Direct high-performance liquid chromatographic separation of unusual secondary amino acids and a comparison of the performances of Chirobiotic T and TAG columns

Two macrocyclic glycopeptide antibiotic-type chiral stationary phases (CSPs) based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and TAG, respectively, were evaluated with regard to the high-performance liquid chromatographic separation of the enantiomers of 10 secondary alpha-amino acids (imino acids). The chromatographic results are given as the retention, separation and resolution factors, together with the enantioselective free energy difference corresponding to the separation of the enantiomers. By application of these two CSPs, excellent resolutions were achieved for the investigated compounds by using reversed-phase mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these particular amino acids ranged between 0.70 and -1.83 kJ mol(-1). It was established that better enantioseparations of the secondary alpha-amino acids were attained in most cases on the aglycone CSP.     

Retention properties of acetone-water mobile phases on a biphenylsiloxane-bonded silica stationary phase in reversed-phase liquid chromatography

The solvation parameter model system constants and retention factors were used to interpret retention properties of 39 calibration compounds on a biphenylsiloxane-bonded stationary phase (Kinetex biphenyl) for acetone-water binary mobile phase systems containing 30–70% v/v. Variation in system constants, phase ratios, and retention factors of acetone-water binary mobile phases systems were compared with more commonly used acetonitrile and methanol mobile phase systems. Retention properties of acetone mobile phases on a Kinetex biphenyl column were more similar to that of acetonitrile than methanol mobile phases except with respect to selectivity equivalency. Importantly, selectivity differences arising between acetone and acetonitrile systems (the lower hydrogen-bond basicity of acetone-water mobile phases and differences in hydrogen-bond acidity, cavity formation and dispersion interactions) could be exploited in reversed-phase liquid chromatography method development on a Kinetex biphenyl stationary phase.     

Acoustic effects in chromatography

An acoustic field effectively affects chromatographic retention in some separation modes and, thus, can be a novel factor controlling retention. After being transmitted into the column, ultrasound energy is mostly converted into heat as a result of absorption by stationary and mobile phases. Thus, ultrasound brings about temperature increases. However, actual temperature increases measured by thermosensors are smaller than those calculated from chromatographic retention changes. In addition, larger ultrasound effects are observed in chromatographic modes involving ionic interactions. These results possibly imply that ultrasound directly influences ionic interactions involved in retention mechanisms.     

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.     

姜黄素键合硅胶固定相的制备、表征及色谱性能

通过γ-［(2,3)-环氧丙氧］丙基三甲氧基硅烷(KH-560)偶联剂将具有抗菌功能的植物有效成分姜黄素键合到硅胶上,制备了姜黄素液相色谱键合硅胶固定相(CCSP)。采用元素分析、红外光谱和热分析对该固定相结构进行了表征。以甲醇和水为二元流动相,不同的中性、酸性和碱性化合物为溶质探针,并用ODS柱作参比,对固定相的色谱性能及保留机理进行了研究。研究结果表明,姜黄素键合固定相不仅具有良好的反相色谱性能,同时由于配体结构中所含有的基团形成了含芳环的共轭体系,从而引入了n-π和π-π作用位点,所含的羟基和β-二羰基与溶质之间存在偶极-偶极和氢键作用,与ODS相比,该固定相在极性化合物分离中占优势,且分析速度较快。     

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.     

Effect of the concentration of organic modifier in an aqueous-ethanol mobile phase on the chromatographic retention and thermodynamic characteristics of the adsorption of enantiomers of α-phenylcarboxylic acids on silica gel with immobilized eremomycin antibiotic

Regularities of the chromatographic retention and thermodynamics of the adsorption of enantiomers of α-phenylcarboxylic acids on a chiral stationary phase with immobilized macrocyclic antibiotic eremomycin under conditions of reversed-phase liquid chromatography with aqueous-ethanol mobile phases are studied. Relationships between the retention characteristics of the acids, the enantioselectivity of their separation, and the concentration of organic modifier in the mobile phase are found. It is shown that the sterical structure of substituents on the chiral atoms of the acids affect the mechanism of retention. The compensation effect in the studied systems is considered.     

Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis

Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100‐5 C18 and LiChrosorb RP‐select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers – acetonitrile or methanol – in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?     

Micellar bile salt mobile phases for the liquid chromatographic separation of routine compounds and optical, geometrical, and structural isomers

Aqueous solutions of bile salts, i.e. sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium taurocholate (NaTC), are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. The separation of the ASTM-recommended RPLC test mix in addition to more than 50 other compounds on a C18 column demonstrates the viability of these bile salts as HPLC mobile phases. The Armstrong-Nome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with these bile salts at low surfactant concentrations. The effect of alcohol additives on chromatographic retention and efficiency was also assessed. Not only are the bile salt molecules rigid and chiral, but they form helical micellar aggregates as well. Consequently, many isomeric compounds can be easily resolved with this mobile phase additive. The base-line resolution of some binaphthyl-type enantiomers with a standard C18 column and the bile salt micellar mobile phases is also demonstrated. In addition, these bile salt mobile phases may be preferable to conventional hydroorganic mobile phase systems for the separation of many classes of routine compounds. A brief prospectus on the future utilization of bile salts in liquid chromatography is presented.     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

磷酸钛离子交换薄层色谱分离无机离子(英文)

The chromatographic behavior of 30 inorganic cations has been studied on thin layers of titanium phosphate ion-exchanger using several aqueous,organic and mixed mobile phases.The separation of one ion from several other ions and also ternary and binary separations have been developed.Some important analytical separations are reported.The effect of pH of the mobile phase on retention factor(Rf)values of the cations in the presence of complex-forming anion along with the separation power of the ion-exchanger were studied.This ion-exchanger exhibits high sorption capacity and varying selectivity towards metal ions and makes it a suitable stationary phase in thin layer chromatography.     

Comparison of four cholesterol‐based stationary phases for the separation of steroid hormones

The chromatographic behavior of steroid hormones on four cholesterol‐bonded stationary phases with different structures in binary methanol/water mobile phases was studied. Of the stationary phases tested, the commercially available stationary phases Cogent UDC cholesterol? and COSMOSIL cholester? provided better separations of steroid hormones in comparison to homemade aminocholesterol and diaminocholesterol stationary phases. The results show that the temperature has a significant influence on the retention and selectivity for steroid hormones separation. The temperature increase may cause changes in the elution order. From the dependences of the retention (ln k) on temperature (1/T), the standard partial molar enthalpy and standard partial molar entropy were calculated and their enthalpic and entropic contributions to the retention were compared. The enthalpic effects principally control the retention mechanism.     

Nonlinear chromatography Recent theoretical and experimental results

The theory of nonlinear chromatography has been advanced by the incorporation of recent results obtained by the theory of partial differential equations. The system of equations of the ideal model has been solved analytically in the case of a single component for which the equilibrium isotherm between the mobile and the stationary phases is given by a Langmuir equation. A series of computer programs has been written which permits the calculation of numerical solutions of the semi-ideal model. The properties of the solutions obtained are described and discussed for a one-component system (profile of high concentration bands of a pure compound eluted by a pure solvent), several two-component systems (elution of a pure compound band by a binary mobile phase, separation of a binary mixture eluted by a pure mobile phase), and three-component systems (separation of a binary mixture eluted by a binary solvent, displacement and separation of a binary mixture). Experimental results are reported which validate the conclusions derived from the numerical integration of the model. The conclusions of the work apply to all high-performance chromatographic procedures, i.e., to those where the kinetics of mass transfer are fast enough for the mobile and stationary phases always to be near equilibrium. More specifically, the contribution from the kinetics of the retention mechanism to the mass transfer resistance must itself be negligible. This clearly excludes affinity chromatography.     

A unified classification of stationary phases for packed column supercritical fluid chromatography

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.     

TLC determination of selected macrocyclic antibiotics using normal and reversed phases

Summary The chromatographic behavior of some macrocyclic antibiotics (erythromycin, troleandomycin, tylosin, vancomycin, rifamycin B, rifampicin) has been compared on normal and reversedphase TLC plates using water, methanol, 1-propanol and binary solvent mixtures (methanolwater, 1-propanol-water) as mobile phases in which the concentration of organic modifier ranged from 0–100%, v/v. In some cases sodium chloride was also added to the eluent. Chromatographic retention data and a possible retention mechanism is discussed.     

一种可控离子比例的两性亲水色谱固定相的制备及性能研究

将不同比例的氨基和巯基的硅烷偶联剂键合到硅胶表面，再利用巯基与乙烯基膦酸之间的点击化学反应将膦酸基团引入到硅胶表面，制备了一种可调节正负离子比例的两性亲水色谱固定相。通过测定固定相中C、H、N、P元素的含量，证明了氨基与膦酸基团已成功键合到固定相的表面，同时通过N元素与P元素的质量分数确定固定相表面氨基与膦酸基团的比例。制备了3种不同电荷比例的氨基膦酸固定相，将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中。以一系列经典的极性小分子作为探针，研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对探针分子在3种色谱柱上的保留的影响，结果表明，分析物在固定相上是多重保留机理。最后通过比较核苷、水溶性维生素、碱性化合物、苯甲酸这几类标准物质在3种色谱柱上的保留行为来对比3种不同电荷比例的固定相的分离选择性与色谱性能。结果表明，对于不同的分析物，3种固定相表现出完全不同的分离选择性和色谱行为。可以根据分析物的特征选取不同电荷比例的固定相，表明此种固定相在极性化合物的分离上具有良好的应用前景。     

Estimation of the degree of self-association of water using a novel retention model

Summary An attempt has been made to gain a semi-quantitative insight into the self-association of water molecules through hydrogen bonds. This was only possible with the use of a new solute retention model for the chromatographic systems by considering intermolecular interactions between the constituents of binary mobile phases. Four different sizes of the average associative aqueous multimer were assumed. By comparing measured and calculated retention values, the existence of associated aqueous multimers consisting of 100 aqueous monomer units is postulated as an average multimer structure.