Separation Of Basic Compounds Research Articles

Synthesis and Chromatographic Evaluation of Perfluorooctyl Stationary Phase for Separation of Basic Compounds

Synthesis and Chromatographic Evaluation of Perfluorooctyl Stationary Phase for Separation of Basic Compounds

Synthesis of a stationary phase based on silica modified with branched octadecyl groups by Michael addition and photoinduced thiol-yne click chemistry for the separation of basic compounds.

A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.

Development of Amine Capillary Column Applied to the Analysis of Basic Compounds by Electrochromatography

An amine capillary column was developed in a fused-silica capillary as stationary phase and applied to separation of basic compounds by capillary electrochromatography (CEC). The functionalized capillary was prepared by inmobilization of 3-Aminopropyl)triethoxysilane (APTES). The CEC conditions including APTES and buffer concentration, pH and applied voltage were investigated to obtain the optimal CEC system for the separation of three anesthetics as basic test molecules. The capillary column provided an efficiency of up to 20,000 plates/m. Lidocaine, ketamine and xilacine were baseline separated under the running conditions with 10 mM Na2HPO4 pH 9.0 as BGE. The applied voltage was 5 kV temperature was set at 25oC and UV detection was performed. The resolutions were 4.97 and 1.53 for ketamine, lidocaine and xilacine, respectively. The column used in CEC mode showed better separation of the anesthetics compared with those used in the capillary zone mode. The comparison with reversed stationary phases used in HPLC in terms of resolution (Rs), sensitivity (LOD), efficiency (N), precision (RSD), asymmetry (T) was also performed. This study provided an alternative way for the CEC separation of basic compounds and demonstrated the improvement in the chromatographic parameters.

Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (β-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab® software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

Preparation of a new stationary phase with s-triazine and amide embedded and its application in separation of basic compounds

A new kind of silica-bonded reserved stationary phase with s-triazine and amide polar groups embedded for HPLC was obtained by step-wise reactions of silica gel with s-triazine, gamma-aminopropyltriethoxysilane, ethanediamine and dodecanoyl chloride. The new stationary phase was characterized by elemental analysis. After that, a column packed with the new stationary phase was used to separate basic compounds. A commercial C18 column was also tested under the same chromatographic conditions for comparison. The results indicated that the ligand was successfully bonded to the surface of the silica and the maximum relative deviations of element contents were within 5% for carbon, nitrogen and hydrogen by three times. Furthermore, in the separation of five basic anilines and four basic pyridines, the excellent selectivity and symmetrical peak shapes provided a reference for advancing the commercialization of the new stationary phase.

Strong cation exchange column allow for symmetrical peak shape and increased sample loading in the separation of basic compounds

Strong cation exchange (SCX) mode, an alternative to reversed-phase (RP) mode, was described in this paper to obtain symmetrical peak shape and high sample loading for the separation of basic compounds. The retention mechanisms on SCX modified silica sorbents have been demonstrated including electrostatic and hydrophobic interactions. The effects of eluent ionic strength, apparent pH on retention were investigated and the cation-exchange capacity was also characterized. Good efficiency (>56,000platem−1) and excellent peak shape (Tf<1.2) for various basic probes were obtained on the SCX column. Furthermore, good peak shapes and separation can be maintained under high concentration injections, indicating good potential in preparative use. These results were also compared with those obtained on two conventional RP stationary phases. The overall results demonstrated that SCX stationary phases can be used as alternatives to RP stationary phases in the separation of basic compounds, especially in the purification of basic compounds.

In-situ Synthesis and Normal Chromatographic Properties of Nonporous SiO2/ZrO2 Core-shell Composite Microspheres

Sub-2 µm core-shell SiO(2)/ZrO(2) composite microspheres with nonporous SiO(2) as the cores and ZrO(2) nanoparticles as the shells were synthesized by sol-gel method in the current work. The fabrication consisted of (1) preparing monodisperse silica spheres; (2) coating the spheres with zirconia nanoparticles in-situ; and (3) calcining the composite microspheres at 823 K. Then uniform, regular, and ordered arrays of nanometer-scale zirconia crystalline particles were formed and dispersed on the surfaces of nonporous silica microspheres. A potential deposition mechanism in the synthesis process was proposed according to isoelectric point theory and to observe microstructural characteristics. The result showed that this stationary phase also has an advantage in the separation of basic compounds in the normal phase.

Comparison of the performance of non‐ionic and anionic surfactants as mobile phase additives in the RPLC analysis of basic drugs

Surfactants added to the mobile phases in reversed-phase liquid chromatography (RPLC) give rise to a modified stationary phase, due to the adsorption of surfactant monomers. Depending on the surfactant nature (ionic or non-ionic), the coated stationary phase can exhibit a positive net charge, or just change its polarity remaining neutral. Also, micelles in the mobile phase introduce new sites for solute interaction. This affects the chromatographic behavior, especially in the case of basic compounds. Two surfactants of different nature, the non-ionic Brij-35 and the anionic sodium dodecyl sulfate (SDS) added to water or aqueous-organic mixtures, are here compared in the separation of basic compounds (β-blockers and tricyclic antidepressants). The reversible/irreversible adsorption of the monomers of both surfactants on the stationary phase was examined. The changes in the nature of the chromatographic system using different columns and chromatographic conditions were followed based on the changes in retention and peak shape. The study revealed that Brij-35 is suitable for analyzing basic compounds of intermediate polarity, using "green chemistry", since the addition of an organic solvent is not needed and Brij-35 is a biodegradable surfactant. In contrast, RPLC with hydro-organic mixtures or mobile phases containing SDS required high concentrations of organic solvents.

Separation performance of 1-μm silica particles as stationary phase for pressurized electrochromatography

Bare nonporous silica particles with diameter around 1 microm were prepared. A 20 cm section of a total length of 45 cm capillary (100 microm i. d.) was packed electrokinetically and the separation performance of basic compounds was investigated in the pressurized capillary electrochromatography (pCEC) system using 1 microm nonporous bare silica spheres as stationary phase and acetonitrile-water as mobile phase. The effects of the composition of the mobile phase, the concentration of the buffer, pH value, applied voltage and so on on the separation performance were investigated. The experimental results demonstrated that the bare silica column showed a typical reversed phase separation mechanism when it was used for separation of basic compounds. The separation performance changed slightly with changing buffer concentration. Since the degree of dissociation of the basic compounds depended greatly on the pH value, the interaction between the compounds and stationary phase changed with the changing of pH value, thereby changing the resolution of the compounds. The separation performance increased with the increase of applied voltage. A column efficiency of 35 000 for o-toluidine was obtained when a voltage of 1 kV was applied.

Electrochromatographic Evaluation of Diol-Bonded Silica Monolith Capillary Column for Separation of Basic Compounds

A diol-bonded silica monolith capillary column as polar stationary phase was successfully prepared for capillary electrochromatography. The preparation of monolithic stationary phase was based on the individual silica matrix forming and subsequent chemical bonding by (3-glycidoxypropyl) trimethoxysilane to produce the desired function. The diol-bonded silica monolith has been successfully employed in the electrochromatographic separation of alkaloids. The effects of experimental parameters, such as the volume fraction of the organic modifier, pH value, and ionic strength of the buffer on the retention behavior of the solutes were investigated. Column efficiencies greater than 110,000 plates/m for separation of basic compounds were obtained. It was observed that retention of alkaloids on the diol-bonded silica monolith was mainly contributed to a reversed-phase and cation-exchange mechanism, and electrophoresis of basic compounds also played a role in separation.

Liophilic Mobile Phase Additives in Reversed Phase HPLC

The separation of basic compounds can be challenging and the use of inorganic mobile phase additives have been successfully used in chromatographic methods development. About fifteen years ago the role of these additives as ion-interaction agents for selectively adjusting retention of ionic analytes was discovered and the theory of chaotropicity was applied to reversed phase chromatography. In the last ten years the studies of the influence of these counterions on the retention of ionizable analytes and the interaction with the stationary phase in various hydro-organic eluents has expanded our knowledge of this phenomenon. The general view and understanding of the process have been significantly updated and the use of these ionic additives (liophilic ions) in the mobile phase has become regular practice in the pharmaceutical industry for optimization and fine tuning of complex separations. This paper reviews the latest developments in the field and discusses the modification and expansion of our theoretical understanding of the process. The paper also describes their application in practical separations for a wide variety of analytes, from small molecules to peptides and even chiral separations.

Comparison of liquid chromatographic behaviors on N-methylimidazolium functionalized ZrO 2/SiO 2-4 and N-methylimidazolium functionalized SiO 2 stationary phases

A new stationary phase N-methylimidazolium functionalized ZrO 2/SiO 2-4 (Zr/SilprMim) has been prepared. The chromatographic property of this stationary phase is investigated by ion chromatography (IC) with inorganic and organic anions, and normal phase HPLC with basic compounds and hydroxybenzenes. The effects of pH and the strength of Lewis base of eluent on separation of anions are studied. This new stationary phase is also compared with a N-methylimidazolium functionalized SiO 2 stationary phase (SilprMim). The results show that this new stationary phase can be used in analysis of inorganic anions with great prospects. At the same time, successful separations of some organic anions can be obtained by using phosphate buffer solution as mobile phase. This new stationary phase also has a distinct advantage in the separation of basic compounds in normal phase. But due to the presence of Lewis acid sites on the surface of ZrO 2/SiO 2-4, Lewis bases such as hydroxybenzenes adsorb very strongly on this new stationary phase, and Lewis acid sites can be masked or modified by the eluent that contain Lewis base groups.

Analysis of basic compounds by reversed-phase liquid chromatography–electrospray mass spectrometry in high-pH mobile phases

The separation of basic compounds in high-pH mobile phases results in extended retention, excellent peak shapes and good chromatographic efficiency. A severe decrease in sensitivity with electrospray mass spectrometric detection in positive ion mode (ESI +-MS) is expected under conditions that suppress analyte ionization in solution. We compared the responses of a large number of various basic drugs covering a wide range of hydrophobic (log P 0.09 to 7.6) and basic character (p K a 6.8–10) in LC–ESI +-MS/MS in 0.1% formic acid in water and acetonitrile, to responses in 10 mM ammonium hydrogencarbonate buffers of different pH (7.8–11), and acetonitrile. Contrary to common expectations, high-pH mobile phases do not affect negatively the responses of basic compounds in ESI +. Analyte responses and limits of detection are comparable, or most often better in high pH compared to acidic mobile phases.

Chromatographic Evaluation of Octadecyl‐Bonded TiO2/SiO2 Stationary Phase with Engelhardt and Tanaka Test Mixtures

Chromatographic performance of octadecyl‐bonded TiO2/SiO2 stationary phase was evaluated with Engelhardt and Tanaka test mixtures. In the two test protocol, some parameters influencing the retention, such as methylene selectivity (αCH2), steric selectivity (αO/T), hydrogen bonding capacity (αC/P), and ion exchange capacity (αB/P) were obtained. By separating several same compounds with different mobile phases, different results were obtained and they were compared with that in the reference, in which octadecyl‐bonded titania packing was prepared and a similar test was carried out. All of the results suggest that the octadecyl‐bonded TiO2/SiO2 stationary phase not only has good methylene selectivity and steric selectivity, but also shows a large potential in the separation of basic compounds.

Separation of basic compounds by capillary electrochromatography on an X-Terra RP18® stationary phase

In this work we demonstrate that the X-Terra RP18® stationary phase, specially designed for the analysis of basic compounds in liquid chromatography, may be successfully used in capillary electrochromatography. Although this packing material does not afford a sufficient electroosmotic flow with classical hydro-organic mobile phases, the addition of a surfactant that adsorbs onto the stationary phase allows to generate a sustainable electroosmosis flow (EOF), the direction of which depends on the charge of the surfactant. The way of manipulating the electroosmotic flow is described (nature and concentration of the added surfactant, proportion of the organic modifier in the mobile phase, pH). It is then demonstrated that high efficiencies can be reached with this packing material (up to 220 000 plates/m with a mean diameter particles of 3.5 μm) when it is operated at high linear velocities. Then the separations of different classes of compounds such as amphenicol antibiotics, macrolide antibiotics or basic test solutes with mobile phases with pH up to 10.8 are described. The influence of the addition of sodium dodcylsulfate (SDS) to the mobile phase on the retention is described and the selectivity of the X-Terra RP18® stationary phase is compared to that of a more traditional phase, i.e. Hypersil C18 stationary phase with SDS added to the mobile phase. However, it is shown that a good repeatability of the retention factors can only be obtained when the ionization of the compounds is totally suppressed since electrolysis of the buffered hydro-organic mobile phase occurs in the buffer reservoirs leading to a variation of the mobile phase pH and consequently to a modification of the ionization degree of the solutes having their pKa close to the mobile phase pH.

Separation of basic compounds by capillary electrochromatography on an X-Terra RP18 ® stationary phase

In this work we demonstrate that the X-Terra RP18 ® stationary phase, specially designed for the analysis of basic compounds in liquid chromatography, may be successfully used in capillary electrochromatography. Although this packing material does not afford a sufficient electroosmotic flow with classical hydro-organic mobile phases, the addition of a surfactant that adsorbs onto the stationary phase allows to generate a sustainable electroosmosis flow (EOF), the direction of which depends on the charge of the surfactant. The way of manipulating the electroosmotic flow is described (nature and concentration of the added surfactant, proportion of the organic modifier in the mobile phase, pH). It is then demonstrated that high efficiencies can be reached with this packing material (up to 220 000 plates/m with a mean diameter particles of 3.5 μm) when it is operated at high linear velocities. Then the separations of different classes of compounds such as amphenicol antibiotics, macrolide antibiotics or basic test solutes with mobile phases with pH up to 10.8 are described. The influence of the addition of sodium dodcylsulfate (SDS) to the mobile phase on the retention is described and the selectivity of the X-Terra RP18 ® stationary phase is compared to that of a more traditional phase, i.e. Hypersil C 18 stationary phase with SDS added to the mobile phase. However, it is shown that a good repeatability of the retention factors can only be obtained when the ionization of the compounds is totally suppressed since electrolysis of the buffered hydro-organic mobile phase occurs in the buffer reservoirs leading to a variation of the mobile phase pH and consequently to a modification of the ionization degree of the solutes having their p K a close to the mobile phase pH.

Mixed Mode of Hydrophilic and Ionic Interaction Pressurized Capillary Electrochromatography for Separation of Basic Compounds

The separation of six basic compounds was obtained using a mixed mode of hydrophilic interaction and strong cation exchange (SCX), pressurized capillary electrochromatography (pCEC) on a novel pCEC instrument. The effects of organic modifier concentration, ionic strength, and pH value in the buffer on retention behaviors of solutes were investigated. The effects of pressure and voltage on separation also were evaluated. Moreover, the separation result was compared with that in reversed phase pCEC, and the separation mechanism was discussed. A satisfactory reproducibility and stability in the separation system was also achieved in our study. This paper demonstrates the mixed mode pCEC is potentially applicable for separation of basic compounds.

A novel silica-based column packing for reversed-phase HPLC of basic compounds

A novel bonded phase for reversed-phase HPLC was synthesized in two steps. Octylamine was first reacted with β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (β-ECTS) and then the intermediate product was coupled onto porous silica. The prepared packing was characterized by elemental analysis, solid-state13C NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes and methanol-water as binary mobile phase. The results showed that the stationary phase has excellent chromatographic properties and is resistant to hydrolysis between pH=2≈8. It can be used efficiently for the separation of basic compounds.

Synthesis and characterization of a polymer-coated C 18 stationary phase with high carbon content for liquid chromatography

High-purity porous silica with a large surface area (particle diameter 5 μm, mean pore diameter 8.4 nm, surface area 350 m 2/g) was coated with a homogeneous silicone polymer film, which was thereafter modified with octadecyl groups to be used as a stationary phase for reversed-phase chromatography. The stationary phase had a high carbon content (18%), and showed a relatively large overall retention for various analytes among other stationary phases available in the market. It also showed preferable characteristics in separation of basic compounds, such as pyridine, and hinokitiol, a substance that easily coordinates with metals, and also had a high durability under an alkaline mobile phase.

HPLC analysis of basic organic compounds in a multi-component ion-interaction system: A mechanistic study

Reversed-phase HPLC separations of basic compounds have been optimized by use of mobile phases containing 1,8-diaminooctane (DAO) as amine modifier and heptanesulfonate as counter-ion. Such studies have demonstrated the high resolution and selectivity of this chromatographic system compared with mobile phases prepared with the more widely used primary amines. With the aim of clarifying the retention mechanism involved, three sets of compounds with different structures and properties were analyzed: sulfamidics and trimethoprim, aromatic diamines and aminophenols, and a standard mixture of lipophilic basic drugs. The conditions investigated were: diaminoalkane modifier concentration, diaminoalkane modifier alkyl-chain length, nature and concentration of the counter-ion, pH of the mobile phase, concentration of the organic modifer, and the nature of the stationary phase as characterized by percentage carbon loading. Analysis of chromatographic behavior as a function of the chromatographic conditions has enabled the proposal of a mechanism of retention of the three classes of compounds.