Effect of the organic modifier concentration on the retention in reversed-phase liquid chromatography II. Tests using various simplified models

Ten simplified expressions for the retention factor, k', that arise from either the adsorption or partition mechanism for retention in reversed-phase chromatographic columns are examined in what concerns the model they express and their performance to fit experimental data. In order to test the simplified expressions, which describe the variation of the retention of a solute with the organic modifier content in the mobile phase, a wide range of solutes in mobile phases modified with three different organic modifiers was used. It is shown that a new three-parameter expression of ln k' works more satisfactorily, since it combines simplicity, high applicability and good numerical behavior. It is also shown that the applicability of a simplified equation does not entail the validity of its model and thus no molecular information can be gained from its use.     

New insights on the retention mechanism of non-polar solutes in reversed-phase liquid chromatographic columns

A clarification of the retention mechanism of non-polar solutes in octadecyl reversed-phase chromatographic columns is attempted based on a systematic comparison of the retention in C18 and C2 columns under the assumption that the retention in C2 columns is due to adsorption. The comparison involves curve fitting procedures and tests based on the properties of special functions suggested in the present paper. For the application of this approach the retention behaviour of six non-polar solutes, benzene, toluene, ethylbenzene, propylbenzene, isopropylbenzene and tert-butylbenzene, is studied from aqueous mobile phases modified with methanol, isopropanol, acetonitrile and tetrahydrofuran using C18 and C2 reversed-phase columns. It was found that the retention mechanism in C18 columns is not the same in the four modifiers. In particular, our results show that the adsorption mechanism has a significant contribution in mobile phases modified by acetonitrile and tetrahydrofuran, the partition mechanism is likely to predominate in isopropanol-water mobile phases provided that the mole fraction of isopropanol is higher than 0.2, whereas the case of MeOH is rather obscure, since the various tests did not give a clear picture about the retention mechanism in methanol-water mobile phases.     

Retention process in reversed phase TLC systems with polar bonded stationary phases

The retention of a solute in RP chromatography is a very complex process which depends on many factors. Therefore, the study of the influence of a mobile phase modifier concentration on the retention in different reversed phase chromatographic systems is very important for understanding the rules governing retention and mechanisms of substance separation in a chromatographic process. Composition changes and the nature of mobile phases enable tuning of the separated analytes' retention over a wide range of retention parameters and optimization of the chromatographic process as well. Optimization of the chromatographic process can be achieved by several different methods; one of them is the so-called interpretative strategy. The key approach adopted in this strategy is the implementation of adequate retention models that couple the retention of solute with the composition of a mixed mobile phase. The use of chemically bonded stationary phases composed of partially non-bonded silica matrix and organic ligands bonded to its surface in everyday chromatography practice leads to questions of the correct definition of the retention model and the dominant retention mechanism in such chromatographic systems. The retention model for an accurate prediction of retention factor as a function of modifier concentration and the heterogeneity of the adsorbent surface should be taken into consideration. In this work the influence of mobile-phase composition on the retention of sixteen model substances such as phenols, quinolines, and anilines used as test analytes in different RP-TLC systems with CN-, NH2-, and Diol-silica polar bonded stationary phases has been studied. The aim of this study is to compare the performance of three valuable retention models assumed as the partition, adsorption/partition, and adsorption mechanism of retention. All the models were verified for different RP-TLC systems by three statistical criteria. The results of investigations presented in this work demonstrate that the best agreement between the experimental and calculated Rf values was obtained by the use of new-generation retention models, which assume heterogeneity of adsorbent surface. The results reported here show that heterogeneity of the adsorbent surface may be important in analysis of the elution process in liquid chromatography. Consideration of the goodness of fit for the experimental data to the examined retention models is in conformity with the adsorption mechanism of retention on all polar bonded stationary phases in most eluent systems for most investigated compounds.     

Deformation of gradient shape as a result of preferential adsorption of solvents in mixed mobile phases

Gradient elution has been studied in typical normal and reversed-phase systems. Deformations of gradient profiles have been evidenced as a result of preferential adsorption of modifiers of the mobile phase. This phenomenon was pronounced in the normal-phase system, for which gradient profiles deviated significantly from those programmed. This influenced the retention and shapes of band profiles of the eluting solute. Hence, in order to predict gradient propagation correctly the adsorption equilibrium of modifiers has been quantified. Moreover, at low modifier content, deformations of band profiles of the solute has been registered as a result of the competitive adsorption in the system solute-modifier. This effect has been predicted by a competitive adsorption model. For the reversed-phase systems the influence of the modifier adsorption on gradient propagation was insignificant for typical mobile phases investigated. Therefore, the work has been focused on gradient predictions in the normal-phase system.     

An Integrated Theory of Adsorption and Partition Mechanism and Eash Contribution to Solute Retention in Reversed Phase Liquid Chromatography

With the combination of the the stoichiometric displacement model for retention (SDM-R) in reversed phase liquid chromatography (RPLC) and the stoichiometric displacement model for adsorption (SDM-A) in physical chemistry,the total number of moles of the re-solvated methanol of stationary phase side.nr,and that of solute side in the mobile phase,q,corresponding the one mole of the desorbing solute,were separately determined and referred as the characterization parameters of the contributions of the adsorption mechanism and partition mechanism to the solute retention,respectively.A chromatographic system of insulin,using mobile phase consisting of the pseudo-homologue of alcohols(methanol,ethanol and 2-propanol)-water and trifluoroacetic acid was employed.The maximum number of the methanol layers on the stationary phase surface was found to be 10.6,only 3 of which being valid in usual RPLC,traditionally referred as a volume process in partition mechanism.However,it still follows the SDM-R.Both of q and nr of insulin were found not to be zero,indicating that the retention mechanism of insulin is a mixed mode of partition mechanism and adsorption mechanism.When methanol is used as the organic modifier,the ratio of q/nr was 1.13,indicating the contribution to insulin retention due to partition mechanism being a bit greater than that due to adsorption mechanism.A linear relationship between q,or nr and the carbon number of the pseudo-homologue in the mobile phase was also found.As a methodology for investigating the retention mechanism retention and behavior of biopolymers.a homologue of organic solvents as the organic modifier in mobile phase has also been explored.     

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

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.     

Laterally attached liquid crystalline polymers as stationary phases in reversed-phase high-performance liquid chromatography. V. Study of retention mechanism using linear solvation energy relationships

A linear solvation energy relationship model was used to characterize the retention behavior of a stationary phase based upon a nematic side-on liquid crystalline polymer (SOLCP) in reversed-phase liquid chromatography. The set of solutes was constituted of a high variety of compounds whose molecular sizes were considerably smaller than the mesogenic unit size. The results showed good statistical fits for these retention data in 65:35, 75:25 and 85:15 (v/v) methanol-water mobile phases. Both the cavity term and excess molar refraction are the most important favorable retention-governing parameters, whereas the solute hydrogen bond acceptor basicity is the most unfavorable retention parameter. Hydrophobicity and pi-pi interactions decrease strongly when the percentage of methanol increases, leading to an important retention decrease despite the fact that the hydrogen bond interaction weakens as the organic solvent is added. The shape recognition ability of this side-on liquid crystalline stationary phase on polycyclic aromatic hydrocarbon solutes is partly explained by the solutes' high polarizability due to the presence of pi-electrons. However, the solute polarizability is not sufficient and a stationary phase's "structure effect" must to be taken into account for the shape discrimination observed. The strong interaction between liquid crystal molecules caused likely a adsorption retention mechanism rather than a partition mechanism.     

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.     

A STOICHIOMETRIC DISPLACEMENT MODEL FOR RETENTION OF SOLUTE IN LIQUID CHROMATOGRAPHY WITH MULTI-COMPONENT SYSTEM

Considering all the kinds of interactions between solute and solvent, solute and stationary phase, solvent and stationary phase molecules as well as the competitional adsorption among various kinds of solvent molecules on the stationary phase, we present a stoichiometric displacement model of solute retention with four sets of parameters in liquid chromatography. This model was tested with data from both literature and experiments done by ourselves. These results show that this model may fit the experimental data for a liquid chromatography system with various kinds of mobile phases consisting of a complete range of multi-components and with different types of stationary phases.     

疏水分配常数用于反相液相色谱保留值的预测

在反相液相色谱保留值基本方程log k_′=a+_cC_B的基础上,描述了采用疏水分配常数及氢键作用能来预测a、c参数的方法,并系统讨论了疏水分配常数对参数a、c的影响,借此对反相液相色谱宽浓度范围内的保留值进行了预测。     

On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths

Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were synthesized in the confines of 100 μm I.D. fused-silica capillaries via a free radical copolymerization of mono and divinyl monomeric precursors in the presence of porogenic diluents. These columns were used in order to determine their suitability for the reversed-phase separation of small molecules in isocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminated polymerization reaction, as well as content of organic modifier in the mobile phase, address the most significant parameters affecting the isocratic performance of these monoliths in the separation of small molecules. We show that the performance of methacrylate-based porous polymer monoliths is strongly affected by the retention factor of the analytes separated. A study of the porous and hydrodynamic properties reveals that the actual nature of the partition and adsorption of the small analyte molecules between mobile and stationary (solvated) polymer phases are most crucial for their performance. This is due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnant mass transfer zones restricting their applicability in the separation of small molecules under conditions of strong retention.     

New equations describing the combined effect of pH and organic modifier concentration on the retention in reversed-phase liquid chromatography

Six equations that express the combined effect of mobile phase pH and organic modifier content on sample retention in reversed-phase liquid chromatography (RPLC) are developed based on either the adsorption or the partition model for retention. The equations are tested against five retention data sets taken from literature. In the tests two pH scales are used, w(w)pH and s(s)pH. It is shown that a new seven-parameter equation works more satisfactorily, because it exhibits good numerical behavior, gives low values of the sum of squares of residuals and represents the experimental retention surfaces successfully. In addition, the danger of overfitting, which leads to the prediction of physically meaningless retention surfaces, is minimized by using the proposed new seven-parameter equation. Finally, the possibility of obtaining reliable pK values of weak acids or bases chromatographically by means of the derived equations is also considered and discussed.     

Adsorption/partition model of liquid chromatography for chemically bonded stationary phases of the aliphatic cyano, reversed-phase C8 and reversed-phase C18 types

A novel approach was introduced to modeling solute retention in the liquid chromatography systems, employing silica-based aliphatic chemically bonded stationary phases of the cyano, reversed-phase C8 and reversed-phase C18 types, and the mixed binary eluents most frequently used in the reversed-phase and normal-phase chromatography modes (i.e. using the methanol-water and the 2-propanol-n-hexane liquid mixtures, respectively). This approach takes notice of the mixed (adsorption/partition) mechanism of solute retention, in which both, the adsorptive and the dispersive forces contribute to the overall energetics of this process. Performance of our new model was compared with that of the widely recognized and on a routine basis applied Schoenmakers approach, and it was found out that both models perform with a practically equal and outstanding accuracy.     

Effect of the concentration of organic modifier in an aqueous-ethanol mobile phase on the chromatographic retention and thermodynamic characteristics of the adsorption of enantiomers of α-phenylcarboxylic acids on silica gel with immobilized eremomycin antibiotic

Regularities of the chromatographic retention and thermodynamics of the adsorption of enantiomers of α-phenylcarboxylic acids on a chiral stationary phase with immobilized macrocyclic antibiotic eremomycin under conditions of reversed-phase liquid chromatography with aqueous-ethanol mobile phases are studied. Relationships between the retention characteristics of the acids, the enantioselectivity of their separation, and the concentration of organic modifier in the mobile phase are found. It is shown that the sterical structure of substituents on the chiral atoms of the acids affect the mechanism of retention. The compensation effect in the studied systems is considered.     

Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector

The adsorption behavior of two amino acids, i.e., l,d-threonine and l,d-methionine has been investigated on the chiral stationary phase (CSP)column packed with teicoplanin bonded to a silica support. The study has been performed under non-linear conditions of adsorption isotherm for various types of organic modifiers (methanol, ethanol, propan-2-ol and acetonitrile) in the reversed-phase mode. A heterogeneous adsorption mechanism of amino acids has been identified that was strongly affected by the nature of organic modifier. Generally, isotherm non-linearity and retention decreased with decrease of the modifier content in the mobile phase exhibiting a minimum at water-rich mobile phases. These trends were suggested to result from a combined effect of the mobile as well as the adsorbed phase composition. To determine the composition of the adsorbed phase the excess adsorption of modifiers in aqueous solutions has been measured and their binary adsorption equilibria have been quantified and compared. Strongly non-ideal behavior of solvents in the mobile phase and the adsorbed phase has been accounted for by activity coefficients. The fraction of the modifiers in the adsorbed phase decreased in the sequence: methanol, ethanol, propan-2-ol and acetonitrile.     

The use of water structure breakers, urea and guanidium chloride, new mobile phase modifiers in reversed-phase partition high-performance liquid chromatography.

Organic solvent-free mobile-phase systems in ion-pair reversed-phase partition high-performance liquid chromatography (IPRP-HPLC) are demonstrated; using urea at 3.0-7.0 molal (mol kg-1) as a modifier in a mobile phase on an octadecylsilanized silica column, four nitrophenolates and metal 4-(2-pyridilazo)resorcinol (PAR) chelates (in PAR chelates system an aqueous mobile phase with 15 wt% methanol was used) were separated rapidly within 6 min at no sacrifice to the separation efficiency. On the addition of urea in the mobile phase, reduced retention times of nitrophenolates and naphthalenesulfonates and also diminution of the height equivalent to a theoretical plate were observed. The addition of urea and guanidium chloride (GuCl) in the mobile phase gave rise to a decrease in the mobile phase volume; in turn, this meant an increased volume of the stationary phase. As the concentration of urea and GuCl in the mobile phase increased, the volume of the mobile phase in the column decreased within about 70% and 40% at 7.0 molal of urea and GuCl, respectively. A decrease in the mobile phase volume suggests an increase in the extent of solvation of the bonded hydrocarbon chain of the stationary phase. The possible explanations for the LC behavior with the urea and GuCl are turned into reduction of hydrophobic interaction in LC processes, solute partitioning and entangling of alkyl chain brushes, with the addition of urea. The water structure breakers, urea and GuCl, most likely affect the solvation states of both solute molecules and the hydrocarboneous stationary phase by changing the nature of the water solvent, which provides a new technique for fine tuning of the LC resolution of the analytes.     

New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

碱性药物在硫醚嵌入苯磺酸硅胶固定相上的色谱行为

混合模式色谱（MMC）在复杂样品的分离分析方面具有独到的优势,相比于单一模式色谱,MMC受到多种作用控制,保留机理更为复杂。利用巯基-烯点击化学方法分别制备了单配体和双配体两种硫醚嵌入苯磺酸硅胶固定相,通过改变pH、离子强度和有机溶剂强度等流动相条件,以4种碱性药物为模型,对其保留机理进行了探讨。结果表明,两种固定相都具有反相和离子交换的混合保留机理。通过改变流动相中盐浓度、考察溶质保留因子与盐浓度倒数的关系,证明了反相、单纯离子交换和反相协同离子交换三种作用形式的保留模型更为合理。定量研究表明,在两个固定相上,由单纯离子交换和反相协同离子交换构成的总离子交换作用占主导,各作用占比与溶质、流动相组成、固定相配体的类型及其比例等密切相关,并且协同作用对溶质的保留和分离选择性影响很大。混合模式色谱保留机理的研究对于新型固定相设计和复杂体系的分离优化具有重要理论指导意义。