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.     

Gas-liquid chromatography with a volatile "stationary" liquid phase

A unique type of gas-liquid chromatography is described in which both mobile and "stationary" phases are composed of synthetic mixtures of helium and carbon dioxide. At temperatures below the critical point of the binary mixture and pressures above the vapor pressure of pure liquid carbon dioxide, helium and carbon dioxide can form two immiscible phases over extended composition ranges. A binary vapor phase enriched in helium can act as the mobile phase for chromatographic separations, whereas a CO2-rich liquid in equilibrium with the vapor phase, but condensed on the column wall, can act as a pseudostationary phase. Several examples of chromatographic separations obtained in "empty" capillary columns with no ordinary stationary liquid phase illustrate the range of conditions that produce such separations. In addition, several experiments are reported that confirm the proposed two-phase hypothesis. The possible consequences of the observed chromatographic phenomenon in the field of supercritical fluid chromatography with helium headspace carbon dioxide are discussed.     

Extrathermodynamic relationships in reversed-phase liquid chromatography

Enthalpy-entropy compensation (EEC) and linear free energy relationships (LFER) are extrathermodynamic correlations frequently used to discuss the mechanistic similarities of chemical equilibria and reaction kinetics. Although empirical, they are widely applied, proving the substantial effectiveness of fundamental studies based on them. Many attempts have been made to interpret theoretically the necessary conditions (or preconditions) of EEC and LFER. LFER is known to rest on the existence of EEC. However, the intimate correlations between EEC on one hand and LFER and the temperature dependence of LFER on the other hand were insufficiently discussed from the viewpoint of molecular structure contributions. We present a simple LFER model relating the slope and intercept of LFER to the compensation temperatures, themselves derived from EEC analyses, and to several parameters characterizing the molecular contributions to the changes in enthalpy and entropy associated with the passage from one phase of the chromatographic system to the other. A theoretical explanation is supplied for the intimate correlation between the two types of extrathermodynamic relationships, EEC and LFER. We demonstrate also that the characteristics of EEC and LFER depend on the structural parameters. This new model allows a proper interpretation of the temperature dependence of LFER. It should permit further progress of fundamental studies of chemical reaction mechanisms based on extrathermodynamic relationships.     

Electrogenerated chemiluminescence detection in reversed-phase liquid chromatography

Electrogenerated chemiluminescence (ECL) has been developed as a detection method for liquid chromatography. The radical cation of tri-p-tolylamine (TPTA) is used as a common electron acceptor for the electrogenerated radical anions of a variety of organic analytes. ECL is accomplished with a high-frequency potential pulse program applied to a microelectrode immersed in the column eluent. ECL detection is demonstrated with reversed-phase liquid chromatography. Selectivity at the ECL detector is shown to be tunable based on differing electrochemical conditions and excited-state energetics. Low minimum detection limits in ECL are attributed to the dependence on the photon detector shot noise, allowing a limit of detection of 0.14 nM for perylene in the presence of 0.1 mM TPTA. A derivatization agent useful for ECL detection is demonstrated by the use of naphthalene-2,3-dicarboxaldehyde. This reagent, which does not itself result in ECL, forms ECL candidates following reaction with primary amines.     

A kinetic study of mass transfer in reversed-phase liquid chromatography on a C18-silica gel

The characteristic features of mass-transfer kinetics in a reversed-phase (RP) column packed with a C18-silica were studied. The relevant information on phase equilibrium thermodynamics and mass-transfer kinetics was obtained by frontal analysis and the pulse method, respectively. The equilibrium isotherm was accounted for by the simple Langmuir model. The ratio of the axial dispersion coefficient to the mobile-phase flow velocity increased almost linearly with increasing solute concentration. Similarly, the mass-transfer rate coefficient (km) showed a linear dependence on the solute concentration. The positive concentration dependence of km resulted from that of the surface diffusion coefficient, which was interpreted with the chemical potential driving force model. The contribution of axial dispersion to band broadening was predominant in the RP column packed with the medium-size packing material used (particle diameter, 12 microns) whereas that of the kinetics of adsorption/desorption was negligibly small. The results of this study demonstrate how an analysis of the dependence of the mass-transfer kinetics on the flow velocity and the solute concentration allows a better understanding of this kinetics.     

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.     

Shape selectivity for constrained solutes in reversed-phase liquid chromatography

In reversed-phase liquid chromatography (RPLC), the separation of compound mixtures of similar polarity can present a significant challenge for the analyst. Examples of such compounds include geometric isomers present in environmental samples (e.g., polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and polychlorinated biphenyl congeners) and compounds of biological significance (e.g., carotenoids and steroids). In general, compounds with rigid, well-defined molecular shape are best separated using a column with enhanced shape selectivity characteristics. This perspective presents an overview of column properties that influence shape selectivity for constrained solutes. Approaches to the characterization of stationary-phase structure are described, and the findings are correlated with chromatographic performance. Finally, retention models of shape discrimination are presented that are consistent with observed retention behavior. An appreciation for shape recognition effects in RPLC will facilitate method development for certain classes of difficult to resolve compounds.     

Overload for ionized solutes in reversed-phase high-performance liquid chromatography

Overloading occurs for submicrogram quantities of ionized solutes particularly when using low ionic strength mobile phases at low pH (e.g., formic acid), even with highly inert silica RP-HPLC columns of normal dimensions. Much higher loads can produce a sharp L-shaped peak with retention above the column void volume, in line with the hypothesis that a small number of high-energy sites fill first and are rapidly overloaded, followed by a much larger number of weaker sites. However, charged acids and bases show identical overloading behavior; overloading is reduced as the mobile-phase ionic strength is increased. These findings raise questions about the physical nature of the strong sites. The rapid overloading of silica and purely polymeric phases could be explained by mutual repulsion of ionic species or their inability to fully penetrate the hydrophobic structure of the phase. However, these alternative hypotheses cannot readily explain the high total saturation capacities obtained using frontal analysis. Ion pairing with trifluoroacetic acid may reduce overload, while the effect is less important for formate or phosphate buffers. A surface layer of acetonitrile is not a prerequisite for rapid overloading, as shown by studies using purely aqueous buffers.     

Chiral separations performed by enhanced-fluidity liquid chromatography on a macrocyclic antibiotic chiral stationary phase

The use of enhanced-fluidity liquid chromatography (EFLC) for chiral separations was demonstrated on a macrocyclic antibiotic column, Chirobiotic-V. This technique was compared to high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) for the separation of chiral compounds in normal-phase mode. The highest resolution was always observed for EFLC condition. Higher efficiency and shorter retention time were also observed for most separations with portions of CO(2) in the range of 0-50 mol %. Larger amounts of CO(2) caused efficiency to decrease and retention time to be prolonged. For some separations, the temperature was elevated to bring the mobile phase to the supercritical condition. Improved efficiency was obtained in SFC, whereas resolution and selectivity were worse. The use of EFLC in reversed-phase chiral separations was also tested. Enantiomer resolution improved under the EFLC condition. For the tested methanol/H(2)O mixture, fluoroform provided more significant improvements in chromatographic performance than CO(2) when used as a fluidity enhancing liquid. The use of EFLC instead of HPLC also caused a markedly lower pressure drop across the column for commonly used flow rates. The low-pressure drop will allow the use of longer columns or multiple columns to increase the total efficiency of the separation. Since chiral columns are often inefficient, this attribute may be very important for chiral separations.     

Molecular-level comparison of alkylsilane and polar-embedded reversed-phase liquid chromatography systems

Stationary phases with embedded polar groups possess several advantages over conventional alkylsilane phases, such as reduced peak tailing, enhanced selectivity for specific functional groups, and the ability to use a highly aqueous mobile phase. To gain a deeper understanding of the retentive properties of these reversed-phase packings, molecular simulations were carried out for three different stationary phases in contact with mobile phases of various water/methanol ratios. Two polar-embedded phases were modeled, namely, amide and ether containing, and compared to a conventional octadecylsilane phase. The simulations show that, due to specific hydrogen bond interactions, the polar-embedded phases take up significantly more solvent and are more ordered than their alkyl counterparts. Alkane and alcohol probe solutes indicate that the polar-embedded phases are less retentive than alkyl phases for nonpolar species, whereas polar species are more retained by them due to hydrogen bonding with the embedded groups and the increased amount of solvent within the stationary phase. This leads to a significant reduction of the free-energy barrier for the transfer of polar species from the mobile phase to residual silanols, and this reduced barrier provides a possible explanation for reduced peak tailing.     

Identification and quantification of 77 pesticides in groundwater using solid phase coupled to liquid-liquid microextraction and reversed-phase liquid chromatography

This paper describes a method for the extraction, separation, identification, and quantification of 77 pesticides (neutral, acidic, and basic) including some s-triazine metabolites. The method is appropriate for organically (e.g. with humic acids) highly loaded groundwater samples. A comparative study of a pH-controlled mixed solid phase (LiChroprep RP18/LiChrolut EN) extraction with different desorption solvents (acetonitrile or acetonitrile and dichloromethane/methanol) is elaborated. A subsequent liquid-liquid microextraction reduces matrix effects. The pesticides in the sample are separated using RP-HPLC, detected, and identified by diode array. The efficiency is illustrated on a natural groundwater sample from a phreatic aquifer.     

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.     

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.