Effect of the net surface charge density of heptakis-6-bromo-6-deoxy-beta-cyclodextrin bonded silica gels on the retention behaviors of neutral cresol isomers in HPLC.

The effect of the surface charge density of heptakis-6-bromo-6-deoxy-beta-cyclodextrin (beta-CD-BR) bonded silica gels, which was used as the stationary phase of a packed capillary column for HPLC, was investigated concerning the retention behaviors of neutral cresol isomers. On the whole, the retention factors of the cresol isomers increased with an increase in the pH values of the mobile phase, although they were slightly smaller at pH 6.1 than at pH 4.7. An investigation on the retention variation using a van't Hoff plot revealed that the increase in the retention factor (k) at a higher pH region could be mainly attributed to the increase in DeltaS, while a partial decrease in k around pH 5 - 6 was caused by a decrease in the -DeltaH/T value. On the other hand, a measurement of the electroosmotic flow velocity under various pH of the mobile phase solutions revealed that the retention variations of the neutral cresol isomers were strongly correlated with the surface charge on the packing materials. The positive charge of secondary ammonium functional groups to bind beta-CD-BR inhibit the insertion of the cresol isomers into the cavity of beta-CD-BR while reducing the retention factor, whereas the negative charge of silanol group enhanced it through a local change in the mobile phase composition.     

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.     

Phenylaminopropyl silica monolithic column for pressure assisted capillary electrochromatography

A novel stationary phase phenylaminopropyl silica (PhA-silica) monolith was successfully prepared for pressure assisted capillary electrochromatography (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by using [3-(phenylamino)propyl]trimethoxysilane as surface modification reagent to produce the phenylaminoporpyl function. The secondary amino groups on the surface of the monolithic stationary phase contributed to the generation of anodic electroosmotic flow (EOF) under acidic conditions. The phenyl group together with the spacer (-(CH(2))(3)-) in PhA-silica provides sufficient hydrophobic properties. To evaluate the column performance, effects of buffer pH and mobile phase composition on the mobile phase linear velocity and the retention factors of alkylbenzenes, phenols and anilines were investigated in pCEC mode. The monolithic stationary phases exhibit typical reversed-phase (RP) electrochromatographic behavior toward neutral solutes. Hydrophobic as well as electrophoretic migration process within the monoliths was observed for the separation of basic solutes such as anilines without peak tailing.     

Migration of neutral solutes by double stepwise gradient elution in capillary electrochromatography

Characteristics of electroosmotic flow (EOF) and the migration of neutral solutes under double stepwise gradient elution in capillary electrochromatography were studied systematically. EOF velocity proved to be the function of operation time changing with the introduction of the second mobile phase. Accordingly, the retention of components also changed. The migration of neutral solutes was studied under the following three situations; A, components eluted when the column was filled only with the first kind of mobile phase; B, solutes eluted still in the first kind of mobile phase while at that time two kinds of mobile phase coexisted in the column and C, samples eluted in the second kind of mobile phase. Equations to describe the retention times of components under these three kinds of conditions were deduced and applied to predict the retention times of 12 aromatic compounds. Relative errors between experimental and calculated values were below 5.0%, which proved the reliability of the equations. In addition, parameters that might affect the retention time of solutes, such as the transferring time of mobile phase vials, the capacity factors of components and EOF velocities two steps were studied systematically.     

Capillary electrochromatography with monolithic-silica columns. II. Preparation of amphiphilic silica monoliths having surface-bound cationic octadecyl moieties and their chromatographic characterization and application to the separation of proteins and other neutral and charged species

Three different synthetic routes have been introduced and evaluated for the preparation of amphiphilic silica-based monoliths possessing surface-bound octadecyl ligands and positively charged groups. The amphiphilic silica monoliths (designated as cationic C18-monoliths) have been designed for use in reversed-phase capillary electrochromatography (RP-CEC) with hydro-organic mobile phases. These amphiphilic stationary phases yielded anodic electroosmotic flow (EOF) over a wide range of mobile phase pH. The magnitude of EOF remained constant up to pH 4.0 and then decreased at pH > 4.0 due to the ionization of silanol groups and the subsequent decrease in the net positive surface charge density of the amphiphilic monoliths. The cationic C18-monoliths exhibited reversed-phase chromatography (RPC) behavior toward non-polar solutes (e.g., alkyl benzenes), which parallels that observed with octadecyl-silica (ODS) monoliths. On the other hand, the amphiphilic stationary phases exhibited both non-polar and polar interactions toward slightly polar solutes such as anilines and PTH-amino acids. CEC retention factor k* and velocity factor k*e, which reflects the contribution of the electrophoretic mobility, were evaluated for charged solutes such as anilines and proteins.     

Electrokinetic flow and electric conduction of salt-free solutions in a capillary

The electrokinetic flow and accompanied electric conduction of a salt-free solution in the axial direction of a charged circular capillary are analyzed. No assumptions are made about the surface charge density (or surface potential) and electrokinetic radius of the capillary, which are interrelated. The Poisson–Boltzmann equation and modified Navier–Stokes equation are solved for the electrostatic potential distribution and fluid velocity profile, respectively. Closed-form formulas for the electroosmotic mobility and electric conductivity in the capillary are derived in terms of the surface charge density. The relative surface potential, electroosmotic mobility, and electric conductivity are monotonic increasing functions of the surface charge density and electrokinetic radius. However, the rises of the relative surface potential and electroosmotic mobility with an increase in the surface charge density are suppressed substantially when it is high due to the effect of counterion condensation. The analytical prediction that the electroosmotic mobility grows with increases in the surface charge density and electrokinetic radius agrees with the experimental results for salt-free solutions in circular microchannels in the literature.     

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of In k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.     

Influence of moderate Joule heating on electroosmotic flow velocity, retention, and efficiency in capillary electrochromatography

The influence of Joule heating on electroosmotic flow velocity, the retention factor of neutral analytes, and separation efficiency in capillary electrochromatography was investigated theoretically and experimentally. A plot of electrical current against the applied electrical field strength was used to evaluate the Joule heating effect. When the mobile phase concentration of Tris buffer exceeded 5.0 mM in the studied capillary electrochromatography systems using particulate and monolithic columns (with an accompanying power level of heat dissipation higher than 0.35 W/m), the Joule heating effect became clearly noticeable. Theoretical models for describing the variation of electroosmotic flow velocity with increasing applied field strength and the change of retention factors for neutral analytes with electrical field strength at higher Tris buffer concentrations were analyzed to explain consequences of Joule heating in capillary electrochromatography. Qualitative agreement between experimental data and implications of the theoretical model analysis was observed. The decrease of separation efficiency in capillary electrochromatography with macroporous octadecylsilica particles at high buffer concentration can be also attributed to Joule heating mainly via the increased axial diffusion of the analyte molecules and dispersion of solute bands by a nonuniform electroosmotic flow profile over the column cross-section. However, within a moderate temperature range, the contribution of the macroscopic velocity profile in the column arising from radial temperature gradients is insignificant.     

Peptide separation in hydrophilic interaction capillary electrochromatography

Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min.     

Separation of acidic and neutral compounds by strong anion-exchange capillary electrochromatography dynamically modified with sodium dodecylsulfate

Summary Acidic and neutral compounds have been separated by strong anion-exchange capillary electrochromatography (SAXCEC) dynamically modified by addition of sodium dodecylsulfate (SDS). It was found that separation of neutral solutes by SAXCEC without addition of SDS is difficult because of the weak interaction of the solutes with the hydrophilic packing surface. The hydrophobicity of the packing surface increases on addition of SDS to the mobile phase, however, and the capacity of separating neutral solutes increases. Acidic solutes are retained, mainly because of the ion-exchange properties of this system. The influence of mobile phase composition, e.g. SDS concentration, ionic strength, and organic modifier fraction, on the retention of acidic and neutral solutes was investigated. Three acids and five neutral solutes were separated in only 5 min under optimized conditions, because the direction of the strong electroosmotic flow (EOF) was the same as that of electrophoretic migration of the acids under the conditions used. The repeatability of this system in terms of migration time relative standard deviation (RSD) is good—less than 0.48% for 10 consecutive runs of all the solutes tested. Column efficiencies for acids were > 125000 plates m⁻¹; those for neutral solutes varied from 25000 to 100000 plates m⁻¹.     

Capillary zone electrophoresis of biological substances with surface-modified fused silica capillaries with switchable electroosmotic flow

A surface modification has been developed which yields fused silica capillaries with switchable electroosmotic flow (anodal/cathodal). The capillary surface is a composite material consisting of unreacted silanol groups, a layer of positively charged quaternary ammonium functions, and a hydrophilic layer of long polyether chains. Because of the presence of positively and negatively charged groups, the net charge of the capillary surface can be varied from positive to negative by changing the pH of the running electrolyte, thus enabling manipulation of the magnitude and direction of the electroosmotic flow. The long polyether chains were effective in shielding biomacromolecules from the charged inner surface of the capillary, thus minimizing electrostatic interaction of the solutes with both unreacted silanols and the quaternary ammonium groups which had been introduced. As a consequence, high separation efficiencies were achieved with proteins, nucleotides, and a series of acidic oligosaccharides.     

一个描述液相色谱体系中溶质进样量与保留值关系的方程

通过考虑溶质和溶剂在液相色谱体系中的相互作用,从理论上给出了一个描述溶质在液相色谱体系中进样量与保留值之间关系的方程。由方程可以证明,当进样量趋于零时,溶质的保留值为一定值,当进样量无限大时,溶质的保留值趋于零,且随着进样量的增加,溶质的保留值必然减小。通过方程的线性形式,可以获得两个描述色谱体系特征的重要参数：一个是溶质与固定相相互作用的平衡常数Ｋ,另一个是假想的分布在固定相表面上的活性点总数。     

Separation of Polar and Basic Compounds in Hydrophilic Interaction Pressurized CEC Using Diethylenetriaminopropyl Silica Monolithic Columns

A novel silica-based monolithic column possessing diethylenetriaminopropyl ligands for hydrophilic interaction pressurized capillary electrochromatography is described. The preparation of monolithic stationary phase was based on the individual silica matrix forming and subsequent chemical bonding. The triamino groups on the surface of the novel stationary phase generated a sustainable anodic electroosmotic flow under acidic conditions. A variety of neutral and basic analytes were used to evaluate the column performance. The monolithic silica stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. For basic tetracycline antibiotics, hydrophilic interaction as well as electrophoretic migration process with the monoliths was observed and peak tailing was avoided.     

Controlling retention, selectivity and magnitude of EOF by segmented monolithic columns consisting of octadecyl and naphthyl monolithic segments--applications to RP-CEC of both neutral and charged solutes

Monolithic capillaries made of two adjoining segments each filled with a different monolith were introduced for the control and manipulation of the electroosmotic flow (EOF), retention and selectivity in reversed phase-capillary electrochromatography (RP-CEC). These columns were called segmented monolithic columns (SMCs) where one segment was filled with a naphthyl methacrylate monolith (NMM) to provide hydrophobic and π-interactions, while the other segment was filled with an octadecyl acrylate monolith (ODM) to provide solely hydrophobic interaction. The ODM segment not only provided hydrophobic interactions but also functioned as the EOF accelerator segment. The average EOF of the SMC increased linearly with increasing the fractional length of the ODM segment. The neutral SMC provided a convenient way for tuning EOF, selectivity and retention in the absence of annoying electrostatic interactions and irreversible solute adsorption. The SMCs allowed the separation of a wide range of neutral solutes including polycyclic aromatic hydrocarbons (PAHs) that are difficult to separate using conventional alkyl-bonded stationary phases. In all cases, the k' of a given solute was a linear function of the fractional length of the ODM or NMM segment in the SMCs, thus facilitating the tailoring of a given SMC to solve a given separation problem. At some ODM fractional length, the fabricated SMC allowed the separation of charged solutes such as peptides and proteins that could not otherwise be achieved on a monolithic column made from NMM as an isotropic stationary phase due to the lower EOF exhibited by this monolith.     

Column wall modifications using polyethylene oxide for capillary electrophoresis of ribonucleotides

Summary In this study, a variety of fused silica capillaries with different combinations and sequences of treatments with HMDS and polyethylene oxide were prepared in order to develop an optimized column modification method for analysis of ribonucleotides. The 12 most common ribonucleotides (UTP, CTP, ATP, GTP, UDP, CDP, ADP, GDP, UMP, CMP, AMP, and GMP) in human cells were used as test solutes. Column performance measurements, including electroosmotic flow (EOF), solute migration speed and retention, column efficiency, peak shape, and resolution were investigated. By analyzing solute migration speed and retention of various hydrophilic/hydrophobic solutes, the column wall effects (EOF and adsorption) can be distinguished. This analysis method can give guidance in optimizing polymer coating properties (hydrophilicity/hydrophobicity) for CE columns. By studying the performance of these columns after various surface treatments, we were able to improve the separation of ribonucleotides from real samples to within 16 minutes with high efficiency and stability (over 300 analyses) using columns first deactivated with hexamethyldisilazane, and then coated with polyethylene oxide.     

Characterisation of retention in micellar high-performance liquid chromatography, in micellar electrokinetic chromatography and in micellar electrokinetic chromatography with reduced flow

The retention (migration) behaviour of various barbiturates, phenylurea and triazine herbicides in micellar electrokinetic chromatography (MEKC) with uncoated fused-silica capillaries was compared with the behaviour in micellar electrokinetic chromatography with reduced electroosmotic flow (RF-MEKC) using capillaries modified with linear polyacrylamide. The error in the values of the retention factors caused by the neglection of the contribution of the electroosmotic flow in RF-MEKC was investigated and a method for correcting this error was suggested. The retention was characterised using the lipophilic and polar indices to characterise and to predict the retention as a function of the concentration of the surfactant (sodium dodecylsulphate) in the running buffer in MEKC and in RF-MEKC. Homologous series of n-alkylbenzenes and of n-alkan-2-ones were compared as the standard sets for the calibration of the retention (migration) index scale. The values of the lipophilic indices of a given solute measured in reversed-phase HPLC, MEKC and RF-MEKC are close to each other. Under ideal MEKC conditions, the values of the polarity indices are close to one for various sample solutes. However, for partially ionised compounds such as weakly acidic barbiturates, where the contribution of the electrophoretic migration is significant, the values of the polarity indices are significantly lower than one. Optimum conditions for separations of mixtures of triazine and phenylurea herbicides and of barbiturates using various techniques tested were compared.     

Reversed-Phase Separation of the Major Deoxyribonucleosides and Their Mononucleotides Using Tetrabutylammontum Hetaerons

Abstract

Simultaneous separation of the four major deoxyribonucleosides and their monophosphate nucleotides was achieved using tetrabutyl ammonium phosphate hetaerons with a reversed-phase (C8) packing material. Baseline resolution for all eight solutes was achieved within 48 minutes, using a 7.5% methanol mobile phase, 2.0 mM in TBA, buffered with 50 mM phosphate at pH 4.8. The effect of methanol and TBA concentrations upon the retention of neutral and anionic solutes was studied in detail. It was determined that changes in solute k' with increasing methanol could be explained by essentially independent phenomena. These are: 1) a decrease in the partition coefficient of the TBA cation with increasing organic concentration, resulting in lower surface charge densities, and 2) a decrease in the hydrophobic interactions of the solutes with the reversed-phase HPLC. The overall effect was a log-linear decrease in k' with increasing methanol concentration. An empirical equation was derived for the above model which was found to be helpful in determining the optimal separation conditions for the nucleosides and nucleotides.     

混合烷基开管电色谱柱的制备和评价

利用溶胶－凝胶（Sol-Gel）技术制备了混合烷基开管毛细管电色谱柱（C8－C13OT－CEC），并考察了其电渗流行为和电色谱性能。研究了流动相中甲醇含量对芳香族中性化合物保留的影响。发现C8－C18OT－CEC柱体现反相分配机理。5种芳香族化合物和4种苯同系物在C8－C13OT－CEC柱上分离良好，同时还考察了分离电压和柱内径对柱效的影响，结果表明高的电压和较小的柱内径能提高柱效。     

Nanofiltration of biogenic amines in acidic conditions: Influence of operation variables and modeling

Biogenic amines are present in some fermented and non-fermented beverages and can cause diseases. This study analyzes the feasibility of separating biogenic amines by nanofiltration in acidic medium. Solutions of chloride salts of three biogenic amines: putrescine, histamine and tyramine were filtered through a nanofiltration membrane with a 1000 Da molecular weight cut-off (MWCO) and a positive electrical charge at pH 3. Increasing the transmembrane pressure or cross flow velocity led to an increase in solute rejection and permeate flow. Moreover, a higher electrical charge or lower concentration of amine cations caused a larger rejection indicating that membrane-solutes repulsion governs the filtration process. Finally, the experimental results were analyzed using the classic Donnan–Steric pore model. Values of 0.83 nm and 5.4 μm were estimated for pore radius and membrane effective ratio thickness-porosity from the filtration of neutral solutes. Membrane volumetric charge density and the proton diffusivity inside the pores were estimated from the experimental results.     

Study of elution behaviour with gradient voltage in CEC using methacrylate monolithic columns

A theoretical study on the retention behaviour and chromatographic performance of neutral solutes using a lauryl methacrylate‐based monolithic column under voltage gradient mode in CEC was carried out. Through a flexible mathematical function based on a modified Gaussian model, the peak shape of compounds was firstly fitted under constant and gradient voltage. Using the peak shape parameters and retention time, the estimation of global chromatographic performance, efficiency and peak capacity under several voltage conditions was performed. The influence of voltage gradient on the separation efficiency is discussed and simple equations are presented to calculate retention and peak widths under voltage gradient conditions. A comparison in terms of chromatographic performance of a test mixture of neutral solutes under constant and gradient voltage modes was also carried out. The experiments carried out under gradient voltage showed better efficiencies (172 000 plates/m) and lower peak widths than those obtained under constant voltage (52 000 plates/m).