Extrathermodynamic study of surface diffusion in reversed-phase liquid chromatography with silica gels bonded with alkyl ligands of different chain lengths

Surface diffusion on adsorbents made of silica gels bonded to C1, C4, C8, and C18 alkyl ligands was studied in reversed-phase liquid chromatography (RPLC) from the viewpoints of two extrathermodynamic relationships: enthalpy-entropy compensation (EEC) and linear free-energy relationship (LFER). First, the values of the surface diffusion coefficient (D(s)), normalized by the density of the alkyl ligands, were analyzed with the modified Arrhenius equation, following the four approaches proposed in earlier research. This showed that an actual EEC resulting from substantial physicochemical effects occurs for surface diffusion and suggested a mechanistic similarity of molecular migration by surface diffusion, irrespective of the alkyl chain length. Second, a new model based on EEC was derived to explain the LFER between the logarithms of D(s) measured under different RPLC conditions. This showed that the changes of free energy, enthalpy, and entropy of surface diffusion are linearly correlated with the carbon number in the alkyl ligands of the bonded phases and that the contribution of the C18 ligand to the changes of the thermodynamic parameters corresponds to that of the C10 ligand. The new LFER model correlates the slope and intercept of the LFER to the compensation temperatures derived from the EEC analyses and to several parameters characterizing the molecular contributions to the changes in enthalpy and entropy. Finally, the new model was used to estimate D(s) under various RPLC conditions. The values of D(s) that were estimated from only two original experimental D(s) data were in agreement with corresponding experimental D(s) values, with relative errors of approximately 20%, irrespective of some RPLC conditions.     

Surface diffusion in reversed-phase liquid chromatography using silica gels bonded with C1 and C18 ligands of different densities

Surface diffusion in reversed-phase liquid chromatography (RPLC) using silica gels bonded with C₁ and C₁₈ alkyl ligands of different densities was studied from the viewpoints of two extrathermodynamic relationships, i.e., enthalpy-entropy compensation (EEC) and linear free energy relationship (LFER). First, according to the four methods proposed by Krug et al., the values of surface diffusion coefficient (D_s) were analyzed to confirm that an actual EEC resulting from substantial physico-chemical effects takes place for surface diffusion. Then, it was also demonstrated that a LFER is observed between surface diffusion and the retention equilibrium. The establishment of EEC and LFER suggests a mechanistic similarity of molecular migration by surface diffusion, irrespective of the alkyl chain length and the densities of C₁ and C₁₈ ligands. Finally, a thermodynamic model for the LFER based on the real EEC was used to estimate D_s values under various RPLC conditions. The D_s values can be estimated with a mean square deviation of about 25–30%. The agreement between the D_s values estimated and those experimentally measured suggests that the total mass flux by surface diffusion consists of the two contributions due to C₁ and C₁₈ ligands and that the contribution of each ligand is proportional to the ligand density.     

Extrathermodynamic Study of Retention Equilibrium in RP-LC Using a C18-Silica Monolithic Stationary Phase

This study is concerned with the explanation of some thermodynamic properties of the retention equilibrium on a C₁₈-silica monolithic column. Pulse response experiments were carried out in a reversed-phase liquid chromatography system using a methanol/water mixture (70/30, v/v) and n-alkylbenzene homologs as the mobile phase and sample compounds, respectively, in the temperature range between 278 and 318 K. The retention equilibrium constant (K _a) was calculated from the first absolute moment of elution peaks. The dependence of K _a on the column temperature was analyzed using the modified van??t Hoff plot proposed by Krug et al. to derive the changes of the Gibbs free energy, the enthalpy and the entropy concerning the retention behavior. First, the presence of a real enthalpy?Centropy compensation (EEC) for the retention equilibrium was demonstrated. Then, a thermodynamic model based on the real EEC was developed to explain the temperature dependence of the linear free energy relationship (LFER) of the retention equilibrium. The model indicates how the slope and intercept of the LFER are correlated with the compensation temperatures and several molecular thermodynamic parameters. The model was effective for explaining the thermodynamic properties of the retention equilibrium of the C₁₈-silica monolithic stationary phase.     

Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C1 phase, 0.42, 1.01, 2.03, and 3.15 micromol/m2 of C18 alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C18 bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density (approximately 2 micromol/m2). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small deltaH) and high saturation capacities (large deltaS) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.     

Retention behaviour of polystyrene oligomers in reversed-phase liquid chromatography

Reversed-phase liquid chromatography (RPLC) was employed to investigate the behaviour of low-molecular-mass polystyrene oligomers with three different end groups, n-butyl, sec-butyl, and tert.-butyl polystyrenes. Exothermodynamic retention studies on the polystyrene oligomers were carried out using a C18 stationary phase column and 100% methanol mobile phase over the temperature range 15 to 60 degrees C. The resulting van't Hoff plots were linear over the entire temperature range for all three end group polystyrenes. Enthalpy-entropy compensation (EEC) showed a linear compensation for the higher-order oligomers, but was non-linear for the lower-order oligomers, indicating a change in the mechanism of retention. Differences in the extent of retention for each of the three end groups were also apparent. The ramifications of these differences are discussed.     

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of temperature

This study represents the first time that both the mobile phase composition and the temperature are simultaneously controlled to examine silica-bonded octadecylsilyl (C18) ligands spectroscopically at typical liquid chromatographic (LC) mobile phase flow-rates and back-pressures. Raman spectroscopy is used to characterize the behavior of the C18 bonded ligands equilibrated at temperatures from 45 to 2 degrees C in neat, single-component, mobile phase solvents including: water, acetonitrile, methanol, and chloroform. In addition, the effect of stationary phase ligand bonding density is examined by using two different monomeric reversed-phase liquid chromatographic (RPLC) stationary phases, a 2.34 and a 3.52 micromol m(-2) Microporasil C18 stationary phase, under identical conditions. The direct, on-column, spectroscopic analysis used in this study allows direct evaluation of the temperature-dependent behavior of the bonded C18 ligands. The temperature-dependent ordering of the stationary phase ligands is examined to determine if the ligands undergo a phase transition from a less-ordered "liquid-like" state at higher temperatures to a more-ordered "solid-like" state at lower temperatures. A discrete phase transition was not observed, but rather a continual ordering as temperature was lowered.     

Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous-organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van't Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows deltaH(o) > 0 and deltaS(o) > 0 while the alkyl chain shows deltaH(o) < 0 and deltaS(o) < 0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van't Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.     

反相高效液相色谱中溶质保留过程的热力学研究4: 焓熵补偿

依据液相色谱中溶质计量置换保留模型, 对溶质在反相液相色谱(RPLC)保留过程及其吸附、解吸附过程中的焓熵补偿进行了研究, 证实了在RPLC中焓熵补偿确实存在。从焓熵补偿的定义出发, 从理论上证明了溶质在保留过程中的焓熵补偿温度本质上为溶质保留值的收敛温度, 其数值为Z对1/T线性作图的斜率与截距之比。与惯常计算焓熵补偿温度的方法相比, 本文的方法所得补偿温度更为合理且不受流动相中强溶剂浓度变化的影响。     

Retention and mass transfer characteristics in reversed-phase liquid chromatography using a tetrahydrofuran-water solution as the mobile phase.

The characteristics of the retention and the mass transfer kinetics in reversed-phase liquid chromatography (RPLC) were measured on a system consisting of a C18-silica gel and a tetrahydrofuran-water (50:50, v/v) solution. These parameters were derived from the first and the second moments of the elution peaks, respectively. Further information on the thermodynamic properties of this system was derived from the temperature dependence of these moments. Some correlations previously established were confirmed for this system, namely, an enthalpy-entropy compensation for both retention and surface diffusion and a linear free-energy relationship. These results are compared with those observed in other similar systems using methanol-water (70:30, v/v) and acetonitnile-water (70:30, v/v) solutions. The contribution of surface diffusion to intraparticle diffusion in C18-silica gel particles was shown to be important. The analysis of the thermodynamic properties of surface diffusion suggests that, in these three RPLC systems, its activation energy is lower than the isosteric heat of adsorption. The nature and the extent of the influence of the mobile phase composition on the parameters describing the retention and the mass transfer kinetics are different but the chromatographic mechanisms involved in RPLC systems appear similar, irrespective of the nature of the organic modifier in the mobile phase.     

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of mobile phase composition

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.     

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C₁₈ columns with different packing materials are obtained from different manufacturers and one octyl-C₈ column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.     

Development of an accelerated low-pH reversed-phase liquid chromatography column stability test

The important experimental design criteria for an accelerated low-pH RPLC column stability test are discussed. The influence of method variables on the amount and rate of retention-loss and the final optimized parameters for the accelerated low-pH RPLC stability test are presented. The retention-loss curves for selected C8 and C18 stationary phases are compared. These studies indicate that ligand chain length, functionality and bonding density play an important role in determining the low-pH stability of a stationary phase. Additionally, elemental analysis data are used to infer the mechanism responsible for the observed retention-loss under low-pH conditions.     

Influence of synthetic routes on the conformational order and mobility of C18 and C30 stationary phases

Silica gels modified with n-alkyl chains (n = 18, 30) are prepared by two different synthetic routes and are examined by variable temperature FTIR and solid-state NMR spectroscopy. HPLC measurements of SRM 869, cis/trans ss-carotene isomers and xanthophylls isomers confirm the dependence of the separation mechanism on the alkyl chain length and the synthetic routes. The determination of the silane functionality and degree of cross-linking of silane ligands on the silica surface is achieved by 29Si CP/MAS NMR measurements. The structural order and mobility of the alkyl chains are investigated by means of variable temperature 13C CP/MAS NMR measurements. Variable temperature FTIR studies are performed where conformational order and flexibility of the alkyl chains in C18 and C30 phases are monitored through conformational sensitive CH2 symmetric, anti-symmetric stretching and wagging modes. In addition, the chromatographic properties of the C18 and C30 phases are determined. The results derived from the FTIR, NMR and HPLC measurements are discussed in the context of the applied synthetic routes and alkyl chain lengths.     

A new thermodynamic model describes the effects of ligand density and type,salt concentration and protein species in hydrophobic interaction chromatography

A new thermodynamic model is derived that describes both loading and pulse-response behavior of proteins in hydrophobic interaction chromatography (HIC). The model describes adsorption in terms of protein and solvent activities, and water displacement from hydrophobic interfaces, and distinguishes contributions from ligand density, ligand type and protein species. Experimental isocratic response and loading data for a set of globular proteins on Sepharose™ resins of various ligand types and densities are described by the model with a limited number of parameters. The model is explicit in ligand density and may provide insight into the sensitivity of protein retention to ligand density in HIC as well as the limited reproducibility of HIC data.     

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C18 bonded silica stationary phase and an acetonitrile-water mixture mobile phase, the sorption process of PEO to the stationary phase showed deltaH(o) > 0 and deltaS(o) > 0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy-entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile-water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: deltaH(o) < 0 and deltaS(o) < 0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy-entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.     

Enhancement of selectivity in reversed-phase liquid chromatography

In an effort to gain insight into the relationship between stationary phase solvation and selectivity, the use of short- and medium-chained-length alcohols (methanol, n-propanol, n-butanol, and n-pentanol) as mobile phase modifiers in reversed-phase liquid chromatography (RPLC) was investigated to determine their impact on chromatographic selectivity. A wide range of mobile phase compositions was evaluated because of the large effect exerted by solvent strength on selectivity. Employing a set of six vanillin compounds as retention probes, evidence is presented to support the view that an increase in the hydrophobicity of the organic modifier used in RPLC can increase the selectivity of the C18 alkyl bonded phase while simultaneously decreasing the retention time of the eluting solutes. Thus, we are presented with an interesting paradox: higher selectivity and shorter retention times, which can be attributed to changes in either solvent selectivity and/or stationary phase solvation by the organic modifier.     

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.     

Inorganic salts as hold-up time markers in C(18) columns

A systematic study of the retention behaviour in a C(18) reversed phase liquid chromatography (RPLC) system of several inorganic salts, namely NaNO(2), NaNO(3), KBr, Bu(4)NBr and K(2)Cr(2)O(7), is presented. The behaviour of the markers in unbuffered mobile phases and the marker retention dependence on the pH and ionic strength of the buffered mobile phases have been analyzed. In addition, a comparison between the retention behaviour of the markers and several ionizable analytes in buffered eluents is presented.