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.     

Chiral stationary phase based on a biostable L-RNA aptamer

An immobilized anti-L-arginine d-RNA aptamer, used as a target-specific chiral stationary phase (CSP), was found to be very quickly degraded by RNases under usual chromatographic utilization and storage. To overcome this severe limitation for a practical use, a CSP based on the L-RNA aptamer, that is, the mirror image of the D-RNA aptamer, was created. It was shown that this mirror-image approach was a very simple and powerful strategy to develop a highly stable stationary phase due to the intrinsic insensitivity of l-RNA to the RNase degradation. In addition, such an approach allowed one to reverse the enantiomer elution order relative to that obtained with the corresponding d-RNA CSP.     

Application of cyclam-capped beta-cyclodextrin-bonded silica particles as a chiral stationary phase in capillary electrochromatography for enantiomeric separations

Two novel types of substituted cyclam-capped beta-cyclodextrin (beta-CD)-bonded silica particles have been prepared and used as chiral stationary phases in capillary electrochromatography (CEC). The two stationary phases have a chiral selector with three recognition sites: beta-CD, cyclam, and the latter's sidearm. They exhibit excellent enantioselectivities in CEC for a wide range of compounds as a result of the cooperative functioning of the anchored beta-CD and cyclam. After inclusion of the metal ion (Ni2+) from the running buffer into the substituted cyclams and their sidearm ligands, the bonded stationary phases become positively charged and can provide extra electrostatic interactions with ionizable solutes and enhance the dipolar interactions with some polar neutral solutes. This enhances the host-guest interaction with some solutes and improves chiral recognition and enantioselectivity. These new types of stationary phases exhibit great potential for fast chiral separations in CEC.     

Improvements in polymer characterization by size-exclusion chromatography and liquid chromatography at the critical condition by using enhanced-fluidity liquid mobile phases with packed capillary columns

Microscale chromatography has found numerous applications in liquid chromatography. The combination of enhanced-fluidity liquid mobile phases with packed-capillary LC is evaluated for polymer characterization using size-exclusion chromatography (SEC) and liquid chromatography at the critical condition (LCCC) phase. Separations of polystyrene polymers and copolymers are completed using liquid chromatography at the critical condition. The critical conditions of polystyrene polymers were approached by changing the concentration of CO(2) in the mixture combined with temperature and pressure variation. Because the solvent strength of enhanced-fluidity liquid mixtures is affected by temperature and pressure variation, the solvent strength could be fine-tuned to accurately find the critical condition. Long packed capillaries could be used in this application because the enhanced-fluidity mobile phases have low viscosities. High efficiencies resulted. The performance of packed-capillary and analytical-scale analytical columns containing the same packing material was compared for a challenging separation at the critical condition.     

Adsorption equilibria of benzodiazepines on a hybrid polymeric chiral stationary phase

The chromatographic behavior of a series of racemic benzodiazepines was evaluated under linear and nonlinear conditions on a new hybrid polymeric (DACH-ACR) chiral stationary phase (CSP). Differently substituted benzodiazepines were employed as probes to make hypotheses concerning possible molecular interaction mechanisms originating between target compounds and active sites on the CSP. Hydrogen bonds were found to be pivotal for chromatographic retention and chiral selectivity. The competitive effect from a mobile-phase (MP) modifier able to interact with the CSP through H-bonds was investigated. The performance of the polymeric DACH-ACR CSP for preparative purposes was also evaluated. The competitive adsorption isotherms of two benzodiazepines, lorazepam and temazepam, were measured at different MP compositions through the so-called inverse method. The adsorption data were fitted with a competitive bi-Langmuir adsorption isotherm. Enantiomeric separations under nonlinear conditions were modeled by using the equilibrium dispersive (ED) model of chromatography. Theoretical overloaded band profiles (obtained by solving the system of partial differential equations described by the ED model) matched, in a significantly accurate way, the profiles experimentally measured.     

Characterization of capillary-channeled polymer fiber stationary phases for high-performance liquid chromatography protein separations: Comparative analysis with a packed-bed column

Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of approximately 1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (alpha), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is < or =1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also < or =1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein separations and preparative-scale protein chromatography.     

Synergistic effects on enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids, bases, and amino acids by HPLC

In an attempt to overcome the limited applicability scope of earlier proposed Cinchona alkaloid-based chiral weak anion exchangers (WAX) and recently reported aminosulfonic acid-based chiral strong cation exchangers (SCX), which are conceptionally restricted to oppositely charged solutes, their individual chiral selector (SO) subunits have been fused in a combinatorial synthesis approach into single, now zwitterionic, chiral SO motifs. The corresponding zwitterionic ion-exchange-type chiral stationary phases (CSPs) in fact combined the applicability spectra of the parent chiral ion exchangers allowing for enantioseparations of chiral acids and amine-type solutes in liquid chromatography using polar organic mode with largely rivaling separation factors as compared to the parent WAX and SCX CSPs. Furthermore, the application spectrum could be remarkably expanded to various zwitterionic analytes such as alpha- and beta-amino acids and peptides. A set of structurally related yet different CSPs consisting of either a quinine or quinidine alkaloid moiety as anion-exchange subunit and various chiral or achiral amino acids as cation-exchange subunits enabled us to derive structure-enantioselectivity relationships, which clearly provided strong unequivocal evidence for synergistic effects of the two oppositely charged ion-exchange subunits being involved in molecular recognition of zwitterionic analytes by zwitterionic SOs driven by double ionic coordination.     

Chiral separations of polar compounds by hydrophilic interaction chromatography with evaporative light scattering detection

The chiral separations of drug substances and underivatized amino acids were demonstrated in this study through the use of hydrophilic interaction chromatography (HILIC). The polar character of the model compounds presented challenges for their analysis by traditional modes of chromatography, but through the employment of multimodal chromatography utilizing the HILIC mechanism and cyclodextrin- or teicoplanin-derivatized stationary phases, effective resolution was achieved. The analytes lacked sufficient ultraviolet chromophores, requiring their determination by evaporative light scattering detection. HILIC was demonstrated to represent a novel technique for the facilitation of chiral chromatography by providing an environment of solubility and retention that could not be achieved through the use of the traditional methods of reversed-phase, normal-phase, or polar organic mode.     

Thermal modulation for multidimensional liquid chromatography separations using low-thermal-mass liquid chromatography (LC)

We report on a proof-of-principle experiment with a novel thermal modulation device with potential use in two-dimensional liquid chromatography (LC × LC) systems. It is based on the thermal desorption concept used in two-dimensional gas chromatography (GC × GC) systems. Preconcentration of neutral analytes eluting from the first dimension column is performed in a capillary "trap" column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes is achieved by rapidly heating the trap column, by applying temperature ramps up to +1200 °C/min. Compared to the nonmodulated signal, the presented thermal modulator yielded narrow peaks, and a concentration enhancement factor up to 18 was achieved. With a thermally modulated LC separation of an epoxy resin, it is shown that when the thermal modulation is applied periodically, the trapped and concentrated molecules can be released periodically and that the modulating interface can both serve as a preconcentration device and as an injector for the second dimension column of an LC × LC setup. Because of the thermal modulation, a high-molecular-weight epoxy resin could be adequately separated and the different fractions were identified with a GPC analysis, as well as an offline second dimension LC analysis.     

Characterization of a thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase in enantioseparation of dihydropyrimidinone acid by quasi-equilibrated liquid chromatography and solid-state NMR

A thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) (i.e., Chiralpak AD) chiral stationary phase (CSP) in the enantioseparation of dihydropyrimidinone (DHP) acid racemate was studied for the first time by quasi-equilibrated liquid chromatography with cyclic van't Hoff and step temperature programs and solid-state ((13)C CPMAS and (19)F MAS) NMR using ethanol and trifluoroacetic acid (TFA)-modified n-hexane as the mobile phase. The conformational transition was controlled by a single kinetically driven process, as evidenced by the chromatographic studies. Solid-state NMR was used to study the effect of the temperature on the conformational change of the solvated phase (with or without the DHP acid enantiomers and TFA) and provided some viable structural information about the CSP and the enantiomers.     

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.     

Molecularly imprinted chiral stationary phase prepared with racemic template

The first example of molecularly imprinted chiral stationary phase prepared using a racemic template is shown. N-(3,5-Dinitrobenzoyl)-α-methylbenzylamine (DNB) was chirally discriminated on the molecularly imprinted stationary phase prepared using racemic DNB as the template. A chiral monomer, (S)-(-)-N-methacryloyl-1-naphthylethylamine, was utilized as the functional monomer toward the racemic template, and its chiral recognition ability was, interestingly, found to be enhanced through racemic molecular imprinting. A thermodynamic discussion briefly suggests that the observed chiral recognition ability of the racemic imprinting was proper value.     

Molecular dynamics study of chiral recognition for the whelk-O1 chiral stationary phase

In this article, we examine the docking of 10 analytes on the Whelk-O1 stationary phase. A proper representation of analyte flexibility is essential in the docking analysis, and analyte force fields have been developed from a series of B3LYP calculations. Molecular dynamics simulations of a representative Whelk-O1 interface, in the presence of racemic analyte and solvent, form the basis of the analysis of chiral selectivity. The most probable docking arrangements are identified, the energy changes upon docking are evaluated, and separation factors are predicted. From comparisons between the analytes, the mechanism of chiral selectivity is divided into contributions from hydrogen bonding, ring-ring interactions, steric hindrance, and molecular flexibility. We find that both hydrogen bonding and ring-ring interactions are necessary to localize the analyte within the Whelk-O1 cleft region. We also identify one docking mechanism that is often dominant and analyze the conditions that lead to alternate docking modes.     

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.     

An inductively coupled plasma carbon emission detector for aqueous carbohydrate separations by liquid chromatography

An inductively coupled plasma atomic emission spectrometer is used to detect carbon-containing compounds following separation by high-performance liquid chromatography. A calcium form ligand exchange column with distilled and deionized water as the mobile phase is used to separate carbohydrates. The eluting species are detected by monitoring the carbon atomic emission line at 193.09 nm. The mass detection limits using a photomultiplier tube for sucrose and glucose are 50 ng, while that for fructose is 60 ng. The carbon emission detector should provide the same detection limit for any compound with a similar mass percent of carbon, whether or not the compound exhibits appreciable absorption characteristics. While the carbon emission detector will universally detect any organic compound, it will discriminate against species with high molar absorptivity that may be present at low concentration. Such species may act as interferences in chromatograms generated with conventional UV-visible absorption detectors. To demonstrate the utility of the carbon emission detector, three sugars (glucose, fructose, sucrose) are determined in apple, crangrape, and orange juice.