Solutes In Reversed-phase Liquid Chromatography Research Articles

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Proof of Concept To Achieve Infinite Selectivity for the Chromatographic Separation of Therapeutic Proteins.

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients.

Effects of large volume injection of aliphatic alcohols as sample diluents on the retention of low hydrophobic solutes in reversed-phase liquid chromatography

Recent studies showed that injection of large volume of hydrophobic solvents used as sample diluents could be applied in reversed-phase liquid chromatography (RP-LC). This study reports a systematic research focused on the influence of a series of aliphatic alcohols (from methanol to 1-octanol) on the retention process in RP-LC, when large volumes of sample are injected on the column. Several model analytes with low hydrophobic character were studied by RP-LC process, for mobile phases containing methanol or acetonitrile as organic modifiers in different proportions with aqueous component. It was found that starting with 1-butanol, the aliphatic alcohols can be used as sample solvents and they can be injected in high volumes, but they may influence the retention factor and peak shape of the dissolved solutes. The dependence of the retention factor of the studied analytes on the injection volume of these alcohols is linear, with a decrease of its value as the sample volume is increased. The retention process in case of injecting up to 200μL of upper alcohols is dependent also on the content of the organic modifier (methanol or acetonitrile) in mobile phase.

Development of Vancomycin-Capped β-CD-Bonded Silica Particles as Chiral Stationary Phase for LC

Vancomycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (VCD-HPS), a new type of substituted β-cyclodextrin-bonded chiral stationary phase (CSP) for liquid chromatography (LC), have been synthesized by treatment of bromoacetate-substituted-(3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (BACD-HPS) with vancomycin in anhydrous methanol. The stationary phase is characterized by elemental analysis. This new CSP has a chiral selector with two recognition sites: vancomycin and β-cyclodextrin (β-CD), which can provide multiple interactions with the solutes. The chromatographic performance of VCD-HPS was studied with several disubstituted benzenes and some chiral drug compounds as solutes in reversed-phase LC. The results show that VCD-HPS has excellent selectivity for the separation of aromatic positional isomers and enantiomers of chiral compounds.

Influence of bonded-phase coverage in reversed-phase liquid chromatography via molecular simulation: II. Effects on solute retention

Particle-based Monte Carlo simulations were employed to examine the molecular-level effects of bonding density on the retention of alkane and alcohol solutes in reversed-phase liquid chromatography. The simulations utilized octadecylsilane stationary phases with various bonding densities (1.6, 2.3, 2.9, 3.5, and 4.2 μ mol/m 2) in contact with a water/methanol mobile phase. In agreement with experiment, the distribution coefficient for solute transfer from mobile to stationary phase initially increases then reaches a maximum with increasing bonding density. A molecular-level analysis of the solute positional and orientational distributions shows that the stationary phase contains heterogeneous regions and the heterogeneity increases with increasing bonding density.

Evaluation of a Semi-Empirical Approach to Correlation of Retention with Octanol-Water Partition Coefficients by Use of Adjustable Hydrogen-Bonding Indicator Variables

An improved model with adjustable hydrogen-bonding (HB) descriptors is evaluated for correlation of LogP with the retention of neutral and ionizable solutes in reversed-phase liquid chromatography (RPLC). This model is developed by adding adjustable secondary HB variables to the main linear Logk-LogP relationship to improve the quality of the correlation. The HB variables are not fixed, as is usual, but determined by nonlinear least-squares optimization from the retention of reference solutes. Retention times obtained for 56 solutes (neutral, acidic, and basic) by use of aqueous-acetonitrile (ACN) mobile phase gradients at different pH on a Zorbax StableBond C18 column are used to evaluate the method and the capability of the model. It is concluded that the simple model, together with the proposed data-processing scheme, describes the retention of both neutral and ionizable compounds very well. Accordingly, the model will simplify prediction of the retention of complex molecules and assist in chromatographic optimization, including selectivity optimization and internal standard selection. The other potential of the model is for simultaneous determination of both LogP and pKa by RPLC.

Statistical tests for the selection of the optimum parameters set in models describing response surfaces in reversed-phase liquid chromatography

An appropriate procedure based on statistical criteria is suggested for the determination of the optimum set of model parameters for a given chromatographic system. The criteria employed are the t-ratio test, the rate of change in the sum of squares of residuals, the standard error of the fit, the F-test, and the CP-test. The suggested procedure has been evaluated using two different models, one based on partition and the other on adsorption mechanisms, which describe the combined effect of pH and organic modifier content on the retention of ionogenic solutes in reversed-phase liquid chromatography. It is shown that all the criteria give almost converged results and therefore we may simply use the F-test, which seems to be the most sensitive and reliable criterion excluding any personal judgement. It is also found that the retention models tested show a different behavior towards their simplification. In particular, the use of a reduced equation of the partition model, selected on the basis of the suggested procedure, is necessary for the prediction of meaningful retention surfaces, whereas the decrease in the number of the adjustable parameters in the adsorption model offers only noise reduction and fitting simplicity, because no version of this model predicts abnormal retention surfaces.

Column selectivity in reversed-phase liquid chromatography: II. Effect of a change in conditions

The isocratic retention of 67 widely-different solutes in reversed-phase liquid chromatography (RP-LC) has been investigated as a function of temperature and mobile phase composition (% B) for three different C 18 columns. Similar studies were also carried out in a gradient mode, where temperature, gradient time and solvent type were varied. These results show that changes in retention with these conditions are similar for each of these three columns. This suggests that relative column selectivity as defined by experiments for one set of experimental conditions will be approximately applicable for other conditions, with the exception of changes in mobile phase pH—which can affect values of the column parameter C (a measure of silanol ionization). Column selectivity as a function of pH was explored for several columns.

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F 13C 9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C 8) and octadecyl (C 18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C 18 phase, F 13C 9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C 8, C 18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C 18 or C 8 phases and was even disadvantageous on F 13C 9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.

Laterally attached liquid crystalline polymers as stationary phases in reversed-phase high-performance liquid chromatography: II. Optimization of the molecular parameter of the polymer

Stationary phases obtained by coating side-chain liquid crystalline polymers (LCPs) with the mesogenic rod like units laterally attached to a polysiloxane backbone via a flexible spacer have been already reported. These phases show excellent planarity and shape recognition for polynuclear aromatic hydrocarbon (PAH) solutes in reversed-phase liquid chromatography. Optimization of these stationary phases in terms of molecular parameters of the polymer is here described. Fifteen stationary phases have been prepared varying different parameters such as the spacer length, the aliphatic tail length, and the proportion of laterally attached mesogenic units along the polymer chain. The results show that the combination of a long spacer and long terminal chains, which generates a smectic phase in the polymer bulk, leads to the best chromatographic performances towards planarity and shape recognition for PAH solutes.

Non-linear least-squares fitting with excel solver and related routines in HPLC modelling of retention

The usefulness of a non-linear least-squares type algorithm such as the Microsoft Excel Solver for response surface modeling of ionogenic solutes in reversed-phase liquid chromatography has been examined. The retention factors of adenosine, a typical ionogenic solute, in buffered mobile phases of different pH modified with methanol and/or acetonitrile were taken from the literature and fitted to a general 9-parameter equation using adequate initial estimates of the fitting parameters and/or ranges of these parameters estimated by a Monte Carlo technique described previously The validity of each fit was tested with a method suggested in the present communication. In addition, the physical meaning and significance of the fitted coefficients is discussed together with the standard errors calculated previously. The possibility of predicting the retention behaviour of adenosine on the basis of a limited number of data was also explored.

Determination of the thermodynamic contribution to peak asymmetry of basic solutes in reversed-phase liquid chromatography

To this day packing materials manufacturers are still trying to develop reversed-phase stationary phases that have silica more completely reacted with bonding ligands to afford more homogeneous particle surfaces. Incomplete bonding causes inhomogeneous effects that are readily observed when separating basic solutes because of the acidic silanols that are unreacted. However, it is still not understood exactly what types of silanol sites are unreacted or if metal impurities are contributing to the resulting peak asymmetry observed. A method is presented which utilizes (1) the frontal analysis method of chromatography to obtain adsorption/partition isotherms, (2) a heterogeneous Langmuir distribution model for the resulting isotherm, (3) an expectation-maximization numerical procedure to solve the mathematical problem to yield the most probable distribution of adsorption parameters, and (4) the equilibrium–dispersive model of chromatography incorporating the fitted isotherm model to check the validity of the sorption model with experimental observations. Correlation of packing materials characterization parameters with results obtained by this procedure will indicate what type of silanols or other sites are responsible for observed tailing behavior. Developers and manufacturers will then be able to more efficiently target their synthetic designs for “base-deactivated” reversed-phase silicas.

Shape Selectivity for Constrained Solutes in Reversed-Phase Liquid Chromatography

In reversed-phase liquid chromatography (RPLC), the separation of compound mixtures of similar polarity can present a significant challenge for the analyst. Examples of such compounds include geometric isomers present in environmental samples (e.g., polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and polychlorinated biphenyl congeners) and compounds of biological significance (e.g., carotenoids and steroids). In general, compounds with rigid, well-defined molecular shape are best separated using a column with enhanced shape selectivity characteristics. This perspective presents an overview of column properties that influence shape selectivity for constrained solutes. Approaches to the characterization of stationary-phase structure are described, and the findings are correlated with chromatographic performance. Finally, retention models of shape discrimination are presented that are consistent with observed retention behavior. An appreciation for shape recognition effects in RPLC will facilitate method development for certain classes of difficult to resolve compounds.

Retention Behavior Modelization of Monoprotic and Diprotic Species in a Hydroorganic Acetonitrile/Water Mixture

The retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) was predicted using retention equations for the monoprotic and diprotic species. In RPLC, with acetonitrile (ACN)/water mobile phases, the influence of pH and ACN cluster fraction can be quantitatively described by two general equations. The cluster solute solvation energies in an ACN/water mixture over a 0.50−0.80 water fraction were calculated. The energetics of the ACN cluster exchange process in the mobile phase was investigated in relation to the difference in pKa (ΔpKa) between a solute used as reference and the other solutes. A linear correlation was found between the Gibbs free energy change of the solvent exchange process and ΔpKa confirming that the solute solvation by ACN clusters was enhanced for the lesser polar solutes.

Ideal model of chromatography applied to charged solutes in reversed-phase liquid chromatography

The ideal model of chromatography assumes that the equilibration of a solute between the mobile and stationary phase is an infinitely rapid process. Under these assumptions, and for a given adsorption isotherm the model can be used to theoretically describe the evolution of a solute zone (peak), as it migrates along the chromatographic column. In this paper hydrophobic charged solutes are injected on a RP-18 column and the ideal model of chromatography is used to calculate the eluting peak shape. The advantage of using charged hydrophobic solutes is that the no linearity of the adsorption isotherm occurs at very low mobile phase concentrations of the solute. Another advantage is that from a theoretical point of view the adsorption isotherm is very well described by the electrostatic surface potential modified linear adsorption isotherm and the physical origin of the non-linearity is well understood. The application of this adsorption isotherm to the ideal model of chromatography is, however, mathematically more complex than for the Langmuir or Freundlich adsorption isotherms which previously have been combined with the ideal model of chromatography. It is shown that for negatively charged solutes the ideal model of chromatography combined with the electrostatically modified linear adsorption isotherm describes well the peak shape of the eluting peak. Since the non-linearity of the used adsorption isotherm is physically well defined, the experimental and theoretical study of eluting peak shapes of changed analytes may be a tool for the characterisation of the chromatographic column and also for the study of the interaction between charged solutes and the stationary phase surface.

Description of the partitioning behaviour of solutes and data treatment in micellar liquid chromatography with modifiers

The physico-chemical meaning of an empirical model, previously reported for describing the retention of solutes in reversed-phase liquid chromatography with micellar mobile phases of surfactant and alcohol, is given. The partition coefficient between stationary phase and water, and the solute-micelle association constant are considered as conditional parameters that depend on the concentration of modifier. An extension of the elution model is proposed for solutes having a strong hydrophobic character, that takes the strong interaction of these solutes with the stationary phase into account. Several fitting procedures (non-linear regression, and non-weighted and weighted linear regression) of the experimental data to the model are compared. The effect of errors in the value of the critical micellar concentration during the calculation of the retention physico-chemical parameters and prediction of the capacity factors, is also studied.

Uniform-sized polymer-based separation media prepared using vinyl methacrylate as a cross-linking agent Possible powerful adsorbent for solid-phase extraction of halogenated organic solvents in an aqueous environment

Uniform-sized macroporous polymer-based separation media were prepared through a typical two-step swelling method utilizing vinyl methacrylate or other ordinary cross-linking agents to investigate their properties as packing materials in HPLC. A simple and less hydrophobic cross-linking agent, vinyl methacrylate, afforded stable macroporous packing materials unexpectedly affording the largest retention volume towards hydrophobic solutes in reversed-phase liquid chromatography with good column efficiency. The retention ability towards seven halogenated organic solvents from chloroform to tetrachloroethylene was found to be much higher than that with a typical hydrophobic polymer-based packing material, poly(styrene-divinylbenzene) particles, and also a typical C 18 silica-based hydrophobic station phase.

Convergence of retention for small solutes in reversed-phase liquid chromatography

It is shown theoretically that when the concentration of organic solvent in the mobile phase increases, or solute size decreases, log k′ values of small solutes in reversed-phase liquid chromatography (RPLC) will tend to have a minimum value called the convergence point. A theoretical model for evaluating the convergent coordinates of small solutes is presented by using a stoichiometric displacement model for retention (SMDR). The physical meaning of the coordinates of each kind of convergence are also elucidated. The convergence points have either two-dimensional coordinates with a common ordinate (the logarithm of the phase ratio of the column, log φ) or threedimensional corrdinates with two common axes: — log φ and the logarithm of the molar concentration of the pure displacing agent in mobile phase, log aD. The other axis relates to the nature of the solutes, such as carbon number of a homolog, van der Waal's surface area, hydrophobic fragment constant etc. for the latter and those and/or concentration axis for the former. The model was tested with published data and found to give a good fit.

Modelling chromatographic behaviour as a function of pH and solvent composition in RPLC

In this work we establish the basic layout of IONICS, an expert system for optimizing the separation of ionogenic solutes in Reversed-Phase Liquid Chromatography, using the pH and the organic-modifier concentration of the mobile phase as parameters. We also present REMO, a front-end system that automates the retention modelling stage, based on a 9-parameter model. This system uses a scale transformation to suppress several numerical problems previously observed and features a strategy for automatic calculation of an initial approximation to the model optimum. The successful application of this system to a set of seven drugs is described. The final models are accurate and have smaller numerical problems. We also describe the use of a genetic algorithm instead of classical non-linear least-squares for fitting the model to the experimental data. Results indicate that genetic algorithms are a valuable, complementary tool for retention modelling.

Role of π-π interactions in reversed-phase liquid chromatography

The retention of various saturated and unsaturated solutes in reversed-phase liquid chromatography (RPLC) was investigated for various saturated and unsaturated packings in mobile packings in mobile phase mixtures of various compositions of methanol and acetonitrile. Non-aqueous eluents containing acetonitrile have a lower solvent strength than eluents containing methanol for saturated alkanes, alcohols and triglycerides, as expected from the solvophobic theory of retention. Very peculiar retention behaviours are observed when unsaturated solutes are analysed on aromatic stationary phases and/or when mobile phase containing acetonitrile is used with either an aromatic stationary phase or with unsaturated solutes or both. All these observations can be rationalized by taking into account the effect of π-π interactions between the π-electron systems of the solute, the stationary phase and the mobile phase. The influence of the residual silanols on silica-based RPLC supports on the retention of alcoholic solutes in pure acetonitrile is demonstrated. The influence of the concentration of a charge-transfer complexing agent (silver nitrate) in the mobile phase on the retention of unsaturated triglycerides was studied in a methanolic eluent and compared with that in a mobile phase containing acetonitrile.