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.     

Influence of bonded-phase coverage in reversed-phase liquid chromatography via molecular simulation I. Effects on chain conformation and interfacial properties

Particle-based Monte Carlo simulations were employed to examine the effects of bonding density on molecular structure in reversed-phase liquid chromatography. Octadecylsilane stationary phases with five different bonding densities (1.6, 2.3, 2.9, 3.5, and 4.2 mumol/m(2)) in contact with a water/methanol (50/50 mol%) mobile phase were simulated at a temperature of 323 K. The simulations indicate that the alkyl chains become more aligned and form a more uniform alkyl layer as coverage is increased. However, this does not imply that the chains are highly ordered (e.g., all-trans conformation or uniform tilt angle), but rather exhibit a broad distribution of conformations and tilt angles at all bonding densities. At lower densities, significant amounts of the silica surface are exposed leading to an enhanced wetting of the stationary phase. At high densities, the solvent is nearly excluded from the bonded phase and persists only near the residual silanols. An enrichment in the methanol concentration and a disruption in the mobile phase's hydrogen bond network are observed at the interface as bonding density is increased.     

Thermodynamics and kinetics of solute transfer in reversed-phase liquid chromatography

In this study, the thermodynamic and kinetic behavior of a homologous series of fatty acids is examined using a polymeric octadecylsilica stationary phase and a methanol mobile phase. The zone profiles are evaluated as the temperature is varied from 20 to 60 degrees C and the average pressure from 400 to 4570 p.s.i. (1 p.s.i.=6894.76 Pa). The rate constant for solute transfer from mobile to stationary phase (k(ms)) appears to be relatively constant with carbon number. In contrast, the rate constant from stationary to mobile phase (k(sm)) decreases logarithmically with increasing carbon number. This suggests that the mass transport processes become progressively slower, owing to the smaller diffusion coefficients of the larger solutes in the stationary phase. The activation energy decreases slightly in the mobile phase and increases slightly in the stationary phase with increasing carbon number. The activation energy in the stationary phase ranges from 41.6 to 55.9 kcal/mol, while the thermodynamic change in internal energy ranges from -9.8 to -29.0 kcal/mol for C10 to C22, respectively (1 cal=4.184 J). The activation volume increases with increasing carbon number in both the mobile and stationary phase. The activation volume in the stationary phase ranges from 31.7 to 211 cm3/mol, while the thermodynamic change in molar volume ranges from -27.1 to -104 cm3/mol for C10 to C22, respectively. These large changes in activation energy and volume suggest that the solutes do not enter and leave the stationary phase in a single step, but in a stepwise or progressive manner.     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases

In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.     

Insights into the Retention Mechanism on a Pentafluorophenylpropylsiloxane-Bonded Silica Stationary Phase (Discovery HS F5) in RP-LC

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.     

Interactions between 33 solutes and four cyano-containing stationary phases: gas chromatographic activity coefficients and the solvation parameter model

Infinite-dilution gas–liquid chromatographic activity coefficients at 393.15 K (with their thermal and athermal components) and derived excess partial molar Gibbs energies, enthalpies, and entropies have been determined for each of 33 solutes of different polarity on four stationary phases with cyano groups, using retention data taken from the literature. The strongest interactions predicted by the solvation model are the dipolarity/polarizability, the acidic solute–basic stationary phase interaction, and nonpolar cavity formation and dispersion. These interactions were compared with those evaluated from the solute activity coefficients; the effect of the solute connectivity index and dipole moment on nonpolar and polar interactions, respectively, is discussed. The dependence of the thermal activity coefficient on nonpolar interactions, and the influence of stationary phase polarity on the four solute–stationary phase interactions, were evaluated. The nonpolar interaction increases with increasing connectivity and with increasing athermal activity coefficient. The dipolarity/polarizability interaction increases with increasing solute dipole moment. Finally, polar interactions increase with increasing stationary phase polarity whereas the nonpolar interaction is independent of stationary phase polarity.     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model IV. Aromatic stationary phases

The purpose of the present work was to systematically study the chromatographic behaviour of different aromatic stationary phases in a subcritical fluid mobile phase. We attempted to assess the chemical origin of the differences in retention characteristics between the different columns. Various types of aromatic stationary phases, all commercially available, were investigated. The effect of the nature of the aromatic bonding on interactions between solute and stationary phases and between solute and carbon dioxide-methanol mobile phase was studied by the use of a linear solvation energy relationship (LSER): the solvation parameter model. This study was performed to provide a greater knowledge of the properties of these phases in subcritical fluid chromatography, and to allow a more rapid and efficient choice of aromatic stationary phase in regard of the chemical nature of the solutes to be separated. Charge transfer interactions naturally contribute to the retention on all these stationary phases but are completed by various other types of interactions, depending on the nature of the aromatic group. The solvation vectors were used to compare the different phase properties. In particular, the similarities in the chromatographic behaviour of porous graphitic carbon (PGC), polystyrene-divinylbenzene (PS-DVB) and aromatic-bonded silica stationary phases are evidenced.     

Contribution of Steric Repulsion to Retention on an Octadecylsiloxane-Bonded Silica Stationary Phase in Reversed-Phase Liquid Chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsilioxane-bonded silica stationary phase, Ascentis ^TM C18, with acetonitrile-water and methanol-water mobile phase compositions containing 10–70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (1), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent compositions with a discontinuity at low organic solvent mobile phase compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes excluded from the models. The overwhelming number of residual values, here defined as the difference between experimental and model predicted retention factors for the excluded solutes, could be explained by contributions from steric repulsion. The latter defined as the inability of solutes to fully insert themselves into the solvated stationary phase because of their size or conformation. Steric repulsion resulted in a systematic reduction in retention compared with predicted values for the fully inserted solute. The bonding density of the stationary phase; the type and composition of the mobile phase; and the size, conformation, type and number of functional groups on the solute are shown to affect the contribution of steric repulsion to the retention mechanism.     

Separation Mechanism of Copolymers from Styrene and Methyl Methacrylate According to Composition by Liquid Adsorption Chromatography

Abstract

Silica gel and a mixture of chloroform and ethanol were used as the stationary and the mobile phases, respectively. At the isocratic elution mode, the copolymers tended to adsorb on the column with increasing MMA content in the copolymers, with increasing column temperature, and with decreasing ethanol content in the mobile phase. The copolymers appeared always at the position proportional to the interstitial volume in the column, otherwise they adsorbed on the column. The equilibrium distribution of the copolymers between the mobile phase and the stationary phase cannot be considered in this system. Hydrogen bonding of carbonyl groups in MMA units of the copolymers to silanol groups on the surface of silica gel was considered to be the main interaction between the copolymers and the adsorbent. The hydrogen bonding increases with increasing the number of carbonyl groups per unit surface of copolymer coil in solution and with increasing free silanol groups on the silica surface. Ethanol in the stationary phase controls the content of the free silanol groups on the surface and is controlled by the content of ethanol in the mobile phase in addition to column temperature. This phenomena can be explained by introducing the term of the adsorption energy of a copolymer segment with the silica surface. The adsorption energy is proportional to the strength of the hydrogen bonding. Above the critical adsorption energy which is defined to be equal to the dissolution energy of the copolymer to the mobile pahse, the copolymers adsorb on the surface of silica gel and the desorption of the copolymers advances when the adsorption energy approaches to the critical adsorption energy. No adsorption of the copolymers is possible when the adsorption energy is equal or below the critical adsorption energy.     

Influence of the pressure on the properties of chromatographic columns .III. retention volume of thiourea, hold-up volume, and compressibility of the C18-bonded layer

The influence of the average column pressure (ACP) on the elution volume of thiourea was measured on two RPLC columns, packed with Resolve-C18 (surface coverage 2.45 micromol/m2) and Symmetry-C18 (surface coverage 3.18 micromol/m2), and it was compared to that measured under the same conditions on an underivatized silica (Resolve). Five different methanol-water mixtures (20, 40, 60, 80 and 100% methanol, v/v) were used. Once corrected for the compressibility of the mobile phase, the data show that the elution volume of thiourea increases between 3 and 7% on the C18-bonded columns when the ACP increases from 50 to 350 bar, depending on the methanol content of the eluent. No such increase is observed on the underivatized Resolve silica column. This increase is too large to be ascribed to the compressibility of the stationary phase (silica + C18 bonded chains) which accounts for less than 5% of the variation of the retention factor. It is shown that the reason for this effect is of thermodynamic origin, the difference between the partial molar volume of the solute in the stationary and the mobile phase, Delta V, controlling the retention volume of thiourea. While Delta V is nearly constant for all mobile phase compositions on Resolve silica (with Delta V approximately equal to -4 mL/mol), on RPLC phases, it significantly increases with increasing methanol content, particularly above 60% methanol. It varies between -5 mL/mol and -17 mL/mol on Resolve-C18 and between -9 mL/mol and -25 mL/mol on Symmetry-C18. The difference in surface coverage between these two RP-HPLC stationary phases increases the values of Delta V by about 5 mL/mol.     

Effects of modifiers in subcritical fluid chromatography on retention with porous graphitic carbon

The effect of different modifiers in subcritical fluid chromatography (SubFC) on interactions between solute and porous graphitic carbon (PGC) and between solute and carbon dioxide-modifier mobile phases was studied by the use of linear solvation energy relationships (LSERs). This study was performed to allow efficient optimization of the composition of the carbon dioxide-modifier mobile phase in regard of the chemical nature of the solutes to be separated. With all modifiers tested (methanol, ethanol, n-propanol, isopropanol, acetonitrile, tetrahydrofuran and hexane), the solute/stationary phase interactions are greater than the solute/mobile phase ones. Dispersion interactions and charge transfer between electron donor solute and electron acceptor PGC mainly explain the retention on this surface, whatever the modifier. These interactions are quite constant over the range of modifier percentage studied (5-40%). For acidic compounds, the retention variation is mainly related to the change in the basic character of mobile and stationary phase due to the variation of modifier percentage. Changes in eluting strength are mostly related to adsorption of mobile phase onto the PGC with methanol and acetonitrile, and to the increase of dispersion interactions between the solute and the mobile phase for other modifiers. Relationships between varied selectivities and solvation parameter values have been studied and are discussed in this paper.     

Chain conformation and solvent partitioning in reversed-phase liquid chromatography: Monte Carlo simulations for various water/methanol concentrations

Many structural models for the stationary phase in reversed-phase liquid chromatography (RPLC) systems have been suggested from thermodynamic and spectroscopic measurements and theoretical considerations. To provide a molecular picture of chain conformation and solvent partitioning in a typical RPLC system, a particle-based Monte Carlo simulation study is undertaken for a dimethyl octadecyl (C(18)) bonded stationary phase on a model siliceous substrate in contact with mobile phases having different methanol/water concentrations. Following upon previous simulations for gas-liquid chromatography and liquid-liquid phase equilibria, the simulations are conducted using the configurational-bias Monte Carlo method in the Gibbs ensemble and the transferable potentials for phase equilibria force field. The simulations are performed for a chain surface density of 2.9 micromol/m(2), which is a typical bonded-phase coverage for mono-functional alkyl silanes. The solvent concentrations used here are pure water, approximately 33 and 67% mole fraction of methanol and pure methanol. The simulations show that the chain conformation depends only weakly on the solvent composition. Most chains are conformationally disordered and tilt away from the substrate normal. The interfacial width increases with increasing methanol content and, for mixtures, the solvent shows an enhancement of the methanol concentration in a 10 Angstrom region outside the Gibbs dividing surface. Residual surface silanol groups are found to provide hydrogen bonding sites that lead to the formation of substrate bound water and methanol clusters, including bridging clusters that penetrate from the solvent/chain interfacial region all the way to the silica surface.     

Intermolecular interactions in normal-phase HPLC systems with alcohol hydrocarbon eluents

Summary We apply our new retention model introduced elsewhere to the studies of intermolecular interactions between the solute and the remaining components of a normal-phase HPLC system using a binary mobile phase consisting of an alcohol and a hydrocarbon. This can be achieved through the analysis of the regressional parameters A, B, and C appearing in the relationship of solute retardation (R_F) vs. mobile phase composition. Negative values of these parameters indicate a greater affinity of the solute to the stationary phase than to a given mobile phase moiety, whereas positive values have a reverse meaning. According to the absolute values of A, B, and C solute groups can be arranged according to the affinity toward a given mobile phase moiety, which is a detailed and important information regarding their separation, the stationary phase and with each individual constituent of the mobile phase.     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

A New Equation for Solute Retention in Liquid Chromatography

In consideration of the adsorption of solvent, diluent and solute molecules on the surface of a stationaryphase, a new equation for solute retention in liquid chromatography is presented. This equation includesthree parameters: the displacement equilibrium constant (Ksd) between the solvent and diluent molecules onthe surface of the stationary phase, the total number(N) of the solvent and diluent molecules released fromthe stationary phase after one solute molecule being adsorbed, and the parameter (I) related to the thermody-namic equilibrium constant for the solute adsorption on the stationary phase. Over the whole concentrationrange of the solvent in the mobile phase, the experimental retention data can be well described by this equa-tion, parameters K~, N and I can be obtained by the regression analysis of the experimental retention data,and consequently the number of the solvent and the diluent molecules displaced by one solute molecule fromthe stationary phase can also be derived at different solvent concentrations in the mobile phase,     

Retention mechanism of the multifunctional solute on columns with different coverage densities using highly aqueous reversed‐phase conditions

A series of 11 homemade octadecyl bonded phases with different coverage densities were tested to determine the influence of the stationary phase on the retention in highly aqueous mobile phases. The concentrations of the organic modifiers (methanol and ACN) were in the range of 0–20%_v/v. The coverage density of bonded ligands and the presence of the end‐capping have strong influence on the solute retention. Amoxicillin (AMO) was chosen as the test compound. Dual properties of AMO, which contain hydrophobic skeleton and polar groups (amino, hydroxyl and carbonyl), cause irregular changes of the retention over the stationary phase hydrophobicity and silanol activity at given mobile phase composition. Presented data show that application of non‐standard low coverage density C18 phases allow to determine AMO in the RPLC condition with high retention.     

Supercritical fluid solid chromatography using novel porous glassy carbon. Evaluation of retention mechanisms

The use of a porous glassy carbon (PGC) material as a packed-column SFC stationary phase has been previously demonstrated [1]. The material is further characterized in terms of its retention characteristics. The effects of variations in mobile phase composition, pressure, and temperature conditions are evaluated. Variation of temperature and pressure yielded expected results, specifically, decreased solute capacity factors with increased mobile phase density. The choice of supercritical fluid mobile phase allows the most notable control of solute retention; this was evaluated by adding low percentages of organic modifiers of varying molecular weights to the supercritical carbon dioxide mobile phase. PGC-SFC provides reversed phase characteristics similar to those found for PGC-HPLC. Porous glassy carbon has selectivity characteristics previously unavailable in supercritical fluid chromatography. Use of porous glassy carbon in supercritical fluid chromatography may provide distinct advantages in difficult analytical separations, allowing separations of molecules with only slight structural differences.     

非理想状态下的塔板理论模型

 在过程中 ,将流动相看成是由许多连续的塔板组成 ,每一塔板的高度与固定相塔板的高度相同。初始浓度的溶质被认为全部集中在流动相的第一塔板中 ;溶质在流动相和固定相之间动态分布。由于动力学因素的影响 ,当流动相流过一个塔板距离时 ,溶质不能够迅速地从固定相释放到流动相中 ,因此溶质在流动相和固定相中的分布浓度受到两个因素即反映系统热力学性质的分配系数P和反映系统动力学性质的释放概率因子α的影响。这一过程被认为是非理想状态下的过程。