Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition

A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC, both modifiers (surfactant and acetonitrile) were observed to decrease or improve the efficiencies in the same percentage, at least in the studied concentration ranges. The study also revealed that the problem of achieving smaller efficiencies in this chromatographic mode, compared to hydro-organic RPLC, is not only related to the presence of surfactant covering the stationary phase, but also to the smaller concentration of organic solvent in the mobile phase.     

Study of peak shape and efficiency in butyl acrylate-based monolithic columns for capillary electrochromatography

The study and modelling peak shape of in capillary electrochromatography (CEC), obtained using butyl acrylate (BA)-based monolithic columns, is described in this article. A modified-Gaussian model, which is a function of conventional experimental parameters: retention time (t_R), peak height (H₀) and standard deviation (σ₀) at the peak maximum, and left (A) and right (B) halfwidths, was used to describe the peaks of thiourea and several polyaromatic hydrocarbons compounds, which were eluted under several applied voltages. A mean relative error below 2% in the prediction of peak shape is obtained. Based on peak parameters, an easy and reliable estimation of global chromatographic performance, efficiency and peak capacity of BA-based monoliths was also considered. A comparison in terms of chromatographic performance of BA-based monoliths in CEC with CE mode and RPLC microparticulate columns was also performed.     

Deconvolution of overlapped peaks based on the exponentially modified Gaussian model in comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) has attracted much attention for the analysis of complex samples. Even with a large peak capacity in GC x GC, peak overlapping is often met. In this paper, a new method was developed to resolve overlapped peaks based on the mass conservation and the exponentially modified Gaussian (EMG) model. Linear relationships between the calculated sigma, tau of primary peaks with the corresponding retention time (tR) were obtained, and the correlation coefficients were over 0.99. Based on such relationships, the elution profile of each compound in overlapped peaks could be simulated, even for the peak never separated on the second-dimension. The proposed method has proven to offer more accurate peak area than the general data processing method.     

葛根提取物高效液相色谱分析方法的优化

针对葛根提取物的复杂体系,分别从色谱模式、流动相、添加剂以及梯度洗脱条件几个方面对其高效液相色谱分析 方法进行了优化。其中梯度洗脱条件的优化是根据各组分在5次线性梯度下的保留时间并借助于CSASS软件快速准确地计 算出各组分的液相色谱保留参数a,c值以及峰形参数σ和τ,在此基础上使用CSASS软件对葛根提取物的色谱分离情况进行 高精度仿真预测,从而优化了梯度洗脱条件。考察了优化条件下的分析方法的精密度和重现性,结果表明该方法稳定、可 靠、重现性好。     

Optimized stationary phases for the high-performance liquid chromatography-electrospray ionization mass spectrometric analysis of basic pharmaceuticals

Stationary phases were investigated for HPLC coupled with electrospray ionization mass spectrometry (ESI-MS) for the analysis of basic drugs. Tricyclic antidepressants (TCAs) and beta-blockers were used as model solutes. The functional groups, pentafluorophenyl (PFP), OH, CN or CH3 were attached to the silica via a propyl chain. The effects of these stationary phases as well as C8 and C18 phases on retention and peak shape of the basic drugs were studied. The CN and PFP phases adequately retained (tR of 2 to 6 min) the basic drugs when the mobile phase was composed of 90% acetonitrile, whereas with the C4, C8 and C18 phases, less than 40% acetonitrile had to be used to provide adequate retention of the basic drugs. Because acetonitrile provides better desolvation in ESI than an aqueous solvent, it produces an increased MS signal. As an example of the HPLC-ESI-MS analysis of the beta-blocker, pindolol, on a CN phase, the use of 90% acetonitrile in the mobile phase increased the ESI-MS signal by 790% when compared to a C18 phase which could use only 5% acetonitrile in the mobile phase for retention of the solute. In addition, the CN and PFP phases provided better peak shape than the OH phase and the hydrophobic phases (C4, C8 and C18) and ion-pairing or ion-suppressing agents were not required. The retention behavior of the TCAs and beta-blockers on each of the phases is described.     

New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.     

液相色谱梯度洗脱中的谱带压缩效应

谱带压缩效应是梯度洗脱区别于等度洗脱的重要特征。经典的范德姆特(van Deemter)理论塔板高度方程基于等度洗脱推导得到,因此不能对谱带压缩效应进行描述。在梯度洗脱中,保留因子(k)会随流动相组成(φ)的改变而发生变化,这就使得对梯度洗脱机理的研究要比等度洗脱复杂许多。该文对近10年来谱带压缩效应的研究进展,特别是溶剂强度模型(即描述ln k与φ关系的数学表达式)的非线性特征对谱带压缩因子(G)的影响进行了述评,指出为了更好地认识谱带压缩效应需要将这种非线性因素考虑在内。     

钴-60辐照对川贝活性成分的影响

考查不同Co-60辐照剂量对川贝活性成分的影响,为川贝的灭菌贮存和质量评价提供参考。采用HPLC法分析成分的变化。在不同辐射剂量照射后,川贝的化学成分的组成基本不变(各组分的保留时间tR不受辐照影响),但钴-60辐照剂量会对川贝药材某些组分的含量有不同程度的影响,在川贝的贮存和质量评价中应引起注意。     

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide‐ and cyclofructan‐based stationary phases. The effects of buffer concentration, pH and acetonitrile‐to‐aqueous‐part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan‐based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan‐based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.     

径向基函数神经网络用于重叠色谱峰解析

 在高斯基径向基函数神经网络 (RBFNN)学习算法中引入了鲁棒性和随机全局寻优的两阶段遗传算法 :结构学习和参数优化。通过两阶段学习算法的交替使用 ,使网络具有结构自学习和参数优化的能力 ,而后将网络应用于组分数未知的重叠色谱峰解析。该方法具有不需人为干预 ,可自动确定网络结构即组分数的优点 ;并且解析精度较高 ,适用于多组分重叠色谱峰的解析 ;对完全重叠色谱峰也具有良好的解析能力。     

Effect of short-chain alcohols on surfactant-mediated reversed-phase liquid chromatographic systems

The behaviour of β-blockers in a reversed-phase liquid chromatographic (RPLC) column with mobile phases containing a short-chain alcohol (methanol, ethanol or 1-propanol), with and without the surfactant sodium dodecyl sulphate (SDS), was explored. Two surfactant-mediated RPLC modes were studied, where the mobile phases contained either micelles or only surfactant monomers at high concentration. Acetonitrile was also considered for comparison purposes. A correlation was found between the effects of the organic solvent on micelle formation (monitored by the drop weight procedure) and on the nature of the chromatographic system (as revealed by the retention, elution strength and peak shape of β-blockers). When SDS is added to the mobile phase, the free surfactant monomers bind the C18 bonded chains on the stationary phase, forming an anionic layer, which attracts strongly the cationic β-blockers. The retention is modified as a consequence of the solving power of the organic solvent, micelles and surfactant monomers. The molecules of organic solvent bind the micelles, modify their shape, and may avoid their formation. They also bind the monomers of surfactant, desorbing them from the stationary phase, which affects the retention. The remaining surfactant covers the free silanols on the siliceous support, avoiding the interaction with the cationic solutes. The retention of β-blockers results from a combination of electrostatic and hydrophobic interactions, the latter being weaker compared to the hydro-organic system. The peak efficiencies and asymmetries are excellent tools to probe the surfactant layer on the stationary phase in an SDS/organic solvent system. The peaks will be nearly symmetrical wherever enough surfactant coats the stationary phase (up to 60% methanol, 40% ethanol, 35% 1-propanol, and 50% acetonitrile).     

反相液相色谱操作参数最优化及水溶性维生素的分离

用最小二乘法拟合的多项式作为描述保留值或峰宽与流动相组成间关系的数学模型, 以影响水溶性维生素分离的主要因素(流动相中有机调节剂甲醇的浓度)作为主变量, 分析时间最短为目标, 各最难分离物质对的分离度≥1.5为约束条件, 在一定变量范围内对流动相组成进行了优化, 并运用此优化方法选择了维生素C、烟酸、烟酰胺、维生素B1、维生素B2和维生素B6六种水溶性维生素的最佳分离条件.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

高效液相色谱法分离测定3-取代-(R,S)-β-丙氨酸对映异构体

以1-氟-2,4-二硝基-5-L-缬氨酰胺(Marfey试剂)为衍生试剂,采用反相高效液相色谱法分离了3-取代-(R,S)-β- 丙氨酸对映异构体。采用梯度洗脱(流动相 A:体积分数为0.1%的三氟乙酸乙腈溶液;流动相B:体积分数为0.1%的三氟乙 酸水溶液)成功分离了 32种3-取代-(R,S)-β-丙氨酸衍生物,所有化合物都是R型异构体衍生物(R-L)较S型异构体衍生物 (S-L)先洗脱。除3-羟基苯和4-羟基苯的疏水参数较小,但其取代的β-丙氨酸衍生物的保留时间较长、分离因子较小外, 其他疏水参数大的取代基的β-丙氨酸衍生物保留时间都长于疏水参数小的取代基的β-丙氨酸衍生物。该文同时测定了R -和S-β-丙氨酸的对映体过剩值。     

Measurement of the elution strength and peak shape enhancement at increasing modifier concentration and temperature in RPLC

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile–water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25?°C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile–water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80?°C the values obtained at equilibrium conditions, already reached with acetonitrile at 25?°C.     

Modeling of ion-exclusion and vacancy ion-exclusion chromatography in analytical and concentration overload conditions

Ion-exclusion chromatography (IEC) finds applications in various different analytical separations of weak acids. Pure, deionized water or a diluted, aqueous solution of a strong mineral acid (such as, e.g., sulphuric acid) is used as the mobile phase, whereas a typical stationary phase is a strongly acidic resin in the H(+) form (e.g., the sulfonated polystyrene-divinylbenzene resin with a high ion-exchange capacity, provided by the sulfonic acid groups). When pure water is used as the mobile phase, then the characteristic leading (i.e., frontally tailing) peaks are obtained, and the retention depends mainly on the concentration of the analyte. An alternative technique is vacancy ion-exclusion chromatography (v-IEC), in which the column is equilibrated with the sample solution, flowing as the mobile phase through the system, and pure water is injected as the sample. In this case, the symmetrical vacant peaks are obtained. The aim of this paper is to describe the retention mechanism in IEC and v-IEC for the adsorptive and nonadsorptive acids in analytical and concentration overload conditions, with pure water and the diluted sulphuric acid solution as the two different mobile phases. The retention times and the peak shapes predicted by the derived equations remain in a good qualitative and quantitative agreement with the experimental data. The model proposed in this paper predicts the new features characteristic of IEC for the adsorptive acids. These are, namely, an increase in the retention time of the peak apexes (up to a certain level and concurring with an increase in the acid concentration), followed by a subsequent decrease of the retention time (with the further growth of the acid concentration in the eluent). Similar changes in the retention time observed for v-IEC in the specific adsorption conditions were also correctly predicted by the model.     

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?     

Effect of solute association on the apparent adsorption isotherm. A model of the separation of non-racemic mixtures of enantiomers in achiral chromatographic systems

Summary A computer spreadsheet application has been developed for the optimization of step-gradient elution conditions as applied in coupled-column RPLC for online clean-up and separation in the analysis of pesticide residues. The procedure is based on the experimentally determined retention behaviour of the analytes as a function of mobile phase composition. Retention and peak volumes of the analytes eluting under isocratic and one- and/or two-step gradient conditions can be calculated. For a given set of analytes, the spreadsheet systematically calculates the resolution of the least efficiently separated pair of peaks in the space defined by the eluotropic strengths of the mobile phases and the time at which the step-gradient takes place.The performance of the proposed procedure was tested and evaluated for the analysis of nine polar pesticides for which in a previous study adequate conditions had been found by computer-aided chromatogram simulation. the optimum conditions predicted by the procedure were in good agreement with those established experimentally and by simulation. The simultaneous production of data on retention, peak volume and resolution for a group of analytes under selected one- and/or two step-gradient elution conditions makes the calculation procedure highly productive and flexible in searching for and/or adjusting appropriate separation conditions.