Insights into the retention mechanism on an octadecylsiloxane-bonded silica stationary phase (HyPURITY C18) 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 octadecylsiloxane-bonded silica stationary phase HyPURITY C18 with methanol-water and acetonitrile-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. (2), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent composition with a discontinuity at low organic solvent 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 solutes 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 not included in the models. The overwhelming number of residual values [log k (experimental) - log k (model predicted)] were negative and could be explained by contributions from steric repulsion, defined as the inability of the solute to insert itself fully into the stationary phase because of its bulkiness (i.e., volume and/or shape). Steric repulsion is shown to strongly depend on the mobile phase composition and was more significant for mobile phases with a low volume fraction of organic solvent in general and for mobile phases containing methanol rather than acetonitrile. For mobile phases containing less than about 20 % (v/v) organic solvent the mobile phase was unable to completely wet the stationary phase resulting in a significant change in the phase ratio and for acetonitrile (but less so methanol) changes in the solvation environment indicated by a discontinuity in the system maps.     

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.     

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.     

System Maps for XTerra MS C18: Effect of Solvent Type on Selectivity in Reversed-Phase Liquid Chromatography

The solvation parameter model is used to establish the contribution of cohesion, dipole-type and hydrogen-bonding interactions to the retention mechanism on an XTerra MS C₁₈ stationary phase with acetonitrile-water, methanol-water and tetrahydrofuran-water mobile phases containing from 10 to 70% (v/v) organic solvent. Solute size and electron lone pair interactions are responsible for retention while dipole-type and hydrogen-bonding interactions result in lower retention. The volume fraction of water in the mobile phase plays a dominant role in the retention mechanism. However, the change in values of the system constants of the solvation parameter model cannot be explained entirely by assuming the principle role of the organic solvent is to act as a diluent for the mobile phase. Selective solvation of the stationary phase by the organic solvent and the ability of the organic solvent to extract water into the stationary phase, and/or the absorption of water-organic solvent complexes by the stationary phase, are important in accounting for the details revealed about the retention mechanism by the solvation parameter model. A qualitative picture of the above solvent effects, compatible with current knowledge of solvent and stationary phase properties, is presented.     

Insights into the Retention Mechanisms on Perfluorohexylpropylsiloxane-Bonded (Fluophase-RP) and Octadecylsiloxane-Bonded (Betasil C18) Stationary Phases Based on the Same Silica Substrate in RP-LC

The solvation parameter model is used to elucidate the retention mechanism on a perfluorohexylpropylsiloxane-bonded (Fluophase RP) and octadecylsiloxane-bonded (Betasil C18) stationary phases based on the same silica substrate with acetonitrile–water and methanol–water mobile phase compositions. Dewetting affects the retention properties of Fluophase RP at mobile phase compositions containing less than 20% (v/v) acetonitrile or 40% (v/v) methanol. It results in a loss of retention due to an unfavorable change in the phase ratio as well as changes in specific intermolecular interactions. Steric repulsion reduces retention of bulky solutes on fully solvated Betasil C18 with methanol–water (but not acetonitrile–water) mobile phase compositions but is not important for Fluophase RP. The retention of weak bases is affected by ion-exchange interactions on Fluophase RP with acetonitrile–water, and to a lesser extent, methanol-water mobile phases but these are weak at best for Betasil C18. The system constants of the solvation parameter model and retention factor scatter plots are used to compare selectivity differences for Fluophase RP, Betasil C18 and a perfluorophenylpropylsiloxane-bonded silica stationary phase Discovery HS F5 for conditions where incomplete solvation, steric repulsion and ion-exchange do not significantly contribute to the retention mechanism. Lower retention on Fluophase RP results from weaker dispersion and/or higher cohesion moderated to different extents by polar interactions since solvated Fluophase RP is a stronger hydrogen-bond acid and more dipolar/polarizable than Betasil C18. Retention factors for acetonitrile–water mobile phases are highly correlated for Fluophase RP and Betasil C18 except for compounds with a large excess molar refraction and weak hydrogen-bonding capability. Selectivity differences are more significant for methanol–water mobile phases. Retention factors on Fluophase RP are strongly correlated with those on Discovery HSF5 for acetonitrile–water mobile phases while methanol–water mobile phases retention on Fluophase RP is a poor predictor of the retention order on Discovery HS F5.     

Comparison of the Separation Characteristics of the Organic–Inorganic Hybrid Stationary Phases XBridge C8 and Phenyl and XTerra Phenyl in RP-LC

Differences in the system constants of the solvation parameter model and retention factor correlation plots for varied solutes are used to study the retention mechanism on XBridge C₈, XBridge Phenyl and XTerra Phenyl stationary phases with acetonitrile–water and methanol–water mobile phases containing from 10 to 70% (v/v) organic solvent. These stationary phases are compared with XBridge C₁₈ and XBridge Shield RP₁₈ characterized in an earlier report using the same protocol. The XBridge stationary phases are all quite similar in their retention properties with larger difference in absolute retention explained by differences in cohesion and the phase ratio, mainly, and smaller changes in relative retention (selectivity) by the differences in individual system constants and their variation with mobile phase type and composition. None of the XBridge stationary phases are selectivity equivalent but XBridge C₁₈ and XBridge Shield RP₁₈ have similar separation properties, likewise so do XBridge C₈ and XBridge Phenyl, while the differences between the two groups of two stationary phases is greater than the difference within either group. The limited range of changes in selectivity is demonstrated by the high coefficient of determination (>0.98) for plots of the retention factors for varied compounds on the different XBridge phases with the same mobile phase composition.     

Factors Affecting the Interpretation of Selectivity on Synergi Reversed-Phase Columns

The solvation parameter model is used to establish the contribution of cohesion, dipole-type, and hydrogen-bonding interactions to the retention mechanism on Synergi Hydro-RP, Fusion-RP, and Polar-RP reversed-phase columns with methanol–water mobile phases containing from 10–70% (v/v) methanol. Large changes in relative retention on the compared columns can result from steric resistance, differences in the phase ratios, and from dewetting at low methanol compositions while changes in intermolecular interactions are responsible for smaller changes at a fixed mobile phase composition. For Synergi Hydro-RP and Polar-RP changing methanol for acetonirile is more powerful for affecting changes in retention order than changing the stationary phase. The three Synergi columns show useful selectivity differences for method development when compared with 13 other modern reversed-phase columns representing a selection of different stationary phase chemistries. The results from this study indicate the limitations of classifying reversed-phase columns by the retention of prototypical compounds to define specific retention mechanisms.     

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.     

Comparison of the Separation Characteristics of the Organic–Inorganic Hybrid Octadecyl Stationary Phases XTerra MS C18 and XBridge C18 and Shield RP18 in RPLC

Differences in the system constants of the solvation parameter model, discontinuities in retention factor plots (log k against volume fraction of organic solvent) and retention factor correlation plots are used to study the retention mechanism on XTerra MS C₁₈, XBridge C₁₈ and XBridge Shield RP₁₈ stationary phases with acetonitrile–water and methanol–water mobile phases containing from 10 to 70% (v/v) organic solvent. Wetting of XBridge C₁₈ at 10 and 20% (v/v) acetonitrile is incomplete and is responsible for small changes in the retention mechanism. The intermolecular interactions responsible for retention on XTerra MS C₁₈ and XBridge C₁₈ are similar with minor differences in cavity formation and hydrogen-bonding interactions responsible for small selectivity differences. On the other hand, for bulky solutes there are large changes in retention at low volume fractions of organic solvent (<40% v/v) associated with steric repulsion on the XTerra MS C₁₈ stationary phases that is absent for XBridge C₁₈. Selectivity differences are more apparent for XBridge C₁₈ and XBridge Shield RP₁₈. For acetonitrile-water mobile phases cavity formation in the solvated XBridge Shield RP₁₈ is slightly more difficult and hydrogen-bond acid and base interactions are more important than for XBridge C₁₈. With methanol–water mobile phases it becomes slightly easier to form a cavity in the solvated XBridge RP₁₈ compared with XBridge C₁₈. In addition, the methanol-water solvated XBridge RP₁₈ is a stronger hydrogen-bond base and more dipolar/polarizable than XBridge C₁₈. These variations in selectivity justify the use of XBridge C₁₈ and XBridge Shield RP₁₈ as complementary stationary phases for method development.     

Influence of solvent effects on retention in reversed-phase liquid chromatography and solid-phase extraction using a cyanopropylsiloxane-bonded,silica-based sorbent

Summary The solvation parameter model is used to characterize the retention properties of a cyanopropylsiloxanebonded, silica-based sorbent with methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as mobile phases. The system constants over the composition range 1 to 50 % (v/v) organic solvent indicate that retention occurs because of the relative ease of cavity formation in the solvated stationary phase compared to the same process in the predominantly aqueous mobile phase as well as from more favorable stationary phase interactions with solutes containing π- and n-electrons. The capacity of the solute for dipole-type interactions is not important whereas all hydrogen-bond-type interactions result in reduced retention. Graphing the system constants as a function of mobile phase composition provides a simple mechanism for interpreting the change in capacity of the chromatographic system for retention in terms of changes in the relative weighting of fundamental intermolecular interactions. A comparison is also made with the retention properties of an octadecylsiloxane-bonded, silica-based sorbent with 30 % (v/v) methanol in water as the mobile phase and the extraction characteristics of a porous polymer sorbent with 1 % (v/v) methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as the sample processing solvent. Changes in sorbent selectivity due to selective uptake of the processing solvent are much smaller for the cyanopropylsiloxane-bonded sorbent than the results found for a porous polymer sorbent.     

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.     

Reversible solvent and temperature induced “monomeric-like” —“Polymeric-like” transitions in alkyl bonded silica

Summary The study of behaviour of homologous series (and more precisely, the plots of methylene selectivity versus the carbon number, n, of the alkyl chain of the homologue) on monomeric and polymeric phases in liquid chromatography enabled us to characterize two kinds of retention mechanism. These mechanisms are not independent; changes in the temperature or mobile phase composition allow passage from one to the other. The first, for which the methylene selectivity versus n decreases with a discontinuity, defines a retention mechanism where solutes are inserted between the grafted chains. It occurs for monomeric phases at ambient temperature, for polymeric phases, at high temperature or with a strong solvent like THF, CH₂Cl₂, AcMe or CHCl₃. The second gives a plot of methylene selectivity versus n with a maximum. It is consistent with a partition mechanism with total immersion of the solute in the bed of the stationary phase. It occurs for a polymeric phase at ambient temperature or for a monomeric one at low temperature.     

反相液相色谱中溶剂强度规律的研究

 以溶质计量置换保留模型 (SDM R)为依据 ,通过研究 3种正链醇同系物溶剂置换剂对 14种正链醇同系物溶质色谱保留行为的影响 ,发现计量置换参数Z(对应 1mol溶质被吸附时从溶质与固定相接触处释放出的溶剂的总摩尔数 )、lgI(与 1mol溶质对固定相亲和势有关的常数 )和 j(与 1mol溶剂对固定相亲和势有关的常数 )均随着同系物置换剂相对分子质量的增大而减小 ,并呈现出线性变化 ,表明溶剂强度与溶剂分子的大小有关 ,分子愈大 ,溶剂洗脱能力愈强 ,并遵循同系物规律。     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.     

Characterization of stationary/mobile phase combinations by using markers : Part 1. Preliminary results for the prediction of reversed-phase retention on various stationary phases

The characterization of stationary/mobile phase combinations can be done in a phenomenological way by measuring the k′ values of specific solutes, the markers. These markers can be chosen optimally from a set of test solutes with the use of multivariate techniques. When retention data of solutes on different stationary phases, with varying mobile-phase compositions, are available, a procedure is given to predict the retention of those solutes on new stationary phases. This procedure uses markers to characterize the new stationary/mobile phase combinations.     

HPLC retention behavior on hydride-based stationary phases

Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.     

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.     

Influence of solvent effects on retention in reversed-phase liquid chromatography and solid-phase extraction using a cyanopropylsiloxane-bonded,silica-based sorbent

Summary The solvation parameter model is used to characterize the retention properties of a cyanopropylsiloxane-bonded, silica-based sorbent with methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as mobile phases. The system constants over the composition range 1 to 50% (v/v) organic solvent indicate that retention occurs because of the relative ease of cavity formation in the solvated stationary phase compared to the same process in the predominantly aqueous mobile phase as well as from more favorable stationary phase interactions with solutes containing - and n-electrons. The capacity of the solute for dipole-type interactions is not important whereas all hydrogen-bond-type interactions result in reduced retention. Graphing the system constants as a function of mobile phase composition provides a simple mechanism for interpreting the change in capacity of the chromatographic system for retention in terms of changes in the relative weighting of fundamental intermolecular interactions. A comparison is also made with the retention properties of an octadecylsiloxane-bonded, silica-based sorbent with 30% (v/v) methanol in water as the mobile phase and the extraction characteristics of a porous polymer sorbent with 1% (v/v) methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as the sample processing solvent. Changes in sorbent selectivity due to selective uptake of the processing solvent are much smaller for the cyanopropylsiloxane-bonded sorbent than the results found for a porous polymer sorbent.