Retention mechanisms in reversed-phase liquid chromatography. Stationary-phase bonding density and partitioning

The partitioning model of retention for reversed-phase liquid chromatography, described by mean-field statistical thermodynamic theory, asserts that one principal driving force for solute retention is the creation of a solute-sized cavity in the stationary phase. Beyond a critical stationary phase bonding density, increased grafted chain density should result in enhanced chain ordering, which will increase the energy necessary for solute cavity formation and result in decreased chromatographic partition coefficients. We have evaluated chromatographic partition coefficients over an octadecyl bonding density range of 1.6-4.1 mumol/m2 and have found a maximum in partition coefficient at approximately 3.1 mumol/m2. Retention, however, approximately plateaus due to compensating changes in the partition coefficient and stationary phase volume. This provides unequivocal evidence that partitioning is the dominant form of retention for small nonpolar solutes.     

A study of the enthalpy and entropy contributions of the stationary phase in reversed-phase liquid chromatography

The goal of this study was to elucidate the roles played by the stationary and mobile phases in retention in reversed-phase liquid chromatography (RPLC) in terms of their individual enthalpic and entropic contribution to the Gibbs free energy of retention. The experimental approach involved measuring standard enthalpies of transfer of alkylbenzenes from typical mobile phases used in RPLC (methanol/water and acetonitrile/water mixtures), as well as from n-hexadecane (a simple analogue of the stationary phase) to the gas phase, using high-precision headspace gas chromatography. By combining the measured enthalpies with independently measured free energies of transfer, the entropies of transfer were obtained. This allowed us to examine more fully the contribution that each phase makes to the overall retention. It was found that the standard enthalpy of retention in RPLC (i.e., solute transfer from the mobile phase to the stationary phase) is favorable, due to the large and favorable stationary-phase contribution, which actually overcomes an unfavorable mobile-phase contribution to the enthalpy of retention. Further, the net free energy of retention is favorable due to the favorable enthalpic contribution to retention, which arises from the net interactions in the stationary phase. Entropic contributions to retention are not controlling. Therefore, to a great extent, retention is due to enthalpically dominated lipophilic interaction of nonpolar solutes with the stationary phase and not from solvophobic processes in the mobile phase. Further, our enthalpy data support a "partition-like" mechanism of retention rather than an "adsorption-like" mechanism. These results indicate that the stationary phase plays a very significant role in the overall retention process. Our conclusions are in direct contrast to the solvophobic model that has been used extensively to interpret retention in RPLC.     

Modeling retention and selectivity as a function of pH and column temperature in liquid chromatography

In reversed-phase liquid chromatography (RPLC), the retention of weak acids and bases is a sigmoidal function of the mobile-phase pH. Therefore, pH is a key chromatographic variable to optimize retention and selectivity. Furthermore, at an eluent pH close to the pKa of the solute, the dependence of ionization of the buffer and solute on temperature can be used to improve chromatographic separations involving ionizable solutes by an adequate handling of column temperature. In this paper, we derive a general equation for the prediction of the retentive behavior of ionizable compounds upon simultaneous changes in mobile-phase pH and column temperature. Four experiments, two limiting pH values and two temperatures, provide the input data that allow predictions in the whole range of these two variables, based on the thermodynamic fundamentals of the involved equilibria. Also, the study demonstrates the significant role that the choice of the buffer compound would have on selectivity factors in RPLC at temperatures higher than 25 degrees C.     

Retention mechanism in reversed-phase liquid chromatography: a molecular perspective

A detailed, molecular-level understanding of the retention mechanism in reversed-phase liquid chromatography (RPLC) has eluded analytical chemists for decades. Through validated, particle-based Monte Carlo simulations of a model RPLC system consisting of dimethyloctadecylsilanes at a coverage of 2.9 micro mol/m2 on an explicit silica substrate with unprotected residual silanols in contact with a water/methanol mobile phase, we show that the molecular-level retention processes for nonpolar and polar analytes, such as alkanes and alcohols, are much more complex than what has been previously deduced from thermodynamic and theoretical arguments. In contrast to some previous assumptions, the simulations indicate that both partitioning and adsorption play a key role in the separation process and that the stationary phase in RPLC behaves substantially different from a bulk hydrocarbon phase. The retention of nonpolar methylene segments is dominated by lipophilic interactions with the retentive phase, while solvophilic interactions are more important for the retention of the polar hydroxyl group.     

Temperature dependence of retention in reversed-phase liquid chromatography. 2. Mobile-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been examined over a wide temperature range with emphasis on the role of the mobile phase. van't Hoff plot shapes were used to assess the retention mechanism, and the data showed evidence of the hydrophobic effect when water-rich and/or hydrogen-bonded mobile phases such as methanol/water were used. However, different van't Hoff plot shape was observed with acetonitrile/water mobile phases, indicating a change in the retention mechanism. These data showed that the hydrophobic effect, which had previously been proposed as the driving force for retention, is not a satisfactory explanation for the retention process in all RPLC systems.     

Covalently bound ionene polyelectrolyte-silica gel stationary phases for HPLC

Micelle-mimetic ionene-based stationary phases for high-performance liquid chromatography (HPLC) are prepared by attaching [3,16]- and [3,22]-ionenes to aminopropyl silica through a carbon-nitrogen bond. These [x,y]-ionenes are polyelectrolytic molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups, some of which can form aggregate species whose properties mimic those of conventional surfactant micelles. These ionene-bonded stationary phases were characterized using different recommended HPLC test mixtures. Test solute chromatographic behavior on the ionene phases was found to be similar to that of intermediate oligomeric or polymeric C-18 and/or phenyl phases, depending upon the specific test mixture employed. In addition, the phases exhibit significant solute shape recognition ability. The ionene stationary phases were successfully employed for the separation of the components of the recommended ASTM reversed-phase test mixture, as well as for ortho-, meta- and para-disubstituted benzenes and other positional or geometric isomeric compounds. The ionene materials allow for chromatographic separations under either reversed-phase or ion-exchange conditions. The retention mechanism on these multimodal phases can occur by hydrophobic partitioning or electrostatic interactions, depending upon the characteristics of the components of the analyte mixture (neutral or anionic). The effects of alteration of the percent organic modifier, flow rate and temperature of the mobile phase on chromatographic retention and efficiency on these phases were briefly examined.     

Reduction of reequilibration time following gradient elution reversed-phase liquid chromatography

A simple and convenient method for the reduction of column reequilibration time following gradient elution reversed-phase liquid chromatography is described. This method utilizes the addition of a constant volume of 3% 1-propanol to the mobile phase throughout the solvent gradient to provide consistent solvation of the reversed-phase stationary phase. Reductions in reequilibration time of up to 78% have been observed. The effect of alkyl chain bonding density on reequilibration volume is also examined. A maximum in the mobile phase volume necessary to reequilibrate the column is found at a bonding density of about 2.9 mumol/m2. The relationship of reequilibration volume to bonding density supports the partitioning model of retention for reversed-phase liquid chromatography.     

Simulation studies on the effects of mobile-phase modification on partitioning in liquid chromatography

Various driving forces have been suggested to explain retention and selectivity in reversed-phase liquid chromatography (RPLC). To provide molecular-level information on the retention mechanism in RPLC, configurational-bias Monte Carlo simulations in the Gibbs ensemble were carried out for model systems consisting of three phases: an n-hexadecane retentive phase, a mobile phase with varying water-methanol composition, and a helium vapor phase as reference state. Liquid n-hexadecane functions as a model of a hydrophobic stationary phase, and a wealth of experimental data exists for this system. Gibbs free energies for solute transfers from gas to retentive phase, from gas to mobile phase, and from mobile to retentive phase were determined for a series of short linear alkanes and primary alcohols. Although the magnitude of the incremental Gibbs free energy of transfer for a methylene segment is always larger for the gas- to retentive-phase transfer than the gas- to mobile-phase transfer, it is found that the partitioning of alkanes and alkyl tail groups is mostly affected by the changes in the aqueous mobile phase that occur when methanol modifiers are added. In contrast, the partitioning of the alcohol headgroup is sensitive to changes in both the n-hexadecane and the mobile phases. In particular, it is found that hydrogen-bonded aggregates of methanol are present in the n-hexadecane phase for higher methanol concentrations in the mobile phase. These aggregates strongly increase alcohol partitioning into the retentive phase. The simulation data clearly demonstrate that due to modification of the retentive-phase hydrocarbons by solvent components, neither the solvophobic theory of RPLC, advocated by Horvath and co-workers, nor the lipophilic theory of RPLC, advocated by Carr and co-workers, can adequately describe the separation mechanism of the hexadecane model system of a retentive phase studied here nor the more complex situation present in actual RPLC systems.     

Control of ice chromatographic retention mechanism by changing temperature and dopant concentration

A liquid phase coexists with solid water ice in a typical binary system, such as NaCl-water, in the temperature range between the freezing point and the eutectic point (t(eu)) of the system. In ice chromatography with salt-doped ice as the stationary phase, both solid and liquid phase can contribute to solute retention in different fashions; that is, the solid ice surface acts as an adsorbent, while a solute can be partitioned into the liquid phase. Thus, both adsorption and partition mechanisms can be utilized for ice chromatographic separation. An important feature in this approach is that the liquid phase volume can be varied by changing the temperature and the concentration of a salt incorporated into the ice stationary phase. Thus, we can control the relative contribution from the partition mechanism in the entire retention because the liquid phase volume can be estimated from the freezing depression curve. Separation selectivity can thereby be modified. The applicability of this concept has been confirmed for the solutes of different adsorption and partition abilities. The predicted retention based on thermodynamics basically agrees well with the corresponding experimental retention. However, one important inconsistency has been found. The calculation predicts a step-like discontinuity of the solute retention at t(eu) because the phase diagram suggests that the liquid phase abruptly appears at t(eu) when the temperature increases. In contrast, the corresponding experimental plots are continuous over the wider range including the subeutectic temperatures. This discrepancy is explained by the existence of the liquid phase below t(eu). A difference between predicted and measured retention factors allows the estimation of the volume of the subeutectic liquid phase.     

Molecular dynamics simulations of alkylsilane stationary-phase order and disorder. 2. Effects of temperature and chain length

In an effort to elucidate the molecular-level structural features that control shape-selective separations, we have investigated the molecular dynamics of chromatographic models that represent both monomeric and polymeric stationary phases with alkylsilane length and temperature conditions analogous to actual materials of low to high shape selectivity. The structural characterization of these models is consistent with previous experimental observations of alkyl chain order and disorder: alkyl chain order increases both with alkyl chain length and with reduced temperature. Models that represent shape-selective reversed-phase liquid chromatography (RPLC) phases possess a significant region of distal end chain order with primarily trans dihedral angle conformations; the extension of these ordered regions into the phase increases with an increase in chain length. Models with extended chain length (C30) possess a higher degree of conformational order and are relatively insensitive to changes in surface coverage, bonding chemistry, and temperature. Chromatography models of various chain lengths and over a temperature range that represents highly shape-selective RPLC stationary phases all contain a series of well-defined and rigid cavities; the size and depth of these "slots" increase for the C30 models, which may promote the enhanced separations of larger size shape-constrained solutes, such as carotenoids.     

Characterization of the polarity of reversed-phase liquid chromatographic stationary phases in the presence of 1-propanol using solvatochromism and multivariate curve resolution

This work characterizes solvation effects in reversed-phase liquid chromatography in the presence of 1-propanol. The solvatochromic method combined with a multivariate curve resolution-alternating least-squares analysis method has been used to characterize two modified silica surfaces--phenyl bonded and C18 bonded silica in mobile-phase mixtures of methanol--water and acetonitrile--water in the presence of 1-propanol. The presence of a small amount of 1-propanol has been shown to affect mainly the polarity properties of the stationary phases while the mobile-phase properties are largely unaffected. The chain collapse mechanism for the C18 stationary phase at higher concentrations of water seems to be inhibited in the presence of 1-propanol, and partitioning is the predominant solute retention mechanism. The phenyl-based phase shows considerably different behavior from that of the C18 phase, and propanol appears to disrupt the pi-stacking interactions between the solute and the phenyl rings anchored to the silica support.     

Theoretical and experimental studies of the effect of pressure on solute retention in liquid chromatography

Pressure is often assumed to have a negligible influence on solute retention in liquid chromatography because of the small compressibility of the mobile and stationary phases. The range of pressures commonly encountered in reversed-phase separations is considerable, however, and may give rise to significant changes in solute capacity factor. In this study, the retention of model solutes is measured directly along the chromatographic column as a function of the local pressure. The model solutes, a homologous series of derivatized fatty acids, exhibit a significant increase in capacity factor ranging from +9.3% for n-C(10) to +24.4% for n-C(20) for inlet pressures from 1500 to 5000 psi. These experimental results are compared with a thermodynamic model derived from regular solution theory. This model suggests that state effects alone are not sufficient to describe the measured change in solute retention and that variations in interaction energy with density must also be considered. By using the simple relationship of van der Waals for the interaction energy (E ∝ 1/V), the change in capacity factor with density is slightly underestimated. However, by using an extended relationship that better describes polar fluids (E ∝ 1/V(2)), good agreement is observed. Finally, the correlation of experimental results with this thermodynamic model reveals that all components in the chromatographic system, including the solute, mobile phase, and stationary phase, must be considered compressible. The results of this study have clear implications for the determination of fundamental physicochemical parameters, as well as for the everyday practice of liquid chromatography.     

Loss of bonded phase in reversed-phase liquid chromatography in acidic eluents and practical ways to improve column stability

Silica-based, reversed-phase liquid chromatographic (RPLC) stationary phases are very widely used to separate basic compounds in acidic eluents due to their high efficiency, good mechanical strength, and the versatile selectivity offered by different functional groups and the chemistry on the silica surface. However, the stability in acid of most silica-based stationary phases is poor, especially at elevated temperatures, due to hydrolysis of the siloxane bonds, which hold silanes on the silica substrate. This hydrolysis is commonly believed to be solely the result of catalysis by protons. However, we show that various metal cations (principally Fe3+/Fe2+, Ni2+, and Cr3+) released from acid corrosion of the stainless steel inlet frit greatly accelerate the hydrolysis of the siloxane bond. Furthermore, these metal cations, and not the high acidity per se, are mainly responsible for column instability. We show that removing the stainless steel inlet frit, or use of a titanium frit, greatly reduces or totally eliminates corrosion of the inlet frit and radically improves retention stability. The effects of various acids and types of organic modifier were also studied. These observations suggest a number of practical approaches that can significantly extend the lifetime of any RPLC stationary phase in acidic media at elevated temperature.     

Measurement of mobile-phase volume in reversed-phase liquid chromatography and evaluation of the composition of liquid layer formed by solvation of packing materials

The mobile-phase volumes (Vm) in reversed-phase liquid chromatography (RPLC) with alkyl-bonded silica, defined as the difference between the total volume of eluent in the column (V0) and the volume of the eluent solvent layer formed by solvation of the bonded phase (VL), are determined by the method derived from the eluent electrolyte effect on the retention of ionic analytes. The validity of the Vm values obtained is evaluated by comparing them with the retention volumes of various organic compounds and inorganic ions, which have been suggested as unretained markers, and those obtained from a linear dependence of the logarithmic retention factor on the carbon numbers of homologous series. From the results obtained, it has been concluded that the solvated liquid phase on a column packing material should be assigned to a part of the stationary phase and the method developed for determination of the Vm value based on the ion partition model gives the most reasonable value as the mobile-phase volume in RPLC. The volume and the solvent composition of the solvated liquid phase on C1, C8, and C18 bonded silica are estimated, and the effects of organic modifiers and the physicochemical structures of the packing materials on these values are discussed.     

C60 and C70 HPLC retention reversal study using organic modifiers

A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes.     

Degree of solute inclusion in native beta-cyclodextrin: chromatographic approach

The reversed-phase liquid chromatography retention and separation of a series of D,L dansyl amino acids were investigated over a wide range of salting-out agent (sucrose) concentrations using native beta-cyclodextrin as a chiral stationary phase. An original treatment was developed to determine the number of sucrose molecules (n) excluded from the solute-beta-cyclodextrin cavity interface when the analyte transfer occurred. Using the n values, the relative degrees of compound inclusion were calculated and correlated to the steric bulkiness of the solute. Thermodynamic parameter variations are discussed in relation to the inclusion degree of the dansyl amino acids. This numerical approach is a valuable tool to explore the steric effects implied in the host-guest complex formation.     

Colloid-imprinted carbons as stationary phases for reversed-phase liquid chromatography

A novel colloid-imprinting method is employed for the preparation of carbonaceous stationary phases for reversed-phase liquid chromatography (RPLC). This colloid-imprinting method combined with oxidative stabilization treatment affords carbons with a porous shell/nonporous core structure. The particle morphology, pore size, pore shape, and Brunauer-Emmett-Teller surface area of these carbons can be finely tuned by selecting proper experimental conditions. Although their surface area and pore volume decrease noticeably after graphitization, their primary pore structure is maintained. In addition, the graphitization process eliminates the high-energy sites and substantially reduces structural heterogeneity, making colloid-imprinted carbons attractive stationary phases for reversed-phase liquid chromatography. The colloid-imprinted graphitic carbons with surface mesoporosity appeared to be attractive for chromatographic separations of alkylbenzenes under reversed-phase conditions.     

Influence of the hofmeister series on the retention of amines in reversed-phase liquid chromatography

The chromatographic behavior of protonated amines in reversed-phase liquid chromatography (RPLC) is influenced markedly by the identity of the mobile-phase anion. For example, retention factor values, k, obtained from protonated nordoxepin, nortriptyline, and amitriptyline increase almost 1 order of magnitude across the following series of anions employed as mobile-phase modifiers: H2PO4- < HCOO- < CH3SO3- < Cl- < NO3- < CF3COO- < BF4- < ClO4- < PF6-. Early eluting primary, secondary, and tertiary benzylamines are retained and resolved using BF4-, ClO4-, and PF6- but elute in or very near the void using all other mobile-phase anions tested. In contrast, a neutral hydrophobic marker, acenaphthene, shows no significant changes in retention with mobile-phase anion identity. Such large differences in amine retention with anion identity can be rationalized via both an ion-pairing model and the Hofmeister effect. Two key findings are reported. First, the dependence of amine retention on mobile-phase anion identity is attributed unambiguously to the Hofmeister effect and is quantified using a simple equation based solely on differences in the solvation of anions. Accurate prediction of k values from the excess chemical potential of anions in water suggests that anion-solvent interactions dominate the retention of amines in RPLC. Thus, controlling amine retention depends critically on judicious selection of mobile-phase anion (in addition to the usual experimental parameters such as organic modifier, temperature, pH, and stationary phase). Second, more lipophilic molecular anions can provide retention and tailing properties comparable to those obtained from traditional amphiphilic ion-pairing reagents such as octanesulfonate, but with the benefit of a superior gradient background and solubility at high concentrations of organic modifier.     

Molecular dynamics simulations of alkylsilane stationary-phase order and disorder. 1. Effects of surface coverage and bonding chemistry

"Shape-selective" polymeric alkylsilane stationary phases are routinely employed over the more common monomeric phases in reversed-phase liquid chromatography (RPLC) to improve the separation of geometric isomers of shape-constrained solutes. We have investigated the molecular dynamics of chromatographic models that represent both monomeric and polymeric stationary phases with alkylsilane surface coverages and bonding chemistries typical of actual materials in an effort to elucidate the molecular-level structural features that control shape-selective separations. The structural characterization of these models is consistent with previous experimental observations of alkyl chain order and disorder: (1) alkyl chain order increases with increased surface coverage; and (2) monomeric and polymeric phases with similar surface coverages yield similar alkyl chain order (although subtle differences exist). In addition, a significant portion of the alkyl chain proximal to the silica surface is disordered (primarily gauche conformations) and the distal end is most ordered. Models that represent shape-selective RPLC phases possess a significant region of distal end chain order with primarily trans dihedral angle conformations. This is consistent with the view that the alkyl chains comprising polymeric stationary phases contain a series of well-defined and rigid voids in which shape-constrained solutes can penetrate and hence be selectively retained.     

Chromatography with two mobile phases

Experimental results for the investigation of chromatographic columns containing two mobile phases are presented. The eluent was composed of mixtures of methanol and carbon dioxide. The column was an uncoated fused-silica-lined stainless steel capillary column. At certain experimental conditions, the eluent divided into two phases, both of which moved through the column. The predominant component of the liquid phase was methanol whereas the gas phase was composed of at least 93 mol % CO2. The columns were studied over a range of feed compositions (45-95 mol % CO2), pressures (61-101 bar), and temperatures (30-100 degrees C). The compositions and densities of each phase were calculated from the Peng-Robinson equation of state. The residence times of the two mobile phases were determined by tracer pulse chromatography. The partition coefficients of a probe solute, benzene, were measured along with the retention times of neon and the total volume of the chromatographic column as a function of temperature, pressure, and stoichiometric feed composition. The calculated column volumes, that is the volume of the liquid and gas, were constant over the full range of feed composition. The partition coefficient of benzene was constant at fixed pressure and temperature, varied logarithmically with density at fixed temperature and feed composition, and displayed a maximum at intermediate temperatures at fixed pressure and feed composition. The measured retention times of neon were consistently equivalent to the calculated residence times of the gas phase, indicating that neon did not dissolve in the liquid phase and could thus serve as an accurate dead time marker. The implementation of chromatography with two mobile phases produces a chromatographic "window". There is a lower limit for the retention volume of all solutes, viz., the residence time of the gas phase, exactly the same as normal chromatography. However, elimination of the stationary phase produces an upper limit to the retention volumes of solutes. This upper limit is the residence time of the liquid phase, so there is a retention window such that tG < or = ti < or = tL for all solutes.