Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error, respectively).     

Retention controlling and peak shape simulation in anion chromatography using multiple equilibrium model and stochastic theory

The stochastic theory of chromatography and an equilibrium based approach were used for the prediction of peak shape and retention data of anions. This attempt incorporating the potential advantages of two different chromatographic phenomena for analytical purposes. It is an integrated method to estimate kinetic and thermodynamic properties for the same chromatographic run of ions. The stochastic parameters of eluted anions, such as the residence time of the molecule on the surface of the stationary phase, and the average number of adsorption steps were determined on the basis of a retention database of organic and inorganic anions (formate, chloride, bromide, nitrate, sulphate, oxalate, phosphate) obtained by using carbonate/bicarbonate eluent system at different pHs (9-11) and concentrations (7-13 mM). In the investigated IC system the residence times are much higher and the average number of sorption steps is somewhat smaller than in RP-HPLC. The simultaneous application of the stochastic and the multispecies eluent/analyte model was utilized to peak shape simulation and the retention controlling of various anions under elution conditions of practical importance. The similarities between the measured and the calculated chromatograms indicates the predictive and simulation power of the combined application of the stochastic theory and the multiple species eluent/analyte retention model.     

离子色谱线性梯度淋洗分离阴离子的计算机模拟

建立了使用氢氧化钠淋洗液时用计算机模拟离子色谱梯度淋洗分离阴离子的新方法,研究了在线性梯度淋洗过程中不同时间阴离子在色谱柱中的位置及所在位置的淋洗液浓度,得出阴离子在不同时间的容量因子、迁移速度,通过积分得到保留时间。再根据离子色谱峰峰形变化的规律,得到色谱峰峰形的参数。模拟色谱图保留值与实验值的相对误差小于5%,模拟色谱图的色谱峰峰形与实验所得到的色谱图的色谱峰峰形也非常接近。     

离子色谱测定口腔含漱剂中的多种成分

建立了离子色谱法对口腔含漱剂中抗龋剂如氟离子、单氟磷酸根、甘油磷酸根、防腐剂山梨酸、甜味剂糖精以及氯离子、硫酸根、磷酸根等同时测定的方法。采用AS11色谱分析柱和KOH梯度淋洗,抑制电导检测。被测物质的保留时间和峰面积重现性分别小于1.51%和6.02%;被测物的线性相关系数为0.9990~0.9999;加标回收率为89.4%~102.5%。本方法可用于商品化含漱剂和牙棒液体多种组分的同时测定。     

Novel criteria for fast searching for optimal method in gradient ion chromatography: an integrated approach

In this article, an integrated approach for prediction and optimization in ion chromatography (IC) was presented. The approach provides a fast and reliable insight in the elution behavior of an IC system. The predictions are based on a mathematical model that predicts ion retentions (for both isocratic and gradient modes) by using an empirical isocratic model. Other chromatographic values significant for the optimal elution conditions (resolution, peak asymmetry) are calculated quickly and easily from the predicted retention values of characteristic points of a chromatographic peak. Every day, IC users might find this approach a suitable tool for finding optimal IC elution conditions in a given system.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

Optimization of artificial neural networks used for retention modelling in ion chromatography

The aim of this work is the development of an artificial neural network model, which can be generalized and used in a variety of applications for retention modelling in ion chromatography. Influences of eluent flow-rate and concentration of eluent anion (OH-) on separation of seven inorganic anions (fluoride, chloride, nitrite, sulfate, bromide, nitrate, and phosphate) were investigated. Parallel prediction of retention times of seven inorganic anions by using one artificial neural network was applied. MATLAB Neural Networks ToolBox was not adequate for application to retention modelling in this particular case. Therefore the authors adopted it for retention modelling by programming in MATLAB metalanguage. The following routines were written; the division of experimental data set on training and test set; selection of data for training and test set; Dixon's outlier test; retraining procedure routine; calculations of relative error. A three-layer feed forward neural network trained with a Levenberg-Marquardt batch error back propagation algorithm has been used to model ion chromatographic retention mechanisms. The advantage of applied batch training methodology is the significant increase in speed of calculation of algorithms in comparison with delta rule training methodology. The technique of experimental data selection for training set was used allowing improvement of artificial neural network prediction power. Experimental design space was divided into 8-32 subspaces depending on number of experimental data points used for training set. The number of hidden layer nodes, the number of iteration steps and the number of experimental data points used for training set were optimized. This study presents the very fast (300 iteration steps) and very accurate (relative error of 0.88%) retention model, obtained by using a small amount of experimental data (16 experimental data points in training set). This indicates that the method of choice for retention modelling in ion chromatography is the artificial neural network.     

Simultaneous determination of peroxydisulfate and conventional inorganic anions by ion chromatography with the column‐switching technique

The application of ion chromatography with the column‐switching technique for the simultaneous analysis of peroxydisulfate and conventional inorganic anions in a single run is described. With this method, conventional inorganic anions were separated by consecutive elution through both the guard column and separation column, but peroxydisulfate that only passed through the guard column had a good peak shape and short retention time. A series of standard solutions consisting of target anions of various concentrations from 0.01 to 75 mg/L were analyzed, with a correlation coefficient (r) ≥ 0.9990. The limits of detection were in the range of 0.49–9.84 μg/L based on the S/N of 3 and a 25 μL injection volume. RSDs for retention time, peak area, and peak height were all <1.77%. A spiking study was performed with satisfactory recoveries between 97.6 and 103.4% for all anions. The quantitative determination of peroxydisulfate and conventional inorganic anions in surface waters was accomplished within 18 min by this column‐switching technique.     

Development of an inorganic cations retention model in ion chromatography by means of artificial neural networks with different two-phase training algorithms

This paper describes development of artificial neural network (ANN) retention model, which can be used for method development in variety of ion chromatographic applications. By using developed retention model it is possible both to improve performance characteristic of developed method and to speed up new method development by reducing unnecessary experimentation. Multilayered feed forward neural network has been used to model retention behaviour of void peak, lithium, sodium, ammonium, potassium, magnesium, calcium, strontium and barium in relation with the eluent flow rate and concentration of methasulphonic acid (MSA) in eluent. The probability of finding the global minimum and fast convergence at the same time were enhanced by applying a two-phase training procedure. The developed two-phase training procedure consists of both first and second order training. Several training algorithms were applied and compared, namely: back propagation (BP), delta-bar-delta, quick propagation, conjugate gradient, quasi Newton and Levenberg-Marquardt. It is shown that the optimized two-phase training procedure enables fast convergence and avoids problems arisen from the fact that every new weight initialization can be regarded as a new starting position and yield irreproducible neural network if only second order training is applied. Activation function, number of hidden layer neurons and number of experimental data points used for training set were optimized in order to insure good predictive ability with respect to speeding up retention modelling procedure by reducing unnecessary experimental work. The predictive ability of optimized neural networks retention model was tested by using several statistical tests. This study shows that developed artificial neural network are very accurate and fast retention modelling tool applied to model varied inherent non-linear relationship of retention behaviour with respect to mobile phase parameters.     

Application of retention modelling to the simulation of separation of organic anions in suppressed ion chromatography

The ion-exchange separation of organic anions of varying molecular mass has been demonstrated using ion chromatography with isocratic, gradient and multi-step eluent profiles on commercially available columns with UV detection. A retention model derived previously for inorganic ions and based solely on electrostatic interactions between the analytes and the stationary phase was applied. This model was found to accurately describe the observed elution of all the anions under isocratic, gradient and multi-step eluent conditions. Hydrophobic interactions, although likely to be present to varying degrees, did not limit the applicability of the ion-exchange retention model. Various instrumental configurations were investigated to overcome problems associated with the use of organic modifiers in the eluent which caused compatibility issues with the electrolytically derived, and subsequently suppressed, eluent. The preferred configuration allowed the organic modifier stream to bypass the eluent generator, followed by subsequent mixing before entering the injection valve and column. Accurate elution prediction was achieved even when using 5-step eluent profiles with errors in retention time generally being less than 1% relative standard deviation (RSD) and all being less than 5% RSD. Peak widths for linear gradient separations were also modelled and showed good agreement with experimentally determined values.     

Determination of glycerophosphate and other anions in dentifrices by ion chromatography

Simple, reliable and sensitive analytical methods to determine the anions, such as fluoride, monofluorophaosphate, glycerophosphate related to anticaries are necessary for basic investigations of anticaries and quality control of dentifrices. A method for the simultaneous determination of organic acids, organic anions and inorganic anions in the sample of commercial toothpaste is proposed. Nine anions (fluoride, chloride, nitrite, nitrate, sulfate, phosphate, monofluorophaosphate, glycerophosphate and oxalic acid) were analyzed by means of ion chromatography using a gradient elution with KOH as mobile phase, IonPac AS18 as the separation column and suppressed conductivity detection. Optimized analytical conditions were further validated in terms of accuracy, precision and total uncertainty and the results showed the reliability of the IC method. The relative standard deviations (RSD) of the retention time and peak area of all species were less than 0.170 and 1.800%, respectively. The correlation coefficients for target analytes ranged from 0.9985 to 0.9996. The detection limit (signal to noise ratio of 3:1) of this method was at low ppb level (<15 ppb). The spiked recoveries for the anions were 96-103%. The method was applied to toothpaste without interferences.     

Probing the kinetic performance limits for ion chromatography. II. Gradient conditions for small ions

A gradient kinetic plot method is used for theoretical characterisation of the performance of polymeric particulate anion exchange columns for gradient separations of small inorganic anions. The method employed requires only information obtained from a series of isocratic column performance measurements and in silico predictions of retention time and peak width under gradient conditions. Results obtained under practically constrained conditions provide parameters for the generation of high peak capacities and rapid peak production for fast analysis to be determined. Using this prediction method, a maximum theoretical peak capacity of 84 could be used to achieve separation of 26 components using a 120 min gradient (R_s > 1). This approach provides a highly convenient tool for development of both mono- and multidimensional ion chromatography (IC) methodologies as it yields comprehensive understanding of the influence of gradient slope, analysis time, column length and temperature upon kinetically optimised gradient performance.     

Simple and Efficient Solution for Robustness Testing in Gradient Elution Liquid Chromatographic Methods

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.     

人参皂甙的反相高效液相色谱多台阶梯度优化方法

建立了一种反相高效液相色谱多台阶梯度分离人参皂甙的方法.该方法以乙腈-水溶液为流动相,通过一系列等度实验,获得了8种人参皂甙Rg1,Re,Rf,Rg2,Rb1,Rc,Rb2和Rd的色谱保留参数,发现两参数保留方程不适合用于人参皂甙这种天然产物的分离条件的优化,而三参数保留方程的高精度才可满足预测的要求.在三参数保留方程的基础上,通过计算确定了8种人参皂甙(包括3台阶梯度)的液相色谱分离条件.通过实验对此优化条件进行了验证,实验结果显示了较好的预测精度和分离度.将本方法用于分离人参皂甙,分析时间短且分离度高,显示了等度台阶梯度优化方法对确定色谱分离条件的优越性.     

From Isocratic Data to a Gradient Elution Retention Model in IC: An Artificial Neural Network Approach

Gradient elution is used in ion chromatography to achieve rapid analysis with reasonable separation. Optimization and prediction of the gradient is clearly a multidimensional problem, however. One approach to prediction of gradient retention behavior is based on isocratic experimentation. In this work, a gradient model for simultaneous prediction of the retention behavior of fluoride, chlorite, chloride, chlorate, nitrate, and sulfate ions, on the basis of isocratic experimental data, is proposed. An artificial neural network was used to predict isocratic results; the network was optimized with regard to the number of data in the training set (25) and number of neurons in the hidden layer (6). A slight systematic error was observed in the isocratic prediction, but this did not effect gradient prediction. Good predictions were achieved for all the anions investigated (average error 1.79%). Deviations were somewhat higher for prediction of sulfate retention than for the other anions, probably because of the higher charge and larger size of sulfate in comparison with the other ions examined.     

Determination of monovalent inorganic anions in high-ionic-strength samples by electrostatic ion chromatography with suppressed conductometric detection

A new ion chromatographic (IC) system has been established by using micelles of 3-(N,N-dimethylmyristylammonio)propanesulfonate (Zwittergent 3-14) loaded onto a reversed-phase packed column as the separation column with an electronic rotary switching valve packed-bed suppressor for conductometric detection of inorganic anions. An aqueous H3BO3-Na2B4O7 solution has been demonstrated to be the most desirable eluent for this IC system. The relationship between retention time and the concentration of the borate eluent was determined for a series of model anionic analytes and this relationship was found to be opposite to that exhibited in a conventional anion-exchange IC system. The rapid elution and complete separation of monovalent inorganic anions were obtained by initially using a high-concentration borate solution as the eluent for a short-period, and then switching to a lower-concentration borate eluent to complete the separation. Detection limits for nitrite, bromide, nitrate, and chlorate were 0.85, 0.88, 0.95 and 4.8 microM, respectively, when a 7.0 mM Na2B4O7 eluent was used. Moreover, the ability to directly detect these monovalent anions in samples containing high concentrations of sulfate and/or chloride ions provided a major advantage of this approach.     

Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation

Chromatographers are cautioned to avoid gradient elution when isocratic elution will do. In this work, we compared the analytical properties of gradient and isocratic separations of a sample which can be done quite readily under isocratic conditions. We found that gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height or linearity of the calibration curve. We also obtained acceptable repeatability in peak area/height and linearity of calibrations curves for a sample that required gradient elution using a practical baseline subtraction technique. Based on these results and related work which show that columns can be reequilibrated by flushing with less than two column volumes of the initial eluent, we conclude that many of the reasons given to avoid gradient elution deserve serious reconsideration, especially for those samples which are easily separated isocratically. However, we believe isocratic elution will remain preferable when: (1) the sample contains less than 10 weakly retained components (i.e. the last peak elutes with k' < 5) or (2) the gradient baseline impedes trace analysis.     

Evaluation of separation in gradient elution ion chromatography by combining several retention models and objective functions

In this work, three different methods for modeling of gradient retention were combined with several optimization objective functions in order to find the most appropriate combination to be applied in ion chromatography method development. The system studied was a set of seven inorganic anions (fluoride, chloride, nitrite, sulfate, bromide, nitrate, and phosphate) with a KOH eluent. The retention modeling methods tested were multilayer perceptron artificial neural network (MLP-ANN), radial-basis function artificial neural network (RBF-ANN), and retention model based on transfer of data from isocratic to gradient elution mode. It was shown that MLP retention model in combination with the objective function based on normalized retention difference product was the most adequate tool for optimization purposes.     

Columns and optimum gradient conditions for fast second-dimension separations in comprehensive two-dimensional liquid chromatography

Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two-dimensional liquid chromatography (LC). We compared suitability of five commercial core-shell columns and one monolithic column for fast gradients in the second LC dimension, where the time of separation is strictly limited by the fraction cycle time. In two-dimensional reversed-phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second-dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second-dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second-dimension gradients with single-step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first-dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two-dimensional retention plane. The approach was applied to the two-dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.     

Prediction of peak shape as a function of retention in reversed-phase liquid chromatography

Optimisation of the resolution of multicomponent samples in HPLC is usually carried out by changing the elution conditions and considering the variation in retention of the analytes, to which a standard peak shape is assigned. However, the change in peak shape with the composition of the mobile phase can ruin the optimisation process, yielding unexpected overlaps in the experimental chromatograms for the predicted optimum, especially for complex mixtures. The possibility of modelling peak shape, in addition to peak position, is therefore attractive. A simple modified-Gaussian model with a parabolic variance, which is a function of conventional experimental parameters: retention time (tR), peak height (H0), standard deviation at the peak maximum (sigma0), and left (A) and right (B) halfwidths, is proposed. The model is a simplification of a previous equation proposed in our laboratory. Linear and parabolic relationships were found between the peak shape parameters (sigma0), A and B) and tR, with a mean relative error of 1-5% in most cases. This error was partially due to variations in peak position and shape among injections, which in some cases were above 2%. Correlations between (sigma0, A and B) and the retention time, which is easily modelled as a function of mobile phase composition, allowed a simple and reliable prediction of chromatographic peaks. A parameter that depends on the slopes of the linear relationships for A and B versus tR is also proposed to evaluate column efficiency. The modified-Gaussian model was used to describe the peaks of six diuretics of diverse acid-base behaviour and polarity, which were eluted with 15 mobile phases where the composition was varied between 30 and 50% (v/v) acetonitrile and the pH between 3 and 7.