Simultaneous separation of acidic, basic, and neutral organic compounds, including strong and moderate acids and bases, by capillary electrochromatography

The separation of strongly basic, moderately basic, weakly basic, strongly acidic, moderately acidic, weakly acidic, and neutral compounds in a single run using capillary electrochromatography (CEC) is presented. This is accomplished using a 3-μm CEC Hypersil C8 capillary with high organic content acetonitrile/phosphate (pH 2.5) mobile phases containing hexylamine. Fifteen basic, acidic, and neutral drugs of forensic interest are resolved using a step gradient. Strong and moderately basic drugs separate before t(o), apparently by a combination of free zone electrophoresis (CZE) and chromatographic phenomena. Weak bases separate after t(o), also by a combination of CZE and chromatographic processes. Due to large selectivity differences between CEC and CZE for bases, there is evidence that the stationary phase is playing a significant role in the separation of these solutes. The CEC approach presented offers unique selectivity, expanded peak capacity, and the ability to solubilize both hydrophilic and hydrophobic solutes in an injection solvent that is compatible with the chromatographic system.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Reversed phase liquid chromatography of organic hydrocarbons with water as the mobile phase

Reversed phase high-performance liquid chromatography (RP-HPLC) is demonstrated for hydrophobic analytes such as aromatic hydrocarbons using only water as the mobile phase. Achievement of reasonable capacity factors for these types of compounds without the need for toxic and costly organic modifiers in the mobile phase is accomplished by substantially decreasing the phase volume ratio of stationary phase relative to the mobile phase volume and by increasing the polarity of the stationary phase relative to stationary phase materials commonly used for RP-HPLC. Applying a stationary phase of trifluoropropylsiloxane, which is a common gas chromatographic stationary phase material, to nonporous glass microspheres yields a stationary phase with a phase volume ratio reduced by about 2 orders of magnitude as compared to common liquid chromatographic packing materials. As a result, a separation was obtained for hydrophobic organic analytes such as benzene, toluene, ethylbenzene, and isopropylbenzene using a water mobile phase at ambient temperature. A separation of sodium benzoate, benzaldehyde, benzene, and butyrophenone is shown in less than 3 min using a water mobile phase and UV/visible absorbance detection. Additionally, the separation of the ionic surfactant species octyl sulfate and dodecyl sulfate in water in less than 3 min, using unsuppressed conductivity detection, is achieved with a separation mechanism based on interactions with the hydrophobic portion of the surfactant. A water mobile phase offers many potential advantages over traditional mixed aqueous/organic solvent systems. In addition to saving on the cost and expense of buying and disposing of toxic solvents and waste, there is less exposure of the operator to potentially harmful solvents. Increased consistency in reproducing retention times can be expected, since there will not be any variability in solvent strength due to slight variations in mobile phase composition. A water mobile phase produces an environment that should provide an inherent advantage of increased signal-to-noise ratio for detection. Additionally, excellent predictions of the octanol/water partitioning coefficient and aqueous solubility for hydrophobic analytes are obtained from a single measurement of the capacity factor in the water mobile phase.     

Polymeric porogens used in the preparation of novel monodispersed macroporous polymeric separation media for high-performance liquid chromatography.

A novel approach to monosized macroporous polymeric separation media with vastly enhanced pore size distributions and chromatographic properties has been developed. Key to this approach is the combined use of monodispersed polymeric particles and suitable solvents as porogens in the copolymerization of styrene and divinylbenzene. Following polymerization, the polymeric porogen is dissolved, leaving behind the monosized beads with a controlled pore structure. The exact pore size and pore size distribution of the final beads are largely controlled by the amount of soluble polymer in the polymerizing mixture: the larger the proportion of soluble polymer in the system, the larger the pores. The uniformly sized macroporous beads prepared with an optimized ratio of polymeric and low molecular weight porogens proved to be very efficient even in short columns for the separation of polystyrene standards in the SEC mode and the separation of proteins in the reversed-phase mode. The relationship between pore size and specific surface area, on one hand, and chromatographic properties of the stationary phase, on the other, have been clearly documented.     

Evaluation of a vancomycin chiral stationary phase in capillary electrochromatography using polar organic and reversed-phase modes

A vancomycin chiral stationary phase (CSP) was fully evaluated in capillary electrochromatography (CEC) in reversed-phase and polar organic modes for a number of racemic pharmaceutical compounds. High efficiency and resolution values were obtained for a number of compound classes including thalidomide in both the polar organic mode (190000 plates meter(-1) and Rs = 13.8) and reversed-phase mode (125000 plates meter(-1) and Rs = 13.0). Experimental parameters, including organic modifier, organic solvent ratio, ionic strength, pH, temperature, and voltage, were examined in both the aqueous and nonaqueous modes to deduce their effect on the resultant EOF, retention times, resolution, and efficiency of chiral separations. All results were consistent with and found to be a combination of what is known from existing literature on CEC theory and experience obtained with macrocyclic antibiotic CSPs in LC. Column stability was excellent, and each column packed was found to offer repeatable separations even when switching from the aqueous to the nonaqueous mode.     

Electrochromatography in microchips: reversed-phase separation of peptides and amino acids using photopatterned rigid polymer monoliths

A microfabricated glass chip containing fluidic channels filled with polymer monolith has been developed for reversed-phase electrochromatography. Acrylate-based porous polymer monoliths were cast in the channels by photopolymerization to serve as a robust and uniform stationary phase. UV light-initiated polymerization allows for patterning of polymer stationary phase in the microchip, analogous to photolithography, using a mask and a UV lamp for optimal design of injection, separation, and detection manifolds. The monoliths are cast in situ in less than 10 min, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer enables electroosmotic flow, allowing the separation channel to be conditioned without need for high-pressure pumps. The microchip was used for separation of bioactive peptides and amino acids labeled with a fluorogenic dye (naphthalene-2,3-dicarboxaldehyde) followed by laser-induced fluorescence detection using a Kr+ ion laser. The microchip-based separations were fast (six peptides in 45 s), efficient (up to 600,000 plates/m), and outperformed the capillary-based separations in both speed and efficiency. We have also developed a method for complete removal of polymer from the channels by thermal incineration to regenerate the glass chips.     

Capillary electrochromatography with fused-silica capillaries packed with copolymeric reversed-phase adsorbent and ion exchangers

Macroporous poly(styrene-divinylbenzene) (PSDVB), PRP-1, a reversed-phase adsorbent, and PSDVB-based strong acid cation exchangers and strong base and weak base anion exchangers were evaluated as stationary phases for capillary electrochromatography (CEC). Electroosmotic flow (EOF) for adsorbent and exchanger packed fused-silica capillaries for acetone as the marker increases with increasing ion exchange capacity, buffer organic solvent concentration, and applied voltage, is nearly independent of pH, and decreases with increased buffer ionic strength. For anion exchangers, EOF is reversed. Thiourea, acetone, acrylamide, nitromethane, propanal, and acetic acid were evaluated as EOF markers and undergo weak interaction with the PSDVB-based stationary phases. EOF in a basic buffer is greater than or equal to silica-based C-18 and cation exchanger packed capillaries. For an acidic buffer, EOF for a PRP-1 capillary is almost twice the C-18 packed capillary. As analyte hydrophobicity increases, retention and migration time increases for the PSDVB-based stationary phases. As exchange capacity increases, availability of the polymeric matrix for analyte partitioning decreases, causing analyte migration time to decrease. Increasing buffer organic solvent concentration decreases analyte retention. The PSDVB-based stationary phases provide good resolving power and reproducibility and are applicable to the CEC separation of neutral, weakly acidic, and basic analytes. Efficiency, however, is less than obtained with silica-based stationary phases. Because of stability in a strong acid buffer, the CEC separation of weak acids, where dissociation is suppressed, and weak bases as cations is possible. Separations of short-chain alkyl aldehydes, methyl ketones, aromatic hydrocarbons, substituted benzene derivatives, and short-chain carboxylic acids are described.     

Tailoring the morphology of methacrylate ester-based monoliths for optimum efficiency in liquid chromatography

Methacrylate ester-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The influence of the composition of the polymerization mixture on the morphology was studied with mercury intrusion porosimetry, scanning electron microscopy, and nitrogen adsorption measurements. A high-density porous polymeric material with a unimodal pore-size distribution was prepared with 40 wt % monomers and 60 wt % solvent in the mixture. A low-density material, prepared with a 20:80 ratio of monomers versus pore-forming solvent, showed a bimodal pore-size distribution and a much finer structure than the high-density monolith. The characteristic pore size could be controlled by changing the ratio of pore-forming solvents. With increasing solvent polarity, both the pore size and the dimension of the globules increased. The best efficiency in the CEC mode was obtained with an average pore size of 600 nm. Low-density monoliths exhibited lower A- and C-terms than high-density monoliths. With the optimal monolithic material, a minimum plate height of 5 mum could be obtained. The low-density monolith also performed better in the HPLC mode, giving a minimum plate height of 15 mum and a much higher flow permeability than that of the high-density material.     

Polar polymeric stationary phases for normal-phase HPLC based on monodisperse macroporous poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads

The effect of variables such as shape template size, porogen composition and percentage, content of cross-linking monomer, and polymerization temperature on the properties of uniformly sized 3-microm porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads prepared by the staged templated suspension polymerization technique has been studied. The porous properties of the beads including surface morphology, pore size distribution, and specific surface area have been optimized to obtain highly efficient stationary phases for normal-phase HPLC. A column packed with diol stationary phase obtained by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads affords an efficiency of 67,000 plates/m for toluene using THF as the mobile phase. The retention properties and selectivity of the diol beads are easily modulated by changes in the composition of the mobile phase. The performance of these beads is demonstrated with the separations of a variety of polar compounds including positional isomers, aniline derivatives, and basic tricyclic antidepressant drugs.     

Capillary electrochromatography using a strong cation-exchange column with a dynamically modified cationic surfactant

A novel mode of capillary electrochromatography (CEC), called dynamically modified strong cation-exchange CEC (DMSCX-CEC), is described in this paper. A column packed with a strong cation-exchange (SCX) packing material was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX packing material, and the resulting hydrophobic layer on this packing was used as the stationary phase. Using the dynamically modified SCX column, neutral solutes were separated with the CEC mode. The highest number of theoretical plates obtained was about 190,000/m, and the relative standard deviations (RSD's) for migration times and capacity factors of alkylbenzenes were less than 1.0% and 2.0% for five consecutive runs, respectively. The effects of CTAB and methanol concentrations and the pH value of the mobile phase on the electroosmotic flow and the separation mechanism were investigated. Excellent simultaneous separation of the basic and neutral solutes in DMSCX-CEC with a high-pH mobile phase was obtained. A mixture containing the acidic, basic, and neutral compounds was well separated in this mode with a low-pH mobile phase; however, peak tailing for basic compounds was observed in this mobile phase.     

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.     

Stepwise Crosslinking: A Facile Yet Versatile Conceptual Strategy to Nanomorphology‐Persistent Porous Organic Polymers

Both high surface areas and well‐orchestrated nanomorphologies are important for porous organic polymers (POPs). However, the two key characteristics are generally difficult to be satisfied simultaneously, because the common pore‐making procedures usually produce ill‐defined nanomorphologies or give rise to damage of precustomized nanomorphologies. Herein, a facile yet versatile stepwise crosslinking strategy for fabrication of POPs with an unusual nanomorphology‐persistent characteristic during pore‐making is reported. Polystyrene nanofibers and poly(styrene‐co‐divinylbenzene) nanosphere arrays are utilized as building blocks, and then transformed into nanofibrillar morphology‐persistent and ordered array morphology‐persistent POPs via stepwise crosslinking, respectively. The stepwise crosslinking strategy includes pre‐crosslinking and hypercrosslinking; the pre‐crosslinking in a carefully selected poor solvent of polystyrene forms a lowly crosslinked structure, which guarantees the stability of nanomorphology during the subsequent pore‐making via hypercrosslinking. The as‐obtained POPs can be used as precursors for novel well‐defined hyperporous carbon nanofibers and ordered carbon nanosphere arrays with excellent adsorption performances.     

Capillary Electrokinetic Chromatography Employing p-(Carboxyethyl)calix[n]arenes as Running Buffer Additives

Calixarenes, a class of macrocyclic phenolic compounds with a basket-like shape, are used as capillary electrophoresis reagents for separations of native and substituted polycyclic aromatic hydrocarbons. The p-(carboxyethyl)calix[n]arenes reported herein are a series of charged, moderately water soluble macrocyclic molecules that can form complexes with neutral molecules. Electrokinetic chromatographic separations are based on the differential distribution of molecules between a running buffer phase, which is transported by electroosmotic flow, and an electrophoretically mediated calixarene. The size of the calixarene influences separation performance, illustrating the importance of cavity size and geometry in the complexation process. p-(Carboxyethyl)calix[7]arene provides the best efficiency (>10(5) plates/m) and selectivity in these studies. The influences of pH, organic solvent, and field strength on elution range, capacity factors, efficiency, and selectivity are also reported. In general, capacity factors are rather low, but the high charge-to-mass ratios of certain calixarenes produce relatively wide elution ranges. Molecular modeling data and solubility data are used to interpret the observed selectivity.     

Fabrication of graphene oxide nanosheets incorporated monolithic column via one-step room temperature polymerization for capillary electrochromatography

Graphene oxide (GO) has received great interest for its unique properties and potential diverse applications. Here, we show the fabrication of GO nanosheets incorporated monolithic column via one-step room temperature polymerization for capillary electrochromatography (CEC). GO is attractive as the stationary phase for CEC because it provides not only ionized oxygen-containing functional groups to modify electroendoosmotic flow (EOF) but also aromatic macromolecule to give hydrophobicity and π-π electrostatic stacking property. Incorporation of GO into monolithic column greatly increased the interactions between the tested neutral analytes (alkyl benzenes and polycyclic aromatics) and the stationary phase and significantly improved their CEC separation. Baseline separation of the tested neutral analytes on the GO incorporated monolithic column was achieved on the basis of typical reversed-phase separation mechanism. The precision (relative standard deviation (RSD), n = 3) of EOF was 0.3%, while the precision of retention time, peak area, and peak height for the tested neutral analytes were in the range of 0.4-3.0%, 0.8-4.0%, and 0.8-4.9%, respectively. In addition, a set of anilines were well separated on the GO incorporated monolith. The GO incorporated monolithic columns are promising for CEC separation.     

Shielded stationary phases based on porous polymer monoliths for the capillary electrochromatography of highly basic biomolecules

A novel stationary phase for capillary electrochromatography has been prepared via photoinitiated grafting of two layers of polymer chains onto the pore surface of a porous polymer monolith. To achieve the desired retention, the original monolith with optimized porous properties was grafted with an "interior" layer consisting of the ionizable monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid, followed by a "covering" layer of hydrophobic polymer chains. This technique affords monolithic CEC columns that facilitate electroosmotic flow (EOF) while preventing ionized analytes from interacting with the charged surface functionalities. Grafting of the second layer does not adversely affect the EOF. Grafting times of 30 and 60 s for AMPS and butyl acrylate, respectively, enabled the preparation of a monolith with full shielding of the analytes from the ionizable functionalities and excellent chromatographic performance. This approach allows for the first time the independent optimization of both electroosmotic flow and retention properties in CEC columns. The efficient isocratic separations of mixtures of peptides, including some that are highly basic and would be affected by unshielded charges, were routinely achieved in 40-90 s using a simple MS compatible mobile phase consisting of 20 mmol/L ammonium acetate in a 1:1 water-acetonitrile mixture.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 1. Optimization of polymerization conditions, porous properties, and chemistry of the stationary phase

Monolithic columns for chiral capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine , ethylene dimethacrylate, and glycidyl methacrylate or 2-hydroxyethyl methacrylate in the presence of mixture of cyclohexanol and 1-dodecanol as a porogenic solvent. The porous properties of the monolithic columns can easily be controlled through changes in the composition of the binary porogenic solvent. Although both thermal- and UV light-initiated polymerizations afford useful capillary columns, monoliths prepared using the former approach exhibit better chromatographic properties. The ability to control pore size independently of the polymerization mixture composition enables the preparation of monoliths with varying percentages of the chiral monomer and cross-linker, as well as the optimization of their separation properties. Very good separations of model racemate (R,S)-N-3,5-dinitrobenzoylleucine were achieved using an optimized monolithic CEC column, with high efficiencies of up to 74000 plates/m for the retained peaks.     

Ion-pair chromatographic separation of water-soluble gold monolayer-protected clusters

We demonstrate the efficacy of ion-pair chromatography for separations of samples of charged, polydisperse, water-soluble gold nanoparticles protected by monolayers of N-acetyl-l-cysteine and of tiopronin ligands. These nanoparticle mixtures have 1-2-nm-diameter Au core sizes as estimated from UV-visible spectra of the separated components. This size range encompasses the transition from bulk metal to molecular properties. The nanoparticle mixtures were resolved, the smallest nanoparticles eluting first, on an octadecylsilyl (C18) column using isocratic elution with a methanol/water mobile phase containing tetrabutylammonium fluoride (Bu4N+F-) and phosphate buffer. The column retention increases with Bu4N+F- concentration, lowered pH, and decreasing methanol volume fraction. The retention mechanism is dominated by ion-pairing in either the mobile phase or at the stationary/mobile-phase interface. Size exclusion effects, used in many previous nanoparticle separations, are insignificant.     

原位生成自引发单体合成超支化聚合物研究

以α-溴代苯乙烷(BEB)为引发剂,CuB r/2,2′-联二吡啶(BPY)为催化体系,对二乙烯苯(DVB)进行原子转移自由基聚合(ATRP)原位生成自引发单体合成超支化聚合物进行了研究。用1H-NM R、GPC、M ALLS分别对聚合反应过程和聚合物进行了表征和分析。结果表明,可以由双烯化合物原位生成自引发单体合成超支化聚合物,聚合物分子质量M-n.GPC在104以下,分子质量分布为2~4,表现出较宽的分子质量分布,以光散射法测定的聚合物绝对分子质量M-w.MALLS达105以上。     

Stochastic theory of size exclusion chromatography: peak shape analysis on single columns

The stochastic theory of size exclusion chromatography (SEC) was applied to analyze the peak shape of chromatograms obtained with a wide range of polystyrene standards on various columns. The columns were packed with stationary phases of different pore sizes. The stochastic-dispersive model of SEC results in a peak shape model that fits well the symmetrical and asymmetrical peaks observed in SEC. From the peak shape parameters obtained after nonlinear parameter estimation, information can be gained regarding the fundamental characteristics of the size exclusion process. When a series of polymer standards are analyzed on one column-in a manner similar to other methods of inverse chromatography-the stationary phase can be characterized. The dependence of the ingress and the egress processes on the relative size of the macromolecule to the pore size was determined. We found that for small molecules the selectivity in SEC arises from the ingress process, while when the size of the macromolecule is comparable to that of the pore-i.e., close to the exclusion limit-the egress process will also strongly affect the selectivity.     

Ultrafast gas chromatography on single-wall carbon nanotube stationary phases in microfabricated channels

The key to rapid temperature programmed separations with gas chromatography are a fast, low-volume injection and a short microbore separation column with fast resistive heating. One of the major problems with the reduction of column dimensions for micro gas chromatography is the availability of a stationary phase that provides good separation performance. In this report, we present the first integration of single-wall carbon nanotubes (SWNTs) as a stationary phase into 100 mum x 100 mum square and 50-cm-long microfabricated channels. The small size of this column with integrated resistive heater and the robustness of the SWNT phase allow for fast temperature programming of up to 60 degrees C/s. A combination of the fast temperature programming and the narrow peak width of small-volume injections that can be obtained from a high-speed, dual-valve injection system allows for rapid separations of gas mixtures. We demonstrate highly reproducible separations of four-compound test mixtures on these columns in less than 1 s using fast temperature programming.