Phases In Reversed-phase Liquid Chromatography Research Articles

Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia–zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia–zirconia reversed stationary phases (C 18PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia–zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9–12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C 18PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the p K a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.

Review on the chemical and thermal stability of stationary phases for reversed-phase liquid chromatography

Review on the chemical and thermal stability of stationary phases for reversed-phase liquid chromatography

Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol–water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.

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.

Comparison of the Characteristics of Adsorption Equilibrium and Surface Diffusion in Liquid−Solid and Gas−Solid Adsorption on C18-Silica Gels

Four parameters characterizing the adsorption equilibrium, surface diffusion, and related thermodynamic properties were derived from pulse-response experiments in various reversed-phase liquid chromatography (RPLC) systems using C18-silica gels and aqueous solutions of three different organic modifiers, methanol, acetonitrile, and tetrahydrofuran. The results were compared with corresponding data similarly measured by gas−solid chromatography on the same type of surface-modified silica gel, with helium. Information on the solvent effect on the adsorption characteristics was provided by the comparison of these experimental results. While the adsorption equilibrium constant and the heat of adsorption at infinite dilution were much larger in the gas−solid than in the RPLC system, the surface diffusion coefficient (Ds) and the activation energy of surface diffusion (Es) were of the same order of magnitude in both systems. Regarding surface diffusion, the logarithm of the frequency factor was linearly correlated with Es by the same straight line, suggesting the fundamental similarity of the surface diffusion mechanism in the gas−solid and liquid−solid systems. Calculations made on the basis of a surface-restricted diffusion model provide an explanation for the comparable values of Ds and Es in the two systems. In conclusion, the liquid phase in RPLC influences the thermodynamic parameters of surface diffusion as well as those of adsorption equilibrium, but similar values of Ds and Es are observed in both the two adsorption systems. A quantitative explanation of the similarities and differences of the characteristics and the mechanism of surface diffusion in gas−solid and liquid−solid adsorption systems is proposed.

Stationary-phase effects in gradient high-performance liquid chromatography

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample polarity range that can be covered using gradients of organic solvents in water. The polarity range available for gradient separations of samples containing compounds differing in the lipophilic parts of the molecules can be characterized by “gradient lipophilic capacity”, Pl, based on the retention of standard compounds with a repeat lipophilic structural unit, such as a methylene group. The gradient lipophilic capacity is also suitable to characterize the separation possibilities of the columns in non-aqueous reversed-phase gradient elution of strongly non-polar compounds, such as triacylglycerols. In the same way, the suitability of various columns for reversed-phase gradient separations of oligomers can be characterized by “gradient oligomer capacity”, as demonstrated in the example of oligo(ethylene glycols). To enable a comparison of the properties of stationary phases independent of column efficiency and dimensions, the gradient lipophilic capacity or the gradient oligomer capacity should be normalized for a “standard” column plate number, gradient range and volume (in column hold-up volume units). The gradient lipophilic capacity or the gradient oligomer capacity and the number of compounds that can be resolved during a gradient run decrease as the initial concentration of the strong solvent in the mobile phase increases and (or) the gradient time decreases. These quantities can be used to select a suitable column and to adjust the optimum gradient profile (the initial composition of the mobile phase and the gradient steepness) with respect to the time of analysis and the number of oligomers or other compounds with regular repeat structural groups that can be resolved during the gradient run.

An Ultra Acid Stable Reversed Stationary Phase

We report a new reversed phase liquid chromatography (RPLC) phase with remarkable acid stability using dimethyl(ethylphenylchloromethyl)chlorosilane (1), oligomeric polystyrene (PS), and octylbenzene (C8). This phase Si-1-PS-C8 was prepared using silica modification processes and Friedel-Crafts alkylation chemistry. Under highly aggressive mobile phase conditions, Si-1-PS-C8 exhibited remarkable stability as evinced by only minimal reduction in retention factor (k') after 1400 column volumes at pH = 0.5 and 150 degrees C. The peak shapes for a variety of basic solutes were symmetric using Si-1-PS-C8. Evidence for a highly cross-linked coating of the silica particles was observed using scanning electron microscopy. The remarkable stability of this phase is unparalleled as compared to all other RPLC phases reported to date.

Effect of phase ratio on van’t Hoff analysis in reversed-phase liquid chromatography, and phase-ratio-independent estimation of transfer enthalpy

In analysis of the thermodynamics of the transfer of a solute from the mobile phase to the stationary phase in reversed-phase liquid chromatography, it is nearly always assumed that the phase ratio is constant. This type of analysis is typically performed by applying a form of the van’t Hoff equation, which relates the retention factor to temperature via the enthalpy and entropy of transfer. When non-linear van’t Hoff plots are observed, it is often assumed that the enthalpy and entropy of transfer change with temperature. However, when the possibility of a change in the phase ratio is considered, it becomes apparent that non-linear van’t Hoff behavior may or may not be due to changes in enthalpy or entropy. In this work, we present mathematical evidence that phase ratio changes, if they occur, can cause deviations from linearity in a van’t Hoff plot. We also show that the phase ratio influence can be eliminated by considering the molecular difference between two solutes instead of the solutes themselves. The resulting selectivity van’t Hoff plots may be linear, even when the van’t Hoff plots of the two solutes are non-linear. In such cases, temperature-dependent phase ratio changes, and not necessarily changes in the transfer enthalpy, may be responsible for the curved van’t Hoff plots of the individual solutes. In addition, we present chromatographic evidence that different solutes may “see” different thermodynamic phase ratios. It is clear that the concept of a phase ratio in reversed-phase chromatography is not nearly as well defined as a phase ratio in a bulk system like a liquid–liquid extraction.

Dependence of Elution Curve and Adsorption Isotherms of Insulin on Composition of Mobile Phase of Frontal Analysis in Reversed Phase Liquid Chromatography

AbstractWith frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent, concentration, the kind and the concentration of ion‐pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RFLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.

Chromatographic evaluation of self-immobilized stationary phases for reversed-phase liquid chromatography

The preparation of stationary phases for HPLC using polymers deposited on silica usually includes an immobilization step involving cross-linking by free radicals induced by ionizing radiation or by other radical initiators. The present paper reports changes which occur at ambient temperature in the character of poly(methyloctylsiloxane) deposited on porous silica particles as a function of the time interval between particle loading and column packing. Column performance and retention factors increase with time and these changes are attributed to rearrangement (self-assembly) which result in “self-immobilization” of the polymer molecules on the silica surface.

Characterization and comparison of HPLC columns for gradient elution

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample polarity range that can be covered using gradients of acetonitrile or of methanol in water. The polarity range available for gradient separations of samples containing compounds differing in the lipophilic parts of the molecules can be characterized by “lipophilic capacity”,Pl, based on the retention of retention standards with regular differences in lipophility, such as homologous alkylbenzenes. “Standard”Pl is defined as a quantity independent of the column dimensions and efficiency. It is controlled mainly by the chemistry of the bonded phase and by the properties of the support materials, but depends significantly also on the nature of the organic solvent in the mobile phase. The gradient lipophilic capacity and the sample polarity range that can be eluted during a “standardized” gradient run were used to compare the gradient-elution performance of more than 20 columns packed with various C8, C18 and other bonded phase materials on polymeric, monolithic, non-porous and superficially porous supports with various shielding of the residual silanol groups, including “aqueous phase” materials designed for the applications in highly aqueous mobile phases. The “lipophilic capacity” approach can be extended to characterize, the suitability of various columns for gradient separations of oligomers and lower polymers, as demonstrated on the example of lower poly(ethylene glycols).

System peaks observed in capillary liquid chromatography with eluents containing triethylamine

Triethylamine is often added to mobile phases in reversed-phase liquid chromatography for dynamic deactivation of free silanol groups of the stationary phase. It has been observed that eluents composed of methanol and triethylamine generate two system peaks in chromatograms obtained with LiChrosorb RP-select B stationary phase, whose retention times correspond to the dead time and to the retention time of triethylamine. It has been demonstrated that the system peaks can be positive or negative depending on the experimental conditions and may be incorrectly interpreted as peaks corresponding to sample components. An approach is outlined to unambiguous identify these system peaks in chromatograms of practical samples.

Characters of the Plateau of Methanol Increment in Frontal Analysis in Reversed Phase Liquid Chromatography

AbstractWith insulin methanol‐water, and the ion‐pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on‐line UV‐spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on‐line UV spectrometries.TFA with high UV absorbance can not be used as an ion‐pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.

Temperature-dependent refractive index issues using a UV–visible detector in high-performance liquid chromatography

While studying the properties of some potential, UV-detection compatible, mobile phase modifiers for reversed-phase liquid chromatography (RPLC) we encountered serious detector baseline problems. The most severe problem was a periodic saw-tooth-shaped drift. This drift was most noticeable when using water–THF and water–dioxolane mobile phases. We demonstrate that the baseline problems are related to temperature fluctuation in a water bath used to maintain a constant column temperature. The temperature fluctuations change the refractive index (RI) of the mobile phase, which in turn affects the amount of light reaching the detector. Our results show that the magnitude of the baseline drift is related to the RI value of the mobile phases. The baseline problems can be alleviated using a different temperature controller whose feedback mechanism assures smoother operation. Since there is no single and complete source of information in the literature concerning the RI values of typical mobile phases in RPLC, we decided to measure them as a function of the % modifier (between 0 and 100%) and the temperature (between 25°C and 50°C). Knowing the RI values allows us to calculate the excess volume of a binary liquid system.

Trends and progress in the characterization of stationary phases for reversed-phase liquid chromatography

The state-of-the-art and progress in the characterization of reversed-phase liquid chromatographic (RPLC) columns are discussed. Existing column tests are compared with tests under development. The validity of several test parameters, e.g. hydrophobicity and silanol activity, is also discussed. Solvent and eluent properties are treated with respect to retention and selectivity. Developments to improve the chemical stability of RPLC phases and thus column lifetime are reviewed. It is shown that substantial progress has been made in the manufacturing of RPLC columns, enabling their use over a wide variety of experimental conditions. In addition, general measures to significantly extend their lifespan are discussed. The substantial improvements in batch-to-batch and column-to-column repeatabilities achieved during the last two decades are also reviewed.

Use of chromatography to investigate biological recognition of a ligand by human serum albumin

Chromatographic temperature studies and differential scanning calorimetry have been used to elucidate the mechanism of retention of dansyl amino acids by human serum albumin (HSA) as stationary phase in reversed-phase liquid chromatography (RPLC) (Guillaume et al.,Anal. Chem. 1997,69, 4979). A novel mathematical development is proposed for description of HSA-dansyl amino acid association when HSA is used as mobile-phase modifier and C18 as stationary phase. The solute retention factor depends on the concentration of HSA in the bulk mobile phase and on the binding constant for the guest-HSA complex. The degree of complexationn c (the amount, %, of guest complexed) was determined. Enthalpy-entropy compensation was also analyzed in relation to this mathematical model to confirm the complexation behavior of the solute with HSA. Variation of the binding constant with the concentration of sodium phosphate, i.e. with the electrostatic force controlling HSA-solute association, is described. This theoretical model enabled estimation of the surface charge density (σ/F) and the accessible solvent surface area of the site II binding cavity; these were found to be approximately 8.7×10−7 mol m−2 and 2 nm2.

Selectivity of amino-, cyano- and diol-bonded silica in reversed-phase liquid chromatography

Amino-, cyano- and diol-bonded silica stationary phases were characterized by estimating their characteristic interaction constants in reversed-phase liquid chromatography (RPLC) based on linear solvation energy relationships. Five characteristic interaction constants of the stationary phases, the hydrophobicity ( v), polarizability ( r), dipolarity ( s), hydrogen bond (HB) acceptor basicity ( a) and HB donor acidity strength ( b) were determined by multiple regression analyses of logarithmic retention factors ( k) for a set of test solutes measured on them in 10% (v/v) methanol–water vs. the solute properties represented by characteristic molecular volume ( V x ), excess polarization ( R 2), dipolarity/polarizability ( π ∗ ), HB donor acidity ( α) and HB acceptor basicity ( β). Magnitudes of the five constants for the phases in RPLC were compared with those in normal-phase LC to see the differences in chromatographic selectivity in the two LC modes.

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F 13C 9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C 8) and octadecyl (C 18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C 18 phase, F 13C 9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C 8, C 18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C 18 or C 8 phases and was even disadvantageous on F 13C 9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.

Vibrational spectroscopy of dimethylchlorooctadecylsilane covalently bonded to ultrathin silica films immobilized on Ag surfaces

FTIR and Raman spectroscopies have been used to characterize the structure and conformational order of dimethylchlorooctadecylsilane (DOS) covalently bonded to ultrathin silica films supported on Ag substrates. Ultrathin silica films of ca. 30 A thickness prepared from sol-gel methods are immobilized on Ag surfaces modified with a self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane (3MPT). This layered structure provides a unique opportunity for acquiring complementary spectral data from both FTIR and Raman spectroscopies, which are useful in elucidating alkylsilane conformation pertaining to stationary phases for reversed-phase liquid chromatography (RPLC). Characterization of octadecyltrichlorosilane (OTS) layers on thin silica films of ca. 800 A thickness on 3MPT-modified Ag surfaces has been reported previously. Differences between the ultrathin silica films used in this study and the thin silica films used in this previous study are considered. The results from both FTIR and Raman spectroscopy presented here suggest that bonded DOS alkyl chains are in a disordered, liquid-like state with close to monolayer surface coverage.

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.