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.     

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.     

Introducing Enantioselective Ultrahigh-Pressure Liquid Chromatography (eUHPLC): Theoretical Inspections and Ultrafast Separations on a New Sub-2-μm Whelk-O1 Stationary Phase

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.     

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.     

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.     

Nonaqueous capillary electrochromatographic separation of synthetic neutral polymers by size exclusion chromatography using polymeric stationary phases

In this paper, we report the separations of large, neutral, synthetic polymers using primarily a nonaqueous mobile phase without the use of a supporting electrolyte. The size- exclusion-based mechanism for separation was achieved on sulfonated polystyrene/divinylbenzene stationary phases. The effect of water, voltage, stationary phase exchange capacity, and pore size were investigated. The stationary phase and solvent interactions were studied by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) and a possible mechanism for the generation of EOF in the THF/water system is provided. Linear calibration curves were obtained for polystyrenes ranging in MW from 5K to 2M, for columns made using a combination of high capacity ion exchanger and a neutral polystyrene/divinylbenzene material of varied pore sizes. Analysis of polyurethane, polystyrene, and other polymer samples using CEC correlated well with results obtained by conventional HPLC. The size exclusion CEC separations provide an alternative mode for determining the relative molecular weights of polymers, with reduced solvent consumption.     

Analysis of bile acids and their conjugates by capillary electrochromatography/electrospray ion trap mass spectrometry

Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Nanoparticle-based continuous full filling capillary electrochromatography/electrospray ionization-mass spectrometry for separation of neutral compounds

Highly efficient reversed-phase capillary electrochromatography (CEC) separations (plate numbers up to 700 000/m), with electrospray ionization mass spectrometry detection were achieved utilizing novel dextran-coated polymer nanoparticles as a pseudostationary phase. A continuous full filling (CFF) technique in which nanoparticles are continuously introduced into the capillary was employed for separation of neutral analytes (dialkyl phthalates), utilizing an orthogonal electrospray interface to prevent nanoparticles from entering the mass spectrometer. CFF-CEC benefits from that an entirely fresh column is employed for every analysis, avoiding carryover effects associated with stationary-phase contamination. The highly efficient separations obtained were accomplished by optimizing the organic modifier concentration in the electrolyte and by using a high nanoparticle concentration (5 mg/mL), to improve interparticle mass transfer and gain sufficient retention. Nanoparticles, with an average diameter of 600 nm, were prepared by polymerization of methacrylic acid and trimethylolpropane trimethacrylate, which in turn were coated with dextran. These nanoparticles formed stable suspensions in electrolytes having broad ranges of polarities, enabling straightforward optimization of the reversed-phase conditions.     

Gold nanoparticle-modified etched capillaries for open-tubular capillary electrochromatography

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.     

High-Performance Membrane Chromatography of Small Molecules

High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.     

Hydrophilic interaction chromatography for mass spectrometric metabonomic studies of urine

High-performance liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly being used for urinary metabonomic studies. Most studies utilize reversed-phase separation techniques, which are not suited to retaining highly polar analytes. Metabonomic studies should encompass a representative "fingerprint" that contains the largest amount of information possible. In this work, we have analyzed human urine samples with LC-MS, comparing traditional reversed-phase separation with hydrophilic interaction chromatography (HILIC), using both positive and negative electrospray ionization modes. The resulting data were analyzed using principal components analysis and partial least-squares-discriminant analysis. Discriminant models were developed for the response variables gender, diurnal variation, and age and were evaluated using external test sets to classify their predictive ability. The developed models using both positive and negative ionization mode data for reversed-phase and HILIC separations were very comparable, indicating that HILIC is a suitable method for increasing the fingerprint coverage for LC-MS metabonomic studies.     

High-performance polymer-based monolithic capillary column

In this report, we introduce a new entry of high-performance polymer-based monolithic capillary column for mainly small molecules. This capillary column was prepared using a newly introduced epoxy monomer with diamines. Simply heat-induced polycondensation in an appropriate porogenic solvent afforded a really homogeneous co-continuous monolithic structure having submicrometer-size skeletons with micrometer-size through-pores. We were also able to prepare chiral monolithic columns using a chiral epoxy monomer as well as a chiral diamine. A 21.5-cm-long, 100-mum-i.d. column afforded up to 40 000 theoretical plate numbers (N) for alkylbenzenes in 60% aqueous acetonitrile as a reversed-phase-mode stationary phase. Due to a quite low column pressure drop, a 150-cm-long column was prepared. This long column afforded up to 200 000 plates for alkylbenzenes with only a 4-MPa column pressure drop. In contrast, in 100% acetonitrile, this column has "HILIC" property to show up to 60 000 plates for methanol with a 17.5-cm-long column. In this mode, we were able to separate nucleic acids. In addition, we have prepared a chiral column with both of the chiral epoxy monomers and an amine. This column was able to chirally discriminate a racemic alcohol in a reversed-phase mode.     

Capillary zone electrophoresis in nonaqueous solvents in the presence of ionic additives

Capillary zone electrophoresis (CZE) in nonaqueous media and in the presence of ionic additives has been successfully applied to the determination of compounds that differ only slightly in their electrophoretic mobilities. Triazine herbicides of environmental interest were chosen as test compounds because they behave as very weak bases. CZE separation of these analytes (especially chlorotriazines) in aqueous solution is difficult due to the low pH required for their conversion into protonated cationic form (HA(+)). However, in mixed nonaqueous solvents, 50% (v/v) acetonitrile-methanol, the acid-base characteristics of these compounds are modified, yielding the protonated ionic species that is susceptible to migration when subjected to an electric field. A noteworthy increase in separation selectivity and resolution can be achieved by using ionic additives. Thus, in this mode of capillary zone electrophoresis, separation is based on ionic interactions between the charged analytes and the ionic additive present in the separation medium. These interactions contribute to enhancing mobility differences and to improving analyte separation. For the separation of chloro- and methylthiotriazines, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol and 20 mM SDS proved to be satisfactory, providing high resolution in short analysis times. The selectivity achieved was found to depend on the degree of association of the analyte with the ionic additive in the nonaqueous medium. This permits manipulation of the selectivity of the electrophoretic separations as a function of the type and concentration of the ionic additive and of the nature of the nonaqueous medium employed.     

圆板轴对称弯曲振动的模态声辐射效率

采用将声强对辐射面积分的方法获得轴对称弯曲振动圆板在固定、简支及自由边界条件下的模态辐射声功率,进而获得其模态声辐射效率.采用高斯-勒让德数值积分法计算出固定、简支及自由边界条件下圆板的模态声辐射效率的数值解,并将同阶模态三种不同边界条件下的圆板声辐射效率进行了对比.结果表明:(1)圆板的模态声辐射效率在全频段内大体呈...     

Silica xerogel as a continuous column support for high-performance liquid chromatography

A preliminary study of the chromatographic performance and permeability of a continuous silica xerogel column under reversed-phase HPLC conditions was performed. A porous chromatographic support was synthesized inside a 0.32 mm i.d. × 13 cm fused silica tube from potassium silicate solution and derivatized with dimethyloctadecylchlorosilane. The plate height at 0.01 cm/s (0.5 μL/min), near the apparent optimum linear velocity, was about 65 μm. The column efficiencies in terms of numbers of plates per meter were 5000 and 13?000 for ethyl benzoate (k = 0.8) and naphthalene (k = 2.0), respectively, at 0.5 μL/min. The major parameter affecting column efficiency was the heterogeneous morphology of the xerogel, modifications to which are expected to improve chromatographic performance. The column provided efficiencies comparable to those reported for continuous polymeric columns but less than that previously reported for a continuous silica column. Gradient elution mode was demonstrated with a mixture of polycyclic aromatic hydrocarbons. The column was highly permeable, exhibiting a linear dependence of pressure to flow rate and a back pressure of only 632 psi at 10 μL/min when a 95% aqueous mobile phase was used.     

Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass spectrometry

In this study, we report a novel procedure for fabricating internally tapered capillary columns suitable for the coupling of capillary electrochromatography (CEC) to electrospray mass spectrometry (ESI-MS). The internal tapers were prepared by slowly heating the capillary end in a methane/O2 flame. Due to continuous self-shrinking of the inner channel of the capillary, the inside diameter of the opening was reduced to 7-10 microm. The procedure is easy to handle, with no requirement for expensive equipment as well as elimination of problematic grinding of the tip. Several advantages of these new internal tapers, as compared to using externally tapered columns, are described. First, the problems of poor durability and tip breakage associated with external tapering were successfully overcome with the internal taper. A comparison of the online CEC/ESI-MS between external versus internal tapers showed that the latter provides enhanced electrospray stability, resulting in significantly lower short-term noise and very short-term noise values. In turn, the more rugged design of internal tapers allows performing CEC/MS utilizing a harsh polar organic mobile phase, which was not previously successful using an external taper due to higher operating current and electrospray arcing. Next, data on the reproducibility of the internally tapered CEC/MS column using warfarin and beta-blockers as model analytes are presented. For example, when comparing the reproducibility for separation of warfarin under reversed-phase conditions, the internal taper demonstrated superior intraday % RSD (1.6-3.4) as compared to the external taper intraday % RSD (5-6). Last, the applicability of performing quantitative CEC/MS with internally tapered capillaries is demonstrated for simultaneous enantioseparation of beta-blockers. Impressive quantitative results include good linearity of calibration curves (e.g., R2 = 0.9940-0.9988) and limit of detection as low as 30 nM. The sensitive detection of a minor impurity of one enantiomer at the 0.1% level in a major chiral entity buttresses the suitability of compliance with FDA guidelines.     

Photoinduced polymerization for entrapping of octadecylsilane microsphere columns for capillary electrochromatography

A novel fritless capillary column for capillary electrochromatography (CEC) has been developed. The ODS microspheres were packed into a capillary and were then immobilized within an organic polymer prepared in situ through a photopolymerization process. The entrapment conditions were investigated to minimize the effect of the polymer matrix on the chromatographic properties of the packing material. The organic polymer matrix in the microsphere-packed column functions to link microspheres at specific sphere-sphere and sphere-capillary contact points. CEC separations of a PAH test mixture using entrapped columns with different UV illumination times were compared in terms of retention factor and separation efficiency. The optimized entrapped column demonstrated better chromatographic performance than similarly packed columns with conventional inlet and outlet frits. The electrochromatographic separations of hormones and peptides were also demonstrated on entrapped ODS columns.     

Amino acid bound surfactants: a new synthetic family of polymeric monoliths opening up possibilities for chiral separations in capillary electrochromatography

By combining a novel chiral amino-acid surfactant containing an acryloyl amide tail, a carbamate linker, and a leucine headgroup of different chain lengths with a conventional cross-linker and a polymerization technique, a new "one-pot" synthesis for the generation of amino-acid based polymeric monolith is realized. The method promises to open up the discovery of an amino-acid based polymeric monolith for chiral separations in capillary electrochromatography (CEC). The possibility of enhanced chemoselectivity for simultaneous separation of ephedrine and pseudoephedrine containing multiple chiral centers and the potential use of this amino-acid surfactant bound column for CEC and CEC coupled to mass spectrometric detection are demonstrated.     

Simultaneous concentration and separation of enantiomers with chiral temperature gradient focusing

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.