Some insights on the description of gradient elution in reversed‐phase liquid chromatography

The so‐called “fundamental equation for gradient elution” has been used for modeling the retention in gradient elution. In this approach, the instantaneous retention factor (k) is expressed as a function of the change in the modifier content (φ(t_s)), t_s being the time the solute has spent in the stationary phase. This approach can only be applied at constant flow rate and with gradients where the elution strength depends on the column length following a f(t?l/u) function, u being the linear mobile phase flow rate, and l the distance from the column inlet to the location where the solute is at time t measured from the beginning of the gradient. These limitations can be solved by using the here called “general equation for gradient elution”, where k is expressed as a function of φ(t,l). However, this approach is more complex. In this work, a method that facilitates the integration of the “general equation” is described, which allows an approximate analytical solution with the quadratic retention model, improving the predictions offered by the “linear solvent strength model.” It also offers direct information about the changes in the instantaneous modifier content and retention factor, and gives a meaning to the gradient retention factor.     

Protonation of polyaniline‐coated silica stationary phase affects the retention behavior of neutral hydrophobic solutes in reversed‐phase capillary liquid chromatography

Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed‐phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH‐dependent charge and structure of polymer chains of the emeraldine‐coated silica stationary phase show a mixed‐mode separation mechanism.     

Quantitative structure‐retention relationships of the chromatographic retentions of phthalic acid ester contaminants in foods

The harmful health effects caused by phthalic acid esters have been supported from the increasing scientific evidence, developing the efficient methodologies to monitor the levels of phthalic acid esters in various foods become especially important from the aspects of human exposure assessment and their migration mechanistic understanding. In this study, quantitative structure‐retention relationship studies on both the gas and liquid chromatographic retention times of 23 phthalic acid esters were performed by genetic function approximation, and the optimal quantitative structure‐retention relationship models (r² > 0.980, r²_CV > 0.960, and r²_pred > 0.865) passed the statistical tests of cross‐validation, randomization, external prediction, Roy′ r_m² metrics, Golbraikh‐Tropsha′ criteria and applicability domain. The established predictive models elucidate the structural requirements for the retention of phthalic acid esters over different chromatographic columns, which were finally used to predict the retention times of 11 new phthalic acid esters. Hopefully, this work could provide useful guidelines for better understanding and accurate prediction of the retention behavior of undetermined phthalic acid esters when lacking standard samples or under poor experimental conditions, and make the simultaneous identification and quantification of numerous phthalic acid esters possible.     

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction–reversed‐phase liquid chromatography mechanism,a review

Hydrophilic interaction liquid chromatography on polar columns in aqueous–organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed‐phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one‐ and two‐dimensional liquid chromatography separations.     

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.     

Extraction,separation and quantitative structure–retention relationship modeling of essential oils in three herbs

The essential oils extracted from three kinds of herbs were separated by a 5% phenylmethyl silicone (DB‐5MS) bonded phase fused‐silica capillary column and identified by MS. Seventy‐four of the compounds identified were selected as origin data, and their chemical structure and gas chromatographic retention times (RT) were performed to build a quantitative structure–retention relationship model by genetic algorithm and multiple linear regressions analysis. The predictive ability of the model was verified by internal validation (leave‐one‐out, fivefold, cross‐validation and Y‐scrambling). As for external validation, the model was also applied to predict the gas chromatographic RT of the 14 volatile compounds not used for model development from essential oil of Radix angelicae sinensis. The applicability domain was checked by the leverage approach to verify prediction reliability. The results obtained using several validations indicated that the best quantitative structure–retention relationship model was robust and satisfactory, could provide a feasible and effective tool for predicting the gas chromatographic RT of volatile compounds and could be also applied to help in identifying the compound with the same gas chromatographic RT.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Applicability of linear and nonlinear retention‐time models for reversed‐phase liquid chromatography separations of small molecules,peptides, and intact proteins

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention‐time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention‐time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution‐strength combinations. The quadratic model provided the most accurate retention‐factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention‐time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention‐time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).     

Retention prediction of large and condensed polycyclic aromatic hydrocarbons in reversed-phase microcolumn liquid chromatography with diphenylsilica stationary phase

The retention behavior of condensed large polycyclic aromatic hydrocarbons has been investigated with diphenylsilica stationary phases in reversed-phase microcolumn liquid chromatography. The results were correlated with two parameters which indicate size and shape of the molecules. Since the resulting equation can be used for retention prediction of large polycyclic aromatic hydrocarbons, computer-assisted “standardless” identification is accomplished for “unknown” compounds contained in the standard.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.