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。     

The ultimate band compression factor in gradient elution chromatography

The equations predicting the ultimate time band compression factor, G=(t(R)-t(F))/t(p) in linear gradient elution chromatography, for an infinitely narrow injection (injection time t(p)-->0) were derived for an ideal-model column (dispersionless chromatography, H=0) assuming the Linear Solvent Strength Model for the retention behavior of the analyte. Numerical solutions can readily be obtained when the LSSM model does not apply. The results can be generalized to any retained organic modifier (k'(A)) in the mobile phase. The stronger the retention of the organic modifier, the more effective the band compression. Dispersion in real chromatographic column (H not equal 0) affects the limits that can be reached in linear gradients but poorly in step gradients. Examples based on a conventional HETP of about 12 microm using a 5 microm particle packed column reveal that the best time compression factor that could be expected is twice the one predicted with an ideal column.     

The bandwidth in gradient elution chromatography with a retained organic modifier

The variance of a chromatographic band is derived in the case of RPLC gradient elution when the organic modifier is significantly retained onto the stationary phase. This derivation is based on the extension of a model due to Poppe et al. [H. Poppe, J. Paanakker, M. Bronckhorst, J. Chromatogr., 204 (1981) 77] which assumes that the gradient front remains unchanged and propagates along the column at the same speed as the mobile phase, following piston flow. Theoretical and experimental results are compared in the case of caffeine on a C(1)-silica stationary phase eluted with an acetonitrile gradient. The actual retention behaviors of caffeine and acetonitrile were implemented in the theoretical calculations. The model predicts compression factors between 0.71 and 0.34 for relatively smooth gradient steepness, betat(0), between 0.009 and 0.054 while the corresponding experimental band compression factors vary between 1.01 and 0.43 for the very same gradient steepness. The model underestimation of these factors arises likely from the strong deviation of the actual retention behavior from the prediction of the Linear Solvent Strength Model (LSSM).     

The prediction of the peak width at half height in HPLC

The prediction of the peak width at half height is an important aspect in the optimization of the chromatographic operating conditions. In this paper, a linear relationship, between the peak widths at half height and the retention values with various isocratic elution is observed. In gradient elution, however, the relationship between the peak widths at half height and the so-called invented retention values that correspond to the mobile phase composition by eluting the solute from the column end is developed. We believe that there is almost the same band width at half height inside the column (in unit of length) for different solutes. The peak width at half height in the chromatogram (in unit of time) is mainly determined by the capacity factor of the solute when it is eluted from the column end. The larger the capacity factor of a solute eluted from the column end, the more slowly will be the solute eluted from the column end and the wider will be the peak width at half height. It is possible to predict the peak width at half height in various isocratic and gradient elutions by using this linear relationship.     

Faster axial band dispersion in a monolithic silica column than in a particle-packed column

The contribution of molecular diffusion to peak broadening was studied in a reversed-phase HPLC system, consisting of a monolithic silica C18 column and methanol-water mobile phase. Study on the band broadening effect of holding a solute in a column or elution at very low linear velocity of mobile phase allowed facile determination of the contribution of the molecular diffusion term. Less obstruction against molecular diffusion, or the faster axial band dispersion in a monolithic silica column than in a particle-packed column, was found both in mobile phase and in stationary phase.     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I—Elution of an unretained tracer

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as “supercritical fluid chromatography” or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Correction of the deviations in the retention times with Chromolith columns associated to the flow rate: implications in the modelling of the retention behaviour

In a previous work (J. Sep. Sci. 2009, 32, 2793-2803), we reported an interpretive optimisation approach to achieve maximal resolution in minimal analysis time, based on models describing the retention and peak shape as a function of mobile phase composition and flow rate. The method was applied to the separation of a group of basic drugs in a Chromolith column. In that work, we found that the retention factors were sensitive to the flow rate. The reason of the observed deviations in retention times is the increase in the column volume at the applied pressure, which decreases the linear velocity inside the column. This behaviour forced to include a correction term in the model that described the retention. We show here how the deviations in retention times can be evaluated, allowing retention models that do not include the flow rate as a variable, similar to isocratic chromatography at fixed flow rate. The logarithm of the deviations in the retention times with flow rate is shown to correlate with the solute polarity. This correlation is compared with similar correlations for the retention factor at fixed mobile phase composition and the extrapolated retention factor in water at fixed flow rate.     

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.     

Numerical modeling of the elution peak profiles of retained solutes in supercritical fluid chromatography

In supercritical fluid chromatography (SFC), the significant expansion of the mobile phase along the column causes the formation of axial and radial gradients of temperature. Due to these gradients, the mobile phase density, its viscosity, its velocity, its diffusion coefficients, etc. are not constant throughout the column. This results in a nonuniform flow velocity distribution, itself causing a loss of column efficiency in certain cases, even at low flow rates, as they do in HPLC. At high flow rates, an important deformation of the elution profiles of the sample components may occur. The model previously used to account satisfactorily for the retention of an unsorbed solute in SFC is applied to the modeling of the elution peak profiles of retained compounds. The numerical solution of the combined heat and mass balance equations provides the temperature and the pressure profiles inside the column and values of the retention time and the band profiles of retained compounds that are in excellent agreement with independent experimental data for large value of mobile phase reduced density. At low reduced densities, the band profiles can strongly depend on the column axial distribution of porosity.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Structural radial heterogeneity of a silica-based wide-bore monolithic column

The radial distribution of the main characteristics (elution time and standard deviation) of the elution profiles of a flat injected band recorded at the exit of a monolithic column were determined. These distributions provide the radial distributions of the average mobile phase velocity, the elution time and the maximum height of the peak of an analyte, the column efficiency and the analyte concentration. The band profiles were measured at the exit of a 10-mm i.d., 100-mm long silica-based monolithic column. An on-column local electrochemical amperometric detector allowed the recording of the elution profiles at different spatial positions throughout the column cross-section. The local spatial distribution of the mobile phase velocity does not follow a piston-flow behavior but exhibits radial heterogeneity. The local efficiency near the wall is lower than that near the column center. The radial distribution of the maximum concentration of the peaks varies throughout the column exit section, partially due to the radial variations of the column efficiency. These results might explain the rather large value of the A term of the Van Deemter or the Knox equations reported previously for monolithic columns.     

Density gradients in packed columns: II. Effects of density gradients on efficiency in supercritical fluid separations

To investigate how fluid compressibility affects efficiency in supercritical fluid separations, band dispersion along a packed capillary column was measured from on-column elution rate profiles obtained under solvating gas chromatography (SGC) conditions; this allowed efficiency to be determined with respect to position along the column. Theoretical efficiency was also modeled. The model indicates that the primary cause of band broadening in SGC is high mobile phase velocity near the column outlet. However, the experimental results show that significant band broadening also occurs near the column inlet in a region that corresponds to high elution rates of the analyte. On-column detection also revealed spatial focusing of the analyte as it moves down the column density gradient.     

分子印迹手性整体柱的制备及对非对映异构体的分离

 采用原位分子印迹技术 ,单步制备了一种辛可宁印迹的手性整体柱。为了提高柱效和选择性 ,选择了相对低极性的甲苯 /十二醇复合致孔体系。在等度及梯度洗脱条件下 ,非对映异构体辛可宁与辛可尼丁被完全分离。等度洗脱中相对较宽的峰可以在梯度洗脱中得到改善。同时考察了流动相中醋酸浓度、流速以及温度对分离的影响。由于柱中存在大的流通孔 ,大大降低了分离过程中的柱压降 ,从而使这种柱能够在相对高的流速下使用。提高温度可以提高分离因子 ,在 60℃获得最大分离因子 5 40。     

Prediction of elution bandwidth for purine compounds by a retention model in reversed-phase HPLC with linear-gradient elution

In reversed-phase liquid chromatography, the retention mechanism of solute has been studied under linearly programmed gradient mobile-phase conditions. The separation of a mixture of four purine compounds (purine, theobromine, theophylline, and caffeine) was considered as a practical case in two binary mobile phase systems, water/methanol and water/acetonitrile. The retention model which describes how the retention factor is related to the mobile-phase composition has been developed in various mathematical forms to predict the retention time in both linear and gradient elutions. For a pulse injection of sample, two important factors, the retention time and the bandwidth of solute, might be computable to predict the elution profiles estimated by the distribution function, such as the Gaussian distribution function. In this work, a prediction method based on the analogue of the retention model was proposed to calculate the bandwidth in linear gradient elutions. Band broadening was caused by the different migration velocities of the front and rear ends of the solute band in a chromatographic column. Therefore, the migration behaviors of the front and rear ends of the solute band were explained with the same retention model which had been used to predict the retention time of solute. For the well retained solutes, theophylline and caffeine, the predicted bandwidth and experimentally obtained bandwidth showed good agreement in both isocratic and gradient elutions.     

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.     

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.     

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.