Development of three end-capped para-benzoyl calix[4,6, or 8]arene bonded stationary phases for HPLC

Three end-capped para-benzoyl calixarene bonded silica gel stationary phases are prepared and characterized by elemental analysis, infrared spectroscopy, and thermal analysis. The comparison and selectivity of these phases are investigated by using PAHs, disubstituted benezene, and naphthalene positional isomers as probes. Possible separation mechanism based on the different interactions between calixarenes and analytes are discussed. The results indicate that the separation for those analytes are influenced by the supramolecular interaction including π-π interaction, π-electron transfer interactions, space steric hindrance, and hydrogen bonding interaction on the calixarene columns. Importantly, the aromatic probes with polar groups such as -OH, -NO(2), and -NH(2) could regulate the selectivity of calixarene-bonded stationary phases.     

Preparation and characterization of six calixarene bonded stationary phases for high performance liquid chromatography

Six calixarene bonded silica gel stationary phases were prepared and characterized by elemental analysis, infrared spectroscopy and thermal analysis. Their chromatographic performance was investigated by using PAHs, aromatic positional isomers and E- and Z-ethyl 3-(4-acetylphenyl) acrylate isomers as probes. Separation mechanism based on the different interactions between calixarenes and analytes were discussed. The chromatographic behaviors of those analytes on the calixarene columns were influenced by the supramolecular interaction including pi-pi interaction, space steric hindrance and hydrogen bonding interaction between calixarenes and analytes. Notably, the presence of polar groups (-OH, -NO(2) and -NH(2)) in the aromatic isomers could improve their separation selectivity on calixarene phase columns. The results from quantum chemistry calculation using DFT-B3LYP/STO-3G* base group were consistent with the retention behaviors of PHAs on calix[4]arene column.     

大黄素键合硅胶高效液相色谱柱的制备和应用

采用中间体法,先将大黄素配体与γ-[（2,3）-环氧丙氧]丙基三甲氧基硅烷（KH-560）偶联剂反应制备含配体的硅氧烷试剂,然后再与硅胶键合,最终制得大黄素键合硅胶液相色谱固定相（以下简称ESP）。通过红外光谱、元素分析和热重分析表征固定相的结构。以萘作为溶质探针,甲醇-水（60：40,v/v）为流动相,流速为0.8 mL/min,测得ESP柱的柱效。采用传统的反相C₁₈和苯基柱作参比,将ESP应用于系列中性、碱性和酸性芳香族化合物以及实际样品风油精的分离分析,并探讨相关的色谱分离机理。结果表明,配体大黄素被成功地键合到球形硅胶表面,测得配体键合量为0.23 mmol/g,ESP柱理论塔板数约为19874 N/m。ESP的偶联剂链和蒽醌环提供了疏水性的结构基础,大黄素配体还能为溶质提供π-π或p-π、电荷转移、氢键、偶极-偶极等作用点。多位点的协同作用使得ESP柱具有独特和优秀的色谱分离选择性,并且无需调节pH值,采用简单而廉价的甲醇-水流动相就能实现胺类、酚类等极性样品的基线分离,实验条件简单、方便。     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

Comparison of column properties in reversed-phase chromatography: monolithic, cholesterolic and mixed bonded stationary phases

A commercial Chromolith C18 column and two new stationary phases with mixed ligands bonded on the Kromasil silica gel support, SG-MIX and SG-Chol, were characterized using simple tests based on the retention of non-polar, basic and acidic compounds. Polar and methylene selectivity tests in acetonitrile-water and methanol-water mobile phases revealed lower hydrophobicities of the SG-MIX and SG-Chol columns in comparison to the Chromolith column. The columns were further characterized using new test criteria - gradient oligomer capacity and isomeric selectivity and peak symmetry of naphthalene di-sulphonic acids in aqueous mobile phases. The cholesterolic column shows greater gradient oligomer selectivity for the separation of oligoethylene glycol samples than the SG-MIX and the Chromolith columns. Increased retention and peak tailing, but decreased isomeric selectivity for naphthalene-di-sulphonic acids was observed with the SG-MIX column, because of interactions with various polar bonded groups.     

Selectivity of calixarene‐bonded silica phases in HPLC: Description of special characteristics with a multiple term linear equation at different methanol concentrations

Retention and selectivity characteristics of different calixarene‐, resorcinarene‐ and alkyl‐bonded stationary phases are examined by analyzing a set of test solutes covering the main interactions (hydrophobic, steric, ionic, polar) that apply in HPLC. Therefore Dolan and Snyder's multiple term linear equation has been adapted to fit the properties of calixarene‐bonded columns. The obtained parameters are used to describe retention and selectivity of the novel Caltrex^® phases and to elucidate underlying mechanisms of retention. Here, differences of stationary phase characteristics at different methanol concentrations in the mobile phases are examined. Both selectivity and retention were found to depend on the methanol content. Differences of these dependencies were found for different stationary phases and interactions. The differences between common alkyl‐bonded and novel calixarene‐bonded phases increase with increasing methanol content.     

Evaluation of porous graphitic carbon as stationary phase for the analysis of fatty acid methyl esters by liquid chromatography

Summary Polyunsaturated fatty acids have been analysed as methyl esters by liquid chromatography on porous graphitic carbon and the results compared with those obtained on octadecyl bonded phases. Chromatographic behaviour on octadecyl bonded phases arises principally as a result of hydrophobic interactions with the bonded phase. Because the retention of analytes is greater on porous graphitic carbon than on octadecyl phases, organic mobile phases are required. When the number of double bonds is low (ca 1–3), the behaviour of porous graphitic carbon is similar to that of octadecyl bonded phases, but when this number increases stronger interactions with the flat surface of the graphite appear, resulting in new selectivity. These two ‘reversed-phase’ systems are considered complementary for separation of different fatty acid methyl esters. An additional advantage of porous graphitic carbon is that it enables isolation of hexadecartrienoic and hexadecadienoic acids, which are not available commercially.     

On-line liquid-chromatography-nuclear magnetic resonance spectroscopy-mass spectrometry coupling for the separation and characterization of secoisolariciresinol diglucoside isomers in flaxseed

The effect of endcapping on an octdecyl bonded phase synthesized by the silanization/hydrosilation method is investigated. The endcapping reagent is a 1:1 molar ratio of trimethylchlorosilane (TMCS) and hexamethyldisilizane (HMDS). Two approaches for endcapping are possible for this synthetic method that produces a silica hydride intermediate: bonding of TMCS-HMDS after silanization (on the hydride intermediate) or after hydrosilation (on the C18 product stationary phase). The use of TMCS-HMDS is designed to eliminate the few remaining silanols on the silica hydride intermediate. The endcapping process is characterized spectroscopically by diffuse reflectance infrared Fourier transform (DRIFT), 29Si cross polarization magic angle spinning nuclear magnetic resonance spectroscopy (CP-MAS-NMR) and 13-C-CP-MAS-NMR. The octadecyl bonded phases are characterized chromatographically by measuring the capacity factors of several hydrophobic and basic test solutes as well as the separation factors among various solute pairs. Finally, long-term stability tests are done on both products at high and low pH.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

Preparation and comparison of three zwitterionic stationary phases for hydrophilic interaction liquid chromatography

Surface‐bonded zwitterionic stationary phases have shown highlighted performances in separation of polar and hydrophilic compounds under hydrophilic interaction chromatography mode. So, it would be helpful to evaluate the characteristics of zwitterionic stationary phases with different arranged charged groups. The present work involved the preparation and comparison of three zwitterionic stationary phases. An imidazolium ionic liquid was designed and synthesized, and the cationic and anionic moieties respectively possessed positively charged imidazolium ring and negatively charged sulfonic groups. Then, the prepared ionic liquid, phosphorylcholine and an imidazolium‐based zwitterionic selector were bonded on the surface of silica to obtain three zwitterionic stationary phases. The selectivity properties were characterized and compared through the relative retention of selected solute pairs, and different kinds of hydrophilic solutes mixtures were used to evaluate the chromatographic performances. Moreover, the zwitterionic stationary phases were further characterized by the modified linear solvation energy relationship model to probe the multiple interactions. All the results indicated that the types and arrangement of charged groups in zwitterionic stationary phases mainly affect the retention and separation of ionic or ionizable compounds, and for interaction characteristics the contribution from n and π electrons and electrostatic interactions displayed certain differences.     

Separation of cis- and trans-isomers of thioxanthene and dibenz[b,e]oxepin derivatives on calixarene- and resorcinarene-bonded high-performance liquid chromatography stationary phases

The chromatographic behavior of six calix[n]arene phases (n=4, 6, 8) and one calix[4]resorcinarene phase is described for the separation of cis- and trans-isomers of three thioxanthene (flupentixol, clopenthixol, chlorprothixene) and one benz[b,e]oxepin derivative (doxepin). The influences of two different organic modifiers (MeOH, MeCN) for the separation of the isomers on every column are described. Different selectivities of the stationary phases exist as a function of the ring size of the calixarenes and their substitution at the "upper rim" with p-tert.-butyl groups. Furthermore, the influence of free phenol groups on the resorcinarene phase is discussed. Relations between structural elements of the analytes and the retention behavior on the stationary phases are found. The selectivity of the calixarene and resorcinarene stationary phases is compared with a RP-C18 phase containing the same base silica. Advantages of the resorcinarene as well as of the calixarene columns exist for the separation of cis- and trans-isomers of three compounds dependent from the substitution in position 2 of the thioxanthenes, respectively the kind of the basic side chain of all substances.     

Preparation of a sulfoxide group and ammonium-salt bonded silica stationary phase for separation of polychlorinated biphenyls from mineral oils

In this study, a silica stationary phase modified with sulfoxide group and ammonium-salt was prepared for the separation of polychlorinated biphenyls (PCBs) from mineral oils, and its properties were investigated. Organic sulfide was attached to a diamino (primary and secondary amino) bonded silica surface by an amide bond, and the bonded sulfide groups were oxidized with periodate to afford sulfoxide groups bonded to the stationary phase. The secondary amino groups in the spacer chain were converted to ammonium-salt by the addition of hydrochloric acid. The sulfoxide group and ammonium-salt bonded stationary phase was tested for their suitability as adsorbent for SPE-type preparative short columns and for an analytical HPLC-type separation. The new stationary phase (1.2 mmol of sulfur bonded per gram) separated PCBs from mineral oils (paraffin-based transformer oils) more efficiently than previously reported stationary phases including sulfoxide group or ammonium-salt bonded ones. The quantitative chromatographic parameters for an aliphatic hydrocarbon (eicosane) and some PCB congeners also indicated strong retention of highly chlorinated biphenyls by the sulfoxide and ammonium-salt bonded silica compared with simple aminopropyl, sulfoxide group or ammonium-salt bonded ones. A cleanup procedure was established for simple determination of PCBs in mineral oil samples using sulfoxide group and ammonium-salt bonded silica packed column fractionation. The analytical method, combination of the cleanup procedure, and measurement with a GC-high resolution (magnetic sector) MS or a GC-quadrupole MS were validated using mineral oil certified reference materials.     

Analysis of non-ionic surfactants used in tertiary oil recovery. Optimisation of stationary phase in normal phase partition chromatography

Summary Non-ionic surfactants, made from ethylene oxide units of various lengths condensed with long chain aliphatic alcohols, which are used extensively in enhanced crude oil recovery have been analysed in the past by both adsorption and normal phase partition chromatography. These methods work satisfactorily for narrow range polymers but are of no use for wide range higher molecular weight materials. We have investigated the performance of a new silica diol bonded phase and compared its chromatographic behaviour with amino and cyano bonded phases for the separation of non-ionic surfactants. After derivatisation with 3,5-dinitrobenzoyl chloride it was found that only the diol phase gave a good evaluation of the distribution versus the number of ethylene oxide units for three surfactants KL 6, KM 11 and KM 20 (the number represent the average number of ethylene oxide units in the molecule). Isocratic elution was used in the case of KL 6 and KM 11 and gradient elution for KM 20. In spite of the presence of over 40 peaks in the chromatogram of KM 20, analysis was accomplished in about 45 minutes. Identification of peaks was achieved by the coelution of a pure standard.     

Novel stationary phases for high-performance liquid chromatography analysis of cyclodextrin derivatives

Novel stationary phases were prepared for separation of cyclodextrins and cyclodextrin derivatives by bonding substituted aromatic groups (phenyl and naphthyl) to the silica gel matrix. Both the electron-withdrawing (nitro) and the hydrogen-donor/acceptor (amide or carbamide) substituents of the phenyl group play essential role in the separation of cyclodextrins and cyclodextrin derivatives. On the basis of the comparison of experimental data obtained on different columns the N-(4-nitrophenyl)-carbamide group bonded silica gel stationary phase was selected as the most effective one for analysis of cyclodextrin derivatives. Improved separation potency was observed for hydroxypropylated, methylated and several other cyclodextrin derivatives compared with the previously and presently used stationary phases. Owing to the strong retention of cyclodextrins and its derivatives on the selected column, detection of their decomposition products was easily achieved. Determination of unsubstituted, natural cyclodextrin as an impurity in the cyclodextrin derivatives was implemented. Identification and characterization of cyclodextrin derivatives in industrial products could also be performed.     

High performance liquid chromatography with cyclodextrin and calixarene macrocycle bonded silica stationary phases for separation of steroids

β-Cyclodextrin, p-tert-butyl-calix[8]arene and chloropropyl bonded silica stationary phases have been prepared and were applied at the same time to develop a chromatographic procedure to separate steroids. In order to select the best type of stationary phase for the analysis, similar preparation processes of the two kinds of macrocycle stationary phases with the same spacer were adopted respectively. The chromatographic behaviors and retention mechanisms of the two kinds of macrocycle stationary phases for steroids were systematically studied and compared with those of chloropropyl bonded silica and ODS. The effect of mobile phase variables, such as methanol content, pH value of buffer, ionic strength and buffer composition on chromatographic behaviors was investigated. The results showed that the retention mechanisms of the four stationary phases for steroids were obviously different, and excellent separation was achieved on β-cyclodextrin bonded silica stationary phase (β-CD-BS), as a consequence of the structure and the properties of the stationary phase. The retention process on β-CD-BS exhibited inclusion complexation, hydrogen-bonding and weak hydrophobic interaction, while for p-tert-butyl-calix[8]arene bonded silica stationary phase (CBS), π-π and hydrogen-bonding besides hydrophobic interaction played an important role.     

Development of a decaaza‐cyclophane stationary phase for high‐performance liquid chromatography

A new stationary phase for high‐performance liquid chromatography was prepared by covalently bonding a heteroatom‐bridged cyclophane onto silica gel using 3‐aminopropyltriethoxysilane as the coupling reagent. The structure of the new material was characterized by infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The linear solvation energy relationship method was successfully employed to evaluate the new phase with a set of 25 solutes, and compared with octadecylsilyl and p‐tert‐butyl‐calix[4]arene bonded stationary phases. The retention characteristics of the new phase are similar to the octadecylsilyl and conventional calixarene phases, and it also has distinctive features. In addition, the chromatographic behavior of the phase was illustrated by eluting alkylbenzenes and inorganic anions in the reversed‐phase mode and anion‐exchange mode, respectively. Thus, multi‐interaction mechanisms and mixed‐mode separation of the new phase can very likely guarantee its promising application in the analysis of complex samples. The column has been successfully employed for the analysis of triazines in milk, and it is demonstrated to be a competitive alternative analytical method for the determination of triazine herbicide residues.     

Liquid chromatography on unmodified and modified Spheron gels III. Phenolic compounds

Summary Chromatographic behaviour of phenolic compounds is studied on the ethyleneglycol methacrylate gel Spheron and on ion-exchangers produced by the chemical modification of this gels (cation exchanger Spheron S and anion exchanger Spheron DEAE) as compared to octadecyl silica. The hydrophobic effects obviously predominate in the retention mechanism on Spherons in aqueous methanolic mobile phases, but a selectivity differring from the behaviour on octadecyl silica was found for a number of phenolic compounds This is due to interactions with the functional groups in the unmodified and modified Spheron materials and may be utilized for the separation of phenols by liquid chromatography.     

Determination of catecholamines in urine using hydrophilic interaction chromatography with electrochemical detection

The determination of catecholamines in urine was investigated using hydrophilic interaction chromatography (HILIC) as an alternative to the commonly used reversed-phase (RP) method. A number of different approaches were explored, including per-aqueous liquid chromatography (PALC), and HILIC using bare silica, bonded amide and zwitterionic phases. The bonded phases gave superior results in terms of both peak shape and selectivity. The mechanism of the HILIC separation was investigated particularly with respect to the contribution of ion exchange to retention. The electrochemical detection of catecholamines was studied and optimised in typical HILIC mobile phases that contain high concentrations of acetonitrile. HILIC offered a number of advantages over the conventional RP approach, giving good retention of the solutes without use of ion pair reagents, the absence of which also would facilitate detection by mass spectrometry. HILIC used in conjunction with solid phase extraction based on RP also gives orthogonal separation mechanisms in the cleanup and analysis steps. Furthermore, good recoveries from the cleanup stage were obtained, as high concentrations of acetonitrile can be used as eluting solvent that are fully compatible with HILIC, and lipophilic impurities are eluted close to the void volume of the HILIC column.     

The effect of chromatographic conditions on the separation of selected alkaloids in RP-HPTLC

Selected alkaloid standards were chromatographed on C18 W layers using various aqueous eluents containing an organic modifier and pH 3 buffer to suppress silanol ionization or an organic modifier and pH 8 buffer to suppress alkaloid ionization. Anionic ion pairs such as sodium dodecyl sulfate, octane-1-sulfonic acid sodium salt, pentane-1-sulfonic acid sodium salt, and bis(2-ethylhexyl)ortho-phosphoric acid are used to improve peak shape, efficiency, and selectivity. Amines (e.g., diethylamine, triethylamine, and tetrabutylamonium chloride) are incorporated into mobile phases to block surface silanols. The effect of chromatographic conditions on the separation of the investigated alkaloids is analyzed by the comparison of particular densitograms, asymmetry factor, or theoretical plate number. The best efficiency, peak symmetry, and separation selectivity of the investigated compounds is obtained through the addition of amine (especially diethylamine) to the mobile phases.     

Temperature effect in separation of fullerene by high-performance liquid chromatography

Summary The effect of column temperature, especially at low temperatures, on the separation of fullerenes on monomeric and polymeric octadecyl silica (ODS) bonded phases has been studied. Decreasing the column temperature induces an increase in selectivity. The best temperature for the separation of fullerenes was determined for both types of ODS phase with n-hexane eluent. The selectivity for higher fullerenes on monomeric phases becomes similar to that on polymeric phases to low temperature. It has been found that as the carbon content of monomeric phases is increased, the selectivity also becomes similar to polymeric phases.