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.     

Liquid chromatographic separations with mobile-phase additives: influence of pressure on coupled equilibria

On the basis of equilibrium thermodynamics, pressure can cause a shift in equilibrium for any interaction that exhibits a change in partial molar volume. This shift in equilibrium can be observed in liquid chromatography as a pressure-dependent shift in solute retention. In this paper, the impact of pressure on liquid chromatographic separations with mobile-phase additives is examined from both theoretical and experimental perspectives. The theoretical development for coupled-equilibria separations shown here is general and can be applied to any separation using mobile-phase additives. Predictions indicate that the coupled nature of these equilibria leads to pressure-induced perturbations in partitioning and complexation that can either compete with or complement one another. Using positional isomers and enantiomers as model solutes, experimental retention observations are fully consistent with these predictions, showing the diminution of individual pressure effects for competing cases and enhanced pressure effects for complementary cases. When pressure-induced changes in capacity or retention factor differ between individual solutes, changes in solute selectivity are predicted and observed. Using a C18 stationary phase with beta-cyclodextrin as the mobile-phase additive, solutes studied here exhibit changes in selectivity ranging from - 7 to + 10% for a change in average pressure of approximately 215 bar. Perhaps the most dramatic change in selectivity is observed for the separation of positional isomers where pressure-induced changes in selectivity actually reverse solute elution order.     

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.     

Temperature dependence of retention in reversed-phase liquid chromatography. 1. Stationary-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been investigated by examining the temperature dependence of retention, with emphasis on the role of the stationary phase in the retention process. Both chromatographic temperature studies and differential scanning calorimetry were used to examine the role of alkyl chain bonding density on the retention mechanism in RPLC. Phase transitions of reversed-phase stationary phases were observed at bonding densities greater than 2.84 mumol/m2. Thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase (delta H degrees and delta S degrees) were calculated for low bonding density columns, and comparison of these values to previously reported values for the partitioning of a nonpolar solute from the bulk organic liquid to water indicated that the chromatographic retention process is not well-modeled by bulk-phase oil-water partitioning processes. In addition, this data showed that the entropic contribution to retention becomes more significant with respect to the enthalpic contribution as the stationary-phase bonding density is increased, providing additional support that partitioning, rather than adsorption, is the relevant model of retention.     

Comparative study of hydrocarbon, fluorocarbon, and aromatic bonded RP-HPLC stationary phases by linear solvation energy relationships.

The retention properties of eight alkyl, aromatic, and fluorinated reversed-phase high-performance liquid chromatography bonded phases were characterized through the use of linear solvation energy relationships (LSERs). The stationary phases were investigated in a series of methanol/water mobile phases. LSER results show that solute molecular size and hydrogen bond acceptor basicity under all conditions are the two dominant retention controlling factors and that these two factors are linearly correlated when either different stationary phases at a fixed mobile-phase composition or different mobile-phase compositions at a fixed stationary phase are considered. The large variation in the dependence of retention on solute molecular volume as only the stationary phase is changed indicates that the dispersive interactions between nonpolar solutes and the stationary phase are quite significant relative to the energy of the mobile-phase cavity formation process. PCA results indicate that one PCA factor is required to explain the data when stationary phases of the same chemical nature (alkyl, aromatic, and fluoroalkyl phases) are individually considered. However, three PCA factors are not quite sufficient to explain the whole data set for the three classes of stationary phases. Despite this, the average standard deviation obtained by the use of these principal component factors are significantly smaller than the average standard deviation obtained by the LSER approach. In addition, selectivities predicted through the LSER equation are not in complete agreement with experimental results. These results show that the LSER model does not properly account for all molecular interactions involved in RP-HPLC. The failure could reside in the V2 solute parameter used to account for both dispersive and cohesive interactions since "shape selectivity" predictions for a pair of structural isomers are very bad.     

Geometrical model for the retention of fullerenes in high-performance liquid chromatography

In high-performance liquid chromatography (HPLC) using a poly(octadecylsiloxane) as a stationary phase, methanol as a mobile phase, C(60) and C(70) fullerenes as solutes, and water as a mobile phase modifier, a study on the surface tension effect of water on fullerene retention was carried out by varying the water concentration [W] and the column temperature T. The thermodynamic parameters for fullerene transfer from the mobile to the stationary phase were determined from linear van't Hoff plots. An enthalpy-entropy compensation revealed that the types of interactions between fullerenes and the stationary phase were independent of the fullerene structure and the mobile phase composition. An analysis of the experimental variations of the retention factor and the selectivity values with [W] was performed using a novel geometrical model. It was shown that the increase in fullerene retention accompanying the water concentration was due to the increased effects of surface tension. This brought about an increase in the interactions between fullerene and the stationary phase, explaining the observed thermodynamic parameter trends over the water concentration range. The theoretical model provided an estimation of the radius of fullerene which was found for C(60) to be equal to 3.3 ? and an activation energy during the transfer equal to 9.8 kJ/mol.     

The effect of stationary-phase pore size on retention behavior in micellar liquid chromatography

One of the limitations that has restricted the applicability of micellar liquid chromatography (MLC) is the weak eluting power of micellar mobile phases compared to conventional hydro-organic mobile phases used in reversed-phase liquid chromatography. This may be the result of Donnan or steric exclusion of the micelles from the pores of the stationary phase, within which nearly all (> or = 99%) of the stationary phase resides and the analytes spend most of their time. To determine whether wide-pore stationary phases would overcome this limitation in MLC, several C8 and C18 stationary phases ranging from 100 to 4000 A were investigated using a diverse set of test solutes and micellar solutions of anionic, neutral, and cationic surfactants as mobile phases. With the larger pore size stationary phases, the eluting power of the MLC mobile phases was enhanced with all surfactant types, the greatest effect being with the neutral surfactant. Differences in retention behavior were observed between various solute types and between the C8 and C18 stationary phases. These differences appear to be related to the relative hydrophobicity of the solutes and to differences in the surfactant-modified stationary phases. Partitioning behavior of representative solutes on the large-pore C8 and C18 columns was shown to follow the three-phase partitioning model for MLC. Methylene group selectivity data showed only minor differences in the stationary-phase characteristics between the small- and large-pore size C18 columns. The true eluting power of micellar mobile phases was revealed with wide-pore stationary phases and was demonstrated by the separation and elution of an extended series of alkylphenones on C18 columns.     

Retention mechanisms of reversed-phase liquid chromatography: determination of solute-solvent interaction free energies

Mean-field statistical thermodynamics theory has recently been developed to account for the partitioning of solutes from aqueous mobile phases into reversed-phase liquid chromatography stationary phases. Several predictions are tested here against an extensive data base of nearly 350 sets of experiments. In agreement with theory, we find that (i) the dependence of retention on mobile phase composition can often be suitably linearized through use of a type of composition plot recently suggested by Dill, (ii) retention measurements can be used to determine the binary interaction constants of solutes with solvents, and (iii) ET-30 solvent probe experiments appear to provide a direct measure of the binary interaction constants. This work suggests that the simple random-mixing approximation for solutes with solvents is often useful even for complex chromatographic solutions.     

Vancomycin dimerization and chiral recognition studied by high-performance liquid chromatography

The retention and separation of D,L-dansylvaline enantiomers (used as test solutes) were investigated using silica gel as stationary phase and vancomycin as chiral mobile-phase additive. A retention model was developed to describe the mechanistic aspects of the interaction between solute and vancomycin in the chromatographic system. It considered the formation of vancomycin dimers both "free" in the mobile phase and adsorbed on silica. By fitting the model equation to experimental data, it appeared clearly that the approach taking into account the vancomycin dimerization described accurately the retention behavior of the compounds. The examination of the model equation parameters showed that the glycopeptide dimerization increased the enantioselectivity by a factor of approximately 3.7. This study demonstrated the preponderant role of the vancomycin dimerization on the chiral recognition process of D,L-dansylvaline. Also, an additional analysis on a vancomycin chiral stationary phase indicated that the addition of vancomycin in the mobile phase promoted a greater enantioselectivity mediated by the formation of dimers in the stationary phase.     

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.     

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.     

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.     

Ice chromatography. Characterization of water-ice as a chromatographic stationary phase

Water-ice has been characterized as a stationary phase for liquid chromatography. Solutes having two or more polar groups are retained on this stationary phase with THF/hexane as the mobile phase, suggesting that multipoint interactions are required for measurable solute retention. Chromatographic separation of phenols or crown ethers on water-ice is possible. The ice surface is expected to provide two different adsorption sites coming from the OH and O dangling bonds. Although the solute partition into the quasiliquid layer is also considered, the dependence of the retention times on the THF concentration implies that the interaction of solutes with the water-ice surface rather than the partition into the quasiliquid layer is responsible for solute retention. A retention model suggests that the number of adsorption sites for a crown ether depends on its ring size, whereas two sites are involved for the retention of phenols having two hydroxyl groups. Although hydroxyl groups can act as both a hydrogen bond donor and an acceptor, the interaction with the ice OH sites, which are exposed to the surroundings in comparison with the ice O sites, is more important. However, when an acyclic polyether is added to the mobile phase, its adsorption onto the water ice surface allows the creation of the O sites that phenols can approach without steric hindrance. In the presence of the polyethers adsorbed on the ice surface, the retention of phenols is enhanced, whereas crown ethers become less retained due to the competitive adsorption of the polyethers.     

Parametric modulation in liquid chromatography: multivariate optimization of mobile phase composition and temperature

The concept and theory of parametric modulation are presented, and the strategy is demonstrated for multivariate optimization of mobile phase composition and temperature in liquid chromatography. Because each parameter to be optimized is maintained in separate and distinct zones along the column, the solutes are able to interact independently within each environment. Under these conditions, solute retention is a simple and rigorously predictable summation of the retention in each environment. Hence, parametric modulation is more accurate and requires fewer preliminary experiments than traditional optimization methods. This approach is demonstrated by application to the separation of isomeric polynuclear aromatic hydrocarbons using a polymeric octadecylsilica stationary phase, with methanol and acetonitrile mobile phases at temperatures from 23 to 45 °C.     

Application of an artificial neural network in chromatography—retention behavior prediction and pattern recognition

Layered feed-forward neural networks are powerful tools particularly suitable for the analysis of nonlinear multivariate data. In this paper, an artificial neural network using improved error back-propagation algorithm has been applied to solve problems in the field of chromatography. In this paper, an artificial neural network has been used in the following two applications: (1) To model retention behavior of 32 solutes in a methanol–tetrahydrofuran–water system and 49 solutes in methanol–acetonitrile–water system as a function of mobile phase compositions in high performance liquid chromatography. The correlation coefficients between the calculated and the experimental capacity factors were all larger than 0.98 for each solute in both the training set and the predicting set. The average deviation for all data points was 8.74% for the tetrahydrofuran-containing system and 7.33% for the acetonitrile-containing system. 2). To classify and predict two groups of different liver and bile diseases using bile acid data analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). The first group includes three classes: healthy persons, choledocholithiasis patients and cholecystolithiasis patients; the total consistent rate of classification was 87%. The second group includes six classes: healthy persons, pancreas cancer patients, hepatoportal high pressure patients, cholelithiasis patients, cholangietic jaundice patients and hepatonecrosis patients; the total consistent rate of classification was 83%. It was shown that artificial neural network possesses considerable potential for retention prediction and pattern recognition based on chromatographic data.     

Nonmolecular solvents in separation methods: dual nature of room temperature ionic liquids

Room temperature ionic liquids (RTIL) are molten salts starting to be used as nonmolecular solvents in separation methods mainly for their extremely low vapor pressure and thermal stability. RTILs are formed by an anion associated to a cation. This intrinsic structure gives them a dual nature. When used as additives in RPLC mobile phases to enhance basic compound separation, RTILs lose their particular physicochemical properties to become just salts. However, a given RTIL is not equivalent to another one made with the same cation. It is shown that both the anion and the cation contribute to solute retention and peak efficiency extending beyond simple "salting-out" or ion-pairing effects. Nine different alkyl-methyl-imidazolium ionic liquids with different alkyl chain length and chloride or BF(4-) or PF(6-) anions were used as additives (50 mM max. conc.) in the liquid chromatography separation of some cationic basic solutes on a Kromasil C18 column. It is shown with sodium salts and an acetonitrile-water 30/70 v/v mobile phase that anions can adsorb on the stationary phase surface according to their lyotropic character. They can also form ion pairs with the cationic basic solutes. Alkyl-imidazolium cations also adsorb on the C18 bonded stationary phase due to hydrophobic character depending on their alkyl chain length. Anion adsorption dramatically increases the cationic solute retention factors when cation adsorption decreases them. The cation adsorption is mainly responsible for peak shape and efficiency enhancements. RTILs are additives that enhance the basic cationic solute peak shape changing peak position. A wise choice of the appropriate combination of anion lyotropy with imidazolium cation hydrophobicity allows playing with solute selectivity and analysis duration.     

Measurement of partition coefficients in waterless biphasic liquid systems by countercurrent chromatography

Countercurrent chromatography (CCC) is a chromatographic separation technique that uses a liquid as a stationary phase. Centrifugal forces are used to immobilize the liquid stationary phase when the liquid mobile phase is pushed through it. In CCC, the solutes are separated according to their liquid-liquid partition coefficients. The solutes studied were the alkylbenzene homologues from benzene to hexylbenzene and some polyaromatic hydrocarbons (PAHs) from naphthalene to coronene. Their liquid-liquid partition coefficients were measured in the five waterless biphasic systems formed by heptane, as the apolar liquid phase of the five biphasic systems, and four dipolar aprotic solvents, dimethyl sulfoxide, dimethylformamide, furfural, and N-methylpyrrolidone, and the polar proton-donor solvent methanol. The coefficients were compared to the corresponding capacity factors obtained by classical liquid chromatography on octadecyl-bonded silica. For the five biphasic solvent systems studied, linear relationships were found between the partition coefficients and the sp(3) and sp(2) hybridized carbon atom number for the alkylbenzene and PAH series, respectively. The sp(2) and sp(3) transfer energies were estimated, and their ratio was used to quantify the solvent selectivity toward aromatic extraction.     

Elution-extrusion countercurrent chromatography. Use of the liquid nature of the stationary phase to extend the hydrophobicity window

Countercurrent chromatography (CCC) is a liquid chromatography technique with a liquid stationary phase. Taking advantage of the liquid nature of the stationary phase, it is possible to perform unique operations not possible in classical liquid chromatography with a solid stationary phase. It is easy to avoid any solute-irreversible absorption in the CCC column. If the retention volumes of solutes become too high, the dual mode will be used. The roles of the phases are reversed. The stationary phase becomes the mobile phase, and the CCC column is started again. The solutes elute rapidly in what was previously the stationary phase. The theoretical basis of the dual-mode method is recalled. The dual-mode method is a discontinuous method. The separation should be stopped when the phase switch is performed. The elution-extrusion procedure is another way to avoid any irreversible adsorption of solutes in the column. The method uses the fact that the liquid volumes occupied by the solutes highly retained inside the column can be orders of magnitude lower than the mobile-phase volume that would be needed to elute them. The elution-extrusion method also has two steps: the first step is a regular CCC chromatogram. Next, the stationary phase containing the partially separated hydrophobic solutes is extruded out of the column in a continuous way using the liquid stationary phase. The theory of the process is developed and compared to the dual-mode theory. Alkylbenzene homologues are experimentally used as model compounds with the heptane/methanol/water biphasic liquid system to establish the theoretical treatment and compare the performance of two types, hydrodynamic and hydrostatic, of CCC columns. It is shown that the method can dramatically boost the separation power of the CCC technique. An apparent efficiency higher than 20 000 plates was obtained for extruded octylbenzene and a 160-mL hydrodynamic CCC column with less than 500 plates when conventionally used.     

Study of interactions in supercritical fluids and supercritical fluid chromatography by solvatochromic linear solvation energy relationships

Linear solvation energy relationships were used to study the retention process in supercritical fluid chromatography (SFC) and to gain a better understanding of intermolecular interactions in supercritical fluids. Correlation of SFC retention data with a set of solute solvatochromic parameters, which are also applicable to gas and liquid chromatography, yields information regarding the relative contributions of dispersion, cavity formation, dipolar, and hydrogen-bonding processes to retention. Dispersion interactions and cavity formation processes dominate retention on an open tubular poly(dimethylsiloxane) stationary phase with pure carbon dioxide as the mobile phase. Dipolar interactions and hydrogen-bonding interactions are of decidedly less importance but do contribute significantly to retention. Based on prior solvatochromic studies of poly(dimethylsiloxane) and carbon dioxide, the changes in the regression coefficients with temperature and pressure are interpreted chemically. The relative importance of these contributions changes with temperature and pressure. As pressure increases, the carbon dioxide becomes more dense, and dispersion interactions between the solute and the mobile phase increase. A temperature increase at constant pressure decreases dispersion interactions with the stationary phase, as in gas chromatography, but also decreases dispersion interactions with the mobile phase, due to a decrease in carbon dioxide density. On the basis of the solvatochromic coefficients, carbon dioxide acts as both a Lewis base and a Lewis acid. The quality of fit for these correlations is very high and compares favorably with similar studies in gas chromatography and liquid chromatography, permitting the prediction of retention behavior from a solute's solvatochromic parameters.