Octadecylsilica Stationary Phase Research Articles

Thermodynamic Insights into the Separation of Carotenoids in Reversed-Phase Liquid Chromatography.

The retention mechanism of four major carotenoids, two xanthophylls (i.e., lutein and zeaxanthin) and two carotenes (i.e., lycopene and β-carotene), was investigated in reversed-phase liquid chromatography with the aim of thermodynamic analysis. The experimental variables considered in this study were the composition of mobile phase (MP) and the temperature. Chromatographic elutions were undertaken under linear, isocratic conditions by using a C18 stationary phase, four different MP compositions (by varying the ratio methanol/acetonitrile from 66.5/28.5 to 47.5/47.5 v/v), and column temperatures in the range 283–313 K. Traditional Van't Hoff analysis has been used to estimate changes of standard enthalpy (ΔH°) and Gibbs free energy (ΔG°) associated with the solute transfer from the mobile to the stationary phase at each mobile phase composition. The thermodynamic quantities have been correlated to the structure of investigated carotenoids and their interaction with the octadecyl silica stationary phase.

Characterization of polyaniline-coated stationary phases by using the linear solvation energy relationship in the hydrophilic interaction liquid chromatography mode using capillary liquid chromatography.

A polyaniline coating was used to modify the surface of bare silica gel and octadecyl silica stationary phases to characterize the properties of altered materials. It was assumed that the mixed-mode retention was established on the basis of the polyaniline chemical structure and its combination with the original sorbents. Polyaniline was deposited onto the original surfaces during the chemical polymerization of aniline hydrochloride. The prepared materials were slurry packed into capillary columns and systematic chromatographic characterization was performed using the linear solvation energy relationship, also employing descriptors that allow inclusion of ionic interactions in the proposed retention mechanism. The retention times of 80 solutes with various chemical structures were measured in the hydrophilic interaction liquid chromatography mode. The obtained results demonstrated the significant contribution of the polyaniline coating to the retention mechanism under the given conditions; the assumed mixed-mode retention was confirmed. The dominant retention interaction for both modified stationary phases was based on the protonation of nitrogen atoms in the polyaniline structure, leading to suitable retention and selectivity for the hydrophilic analytes, especially anionic and zwitterionic species. Thus, especially, the polyaniline-coated bare silica gel sorbent seems to be promising for potential applications related to the separation of polar compounds.

Multivariate assessment of lipophilicity scales—computational and reversed phase thin-layer chromatographic indices

Needs for fast, yet reliable means of assessing the lipophilicities of diverse compounds resulted in the development of various in silico and chromatographic approaches that are faster, cheaper, and greener compared to the traditional shake-flask method. However, at present no accepted “standard” approach exists for their comparison and selection of the most appropriate one(s). This is of utmost importance when it comes to the development of new lipophilicity indices, or the assessment of the lipophilicity of newly synthesized compounds. In this study, 50 well-known, diverse compounds of significant pharmaceutical and environmental importance have been selected and examined. Octanol-water partition coefficients have been measured with the shake-flask method for most of them. Their retentions have been studied in typical reversed thin-layer chromatographic systems, involving the most frequently employed stationary phases (octadecyl- and cyano-modified silica), and acetonitrile and methanol as mobile phase constituents. Twelve computationally estimated logP-s and twenty chromatographic indices together with the shake-flask octanol-water partition coefficient have been investigated with classical chemometric approaches—such as principal component analysis (PCA), hierarchical cluster analysis (HCA), Pearson’s and Spearman’s correlation matrices, as well as novel non-parametric methods: sum of ranking differences (SRD) and generalized pairwise correlation method (GPCM). Novel SRD and GPCM methods have been introduced based on the Comparisons with One VAriable (lipophilicity metric) at a Time (COVAT). For the visualization of COVAT results, a heatmap format was introduced. Analysis of variance (ANOVA) was applied to reveal the dominant factors between computational logPs and various chromatographic measures. In consensus-based comparisons, the shake-flask method performed the best, closely followed by computational estimates, while the chromatographic estimates often overlap with in silico assessments, mostly with methods involving octadecyl-modified silica stationary phases. The ones that employ cyano-modified silica perform generally worse. The introduction of alternative coloring schemes for the covariance matrices and SRD/GPCM heatmaps enables the discovery of intrinsic relationships among lipophilicity scales and the selection of best/worst measures. Closest to the recommended logKOW values are ClogP and the first principal component scores obtained on octadecyl-silica stationary phase in combination with methanol-water mobile phase, while the usage of slopes derived from Soczewinski-Matyisik equation should be avoided.

Lipophilicity Assessment of Ruthenium(II)-Arene Complexes by the Means of Reversed-Phase Thin-Layer Chromatography and DFT Calculations

The lipophilicity of ten ruthenium(II)-arene complexes was assessed by reversed-phase thin-layer chromatography (RP-TLC) on octadecyl silica stationary phase. The binary solvent systems composed of water and acetonitrile were used as mobile phase in order to determine chromatographic descriptors for lipophilicity estimation. Octanol-water partition coefficient, logK OW, of tested complexes was experimentally determined using twenty-eight standard solutes which were analyzed under the same chromatographic conditions as target substances. In addition, ab initio density functional theory (DFT) computational approach was employed to calculate logK OW values from the differences in Gibbs' free solvation energies of the solute transfer from n-octanol to water. A good overall agreement between DFT calculated and experimentally determined logK OW values was established (R 2 = 0.8024–0.9658).

Molecular Shape Selectivity for Polycyclic Aromatic Compounds on a Core–Shell Octadecylsilica Stationary Phase at Subambient Column Temperatures

Molecular shape selectivity for polycyclic aromatic compounds on a core–shell-type octadecylsilica (ODS) phase at subambient column temperatures was studied in reversed-phase liquid chromatography. The plate height on the core–shell ODS column was relatively stable at subambient column temperatures when compared with that of a conventional ODS column. In order to compare the sample diffusivities in the conventional and core–shell ODS columns, van Deemter plots were prepared. The plate height of the core–shell column was significantly lower than that of conventional column, suggesting an advantageous feature of the core–shell-type stationary phase especially at a high flowrate of the mobile phase. An enhanced molecular shape recognition capability of the core–shell ODS phase was also confirmed at subambient column temperature. The result could be consistent with an improved shape selectivity as normally observed on conventional ODS phases at low temperatures, however, the enhanced molecular shape recognition capability of the core–shell phase enables a good separation between benz[a]anthracene and chrysene at subambient column temperatures. Similar improved shape selectivities for terphenyl isomers were also confirmed on the core–shell phase.

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse-phase liquid chromatography at different methanol-water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol-water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.

Synthesis, structure elucidation, determination of the lipophilicity and identification of antitumour activities in vitro of novel 3-(2-furanyl)-8-aryl-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones with a low cytotoxicity towards normal human skin fibroblast cells

Eleven novel 3-(2-furanyl)-8-aryl-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones (12–22) were designed and obtained from appropriate 1-aryl-2-hydrazonoimidazolidines (1–11) by condensation reaction with 2-oxo-2-furanacetic acid and subsequent cyclocondensation of intermediate chain derivatives. IR, 1H NMR and 13C NMR spectra and elemental analyses confirmed the chemical structure of all the synthesized compounds. The reversed-phase HPLC method was optimized and proved to be applicable and reliable for the analysis of these unknown small molecules (12–22). These compounds were chromatographed on octadecyl silica (ODS) stationary phase and their hydrophobic parameters expressed as the logkw values were determined by RP-HPLC, using mixtures of methanol and water as mobile phases with different methanol concentrations. Octane-1-sulfonic acid sodium salt (OSA-Na) and 20% acetate buffer (pH 3.5) was added to the mobile phase (eluent containing 0.01M/L OSA-Na in organic modifier (MeOH)—buffered mobile phase). The high values of regression coefficients (r >0.9841) for all the compounds investigated proved the excellent fit between experimental data and the Snyder–Soczewiński equation. Results obtained from the reversed-phase HPLC were compared both with those theoretically calculated and with those obtained from an ALOGPS 2.1. software by the use of nine different computational methods for estimation of logP. The predicted values of logP by use of AB logP algorithm revealed the best correlation with the experimental logkw values for the investigated solutes, since a good correlation (r=0.7760) between these quantities was found. The majority of novel imidazotriazinones were found to be evidently effective in vitro against human cancerous cells (HeLa and T47D) in an effective concentration of 50μg/mL. Five compounds (13, 15, 16, 18 and 22) revealed remarkable antiproliferative activities and selective cytotoxicities for cancer cells over normal HSF cells. Therefore these ones may be considered as a basis for the design of novel useful non-toxic (13, 15 and 16) and low toxic (18 and 22) anticancer agents.

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.

COMPARISON OF THEORETICAL METHODS FOR EXTRACTING RETENTION FACTORS AND RATE CONSTANTS IN LIQUID CHROMATOGRAPHY

In this research, experimental data are used to evaluate theoretical methods that provide detailed information concerning mass transfer processes in liquid chromatography. To this end, a well-characterized homologous series of fatty acids is separated on a polymeric octadecyl silica stationary phase with a methanol mobile phase. These solutes are detected via laser-induced fluorescence to generate characteristic zone profiles as a function of column length and linear velocity. Three theoretical methods are then applied to extract the thermodynamic and kinetic information from the zone profiles. Each method relies on evaluating the shape of the zone profiles. The statistical moments of the profiles are used to calculate the mean and variance, which are related to the retention factor and rate constant, respectively. The profiles are also fit to the exponentially modified Gaussian equation and the Thomas equation. The fitting parameters from these equations are then used to calculate the retention factors and rate constants. These methods are compared and their inherent advantages and limitations are discussed.

Principal Component Analysis of HPLC Retention Data and Molecular Modeling Structural Parameters of Cardiovascular System Drugs in View of Their Pharmacological Activity

Evaluation of relationships between molecular modeling structural parameters and high-performance liquid chromatography (HPLC) retention data of 11 cardiovascular system drugs by principal component analysis (PCA) in relation to their pharmacological activity was performed. The six retention data parameters were determined on three different HPLC columns (Nucleosil C18 AB with octadecylsilica stationary phase, IAM PC C10/C3 with chemically bounded phosphatidylcholine, and Nucleosil 100-5 OH with chemically bounded propanodiole), and using isocratically acetonitrile: Britton-Robinson buffer as the mobile phase. Additionally, molecular modeling studies were performed with the use of HyperChem software and MM+ molecular mechanics with the semi-empirical AM1 method deriving 20 structural descriptors. Factor analysis obtained with the use of various sets of parameters: structural parameters, HPLC retention data, and all 26 considered parameters, led to the extraction of two main factors. The first principal component (factor 1) accounted for 44–57% of the variance in the data. The second principal component (factor 2) explained 29–33% of data variance. Moreover, the total data variance explained by the first two factors was at the level of 73–90%. More importantly, the PCA analysis of the HPLC retention data and structural parameters allows the segregation of circulatory system drugs according to their pharmacological (cardiovascular) properties as shown by the distribution of the individual drugs on the plane determined by the two principal components (factors 1 and 2).

Time-dependent Changes in Concentration of Two Clinically used Acetylcholinesterase Reactivators (HI-6 and Obidoxime) in Rat Plasma Determined by HPLC Techniques after in vivo Administration

A simple and reliable HPLC method for determination of rat plasma levels of clinically used acetylcholinesterase (AChE) reactivators (HI-6 and obidoxime) is presented in our study. Separation was carried out by HPLC using an octadecyl silica stationary phase and a mobile phase consisting of 24% acetonitrile and containing 5 mM sodium octanesulfonate and 5 mM tetramethylammonium chloride (pH 2.3). Following intramuscular administration of equimolar doses of both oximes (22.23 mg/kg), the maximum of HI-6 concentration in rat plasma was reached in about 20 min giving 15.26 +/- 1.71 microg/mL. The distribution of obidoxime was fast; the single maximum 23.62 +/- 3.563 microg/mL was recorded at about 10 min. HPLC with UV detection presented in our study is a general method which could be applied for quick measurements of bisquaternary AChE reactivators in rat plasma.

Chemometric characterization of (chromatographic) lipophilicity parameters of newly synthesized s‐triazine derivatives

AbstractA (chromatographic) lipophilicity parameters of newly synthesized 2,4‐bis(substituted)‐6‐chloro‐s‐triazines were studied. PCA‐HCA were performed in order to classify and organize various chromatographic lipophilicity parameters that were determined by using different stationary and mobile phases and by various chromatographic techniques. The PCA‐HCA indicates that for the set of investigated compounds, similar lipophilicity scales are given by chromatographic systems with covalently bonded hydrocarbons as a stationary phase and acetonitrile as a mobile phase modifier. Another cluster of lipophilicity scales forms cyano and octadecylsilica stationary phases with dioxane or tetrahydrofuran as a mobile phase modifiers. The general arrangement of investigated compounds along PC1 was according to the increase in lipophilicity forming two clusters (one of lower and the other of higher lipophilicity). PCA study of RM values underline the differences concerning solvent fraction. Also reveal that HPTLC systems on amino plates are distinguishable from the cyano and octadecylsilica supports, which were similar. Copyright © 2008 John Wiley & Sons, Ltd.

Modeling transport effects of perfluorinated and hydrocarbon surfactants in groundwater by using micellar liquid chromatography

The effects of hydrocarbon and perfluorinated surfactants, above their critical micelle concentration (CMC), on the transport of neutral environmental pollutants are compared. Reversed-phase micellar liquid chromatography is used to model the groundwater system. The octadecylsilica stationary phase serves to simulate soil particles containing organic matter, whereas the aqueous surfactant mobile phases serve to simulate groundwater containing a surfactant at varying concentrations. Sodium dodecyl sulfate and lithium perfluorooctane sulfonate are used as representatives of the hydrocarbon and perfluorinated surfactants, respectively. Benzene, mono- and perhalogenated benzenes, and polycyclic aromatic hydrocarbons are used as models for environmental pollutants. Transport effects were elucidated from the retention factor, k, and the equilibrium constant per micelle, K eq, of the model pollutants in the individual surfactants. Based on k values, the transport of the model pollutants increased in both surfactant solutions in comparison to pure water. As the concentration of the surfactants increased, the transport of the pollutants increased as well. Notably, the K eq values of the pollutants in the perfluorinated surfactant were at least an order of magnitude less than those in the hydrocarbon surfactant. Overall, these results suggest that the presence of a perfluorinated surfactant, above its CMC, increases the transport of pollutants in a groundwater system. However, the perfluorinated surfactant exhibits a lesser transport effect than the hydrocarbon surfactant.

Measurement of K-27, an oxime-type cholinesterase reactivator by high-performance liquid chromatography with electrochemical detection from different biological samples

K-27 is a bisquaternary asymmetric pyridinium aldoxime-type cholinesterase reactivator of use in the treatment of poisoning with organophosphorous esterase inhibitors. A sensitive, simple and reliable reverse-phase high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of K-27 concentrations in rat brain, cerebrospinal fluid, serum and urine samples. Male Wistar rats were treated intramuscularly with K-27 and the samples were collected 60 min later. Separation was carried out on an octadecyl silica stationary phase and a disodium phosphate solution (pH 3.7) containing citric acid, octane sulphonic acid and acetonitrile served as mobile phase. Measurements were carried out at 30 °C at E ox 0.65 V. The calibration curve was linear through the range of 10–250 ng/mL. Accuracy, precision and the limit of detection calculated were satisfactory according to internationally accepted criteria. Limit of quantitation was 10 ng/mL. The method developed is reliable and sensitive enough for monitoring K-27 levels from different biological samples including as little as 10 μL of cerebrospinal fluid. The method – with slight modification in the composition of the mobile phase – can be used to measure a wide range of other related pyridinium aldoxime-type cholinesterase reactivators.

Determination of a potent non-competitive AMPA receptor antagonist in rat brain

N-acetyl-1-( p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic–lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 °C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 μg/ml for all studied compounds ( n = 6). In the within-day assay ( n = 4), the accuracy ranged from 87.5% determined with 0.05 μg/ml of PS3 to 110.1% determined with 0.2 μg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 μg/ml of PS3 to 9.7 determined with 0.2 μg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 μg/ml to 94.3 for PS3Ac at 0.5 μg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.

Chromatographic Behavior of C60 and C70 Fullerenes at Subambient Temperature with n-Alkanes Mobile Phases

The influence of temperature on the retention and separation of C60 and C70 fullerenes was studied under HPLC conditions. Particularly, chromatographic experiments were conducted using moderate carbon loaded octadecylsilica stationary phase and homologous series of n-alkanes including n-pentane, n-hexane and n-heptane as the mobile phases. All studies were performed across wide range of subambient temperature from −80 to +20 °C. From practical point of view the best chromatographic conditions for baseline separation of the components of interest were selected. The retention of analytes was strongly affected by temperature and below minus 30 °C strong deviation from van't Hoff behavior was observed. To explore this phenomenon selected thermodynamic parameters including changes of enthalpy (ΔH o ) and changes of entropy (ΔS o ) were estimated. Positive values of the ΔH o and ΔS o at low temperature region may indicate the lack of the interaction with the stationary phase ligands. A possible retention mechanism at different temperatures for C60 and C70 molecules has been discussed.

HPLC and HPLC-MS Analysis of Urinary N -Monomethyl-Lysine

Administration of (14)C-labelled L-deprenyl to rats results in the urinary elimination of a 14C-labelled compound. The 9-fluorenylmethoxycarbonyl chloride-reacted urine sample is fractionated by high-performance liquid chromatography (HPLC) on an octadecyl silica stationary phase. N(epsilon)-Monomethyl-lysine is identified in the fraction containing the majority of the radioactivity. Structural elucidation is carried out using HPLC-mass spectrometry in atmospheric pressure chemical ionization mode. Identification of the 14C-labelled fragment in Ne-monomethyl-lysine is an experimental proof that an N-methylated amino acid is generated by transmethylation from a well-known drug. This type of transmethylation may have basic importance in the positive side effects of certain drugs.

Determination of ochratoxin A in Hungarian wines

Determination of ochratoxin A (OA) in musts and wines is described using high performance liquid chromatography with fluorescence detection (HPLC-FL). The applicability of common solid phase extraction cartridges with octadecyl-silica stationary phase (C 18-SPE) instead of immunoaffinity columns (IAC) for sample clean-up and preconcentration of OA is discussed. Utilizing 100-fold concentration, the detection limit and quantification limit of OA in wine were 0.024 and 0.125 ng/ml, respectively. Mean recovery amounted to 83.5%. Confirmation of the presence of OA was performed by quantitative conversion of OA into its methyl ester with BF 3/methanol. The method was successfully applied to routine analysis of 87 Hungarian must and wine samples. The method proved to be robust during the 2 weeks of routine measurements resulting in a mean recovery of 99.4% (std. dev. 2.4%, n=11) related to the first measurements on the control sample. OA was not detectable in the Hungarian wine and must samples.

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.

Development and validation of a high-performance liquid chromatographic method for the determination of cyproterone acetate in human skin

In the framework of a preliminary study on the transdermal penetration of cyproterone acetate (CPA), a simple and rapid procedure involving an extraction step coupled to a HPLC-UV determination has been developed for the separation and quantification of CPA in the two main skin layers-epidermis and dermis-after local application. The separation of epidermis and dermis layers was carefully carried out by means of a sharp spatula after skin immersion in heated water at 65 °C. The two skin layers were then treated separately according to the same process: (1) sample homogenization by vibration after freezing with liquid nitrogen in a Mikro-Dismembrator ®; (2) CPA extraction with methanol after addition of the internal standard (betamethasone dipropionate); (3) centrifugation; (4) evaporation of a supernatant aliquot; (5) dissolution of the dry residue in methanol and addition of water; (6) centrifugation; (7) injection of a supernatant aliquot into the HPLC system. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of acetonitrile and water (40:60 (v/v)). The method was then validated using a new approach based on accuracy profiles over a CPA concentration range from 33 to 667 ng/ml for each skin layer. Finally, the method was successfully applied to the determination of CPA to several skin samples after topical application of different gel formulations containing CPA.