provide excellent resolution of all peaks.