Retention In Reversed-phase Liquid Chromatography Research Articles

Dependence of reversed-phase liquid chromatographic retention for aripiprazole and some related chemical impurities on the main elution parameters

ABSTRACTThe present study focuses on the chromatographic behavior of a novel atypical antipsychotic drug, aripiprazole, and its five chemical related impurities. The experiments were performed on a Zorbax SB-C18 column (150 mm × 4.6 mm, 5 µm particle size), whereas the detection was provided by an UV-Vis DAD detector set at 254 nm. The influences of the organic modifier, column temperature, and the pH of the mobile phase on the retention of the six compounds were studied. From these dependences some molecular descriptors and thermodynamic parameters that are important in drug characterization were evaluated. Also, the effect of two different acids added to the mobile phase (phosphoric acid and trifluoroacetic acid) on the retention behavior for chosen compounds was studied.

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds.

Organic acids with very low pKa require extremely low pH conditions to achieve adequate retention in reversed-phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed-phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core-shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.

Qualitative and quantitative characterization of monomers of uncured bulk-fill and conventional resin-composites using liquid chromatography/mass spectrometry

ObjectivesThe aim of this study was to assess the resin matrix monomer composition of selected bulk-fill and conventional resin-composite materials using reverse phase liquid chromatography coupled with electron spray ionization mass spectrometry. Material and methodsSix bulk-fill (SureFil SDR, Venus Bulk Fill, X-tra base, Filtek Bulk Fill flowable, Sonic Fill, and Tetric EvoCeram Bulk Fill) and eight conventional resin-composites (Grandioso Flow, Venus Diamond Flow, X-Flow, Filtek Supreme XTE, Grandioso, Venus Diamond, TPH Spectrum, and Filtek Z250) were tested. For assessment of resin composition and relative monomer amounts, uncured resin-composites were analysed with reverse phase liquid chromatography/electron spray ionization mass spectrometry. logP values (a measure of hydrophobicity) of detected compounds were calculated and their correlation to reverse phase liquid chromatography retention time was explored. Data were analysed with one-way ANOVA, Tukey post hoc test, Pearson correlation and regression analyses at α=0.05. ResultsThe main monomers detected were BisGMA, UDMA, TEGDMA, and BisEMA. Monomers were detected at variable combinations in different materials with significantly different relative amounts. Other monomers were detected including HDDMA, DEGDMA, TCD-DI-HEA, and SDR-UDMA in Grandioso flow, X-flow, Venus Diamond, and SureFil SDR respectively. A positive correlation between logP and reverse phase liquid chromatography retention time was detected (r2=0.62, p=0.004). ConclusionsResin composition of bulk-fill resin-composites is comparable to that of conventional materials with the exception of SureFil SDR. The relative hydrophobicity of dental monomers can be determined by their reverse phase liquid chromatography retention time.

Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography

Two tricationic ionic liquids were prepared and then bonded onto the surface of supporting silica materials through “thiol-ene” click chemistry as new stationary phases for high-performance liquid chromatography. The obtained columns of tricationic ionic liquids were evaluated respectively in the reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode, and possess ideal column efficiency of 80,000 plates/m in the RPLC mode with naphthalene as the test solute. The tricationic ionic liquid stationary phases exhibit good hydrophobic and shape selectivity to hydrophobic compounds, and RPLC retention behavior with multiple interactions. In the HILIC mode, the retention and selectivity were evaluated through the efficient separation of nucleosides and bases as well as flavonoids, and the typical HILIC retention behavior was demonstrated by investigating retention changes of hydrophilic solutes with water volume fraction in mobile phase. The results show that the tricationic ionic liquid columns possess great prospect for applications in analysis of hydrophobic and hydrophilic samples.

Separation and identification of oligomeric phenylethoxysiloxanols by liquid chromatography-electrospray ionization mass spectrometry

Liquid chromatography-electrospray ionization mass spectrometry has been applied to qualitative analysis of oligomeric phenylethoxysiloxanols, a class of organosilanols as active intermediates to polyhedral silsesquioxanes. The phenylethoxysiloxanol samples were prepared by controlled acid-catalyzed hydrolysis and condensation of phenyltriethoxysilane at various molar equivalents of water (r1) and characterized by standard spectroscopic techniques. Using a gradient binary water–methanol mobile phase, these reaction products were resolved on octadecylsiloxane silica stationary phase and subsequently identified by online electrospray ionization mass spectrometric detection. Results show that the reaction products are composed of a multitude of linear and monocyclic siloxanol oligomers with various numbers of silicon atoms and hydroxyl groups, depending upon the reaction conditions used. With the r1 value increasing from 0.5 to 2.0, the chain lengths of the oligomers increase slightly but the numbers of hydroxyl groups increase considerably, accompanying by structural evolution from chains to rings. Characterization of the retention behavior of these oligomers indicates that hydrophobic interactions of phenyl and ethoxy groups with the stationary phase are responsible for their retention in reversed-phase liquid chromatography.

Artificial neural network prediction of multilinear gradient retention in reversed-phase HPLC: comprehensive QSRR-based models combining categorical or structural solute descriptors and gradient profile parameters.

A multilayer artificial neural network (ANN) is used to model the reversed-phase liquid chromatography retention times of 16 selected compounds, including purines, pyrimidines and nucleosides. The analysed data, taken from literature, were collected in acetonitrile-water eluents under the application of 16 different multilinear gradients. The parameters describing the gradient profile together with solute descriptors are considered as the independent variables of an ANN-based model providing the retention time as response. Categorical variables or, alternatively, a selected set of molecular descriptors of computational origin are adopted to represent the solutes. Network training, validation and testing are performed preliminarily using data of 12, 2 and 4 gradients, respectively and successively, to investigate model performance under more severe calibration conditions, with data of 9, 2 and 7 gradients. The proposed approach allows a quite accurate prediction of retention times of the target analytes in external multilinear gradients. Categorical variables can successfully represent the target solutes when the model is called to transfer retention data from calibration to external gradients. In particular, using a five-dimensional bit string to represent the analytes, mean errors on retention times are 2 and 3% under the most and less favourable calibration conditions, respectively. A comparable performance is observed if the categorical variables are replaced by five molecular descriptors, selected by a genetic algorithm within a large set of structural variables of computational origin.

Prediction of Reversed-Phase Liquid Chromatography Retention Parameters for Phenylisothiocyanate Derivatives of Amino Acids

Abstract We report our experience with highly polar and charged analyte retention parameter prediction for a reversed-phase high-performance liquid chromatographic method. The solvatic retention model has been used to predict retention of phenylisothiocyanate derivatives of 25 natural amino acids under gradient elution conditions. Retention factors have been calculated from molecular parameters of analyte structures and from the column and eluent characteristics. A step-by-step method which includes the first guess prediction of initial conditions from structural formula and fine tuning of the retention model parameters using data from successive runs can substantially save method development time

RELATIONSHIP BETWEEN HYDROPHOBICITY AND RPLC RETENTION BEHAVIOR OF AMPHOTERIC COMPOUNDS

In this paper, we systematically investigate the regulation of retention behavior of amphoteric compounds, amino acids, and small peptides in the whole pH range in reversed-phase liquid chromatography (RPLC) using methanol-ammonium acetate buffer solution as a binary mobile phase. According to Snyder-Soczewinski equation, logarithm of hypothetical retention factor corresponding to neat aqueous fraction of hydroorganic mobile phase (logk w) is obtained by extrapolation from different compositions of the mobile phase. Then, we establish the relationship between octanol-water partition coefficient (P) and k w of these amphoteric compounds and discuss the dependence of retention behavior on mobile phase pH. The experimental results reveal that the linear correlation between logarithm of apparent octanol-water coefficient (logD) and logk w is better than that between logP and logk w, as well as that between logD and logk, the logarithm of retention factor at a specific organic modifier fraction for common amphoteric compounds. The linear relationships of logD–logk w approximate those of logP–logk w, and they are superior to those of logD–logk for amino acids and small peptides. In addition, we provide an insight into the fact that isoelectric point (pI), dissociation constants (p K a1 and p K a2 ) of amphoteric compounds affect their RPLC retention behavior significantly.

Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (β-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab® software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

The influence of stationary phase on pressure-induced retention, selectivity and resolution changes in RP-LC

This paper reports the influence of a diverse range of stationary phases and differing mobile phase modifiers on pressure-induced retention changes in reversed-phase liquid chromatography (RP-LC). The practical implications of these effects in the Tanaka column characterization using ultra high-performance liquid chromatography (UHPLC) conditions, and implications for HPLC to UHPLC translations in order to increase productivity and resolution are investigated. The stationary and mobile phase combinations responded to a similar degree to elevated pressure; hence, the authors believe that reliable column characterization parameters should be obtainable when UHPLC format columns are evaluated using the Tanaka approach. Analytes exhibited differing pressure-induced retention changes even for only modest increases in pressure (i.e. ΔP(total) 85 bar as shown when one transfers from a 3- to 2-μm particle). The degree of pressure-induced retention changes correlated with the analyte's molar volume and refractivity. The hydrophobicity of the analytes, as measured by logD, only exhibited a weak correlation. Hence, translating a RP-LC methodology from large to smaller particle size material of the same type may result in an increased or decreased selectivity and hence resolution between two analytes depending on their differing response to the pressure-induced retention changes. This potentially has a major impact on LC method development/optimization strategies and LC method translations.

Quantitative in silico Analysis of Organic Modifier Effect on Retention in Reversed-Phase Liquid Chromatography

Retention times in reversed-phase liquid chromatography were quantitatively analyzed in silico using alkanes as standard compounds, much like they have been used for Kovats indices in gas chromatography. The molecular interaction energy was calculated between an analyte and a model hydrophobic phase using a molecular mechanics program. The solvation energy was calculated between an analyte and a model solvent phase. The correlation coefficients between the log k values and the combined molecular interaction and solvation energy values were 0.97 (n = 18) for alkanes, alkanols, alkylbenzenes and polycyclic aromatic hydrocarbons.

An Abnormal Temperature Dependence of Alkylpyrazines’ Retention in Reversed-Phase Liquid Chromatography

Retention behaviors of pyrazine and alkylpyrazines on various stationary phases in reversed-phase liquid chromatography were examined. An abnormal temperature effect on the retention of alkylpyrazines with a mobile phase consisting of acetonitrile and water was observed when changing the column temperature. On the other hand, no similar trend was found with a methanol-water mobile phase. For all the columns investigated in this work, the above tendency to the temperature-dependence was consistently observed, suggesting that the abnormal temperature effect on the retention of alkylpyrazines could be mainly induced by an acetonitrile-based mobile phase.

Simple models for the effect of aliphatic alcohol additives on the retention in reversed-phase liquid chromatography

Four retention models for the effect of aliphatic alcohol additives on the retention of analytes in reversed-phase liquid chromatography have been developed following either a semi-thermodynamic treatment or an empirical approach. Their performance was tested using the experimental retention times of six non-polar analytes (alkylbenzenes) and ten o-phthalaldehyde derivatives of amino acids under different isocratic chromatographic runs when a small amount of ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol was added to methanol/water mixtures containing a constant amount of methanol. It was shown that for the structurally simple alkylbenzenes all the models can be adopted for retention prediction with good results. In contrast, just one out of four models, that with the fewest approximations, predicts satisfactorily the retention properties of amino acids derivatives. However, the most interesting feature is that this model can predict the effect of an alcohol-additive on the retention properties of solutes, even if this additive was not used in chromatographic runs done for the fitting procedure, provided that it belongs to the same homologous series of alkanols. This feature is also observed in all models described the retention of alkylbenzenes.

Mobile phase effects in reversed-phase liquid chromatography: A comparison of acetonitrile/water and methanol/water solvents as studied by molecular simulation

Abstract Molecular simulations of water/acetonitrile and water/methanol mobile phases in contact with a C 18 stationary phase were carried out to examine the molecular-level effects of mobile phase composition on structure and retention in reversed-phase liquid chromatography. The simulations indicate that increases in the fraction of organic modifier increase the amount of solvent penetration into the stationary phase and that this intercalated solvent increases chain alignment. This effect is slightly more apparent for acetonitrile containing solvents. The retention mechanism of alkane solutes showed contributions from both partitioning and adsorption. Despite changes in chain structure and solvation, the molecular mechanism of retention for alkane solutes was not affected by solvent composition. The mechanism of retention for alcohol solutes was primarily adsorption at the interface between the mobile and stationary phase, but there were also contributions from interactions with surface silanols. The interaction between the solute and surface silanols become very important at high concentrations of acetonitrile.

QUANTITATIVE CORRELATIONS BETWEEN REVERSED-PHASE LC DATA AND MOLECULAR PARAMETERS FOR SOME WEAKLY RELATED PESTICIDES

Retention in reversed-phase liquid chromatography for some polar and weakly related pesticides (urea derivatives or containing the thiophosphate group), using two types of stationary phases (C8 and C18), was studied for different compositions of mobile phase in order to estimate extrapolated values of capacity factor for entirely aqueous content of mobile phase. Two organic modifiers were used in composition of mobile phase: methanol or acetonitrile. The inter-correlations between extrapolated values for the two types of stationary phase were very high. The extrapolated values for studied pesticides were correlated by linear and multilinear regressions with molecular parameters, computed by means of different theoretical approaches (PM3 quantum chemical model, and DFT computational scheme). The best correlations were obtained for extrapolated values from retention study using acetonitrile as organic modifier in mobile phase composition.

Investigation of the driving forces for retention in reversed-phase liquid chromatography: Monte Carlo simulations of solute partitioning between n-hexadecane and various aqueous–organic mixtures

With the aim of learning about the solubility characteristics of retentive and mobile phases often encountered in reversed-phase liquid chromatography (RPLC) and to examine the driving forces for retention, configurational-bias Monte Carlo simulations in the Gibbs ensemble were carried out for systems consisting of three phases: an n-hexadecane retentive phase (serving as a model of the hydrophobic RPLC stationary phase), a mobile phase with varying water–acetonitrile or water–methanol composition, and a helium vapor phase (serving as an ideal gas reference state). Gibbs free energies of transfer between each of these phases were computed for a series of small alkane and alcohol solutes and from these the methylene and hydroxyl increments were determined. The results indicate that both nonpolar and polar solutes have favorable interactions with both the mobile and stationary phase thus showing that neither the solvophobic theory nor lipophilic theory alone can explain the driving forces for retention in RPLC. In addition, an analysis of the solvation environments of methylene and hydroxyl groups in the mobile and stationary phases was carried out. The methylene group is shown to be preferentially solvated by the organic component of the aqueous–organic mixture, however, clustering of the organic solvent molecules in the absence of the solute was not observed. For the hydroxyl group, hydrogen bonding was shown to be important in both the mobile and stationary phases.

Prediction of retention in reversed-phase liquid chromatography by means of the polarity parameter model

The polarity parameter model previously developed: log k = (log k) 0 + p( P m N − P s N ) has been successfully applied to study several chromatographic systems involving new generation RPLC columns (Luna C18, Resolve C18, XTerra MSC18, and XTerra RP18). In this model the retention of the solutes (log k) is related to a solute parameter ( p), a mobile phase parameter ( P m N ) and two chromatographic system parameters [ P s N and (log k) 0]. The studied systems have been characterized with different acetonitrile–water and methanol–water mobile phases, using a set of 12 neutral solutes of different chemical nature. The polarity parameter model allows prediction of retention of any solute in any mobile phase composition just using the retention data obtained in one percentage of organic modifier and the polarity parameters established in the characterization of the chromatographic systems. This model also allows the solute polarity data transference between RPLC characterized systems, so it is possible to predict the retention in various RPLC systems working experimentally with just one of them. Moreover, the global solvation parameter model has also been applied to the same chromatographic systems using a wide set of solutes in order to compare its predictive ability with the one of the polarity parameter model. The results clearly show that both models predict retention with very similar accuracy but the polarity parameter model requires much less preliminary experimental measurements to achieve equivalent results than the global solvation approach.

The effects of chain length, embedded polar groups, pressure, and pore shape on structure and retention in reversed-phase liquid chromatography: Molecular-level insights from Monte Carlo simulations

Particle-based simulations using the configurational-bias and Gibbs ensemble Monte Carlo techniques are carried out to probe the effects of various chromatographic parameters on bonded-phase chain conformation, solvent penetration, and retention in reversed-phase liquid chromatography (RPLC). Specifically, we investigate the effects due to the length of the bonded-phase chains (C 18, C 8, and C 1), the inclusion of embedded polar groups (amide and ether) near the base of the bonded-phase chains, the column pressure (1, 400, and 1000 atm), and the pore shape (planar slit pore versus cylindrical pore with a 60 Å diameter). These simulations utilize a bonded-phase coverage of 2.9 μ mol/m 2and a mobile phase containing methanol at a molfraction of 33% (about 50% by volume). The simulations show that chain length, embedded polar groups, and pore shape significantly alter structural and retentive properties of the model RPLC system, whereas the column pressure has a relatively small effect. The simulation results are extensively compared to retention measurements. A molecular view of the RPLC retention mechanism emerges that is more complex than can be inferred from thermodynamic measurements.

Influence of Acetyl and Amide Groups on Peptides RP-LC Retention Behavior

Reversed phase liquid chromatography (RP-LC) is still one of the most efficient analytical techniques for separation of complex biologically active analytes, including proteins and peptides. However, the separation of those analytes is not easy to perform in practice. Therefore, the appropriate optimization of the separation conditions is necessary. The aim of the study was to explore the influence of acetyl and amide groups in peptides on their retention in gradient elution reversed phase LC. Moreover, the investigation regards the influence of temperature and gradient time for such peptides. Those results were suggested in view of their usefulness in predictions of peptides RP-LC retention on the basis of quantitative structure retention relationships for proteomic research.

A robust linear regression based algorithm for automated evaluation of peptide identifications from shotgun proteomics by use of reversed-phase liquid chromatography retention time

BackgroundRejection of false positive peptide matches in database searches of shotgun proteomic experimental data is highly desirable. Several methods have been developed to use the peptide retention time as to refine and improve peptide identifications from database search algorithms. This report describes the implementation of an automated approach to reduce false positives and validate peptide matches.ResultsA robust linear regression based algorithm was developed to automate the evaluation of peptide identifications obtained from shotgun proteomic experiments. The algorithm scores peptides based on their predicted and observed reversed-phase liquid chromatography retention times. The robust algorithm does not require internal or external peptide standards to train or calibrate the linear regression model used for peptide retention time prediction. The algorithm is generic and can be incorporated into any database search program to perform automated evaluation of the candidate peptide matches based on their retention times. It provides a statistical score for each peptide match based on its retention time.ConclusionAnalysis of peptide matches where the retention time score was included resulted in a significant reduction of false positive matches with little effect on the number of true positives. Overall higher sensitivities and specificities were achieved for database searches carried out with MassMatrix, Mascot and X!Tandem after implementation of the retention time based score algorithm.