Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were prepared by a simple one-step dispersion polymerization process. Examination of the polymeric microspheres showed that they had a mean particle diameter of 3 microm and dual pore size distribution with mean pore diameters of 300 and 800 A. The microspheres were functionalized by introducing quaternary ammonium/octadecyl groups to obtain positively charged beads in a wide pH range. The functionalized beads were packed into fused-silica capillary having 50 microm inner diameter and used to separate erythromycin derivatives by capillary electrochromatography (CEC). These samples require gradient elution when separated by high-performance liquid chromatography (HPLC) or micro-HPLC, but with the new columns isocratic elution suffices for their separation by CEC. The column efficiency ranged from 40,000 to 50,000 theoretical plates.     

Capillary electrochromatography of peptides on a column packed with tentacular weak cation-exchanger particles

Silica-based, tentacular weak cation-exchanger particles were prepared for use as the stationary phase in the separation of positively charged sample components by capillary electrochromatography (CEC). Silica beads were first silanized with 3-(trimethoxysilyl) propyl methacrylate that served as a heterobifunctional linker, which reacted with 2-acrylarmidoglycolic acid in a second step by radical polymerization in aqueous solution. Baseline separation of basic peptides with good column efficiency was obtained on packed capillary columns by isocratic elution CEC with NaCl as the mobile phase modulator. The retention mechanism in the electrochromatographic process was studied by examining the effect of salt concentration on the migration behavior of the peptides. The chromatographic retention factor k'(lc) for charged sample components in the electrochromatographic process was estimated on the assumption that the overall migration rate of a charged migrant can be taken as the sum of the rate of chromatographic elution and the rate of electrophoretic migration. The estimated k(lc) values from experimental results were plotted against the molal salt concentration on a double logarithmic scale. The linear correlation is in good agreement with the prediction by the theory on the basis of traditional ion-exchange chromatography. The comparison of CEC results, obtained with open tubular and packed capillary columns having the same retentive functions as the stationary phase, supports the notion that variation of the phase ratio in the column offers an additional means to modulate the electrochromatographic migration behavior.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Electrokinetically-driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography.

This paper examines protein ion-exchange behavior in electrokinetically-driven open-tubular chromatography with columns produced by immobilization of poly(aspartic acid) on capillary walls. Retention and selectivity are similar in the electrokinetic elution mode to that observed in HPLC. The separation mechanism was found to depend on the relationship of mobile phase pH to that of protein pI and ionic strength. Column efficiency in the electrokinetic elution mode was found to be 10-100-times higher than in HPLC. The best separations were achieved at intermediate ionic strength and high pH. The great advantage of these low-phase-ratio, high-efficiency open tubular columns is that isocratic separations in the electrokinetic elution mode were equivalent to gradient elution in the HPLC mode. Low phase ratio has the net effect of collapsing the chromatogram into a narrow elution window while the very high efficiency produces the requisite resolution.     

Comparative study of capillary electroendosmotic chromatography and electrically assisted gradient nano-liquid chromatography for the separation of peptides

Capillary electroendoendosmotic chromatography (CEC), being a hybrid of high-performance liquid chromatography (HPLC) and capillary electrophoresis, offers considerable changes to enhance column efficiency, speed of analysis and additional selectivity as compared to the parent methods. The analytes are driven by the electroendosmotic flow (EOF) and separated by surface-solute interactions as well as by differences in electromigration. In this paper on the separation of peptides on C18 reversed-phase and mixed-mode (sulphonic acid-n-alkyl) packings in CEC and electrically assisted reversed-phase gradient nano-LC are investigated. It is shown that mixed mode packings generate a higher EOF than reversed-phase packings that is scarcely dependent on the pH of the eluent. Applying a potential in gradient elution reversed-phase nano-LC of peptides shortens the analysis time as compared to separations without a potential. Electrically assisted reversed-phase gradient elution nano-LC is a powerful separation tool for analysis of tryptic digests. Peptides can be successfully resolved in acidic organic mobile phase at pH 2-3 with and without trifluoroacid as ion pairing reagent under isocratic conditions. It is demonstrated that CEC with mixed mode packing and an eluent of pH 2.3 with varying acetonitrile content can be applied to monitor impurities in a synthetic peptide.     

Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry

Macroporous, monolithic capillary electrochromatography (CEC) columns, featuring a hydrophobic stationary phase, have been applied to the separations of steroids with good column efficiency. Using isocratic and gradient elution runs, mixtures of neutral or conjugated steroids could be resolved. While dansylated ketosteroids were detectable through laser-induced fluorescence at attomole levels, the CEC columns coupled to electrospray-ion-trap mass spectrometry featured femtomole detection limits.     

Capillary electroendoosmotic chromatography of peptides

This review focuses on the current state of peptide separation by capillary electroendoosmotic chromatography (CEC). When carried out under optimised conditions, peptide separation by CEC methods represents an orthogonal and complementary technique to micro-HPLC (micro-HPLC) and high-performance capillary zone electrophoresis (HPCZE). The origin of the selectivity differences that can be achieved with these three separation techniques (CEC, micro-HPLC and HPCZE), respectively are discussed, and the current limits of performance with CEC methods documented. Peptide separations by CEC methods with n-alkyl bonded silicas or mixed-mode phases are also illustrated. The development of different variants of CEC and pressurised CEC (also commonly referred to in the literature as electrically-assisted micro-HPLC) are examined. The potential of coupling CEC systems to mass spectrometers for real-time analyses of peptides or protein digests has been examined. Several future directions for the application of this technique in phenotype/proteomic and zeomic mapping of naturally occurring peptides and proteins are highlighted.     

Separation of 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine derivatives of carbonyl compounds by reversed-phase capillary electrochromatography

4-Dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) is a novel derivatizing reagent specially designed for the determination of carbonyl compounds. In this work, we describe the separation of DMNTH-derivatized carbonyl compounds by reversed-phase capillary electrochromatography (CEC). After systematic investigations of the effects of experimental conditions viz. pH and concentration of buffer, type of stationary phase, injection volume of sample, organic modifier, and temperature, optimal conditions were found. The sample compounds, which were separated with gradient high performance liquid chromatography (HPLC), were separated by CEC under isocratic elution due to the high efficiency. Comparisons of separations by CEC and micellar electrokinetic chromatography (MEKC) were made.     

Poly(glycidyl methacrylate-divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing-matrix-assisted laser desorption/ionization mass spectrometry (CIEF-MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB)-based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off-line coupling of IPG-CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG-CIEF. Enhanced neuropeptide detection is also observed after coupling IPG-CIEF with MALDI MS.     

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘油酯（glycidyl methacrylate,GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate,EDMA））毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

High temperature and temperature programming in capillary electrochromatography

In electrochromatography, solvent electrophoretic mobility and solute partitioning are temperature dependent processes. If temperature variations are controlled, solute selectivity and analysis times can be tailored. In this study the feasibility of temperature programming in capillary electrochromatography (CEC) was demonstrated using a reversed-phase CEC mode. The outcome of programmed separations was compared with isothermal, isocratic and isorheic (constant flow) separations. The combined effects of column temperature and mobile phase flow-rate changes during the separation run, resulted in up to a 50% reduction in the separation run time, without adversely affecting the quality of separation. For capillary electrochromatography, temperature programming may be a valuable alternative to solvent programming modes because of the great technical difficulties associated with carrying out solvent gradient elution.     

Gradient elution techniques for capillary electrochromatography

Capillary electrochromatography (CEC) is a rapidly maturing technique, but still in need of further instrumental development and in need of unique applications that are not possible by traditional pressure-driven LC. We review the development of gradient elution schemes for CEC, beginning with pH gradients initially developed for capillary electrophoresis. Step gradients are the most easily instrumentally implemented, but provide less flexibility in separation than continuous gradients. Pressure-assisted CEC is easily adapted to gradient elution schemes, but does not offer the advantages of very high column efficiency provided by totally electro-driven mobile phases. The development of flow-injection interfaces allows a true solvent gradient to be generated by micro-LC pumps, with the mobile phase drawn into the separation capillary by pure electroosmotic flow. While requiring both a CEC instrument and a traditional pump or pumps capable of generating the gradient, this method offers advantages of greatly reduced column handling, prolonging column lifetimes, and allows simple autosampling. We also discuss voltage gradients, which provide a mobile phase velocity gradient.     

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

High-pressure liquid chromatography of trace elements: Determination of terbium in terbium doped gadolinium oxide sulphide phosphors

Summary A detailed study of isocratic and gradient elution separations of lanthanides has been carried out. Analyses of industrially and scientifically interesting products such as luminescent phosphors have been carried out by gradient elution with DL-2-hydroxyisobutyric acid. The determination of small amounts of terbium in gadolinium oxide sulphide phosphors is described in which an HCl solution was eluted through a stainless steel column packed with microparticulate silica, with bonded cation-exchange groups. Complete separation of gadolinium and terbium is achieved. Detection is with a variable wavelength detector following post-column complex formation with 4-(2-pyridylazo)-resorcinol monosodium salt. Results obtained on test solutions show good reproducibility and sensitivity and the method may be considered sufficiently reliable to be used in routine quality control procedures.Work financially supported by C.N.R. of Italy.     

Methods to determine the kinetic performance limit of contemporary chromatographic techniques

By combining separation efficiency data as a function of flow rate with the column permeability, the kinetic plot method allows to determine the limits of separation power (time vs. efficiency) of different chromatographic techniques and methods. The technique can be applied for all different types of chromatography (liquid, gas, or supercritical fluid), for different types of column morphologies (packed beds, monoliths, open tubular, micromachined columns), for pressure and electro‐driven separations and in both isocratic and gradient elution mode. The present contribution gives an overview of the methods and calculations required to correctly determine these kinetic performance limits and their underlying limitations.     

Liquid chromatography techniques for characterizing poly(Vinyl Alcohol)

Poly(vinyl alcohol) (PVOH) samples may contain several heterogeneities requiring the development of chromatographic techniques for characterization. Size exclusion separations have been carried out using a number of aqueous eluents, incorporating electrolyte, or electrolyte/organic modifier, or surfactant. The most favourable molecular size separation was obtained using 0.25% w/v sodium lauryl sulfate as eluent. Reasonable values for molecular weights of PVOH samples have been determined. Compositional distributions in copolymer systems can be assessed using high-performance liquid chromatography employing a reversed-phase separation mechanism. For poly(vinyl alcohol), gradient elution with water/tetrahydrofuran (THF) with a wide pore polystyrene-based packing produced separations dependent on degree of hydrolysis and sequence length distribution. The elution results were verified with a column packed with non-porous beads. Partially hydrolysed PVOH samples appeared to have a broad distribution of composition.     

Capillary Electrochromatography as a Method Development Tool for the Liquid Chromatographic Separation of DUP 654 and Related Substances

Capillary Electrochromatography (CEC) offers a rapid, economical, and efficient means for resolving nonionic compounds in the reversed phase mode on octadecylsilane (ODS) columns. A CEC optimization on a Hypersil ODS capillary column was employed to identify a suitable mobile phase for the pressure-driven (reversed phase ODS) separation of the anti-inflammatory 2-phenylmethyl-1-naphthol (DUP 654), and its related substances. The proportions of mobile phase modifiers methanol, acetonitrile, and water as well as pH were employed as variables in a stacked mixture design. Comparable response surface profiles were obtained for the CEC separations at pH 4 and pH 8. However, subtle differences were evident in the quality of separations obtained in the liquid chromatographic (LC) mode when using a specially-prepared column packed with exactly the same stationary phase as used in the CEC experiments. A mapping of the response surface for separations on a commercially available Hypersil ODS LC column revealed obvious differences. The differences indicate that the transfer of ODS based separation methods between CEC and LC involves more than simply transferring the conditions from one mode to the other.