Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Separation of xanthines in hydro‐organic and polar‐organic elution modes on a titania stationary phase

Hydrophilic interaction LC was investigated in hydro‐organic and nonaqueous elution modes on a titania column by using a set of N‐methyl xanthines as neutral polar probes. To get information regarding the mechanisms that are behind the discrimination of these analytes in hydrophilic interaction, we focused our study on the type and amount of organic modifier as a critical yet rarely explored mobile phase parameter. Several alcohols such as methanol, ethanol, and isopropanol were studied as substitutes to acetonitrile in hydro‐organic elution mode. Compared to silica, the investigation of the eluotropic series of these alcohols on titania highlighted a different implication in the retention mechanism of the xanthine derivatives. At low amounts of protic solvents, the adsorption mainly characterized the retention of analytes on bare silica; whereas mixed interactions including adsorption and ligand exchange were identified on native titania. To investigate the peculiar behavior of alcohols on the metal oxide, methanol, ethanol, and ethylene glycol were tested in replacement of water in polar‐organic elution mode. Distinctive effects on the chromatographic retention and selectivity of xanthines were noticed for the dihydric alcohol, which was found to be a stronger eluting component than water on titania.     

Optimum conditions for the separation of nucleobases and nucleosides on unmodified silica by HPLC

Summary The retention behaviour of nucleobases and nucleosides on unmodified silica with dichloromethane-methanol-water mixtures has been systematically investigated. The degree and order of retention can be varied over a wide range by changing the pH, the type and concentration of the acidic additives and by the methanol and water content of the mobile phase. The retention process cannot be considered as simple adsorption but rather as a very complex mixed distribution process of adsorption and absorption including the involvement of ion-pair formation. Further data on the effect of the type of silica (source of supply) on retention behaviour, column efficiency and column reproducibility are reported. The practical application of unmodified silica for the separation of nucleobases and nucleosides is demonstrated by the analysis of a hydrolysate of calf thymus DNA.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

黄酮醇异构体的超临界流体色谱法分离

用超临界流体色谱法进行了黄酮醇异构体的分离研究。考察了温度、压力、流动相组成、柱条件等对分离的影响。在实验的温度范围４０～６０℃和压力范围１５～３０ＭＰａ内,这组化合物都能得到很好的分离;流动相组成是影响色谱分离的最显著的因素,磷酸的加入大大改变了各物质的保留行为;考察了三种硅胶基质键合相对分离的影响,发现苯基柱用于这组异构体的分离最为合适。     

氯丙基键合硅胶固定相色谱性能与应用 I.二取代苯及前列腺素差向异构体的分离

研究了氯丙基键合硅胶固定相的高效液相色谱性能和在分离二取代苯异构体、前列腺素差向异构体方面的应用,探讨了流动相组成、性质、ｐＨ值等对溶质保留和分离的影响,讨论了溶质保留机理和最佳色谱分离条件。对上述异构体进行了良好的分离,而且分离速度比一般的反相色谱体系要好。     

硅胶电色谱分离机理的研究

对硅胶电色谱柱的性能进行了考察,发现在水／有机溶剂流动相条件下,几乎不存在气泡问题,流动相的组成在有机溶剂浓度、电解质浓度、ＰＨ值等方面可以在较大范围变化,选用５种典型样品,对硅胶电色谱的分离机理进行了系统研究,发现有反相分离机理、正相吸附机理、离子交换机理以及电泳机理参与作用。同时考察了有机溶剂浓度、电解质浓度、ＰＨ等对分离的影响。此外,还首次提出了一种全新的电色谱模式－动态改性硅胶电色谱。     

Studies on the retention behaviour of some thiazolylazo derivatives in reversed phase liquid chromatography

The retention behaviour of thiazolylazo derivatives, 4-(2-thiazolylazo) resorcinol (TAR), 4-(2-thiazolylazo)-orcinol (TAO), 2-(2-thiazolylazo)-4-methylphenol (TAC) and 1-(2-thiazolylazo)-2-naphtol (TAN) was studied by reversed phase liquid chromatography. The optimum conditions for the separation of four thiazolylazo derivatives were examined with respect to column, flow rate, mobile phase composition and pH of mobile phase. These derivatives were separated simultaneously on Symmetry C₈ column using composition of acetonitrile/water (60/40, v/v) as mobile phase. The capacity factor (k′) has been decreased at higher pH than pK_a of solute which may due to the increasing concentration of the ionized species as increase the pH of mobile phase. The dependence of log k′ on the volume faction of water in the binary mobile phase and k′ on the liquid–liquid extraction distribution ratio (D_c) in acetonitrile–water (60/40, v/v)/n-octane extraction system for thiazolylazo derivatives were obtained good linear relationship. The results showed that the retention behaviour of these derivatives was mainly affected by the hydrophobic interaction between thiazolylazo as solute and mobile phase.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol‐bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous‐acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show “U‐profile” characteristic of a dual hydrophilic interaction–reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton–donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Mechanism and prediction of retention of oligomers in normal-phase and reversed-phase HPLC

Summary The simultaneous dependence of the retention in oligomeric series on the number of repeat structural units and on the mobile phase composition may be described by very similar equations for reversed-phase and for normal-phase systems.In reversed-phase systems, the separation selectivity of the individual oligomers is determined mainly by the size and by the polarity of the repeat structural unit, but the influence of a bulky and polar structural residue may also become important so that even reversed order of elution may be observed for oligomeric series with the same oligomeric units but significantly different end groups. For example, oligoethylene glycols are eluted in the order of increasing size of the oligomers, whereas ethoxylated nonylphenols are eluted in the order of decreasing size.In normal-phase systems, the separation selectivity in oligomeric series depends on the adsorption energy and on the adsorbed area of the oligomeric unit. If the oligomeric unit is small, the concentration of the polar solvent in the binary organic mobile phase has only a minor effect on retention and selectivity, which may be controlled by taking account of the nature of the adsorbent and of the polar solvent or by varying the proportion of two polar solvents in a ternary mobile phase.     

High performance liquid chromatography of structural isomers using a cyclodextrin-poly(allylamine) coated silica column

Summary High performance liquid chromatography (HPLC) separation of substituted phenols by isocratic elution was investigated using a β-cyclodextrin-bonded column. The HPLC support was prepared in-house. This support was based on silica beads coated with a β-cyclodextrin-containing poly(allylamine). The retention behavior of some substituted phenol isomers and aromatic compounds was studied. The contribution of the amino groups of the polyamine and of the β-cyclodextrin cavity to the separation process is discussed. Two retention mechanisms, inclusion complex formation and acidbase interaction, were found. The effect of the composition and pH of the mobile phase on the separation was also examined.     

High performance liquid chromatography of structural isomers using a cyclodextrin-poly(allylamine) coated silica column

Summary High performance liquid chromatography (HPLC) separation of substituted phenols by isocratic elution was investigated using a β-cyclodextrin-bonded column. The HPLC support was prepared in-house. This support was based on silica beads coated with a β-cyclodextrin-containing poly(allylamine). The retention behavior of some substituted phenol isomers and aromatic compounds was studied. The contribution of the amino groups of the polyamine and of the β-cyclodextrin cavity to the separation process is discussed. Two retention mechanisms, inclusion complex formation and acidbase interaction, were found. The effect of the composition and pH of the mobile phase on the separation was also examined.     

Use of a database of plots of pesticide retention (R F) against mobile-phase composition.Part II. TLC as a pilot technique for transferring retention data to HPLC, and use of the data for preliminary fractionation of a mixture of pesticides by micropreparative column chromatography

Summary Relationships betweenR _F values and mobile-phase composition have been determined for moderately polar pesticides in normal-phase systems (NP) of the type silica-non-polar diluent (heptane)-polar modifier (ethyl acetate, tetrahydrofuran, or dioxane) and in reversed-phase systems (RP) of the type octadecyl silica-water-polar modifier (acetonitrile, methanol, or tetrahydrofuran). These relationships constitute a retention database which has enabled choice of the optimum conditions for preparative column chromatographic separation of pesticides into fractions; these were then applied to a silica plate and chromatographed. The plate was videoscanned, furnishing a real picture of the plate showing complete separation of the pesticide fractions.     

Analysis of the band profiles of the enantiomers of phenylglycine in liquid chromatography on bonded teicoplanin columns using the stochastic theory of chromatography.

The retention behaviour of the enantiomers of underivatized phenylglycine was studied on a Chirobiotic T column packed with amphoteric glycopeptide teicoplanin covalently bonded to the surface of silica gel. The retention and the selectivity of separation of the enantiomers increase with rising concentration of ethanol or of methanol in aqueous-organic mobile phases. The band profiles of the less retained L-phenylglycine are symmetrical, but the band profiles of the more strongly retained D-phenylglycine are tailing in all mobile phases tested. The band broadening does not diminish even at very low concentrations of phenylglycine, so that it cannot be attributed to possible column overload. The analysis of the band profile using the stochastic theory of chromatography suggests that the broadening can be attributed to at least two additional chiral centres of adsorption in the stationary phase contributing to the retention of the more strongly retained enantiomer in addition to the adsorption of the less retained one. This behaviour can be explained by the complex structure of the teicoplanin chiral stationary phase.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

对氨基苯胂酸及其氧化物的色谱研究

用紫外分光光度计分析了对氨基苯胂酸（PABAA）及其氧化物的光谱特征后,在十八烷基键合相硅胶柱上,以甲醇-缓冲液作流动相,研究了二者的容量因子随流动相离子强度、柱温、甲醇含量变化的规律。用季铵盐作离子对试剂,反相离子对色谱法分离PABAA时,分离机理符合高子对机理,在适当条件下,所试验的化合物都可有所保留。对保留值作出贡献的有固定相排阻作用、分配作用以及居次要地位的PABAA与固定相表面剩余硅醇基的相互作用。排阻作用及分配作用的相对重要性与流动相中甲醇和离子对试剂的浓度有关。     

对氨基苯胂酸及其氧化物的色谱研究

 用紫外分光光度计分析了对氨基苯胂酸（PABAA）及其氧化物的光谱特征后,在十八烷基键合相硅胶柱上,以甲醇-缓冲液作流动相,研究了二者的容量因子随流动相离子强度、柱温、甲醇含量变化的规律。用季铵盐作离子对试剂,反相离子对色谱法分离PABAA时,分离机理符合高子对机理,在适当条件下,所试验的化合物都可有所保留。对保留值作出贡献的有固定相排阻作用、分配作用以及居次要地位的PABAA与固定相表面剩余硅醇基的相互作用。排阻作用及分配作用的相对重要性与流动相中甲醇和离子对试剂的浓度有关。