Enantiomeric separations using bovine serum albumin immobilized on ion-exchange stationary phases.

Bovine serum albumin (BSA) can be readily immobilized on ion-exchange stationary phases by frontal analysis of a proper solution. This provides a simple means of adjusting the amount of BSA contained in the column and of measuring it accurately. Although the immobilization is ionic and not covalent, the columns are stable for extensive periods of time. If needed, they can be easily regenerated by the same frontal analysis procedure.     

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.     

Latex-coated polymeric monolithic ion-exchange stationary phases. 2. Micro-ion chromatography

Latex-coated monolithic polymeric stationary phases are used for micro-ion chromatography (mu-IC) of inorganic anions. Monolithic columns were prepared by the in situ polymerization of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid within fused-silica capillaries of varying internal diameters. Introduction of ion-exchange sites was achieved by coating the anionic polymeric monolith with either Dionex AS10 or Dionex AS18 quaternary ammonium functionalized latex particles to give total ion-exchange capacities in the range 9-24 nequiv for a 30-cm column. The resultant mu-IC columns were used for the separation of anionic analytes using chloride or acetate as the eluent-competing ion and direct UV spectrophotometric detection at 195 nm or using hydroxide as the eluent-competing ion and suppressed or nonsuppressed contactless conductivity detection. Separation efficiencies of 13,000 plates/m were observed (for iodate), and separation efficiency was maintained for large increases in flow rate (up to 42 microL/min, corresponding to a linear flow velocity of 18.5 mm/s), enabling highly reproducible, rapid separations to be achieved (seven analyte anions in less than 2 min). Use of a hollow fiber micromembrane suppressor enabled effective suppression of hydroxide eluents over the range 0.5-5.0 mM, thereby permitting suppressed conductivity detection to be performed. However, the relatively large size of the suppressor resulted in reduced separation efficiencies (e.g., 5400 plates/m for iodate). Detection limits obtained with suppressed conductivity detection were in the range 0.4-1.2 microM.     

Covalently bound ionene polyelectrolyte-silica gel stationary phases for HPLC

Micelle-mimetic ionene-based stationary phases for high-performance liquid chromatography (HPLC) are prepared by attaching [3,16]- and [3,22]-ionenes to aminopropyl silica through a carbon-nitrogen bond. These [x,y]-ionenes are polyelectrolytic molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups, some of which can form aggregate species whose properties mimic those of conventional surfactant micelles. These ionene-bonded stationary phases were characterized using different recommended HPLC test mixtures. Test solute chromatographic behavior on the ionene phases was found to be similar to that of intermediate oligomeric or polymeric C-18 and/or phenyl phases, depending upon the specific test mixture employed. In addition, the phases exhibit significant solute shape recognition ability. The ionene stationary phases were successfully employed for the separation of the components of the recommended ASTM reversed-phase test mixture, as well as for ortho-, meta- and para-disubstituted benzenes and other positional or geometric isomeric compounds. The ionene materials allow for chromatographic separations under either reversed-phase or ion-exchange conditions. The retention mechanism on these multimodal phases can occur by hydrophobic partitioning or electrostatic interactions, depending upon the characteristics of the components of the analyte mixture (neutral or anionic). The effects of alteration of the percent organic modifier, flow rate and temperature of the mobile phase on chromatographic retention and efficiency on these phases were briefly examined.     

Light-scattering studies of packed stationary phases for capillary electrochromatography

Multiply scattered light through turbid media, packed particles, or compressed powders will inherently have a significantly longer optical path length than that of light which is not scattered. The concept of using the multiply scattered light potentially generated in the packed stationary phase of a capillary electrochromatography (CEC) column for enhanced detection as a result of its increased optical path length was examined. Ultraviolet (UV) light at 365 nm or laser light at 635 nm was focused to a small spot onto the packed section of a 3 microns spherisorb ODS1 CEC column (100 microns i.d.). The light was transported inside the capillary, and an image of the multiply scattered light several millimeters along the capillary was collected using a charged-couple device detector. Even if the spot size was less than 100 microns in diameter, evidence of light scattering was observed at a detection spatial off-set distance of 1-2 mm from the illumination point. When the calcium channel blocking drug felodipine was flushed through the column, the light intensity value dropped (increase in absorbance) to a greater degree at a spatial off-set (1.5 mm) than at the illumination point. The greater absorbance values at the spatial off-set were examined experimentally when felodipine was eluted from the column in the CEC mode in 6 min using MeCN/50 mM TRIS (pH 8.0) (80:20, v/v) at an applied voltage of 300 V/cm and an injection time of 2 s at 10 kV. A factor of 8.5 increase in absorbance was observed at a spatial off-set of 1 mm compared to the value obtained at the illumination point. An efficiency value of approximately 234,000 plates m-1 was obtained for this higher felodipine peak. Higher noise values, however, were also observed with this increase in absorbance. Using a spectrophotometer or an open capillary to obtain reference values for optical length, it was possible to estimate the average optical path length of light traveled through the packed stationary phase when transmitted at a spatial off-set. It was concluded that, although an increase in absorbance of 8.5 was observed at a spatial off-set, this most likely arises from the light being "redirected" and scattered in a straightforward fashion along the capillary. It was expected that if substantial multiple scattering did occur inside the packed stationary phase, a significantly larger absorbance increase would be attained. A number of proposals are thus given to explain the relatively low degree of multiple scattering in this stationary phase and suggestions offered on means to attain even higher absorbance increases at a spatial off-set. Additional potential applications are also discussed.     

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.     

High-stability ionic liquids. A new class of stationary phases for gas chromatography

Room-temperature ionic liquids are a class of non-molecular ionic solvents with low melting points. Their properties have the potential to be especially useful as stationary phases in gas-liquid chromatography (GLC). A series of common ionic liquids were evaluated as GLC stationary phases. It was found that many of these ionic liquids suffer from low thermal stability and possess unfavorable retention behavior for some classes of molecules. Two new ionic liquids were engineered and synthesized to overcome these drawbacks. The two new ionic liquids (1-benzyl-3-methylimidazolium trifluoromethanesulfonate and 1-(4-methoxyphenyl)-3-methylimidazolium trifluoromethanesulfonate) are based on "bulky" imidazolium cations with trifluoromethanesulfonate anions. Their solvation characteristics were evaluated using the Abraham solvation parameter model and correlations made between the structure of the cation and the degree to which the ionic liquids retain certain analytes. The new ionic liquids have good thermal stability up to 260 degrees C, provide symmetrical peak shapes, and because of their broad range of solvation-type interactions, exhibit dual-nature selectivity behavior. In addition, the ionic liquid stationary phases provided different retention behavior for many analytes compared to a commercial methylphenyl polysiloxane GLC stationary phase. This difference in selectivity is due to the unique solvation characteristics of the ionic liquids and makes them very useful as dualnature GLC stationary phases.     

Structure-function relationships in high-density docosylsilane bonded stationary phases by Raman spectroscopy and comparison to octadecylsilane bonded stationary phases

Raman spectroscopy is used to investigate the effects of temperature, surface coverage, polymerization method (surface or solution polymerized), and nature of the alkylsilane precursor on alkyl chain conformational order in a series of high-density docosylsilane (C22) stationary phases at surface coverages ranging from 3.61 to 6.97 mumol/m(2). The results of this study contribute to an enhanced understanding of the shape-selective retention characteristics of these phases at a molecular level. Conformational order is evaluated using the intensity ratio of the antisymmetric and symmetric nu(CH2) modes as well as the frequency at which these modes are observed. Alkyl chain order is shown to be dependent on surface coverage, alkyl chain length, and polymerization method. In general, alkyl chain order increases with surface coverage. Temperature-induced changes are observed between 250 and 350 K for the three phases with surface coverages between 3.61 and 4.89 mumol/m(2). These changes occur over a broad range of temperatures characteristic of two-dimensional systems, but in general adhere to the behavior predicted for a simple first-order transition. This change is not believed to be an abrupt cooperative disassociation characteristic of a phase transition in a bulk phase, but instead is thought to involve significant changes in conformational order in segments of the surface-tethered molecules, mostly those segments at the outer edge of the alkylsilane. In contrast to the changes observed in coverages below 5 mumol/m(2), a first-order change is not observed for the stationary phase with coverage of 6.97 mumol/m(2). A molecular picture of the temperature-induced disorder is proposed with disorder originating at the distal carbon and propagating only a short distance toward the proximal carbon. A comparison is made between these C22 stationary phases and similar high-density octadecylsilane (C18) bonded phases.     

Highly cross-linked self-assembled monolayer stationary phases: an approach to greatly enhancing the low pH stability of silica-based stationary phases

A new type of silica-based stationary phase with dramatically improved acid stability compared to any currently available silica-based stationary phase has been developed. Superior low pH stability is achieved by first self-assembling a densely bonded monolayer of (chloromethyl)-phenylethyltrichlorosilane (CMPES). The self-assembly step is followed by a Friedel-Crafts cross-linking of the reactive moieties with their neighbors, by addition of secondary, cross-linkable aromatic reagents, or by both. This phase is not endcapped. Elemental analysis data shows that an aluminum chloride catalyst is very effective at bonding aromatic cross-linking reagents, such as styrene heptamer and triphenylmethane, to the reactive CMPES monolayer. The stability of the retention factor of decylbenzene on the cross-linked self-assembled CMPES phases is compared to a sterically protected C18 phase to illustrate its superior resistance to acid-catalyzed-phase loss. Inverse size exclusion chromatography and flow-curve comparisons of the cross-linked self-assembled CMPES and the sterically protected C18 stationary phases illustrate their similar chromatographic efficiency.     

Supercritical fluid generated stationary phases for liquid chromatography and capillary electrochromatography

Chromatographic silica-bonded stationary phases have been prepared using supercritical CO(2) as the reaction medium. (29)Si solid-state NMR spectra of the generated bonded silica phases show unreacted silica species Q(3) and Q(4), alongside important resonances for surface-bound ligands, T(1), T(2), and T(3). Initially, a fluorinated octyl silica (C(8)) phase was produced, by reacting (1)H,(1)H,(2)H,(2)H-perfluorooctyltriethoxysilane with silica particles (3 microm) in sc-CO(2) at 60 degrees C and 450 atm for 3 h. In-house-packed LC columns of this fluorinated sc-C(8) silica phase yielded typical reversed-phase behavior when a standard test mix of benzamide (k' = 1.03), benzophenone (k' = 8.11), and biphenyl (k' = 14.92) was eluted. When packed into fused-silica capillaries for CEC, this fluorinated sc-C(8) silica phase gave linear plots of log k' versus percentage MeOH for benzophenone and biphenyl and, in contrast to octyl or octadecyl silica phases, displayed selectivity for aromatic thioureas when chromatographed among a series of synthetic organic thiourea test solutes. Similarily, an octadecyl silica phase (sc-C(18) silica) was prepared by reaction of n-octadecyltriethoxysilane in sc-CO(2), packed at 9500 psi and examined by LC. The sc-C(18) silica LC column yielded high column efficiency (up to 141 000 N/m (fluorene)) and excellent asymmetry factors (1.06, fluorene) without resource to end-capping. Following a second silylating or end-capping step using hexamethyldisilazane in sc-CO(2), sc-end-capped sc-C(18) silica phases elute N,N-DMA before toluene and the toluidine isomers as a single peak, indicating lowered silanol activity according to the Engelhardt test. A rapid separation of the important pharmaceutical substances, ketoprofen, naproxen, fentoprofen, and ibuprofen, on an sc-end-capped sc-C(18) silica phase is also shown.     

Development of engineered stationary phases for the separation of carotenoid isomers

A variety of bonded phase parameters (endcapping, phase chemistry, ligand length, and substrate parameters) were studied for their effect on column retention and selectivity toward carotenoids. Decisions were made on how each of these variables should be optimized based on the separation of carotenoid and polycyclic aromatic hydrocarbon test probes. A column was designed with the following properties: high absolute retention, enhanced shape recognition of structured solutes, and moderate silanol activity. These qualities were achieved by triacontyl (C30) polymeric surface modification of a moderate pore size (approximately 20 nm), moderate surface area (approximately 200 m2/g) silica, without subsequent endcapping. The effectiveness of this "carotenoid phase" was demonstrated for the separation of a mixture of structurally similar carotenoid standards, an extract of a food matrix Standard Reference Material, and a beta-carotene dietary supplement under consideration as an agent for cancer intervention/prevention.     

Retention of ions on nonporous charged stationary phases

The interactions between ion-exchange resins and counterions consist of several mechanisms, such as ion-pair formation between active sites and counterions, specific adsorption, solvation changes, and double-layer accumulation. The double-layer accumulation of ions, which is a typical nonstoichiometric mechanism, is an important factor governing overall ion-exchange chromatographic retention when a major part of the stationary-phase surface is in contact with eluent flows. Nonporous stationary phases, where solutes are accessible to the surfaces by convection as well as by diffusion, possibly highlight this nonstoichiometric contribution through the coupling of a flow profile with an electrostatic potential function. The retention of ions on nonporous stationary phases has been interpreted by a model derived on the basis of the Poisson-Boltzmann equation including solvation change terms. Unusual retention behaviors have been confirmed only for anions, and can be explained by the model including the assumption that anions undergo solvation changes in a thin layer (approximately 5 nm thickness) at the vicinity of the stationary phase; the thickness should be a function of eluent flow rates. This strongly suggests that there is a difference in solvation nature between cations and anions. It can be inferred that water molecules interacting with polymer domains of the stationary phase behave like single molecules and cannot form a stable hydration shell around an anion as usually seen in bulk solution.     

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.