Compounds In Reversed-phase Liquid Chromatography Research Articles

Fibrous Polyimide Stationary Phase for the Separation of Aromatic Compounds in Reversed-Phase Liquid Chromatography

Fibrous Polyimide Stationary Phase for the Separation of Aromatic Compounds in Reversed-Phase Liquid Chromatography

Preparation and investigation of two-component silica-modified hydrophobic layer for minimizing retention loss of reversed-phase chromatographic columns using fully aqueous mobile phases

Chromatographers often employ fully aqueous mobile phases to retain highly polar compounds in reversed-phase liquid chromatography (RPLC). However, when the flow rate is interrupted, either accidentally or intentionally, a substantial loss in retention occurs due to the spontaneous dewetting of water from the hydrophobic surface of conventional RPLC-C18 particles. Previous studies have shown that maintaining a low C18 surface coverage (approximately 1.5 μmol/m2) can mitigate water dewetting by increasing chain disorder, facilitating the intercalation of water clusters between the C18-bonded chains, and keeping the mesopores wetted. In this research, we explore the potential and additional benefits of using two-component surface bonding materials (C8/C18 and PhenylHexyl (PhHx)/C18) at a constant and low total surface coverage of 1.51 ± 0.15 μmol/m2. We synthesized seven one- and two-component modified silica particles with a volume average particle size of 5.22 μm and an average mesopore size of 104 Å. The surface coverage was increased from 0 to 0.54, 1.00, and to 1.66 μmol2 for C8 chains and from 0 to 0.52, 0.70, and to 1.65 μmol2 for PhHx ligands. To prevent interactions between water and any unreacted silanols, all seven derivatized particles were heavily endcapped with trimethylsilane (TMS) reagent. The fraction of the surface area remaining in contact with water was determined by measuring the retention times of weakly (thiourea) and strongly (thymine) retained compounds at intervals of 1, 2, 4, 8, 16, 32, and 64 minutes following the cessation of flow. Two distinct column temperatures, 24°C and 60°C, were employed in the experiments. Retention losses were found to be minimized in the presence of a small quantity of C8 chains (less than 40% of the total surface coverage). Additionally, it is essential to consider substantial fractions of PhHx chains, as long as the presence of the PhHx ligand does not significantly impact retention and selectivity. Combining mixed RPLC bondings with a low total surface coverage of approximately 1.5 μmol/m2 emerges as a viable solution for further minimizing retention loss in standard C18-bonded RPLC columns, particularly within the surface coverage range of 2.5-3.0 μmol/m2.

Design of β-Alanine-Derived Novel C18 Stationary Phase Containing Embedded Urea and Amide Groups for the Separation of Versatile Analytes.

Novel stationary phases have been emerging recently. A β-alanine-derived embedded urea and amide group-containing C18 phase (Sil-Ala-C18) was prepared for the first time. The media were packed into a 150 × 2.1 mm HPLC column, and the newly designed column was evaluated with the Tanaka and Neue test protocols in reversed-phase liquid chromatography (RPLC) separation mode. Moreover, it was characterized by the Tanaka test protocol in hydrophilic interaction chromatography (HILIC) separation mode. The new phase was characterized by elemental analysis, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and solid-state 13C cross-polarization magic angle spinning (CP/MAS) NMR spectroscopy at variable temperatures. The chromatographic evaluation involved very good separation of nonpolar shape-constrained isomers, polar and basic compounds in RPLC, and highly polar compounds in HILIC compared to the commercial reference columns. The Sil-Ala-C18 phase was able to separate the challenging β- and γ-isomers of tocopherol. The phase was also successfully applied for the separation of the isomers of tocopherol (vitamin E) and capsaicinoids from real samples of chili peppers (Capsicum spp.) in RPLC and ascorbic acid (vitamin C) in HILIC.

Linear free energy relationship models for the retention of partially ionized acid-base compounds in reversed-phase liquid chromatography

The LFER model of Abraham is applied to the retention of the neutral and ionic forms of 94 solutes in a C18 column and 40% v/v acetonitrile/water mobile phase. The results show that polarizability and cavity formation interactions increase retention, whereas dipole and hydrogen bonding interactions favours partition to the mobile phase and thus, they decrease retention. The coefficients of the ionic descriptors measure the effect of the electrostatic interactions and their contribution to partition of the cation or anion between the two mobile and stationary chromatographic phases.A new LFER model for application to the retention of partially dissociated acids and bases is derived averaging the descriptors of the neutral and ionic forms according to their degrees of ionization in the mobile phase. This new LFER model is satisfactorily compared to other literature modified Abraham models for a set of 498 retention data of partially dissociated acids and bases.All tested models require the calculation of the ionization degrees of the compounds at the measuring pH. Calculation of the ionization degrees in the chromatographic mobile phase (i.e. from pH and pKa in the eluent) give good correlations for all tested models. However, estimation of these ionization degrees from pH – pKa data in pure water gives biased estimations of the retention of the partially ionized solutes.

Design of C18 Organic Phases with Multiple Embedded Polar Groups for Ultraversatile Applications with Ultrahigh Selectivity.

For the first time, we synthesized multiple embedded polar groups (EPGs) containing linear C18 organic phases. The new materials were characterized by elemental analysis, IR spectroscopy, (1)H NMR, diffuse reflectance infrared Fourier transform (DRIFT), solid-state (13)C cross-polarization magic angle spinning (CP/MAS) NMR, suspended-state (1)H NMR, and differential scanning calorimetry (DSC). (29)Si CP/MAS NMR was carried out to investigate the degree of cross-linking of the silane and silane functionality of the modified silica. Solid-state (13)C CP/MAS NMR and suspended-state (1)H NMR spectroscopy indicated a higher alkyl chain order for the phase containing four EPGs than for the phase with three EPGs. To correlate the NMR results with temperature-dependent chromatographic studies, standard reference materials (SRM 869b and SRM 1647e), a column selectivity test mixture for liquid chromatography was employed. A single EPG containing the C18 phase was also prepared in a similar manner to be used as a reference column especially for the separation of basic and polar compounds in reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), respectively. Detailed chromatographic characterization of the new phases was performed in terms of their surface coverage, hydrophobic selectivity, shape selectivity, hydrogen bonding capacity, and ion-exchange capacity at pH 2.7 and 7.6 for RPLC as well as their hydrophilicity, the selectivity for hydrophilic-hydrophobic substituents, the selectivity for the region and configurational differences in hydrophilic substituents, the evaluation of electrostatic interactions, and the evaluation of the acidic-basic nature for HILIC-mode separation. Furthermore, peak shapes for the basic analytes propranolol and amitriptyline were studied as a function of the number of EPGs on the C18 phases in the RPLC. The chromatographic performance of multiple EPGs containing C18 HILIC phases is illustrated by the separation of sulfa drugs, β-blockers, xanthines, nucleic acid bases, nucleosides, and water-soluble vitamins. Both of the phases showed the best performance for the separation of shape-constrained isomers, nonpolar, polar, and basic compounds in RPLC- and HILIC-mode separation of sulfa drugs, and other polar and basic analytes compared to the conventional alkyl phases with and without embedded polar groups and HILIC phases. Surprisingly, one phase would be able to serve the performance of three different types of phases with very high selectivity, and we named this phase the "smart phase". Versatile applications with a single column will reduce the column purchasing cost for the analyst as well as achieve high separation, which is challenging with the commercially available columns.

Retention Prediction and Hydrophobicity Measurement of Weakly Basic Compounds in Reversed-phase Liquid Chromatography Using Ammonia and Triethylamine as Ion-suppressors

The purpose of this study is to investigate the retention behavior of weakly ionizable basic compounds in ionsuppression reversed-phase liquid chromatography (IS-RPLC) using ammonia or triethylamine as ion-suppressor in a binary hydroorganic mobile phase. The apparent n-octanol-water partition coefficient (Papp) was proposed to adjust the noctanol- water partition coefficient (Po/w) of weakly basic compound. LogPapp was found to have a better linear correlation with logkw, the logarithm of hypothetical retention factor corresponding to neat aqueous fraction of the mobile phase. This straightforward relationship has been applied to effective prediction of retention behavior of weakly basic compounds, and offered a promising medium for direct determination of Po/w data for these solutes. In addition, the relationship between mobile phase pH and retention behavior of weakly basic compounds was studied in this IS-RPLC mode, and the possible reasons for the biphasic effect of pH on retention have been explained. Keywords: Ion-suppressor, n-Octanol-water partition coefficient (Po/w), retention behavior, reversed-phase liquid chromatography, weakly ionizable basic compound.

Optimization of the Separation and Determination of Nitidine and Chelerythrine in Zanthoxylum nitidum by High-Performance Liquid Chromatography with Fluorescence Detection

A new ion-pair high-performance liquid chromatography method with fluorescence detection was developed for the determination of nitidine and chelerythrine in Zanthoxylum nitidum. To optimize the separation of the two compounds in reversed-phase liquid chromatography, a response surface method (Box-Behnken designs) was used. Three important factors: concentration of ion pair agent, mobile phase composition and buffer pH, were studied for their contribution to the analytes' response, leading to a total of 17 experiments performed on a Kromasil C18 column. The experimental responses were fitted into a second-order polynomial to predict the best conditions. The optimal mobile phase conditions were predicted to be acetonitrile-sodium dodecyl sulphate (17.8 mM, 20 mM citric acid, pH 2.98, 57:43, v/v). The proposed method was validated according to International Conference on Harmonization guidelines and it is suggested to be appropriate for the routine quality control analysis of nitidine and chelerythrine in Zanthoxylum nitidum.

Predicting Retention Times of Naturally Occurring Phenolic Compounds in Reversed-Phase Liquid Chromatography: A Quantitative Structure-Retention Relationship (QSRR) Approach

Quantitative structure-retention relationships (QSRRs) have successfully been developed for naturally occurring phenolic compounds in a reversed-phase liquid chromatographic (RPLC) system. A total of 1519 descriptors were calculated from the optimized structures of the molecules using MOPAC2009 and DRAGON softwares. The data set of 39 molecules was divided into training and external validation sets. For feature selection and mapping we used step-wise multiple linear regression (SMLR), unsupervised forward selection followed by step-wise multiple linear regression (UFS-SMLR) and artificial neural networks (ANN). Stable and robust models with significant predictive abilities in terms of validation statistics were obtained with negation of any chance correlation. ANN models were found better than remaining two approaches. HNar, IDM, Mp, GATS2v, DISP and 3D-MoRSE (signals 22, 28 and 32) descriptors based on van der Waals volume, electronegativity, mass and polarizability, at atomic level, were found to have significant effects on the retention times. The possible implications of these descriptors in RPLC have been discussed. All the models are proven to be quite able to predict the retention times of phenolic compounds and have shown remarkable validation, robustness, stability and predictive performance.

IONIC LIQUIDS AS MOBILE PHASE ADDITIVES IN REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Ionic liquids are compounds of salt structure with melting point below 100°C and even 25°C known as room temperature ionic liquids. They have been widely investigated as new nonmolecular solvents exhibiting a wide range of changing properties, in many fields of analytical chemistry. This work reports on the use of 1-butyl-3-methylimidazolium of varying anion chaotropicity as a mobile phase additive for separation and chromatographic behavior studies of acidic, basic, and amphoteric compounds in reversed-phase liquid chromatography on two hydrophobic columns: Zorbax XDB-C18 and Zorbax SB-Phenyl. Satisfactory separations are achieved by the use of carefully optimized chromatographic systems modified with ionic liquids’ additives. The retention mechanism involving molecular interactions between analytes and additives and partitioning process between mobile phase and interphase created during equilibration process are discussed.

10.2478/s11814-009-0210-y

In reversed-phase liquid chromatography (RP-LC), the separation of ionized compounds at neutral pH of the mobile phase is ineffective because these compounds easily become ionized and too hydrophilic to adsorb on the hydrophobic C18 stationary phase. The common method to separate ionized compounds in RP-LC is the usage of the ion pairing agent and the control of pH in the mobile phase. In this work, [hmim][BF4] ionic liquid was adopted as a mobile phase additive in reversed-phase simulated moving bed chromatography (RP-SMB) to overcome the problem mentioned above. The results showed that when 1.5 ml/l [hmim][BF4] was added to mobile phase, 3-hydroxy benzoic acid (3-HBA) and 4-hydroxy benzoic acid (4-HBA) were separated at neutral pH in the RP-SMB. In particular, purities of 3-HBA and 4-HBA products were 97.62% and 100%, respectively, and had a good agreement with simulation results.

Application of [hmim][BF4] ionic liquid in reversed-phase simulated moving bed for the separation of 3- and 4-hydroxybenzoic acid

In reversed-phase liquid chromatography (RP-LC), the separation of ionized compounds at neutral pH of the mobile phase is ineffective because these compounds easily become ionized and too hydrophilic to adsorb on the hydrophobic C18 stationary phase. The common method to separate ionized compounds in RP-LC is the usage of the ion pairing agent and the control of pH in the mobile phase. In this work, [hmim][BF4] ionic liquid was adopted as a mobile phase additive in reversed-phase simulated moving bed chromatography (RP-SMB) to overcome the problem mentioned above. The results showed that when 1.5 ml/l [hmim][BF4] was added to mobile phase, 3-hydroxy benzoic acid (3-HBA) and 4-hydroxy benzoic acid (4-HBA) were separated at neutral pH in the RP-SMB. In particular, purities of 3-HBA and 4-HBA products were 97.62% and 100%, respectively, and had a good agreement with simulation results.

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes ( ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.

Comparison of the performance of a few packing materials designed to minimize the thermodynamic band tailing of basic compounds in reversed-phase liquid chromatography

The adsorption isotherms of phenol, caffeine, propranolol chloride, and amitriptyline chloride were measured on three new brands of C 18-bonded silica that have been designed to be more resistant than conventional C 18-bonded silica at high pHs ( > 8). These columns were the 4 μ m Bidendate Cogent-C 18 (Microsolv Technology Corporation, Long Branch, NJ, USA), the 3.5 μ m Zorbax Extend-C 18 (Agilent Technologies, Palo Alto, CA, USA), and the 5 μ m XTerra-C 18 (Waters, Milford, MA, USA). The originality of these adsorbents is due to their surface chemistry, which protects them from rapid hydrolysis or dissolution at extreme pH conditions. Their adsorption properties were compared to those of the 3 μ m Luna-C 18 (Phenomenex, Torrance, CA), which is a more conventional monofunctional material. The adsorption data were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) of all systems studied were calculated by the expectation–maximization (EM) method. The experimental results show that neither a simple surface protection (Extend-C 18) nor the elimination of most of the silanol groups (Cogent-C 18) is sufficient to avoid a peak tailing of the basic compounds at pH 8 that is of thermodynamic origin. The incorporation of organic moieties in the silica matrix, which was achieved in XTerra-C 18, the first generation of hybrid methyl/silica material, reduces the silanols activity and is more successful in reducing this peak tailing.

Heterogeneity of the Adsorption Mechanism of Low Molecular Weight Compounds in Reversed-Phase Liquid Chromatography

The retention mechanism in RPLC mode was investigated based on the acquisition of adsorption isotherm data by frontal analysis measurements and their modeling. This work is a review of the results of four years of adsorption data measurements. The data were acquired on a wide variety of brands of C18-silica columns (from Akzo Nobel, Bishoff, Hypersil, Merck, Phenomenex, Supelco, Vydac, and Waters) with several low molecular weight compounds such as phenol (94 g/mol), caffeine (194 g/mol), tryptophan (204 g/mol), sodium 2-naphthalenesulfonate (235 g/mol), and propranololium chloride (295 g/mol). The mobile phase was a mixture of methanol and water at variable composition. The adsorption isotherms were all convex upward (langmuirian), and the degree of heterogeneity of the adsorption system was determined from the calculation of the adsorption energy distribution using the expectation-maximization method. The adsorption isotherm parameters (number of types of adsorption sites, surface concentration of each type of site, and difference between the adsorption energies E(i) - E(j) on sites i and j), obtained from the mathematical fit of the adsorption data to the appropriate multi-Langmuir adsorption isotherm model, were analyzed and compared. The results allow the drawing of general conclusions regarding the relationships between the size of the analyte and the adsorption properties (saturation capacities, adsorption energies) characterizing the retention mechanism in RPLC mode for neutral, anionic, and cationic compounds.

Effect of temperature on the retention of ionizable compounds in reversed-phase liquid chromatography: Application to method development

The analysis of pharmaceutical compounds is often a difficult challenge which requires mathematical tools to improve the quality of the separation method. This work is an attempt to rationalize the anomalous variation of the logarithm of the retention factor with temperature in case of ionizable compounds. The effect of temperature on ionizable compounds was studied within a large range of temperature, ranging from 30 to 130 °C. The determination of the so-called chromatographic p K a and the study of its variation with temperature allow to explain why the forms of the van’t Hoff curves are so different depending on the type of solute, the type of buffer and the type of the mobile phase. A retention model along with a computation procedure is proposed to optimize both temperature and mobile phase composition and to provide good and robust conditions as shown by illustrative examples.

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation-desolvation equilibria in the mobile phase, also known as "chaotropic effect". With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF6, NaClO4, NaBF4, NaH2PO4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF6- > ClO4- approximately BF4- > H2PO4-. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation–desolvation equilibria in the mobile phase, also known as “chaotropic effect”. With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF 6, NaClO 4, NaBF 4, NaH 2PO 4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF 6 − > ClO 4 − ∼ BF 4 − > H 2PO 4 −. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development

Optimizing separation of ionizable compounds in order to find robust conditions has become an important part of method development in liquid chromatography. This work is an attempt to explain the observed variations of retention of acid and basic compounds with the organic modifier content in the mobile phase, according to various factors: the type of modifier, the type of buffer, the temperature and of course the type of solute. This is done by considering the variation of the so-called chromatographic p K a which refers to the pH measured in the aqueous medium and is determined from retention data. A procedure is described that accurately relates, from nine experiments, retention to solvent composition and pH. The limits of such a procedure are evaluated and two examples of optimized separations of basic compounds are given.

Retention of ionizable compounds in reversed-phase liquid chromatography. Effect of the ionic strength of the mobile phase and the nature of the salts used on the overloading behavior.

The retention mechanism of the protonated cation in propranolol chloride on C18-Xterra was investigated using mobile phases of various compositions. Accurate adsorption data were measured by frontal analysis, with a mixture of methanol and water (25% methanol), with no salt, as the mobile phase. The experimental isotherm has at least two inflection points, at concentrations of about 0.2 and 6.0 g/L, respectively. This precludes the modeling of these data with a simple convex-upward isotherm (e.g., Langmuir). The adsorption energy distribution or relationship between the number of sites on the adsorbent surface and the energy of adsorption on these sites was calculated by assuming Moreau isotherm behavior (S-shaped isotherm). This model has never been applied to describe the surface heterogeneity of any RPLC adsorbent. The calculation converged toward a bimodal energy distribution. Accordingly, the bi-Moreau model is the simplest theoretical model accounting for the adsorption data of propranolol from a mobile phase without salt. The complex-overloaded band profiles of propranolol measured in the presence of increasing concentrations of a supporting salt (KCl) in the mobile phase demonstrate that the same isotherm model applies also under these conditions, as was merely assumed in a previous work. The elution band profiles of propranolol calculated with the bi-Moreau isotherm model for solutions of salts of different natures (CaCl2, CsCl, Na2SO4) in the same mobile phase agree very well with the experimental band profiles.