高效液相色谱用线性梯度洗脱求保留值方程

〕提出了采用线性梯度洗脱求取未知样品中溶质保留值方程的原理及其计算方法,用光二极管阵列扫描检测器解决了梯度洗脱条件下色谱峰的归宿。实验证明：用所得的保留值方程预测等度洗脱下容量因子k'的相对误差不大于3%,而用梯度洗脱下的半峰宽规律可以估算未知样品容量因子k的分布范围,以解决对未知样品是采用等度或梯度洗脱方式。最后,借助计算机仿真方法用于样品的优化,获得了满意的结果。     

反相高效液相色谱中复杂体系二元多台阶梯度分离条件快速优化方法

提出了一种反相高效液相色谱中二元多台阶梯度分离条件快速优化方法。通过数次线性梯度初始实验，求得溶质的保留方程。在此基础上，利用重叠分离区域图（OSRM）方法，快速求得复杂样品的最佳多台阶梯度分离条件。该方法只需要几个小时就可以完成对复杂样品分离条件的优化，并通过对中药川芎提取物的分离加以验证，获得了较好的预测精度和分离效果。     

中药指纹图谱的线性梯度分离条件的快速优化方法

提出了一种复杂样品线性梯度分离条件的快速优化方法.先通过4次线性梯度的初始实验,然后利用Excel规划求解工具获得准确的溶质保留方程系数.在此基础上利用网格搜索完成线性梯度的参数优化.整个优化策略通过对中药金银花提取液中各组分的分离得以验证.同时比较了网格搜索、遗传算法、遗传算法与规划求解联用的3种寻优算法.结果表明:3种方法优化结果接近,但运算时间有所差异.     

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.     

色谱累加进样分离实验条件的研究

在采用反相液相色谱或亲和色谱完成蛋白质等大分子分离时,根据溶质保留值随溶剂梯度变化曲线上突变点的差别,可以通过累加进样分离法进行样品制备或直接柱内富集分析,但这一方法并非在任意条件下、对任何样品都适用。该文根据不同形式的保留值方程从理论上探讨了样品保留值与进样时间差、梯度洗脱速率等实验条件之间的关系;结果表明：两次进样的出峰时间差与进样时间间隔成正比关系,也与其在等度情况下的容量因子有关。样品中的两种组分在间隔进样时的流出时间差主要由两组分的容量因子决定,当样品中存在两种以上保留性能相近组分时,若保证指定的分离度,进样时间间隔存在一极大值,超出该范围,分离条件将不能满足     

液相色谱中的半峰宽规律

本文介绍通过相同组分、不同配比冲洗剂等度和梯度冲洗样品的实验,首次发现在液相色谱中,溶质的色谱峰半宽度与保留值间存在同一线性关系;在梯度冲洗时流出组分的色谱峰半宽度与虚拟保留值间同样存在线性关系。该结果证明,我们提出的组分在柱末端有相近的以长度为量纲的谱带半宽度的正确性,同时该结果对梯度最佳分离条件的预测亦很有益。     

反相高效液相色谱中溶质保留值与流动相组成关系的探讨

］本文采用分配理论和共溶剂理论的方法,探讨了反相高效液相色谱中溶质的保留机制,并推导了溶质保留值与流动相组成的关系式。利用该关系式计算溶质的保留值,所得结果与实验值基本吻合。     

一个描述液相色谱体系中溶质进样量与保留值关系的方程

通过考虑溶质和溶剂在液相色谱体系中的相互作用,从理论上给出了一个描述溶质在液相色谱体系中进样量与保留值之间关系的方程。由方程可以证明,当进样量趋于零时,溶质的保留值为一定值,当进样量无限大时,溶质的保留值趋于零,且随着进样量的增加,溶质的保留值必然减小。通过方程的线性形式,可以获得两个描述色谱体系特征的重要参数：一个是溶质与固定相相互作用的平衡常数Ｋ,另一个是假想的分布在固定相表面上的活性点总数。     

毛细管电色谱有机流动相双二元双台阶分离

根据毛细管电色谱中溶质保留值与有机改性剂种类和浓度之间的相互关系，首次实现了毛细管电色谱有机流动相双二元双台阶分离。利用此方法分离含１２种芳香族化合物的混合样品，各组分在六次连续进样中保留时间的ＲＳＤ（％）值均在１．６５％以内，并将样品的分离时间缩短到等度洗脱方式下的三分之一。这说明该方法分离复杂样品快速、可靠。此外还推导了双二元双台阶分离方式下溶质保留值的预测公式，并对该公式的准确性进行了检验，预测值与实测值间的相对误差在５．７３％以内。     

动态磁涂覆混合纳米固定相毛细管电色谱中芳香酸的输运特征研究

推导了芳香酸在动态磁涂覆混合纳米固定相开管毛细管柱中等度洗脱和二元台阶梯度洗脱条件下的输运方程, 考察了其在该色谱柱上的保留特征. 结果表明, 等度洗脱时, 电渗流方向及大小随着两种固定相配比的改变而改变, 溶质的容量因子与弱阴离子交换固定相和反相固定相比例(Fe₃O₄@SiO₂-NH₂︰Fe₃O₄@SiO₂-C₁₈)之间具有良好的线性关系, 拟合后相关系数大于0.9166. 二元台阶梯度洗脱条件下, 溶质的迁移时间可以通过其在等度洗脱条件下的洗脱时间加以预测. 溶质在经磷酸缓冲液浓度、有机调节剂浓度和pH值台阶梯度洗脱后, 其预测保留时间和实际保留时间之间的相对误差分别小于8.0%, 10%和5.8%. 构建的系统在优化的色谱条件下对水提天麻进行了分离, 其主要成分柠檬酸的保留时间预测值与实际值之间相对误差为11%.     

可调移动重叠分离图法用于高效液相色谱多台阶梯度分离条件优化的研究

计算机辅助高效液相色谱（HPLC）分离条件优化可以低成本、快速地得到优化的分离条,因而已较为广泛地用于复杂样品的分离分析。基于移动重叠分离图方法,又发展了一种新型的多台阶梯度分离条件的优化方法可调移动重叠分离图法。该方法通过预测不同流动相条件下各组分的保留时间、峰宽和分离度,绘制出对于样品中各组分的重叠分离区域图。在对当前台阶流动相组成进行优化的同时,考虑其对后面一到两个台阶上流出组分保留的影响,实时地重新绘制对于后面台阶上流出组分的重叠分离区域图。通过观察当前台阶流动相条件对当前台阶和后面台阶上流出组分分离的影响,综合考虑样品中所有组分的分离情况,找到更接近全局最优的分离条件。通过扫描的方法对优化得到的分离条件进行微调,能够进一步提高分离效果。采用文献数据对可调移动重叠分离图法的应用加以说明,在二元流动相体系下,证明了该方法在HPLC方法建立方面的优越性。     

Retention modeling and simultaneous optimization of pH value and gradient steepness in RP‐HPLC using feed‐forward neural networks

A novel approach is proposed for the simultaneous optimization of mobile phase pH and gradient steepness in RP‐HPLC using artificial neural networks. By presetting the initial and final concentration of the organic solvent, a limited number of experiments with different gradient time and pH value of mobile phase are arranged in the two‐dimensional space of mobile phase parameters. The retention behavior of each solute is modeled using an individual artificial neural network. An “early stopping” strategy is adopted to ensure the predicting capability of neural networks. The trained neural networks can be used to predict the retention time of solutes under arbitrary mobile phase conditions in the optimization region. Finally, the optimal separation conditions can be found according to a global resolution function. The effectiveness of this method is validated by optimization of separation conditions for amino acids derivatised by a new fluorescent reagent.     

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.     

Optimisation technique for stepwise gradient elution in reversed-phase liquid chromatography

An optimisation technique of reversed-phase liquid chromatographic separations based on gradient elution with a stepwise variation pattern of the volume fraction phi of the organic modifier in the water-organic mobile phase is presented. It uses a non-linear least-squares programme with a Monte-Carlo search for initial estimates in order to determine the best variation pattern that leads to the optimum separation of a mixture of solutes. The validity of the above methodology was tested by separating eight catechol-related solutes with mobile phases modified by methanol or acetonitrile and variation patterns of two, three or four steps in the psi values. It was found in all cases a very satisfactory accuracy of the predicted gradient elution times, which is of the same order with the accuracy of the retention times predicted under isocratic or linear gradient conditions. In addition, it was shown that the proposed optimisation technique is both effective and flexible but well-shaped chromatograms are obtained under electrochemical detection only if steps with increasing psi are used and the change in psi is programmed to occur at the intermediate of the predicted peaks.     

Multilinear gradient elution optimization in reversed-phase liquid chromatography based on logarithmic retention models: application to separation of a set of purines, pyrimidines and nucleosides

The analytical solutions of the fundamental equation of the multilinear gradient elution are derived in two cases, when the dependence of the logarithm of the solute retention (lnk) upon the volume fraction of organic modifier (φ) is a three-parameter logarithmic expression, and when a simple linear relationship between lnk and lnφ is adopted. The derived theoretical expressions for retention times under multilinear gradient conditions are embodied to simple algorithms for fitting gradient data and especially for resolution optimization. Their performance was examined by using a mixture of 16 model compounds chosen among purines, pyrimidine and nucleosides in eluting systems modified by acetonitrile. It was found that the accuracy of the predicted gradient retention times is very satisfactory even if the simple logarithmic expression for the retention behavior of solutes, i.e. the linear dependence of lnk upon lnφ, is used.     

Determination of retention factors of phospholipids in gradient HPLC

Summary The mechanism of separation of solutes under gradient elution conditions has been studied. Separation of a mixture of soybean phospholipids (phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine) in the ternary mobile phase: methanol, hexane, isopropanol was considered as an example. An analytical expression in terms of the composition of the ternary mobile phase was obtained to calculate retention factors in two-step gradient modes. The following new polynomial equation was derived: logk=A+BF+CF ²+DG+EG ² where A, B, C, D, and E are empirical coefficients; F and G are volume fractions of isopropanol and methanol respectively in the mobile phase based on hexane. The coefficients for phospholipids were obtained under gradient conditions.     

Optimization of stepwise gradient elution in columns chromatography

Summary A general equation for the final retention of a solute chromatographed under conditions of stepwise gradient elution has been derived. The elution process and the distances travelled by solutes as a function of eluent volume were simulated by computer for the optimization of stepwise gradient prorams from isocratic HPLC data. The validity of the equations was experimentally veritied.     

The principle of optimization of binary mobile phase composition of multi-step linear gradient elution

The basic principle of optimal method called “moving overlapping resolution mapping method” to select the optimal binary mobile phase composition of multi-step linear gradient liquid chromatography is discussed with simultaneously considering effects of position of solute inside the column and mobile phase composition on peak resolution and retention value, then a BASIC program based on this principle is developed in IBM-PC computer. The validities of both principle of optimization and BASIC program are confirmed by separation of samples containing bile acids and PAHs in RP-HPLC.     

Multi-segment linear gradient optimization strategy based on resolution map in HPLC

1 Introduction As a powerful tool of separating and analyzing complex samples, high performance liquid chroma- tography (HPLC) is widely applied. The key point of establishing stable and complete method of analysis is the selection of separation conditions. Accompanying the development of computer technology, it is possible to investigate the separation performance of each component in the sample under a wide range of ex- perimental conditions based on the information ob- tained by carrying …     

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of In k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.