Deformation of gradient shape as a result of preferential adsorption of solvents in mixed mobile phases

Gradient elution has been studied in typical normal and reversed-phase systems. Deformations of gradient profiles have been evidenced as a result of preferential adsorption of modifiers of the mobile phase. This phenomenon was pronounced in the normal-phase system, for which gradient profiles deviated significantly from those programmed. This influenced the retention and shapes of band profiles of the eluting solute. Hence, in order to predict gradient propagation correctly the adsorption equilibrium of modifiers has been quantified. Moreover, at low modifier content, deformations of band profiles of the solute has been registered as a result of the competitive adsorption in the system solute-modifier. This effect has been predicted by a competitive adsorption model. For the reversed-phase systems the influence of the modifier adsorption on gradient propagation was insignificant for typical mobile phases investigated. Therefore, the work has been focused on gradient predictions in the normal-phase system.     

Parcel model for peak shapes in chromatography numerical verification of the temporal distortion effect to peak asymmetry

The traditional plate concept has been reassessed and improved to a parcel matrix model, which can be used to imitate the chromatographic behavior of a hypothetic column on a computer worksheet. Under programmed conditions, various peak shapes (nearly Gaussian, and with prolonged or fronting tails) are generated. The peak tailing has been separated into two major fractions: spatial and temporal. The former fraction is caused by the retention nature of a column, whereas the latter is induced by the observer's relative position and the changing of the zone broadening rate. The temporal distortion effect can be identified qualitatively and quantitatively through a normalized peak-overlapping process. In general, a chromatographic peak may carry a prolonged (or normal type) tail under linear isotherms, while both prolonged and fronting tails will appear under non-linear conditions. The temporal distortion is proved to be significant, and may be regarded as the major cause of peak asymmetry in most cases. This is in contrast to the conclusions of many previous studies. The model is also eligible to simulate chromatographic peaks for various injection sizes.     

Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

The purpose of this work was to test the applicability of the current theory to predict the peak retention time and the peak width in the combined pH/organic modifier gradient reversed phase high performance liquid chromatography (RP HPLC). A series of 38 isocratic measurements have been conducted for a wide range of pH and methanol contents for ketoprofen (weak acid) and papaverine (weak base). It served to find the model describing dependence of retention factor and the height equivalent of a theoretical plate (HETP) on pH and organic modifier content. The information gathered in the isocratic mode was used to simulate retention times and peak widths for 30 various methanol gradients, 25 pH gradients, and 3 combined pH/methanol gradients. The simulations were compared with the experimental data. We also proposed a simplified version of this model that was parameterized based on 12 initial organic modifier gradients carried out for different pHs and for the 20 min and 60 min gradient development times. The full and the simplified model described the experimental data very well. In conclusion, the proposed modeling approach allowed predicting analyte retention times and peak width for various pH and organic modifier changes. Its simplified version required only 12 initial experiments and seems to be very promising in the optimization RP HPLC separations for complex samples and for conditions providing peak compression.     

Retention process in reversed phase TLC systems with polar bonded stationary phases

The retention of a solute in RP chromatography is a very complex process which depends on many factors. Therefore, the study of the influence of a mobile phase modifier concentration on the retention in different reversed phase chromatographic systems is very important for understanding the rules governing retention and mechanisms of substance separation in a chromatographic process. Composition changes and the nature of mobile phases enable tuning of the separated analytes' retention over a wide range of retention parameters and optimization of the chromatographic process as well. Optimization of the chromatographic process can be achieved by several different methods; one of them is the so-called interpretative strategy. The key approach adopted in this strategy is the implementation of adequate retention models that couple the retention of solute with the composition of a mixed mobile phase. The use of chemically bonded stationary phases composed of partially non-bonded silica matrix and organic ligands bonded to its surface in everyday chromatography practice leads to questions of the correct definition of the retention model and the dominant retention mechanism in such chromatographic systems. The retention model for an accurate prediction of retention factor as a function of modifier concentration and the heterogeneity of the adsorbent surface should be taken into consideration. In this work the influence of mobile-phase composition on the retention of sixteen model substances such as phenols, quinolines, and anilines used as test analytes in different RP-TLC systems with CN-, NH2-, and Diol-silica polar bonded stationary phases has been studied. The aim of this study is to compare the performance of three valuable retention models assumed as the partition, adsorption/partition, and adsorption mechanism of retention. All the models were verified for different RP-TLC systems by three statistical criteria. The results of investigations presented in this work demonstrate that the best agreement between the experimental and calculated Rf values was obtained by the use of new-generation retention models, which assume heterogeneity of adsorbent surface. The results reported here show that heterogeneity of the adsorbent surface may be important in analysis of the elution process in liquid chromatography. Consideration of the goodness of fit for the experimental data to the examined retention models is in conformity with the adsorption mechanism of retention on all polar bonded stationary phases in most eluent systems for most investigated compounds.     

Conformational transitions in ultrahigh-molecular-mass polymers and their manifestation in chromatography on monolithic columns

For a monolithic capillary column based on polydivinylbenzene, a change in the elution profile of polystyrene standards with variation in the eluent-flow rate is studied. It is shown that, for polymers with molecular masses up to 3 × 10⁶, the elution profile does not depend on the flow rate. For higher molecular mass polymers at low flow rates, there is a single almost Gaussian peak that splits into two peaks that move to different sides from the initial peak with an increase in the flow rate. A peak with a smaller retention time (peak I) rapidly attains the limiting elution time, and later on, its retention is independent of the eluent-flow rate. In contrast, the retention time of the other peak (peak II) continuously increases with an increase in the flow rate of the mobile phase, so that, at high flow rates, this peak is retained for a longer time than the low-molecular-mass marker. The intensity of peak I decreases, while the intensity of peak II increases with an increase in the eluent-flow rate; the ratio of their intensities tends toward a certain limiting value (≤1). The observed profile of elution of ultrahigh-molecular-mass polymers assumes the presence of a dynamic equilibrium similar to that existing in the case of first-order reversible reactions. For the assumed equilibrium, the rate constants of direct and back reactions are determined. It is found that these constants are close to the inverse maximum relaxation time of a polymer molecule. The character of the transformations of the polymer molecule in the chromatographic column is discussed.     

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

Hydrophilic interaction chromatography has been applied for the separation of epirubicin and its analogues using high-purity silica column with aqueous-organic mobile phase. Parameters affecting the chromatographic behavior of the solutes such as organic modifier, buffer pH, ionic strength and sample size, have been investigated. Of utmost importance for successful separation of these analogues is the choice of organic modifier, since it impacts both the solvent selectivity and the ionization of silica silanols as well as buffer solution, and consequently the retention behavior of solutes. Acetonitrile was shown to offer superior separation of these analogues to methanol, isopropanol or tetrahydrofuran. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange. In addition, an irreversible adsorption of these compounds was found on silica in the weakly acidic or neutral mobile phases, and the effect of various factors on irreversible adsorption was also preliminarily discussed. More significantly, these basic compounds have exhibited peaks with a slanted front and a sharp tail, a typical overloading peak profile belonging to the behavior of competitive anti-Langmuir isotherm by increasing the sample size at the experimental conditions.     

Comparison of chromatographic properties of cyanopropyl-, diol- and aminopropyl- polar-bonded stationary phases by the retention of model compounds in normal-phase liquid chromatography systems.

Polar-bonded stationary phases, such as CN-, diol- and NH2-silica, have been characterised by the retention of model solutes (phenols, aromatic amines and quinoline bases) in normal-phase systems using n-heptane--polar modifier (2-propanol, tetrahydrofuran or dioxane) mixtures as eluents. The selectivity of separation for the particular groups of substances has been analysed by the log kI versus log kII relationships for CN- and diol, CN- and NH2- and NH2- and diol phases in examined eluent systems by the plotting of correlation lines. The values of regression coefficient r indicate either the similarity of the retention mechanisms of model solutes in some examined systems where r>0.9, or differences among various systems where r<0.9. The values of slopes of correlation lines show the selectivity of separation for particular group of compounds. The selectivity of separation has also been characterised by deltalog k values. The effect of modifier (2-propanol, tetrahydrofuran and dioxane) on selectivity of model solutes on these phases has also been discussed.     

Deformation of a sheet with coupling between elasticity and concentration

We investigate a simple model for equilibrium deformation of a sheet with concentration-dependent elasticity. The model is motivated by several physical situations where deformation of a sheet is modulated by concentration of a mobile species, for example, a quasi-2D array of carbon nanotubes. Elasticity of the sheet is modeled using a free energy functional that includes concentration-dependent potential energies, and a free energy of mixing. We show that the sheet responds to imposed distortion by rearranging its constituents and can do so in a smooth and stable manner only for sufficiently small distortion. For larger distortions, through phase plane and numerical analysis, we consider how the system can meet boundary conditions through nonsmooth solutions.     

Theory of glassy dynamics in conformationally anisotropic polymer systems

A mode coupling theory for the ideal glass transition temperature, or crossover temperature to highly activated dynamics in the deeply supercooled regime, T(c), has been developed for anisotropic polymer liquids. A generalization of a simplified mode coupling approach at the coarse-grained segment level is employed which utilizes structural and thermodynamic information from the anisotropic polymer reference interaction site model theory. Conformational alignment or/and coil deformation modifies equilibrium properties and constraining interchain forces thereby inducing anisotropic segmental dynamics. For liquid-crystalline polymers a small suppression of T(c) with increasing nematic or discotic orientational order is predicted. The underlying mechanism is reduction of the degree of coil interpenetration and intermolecular repulsive contacts due to segmental alignment. For rubber networks chain deformation results in an enhanced bulk modulus and a modest elevation of T(c) is predicted. The theory can also be qualitatively applied to systems that undergo nonuniversal local deformation and alignment, such as polymer thin films and grafted brush layers, and large elevations or depressions of T(c) are possible. Extension to treat directionally dependent collective barrier formation and activated hopping is possible.     

一种变压器油色谱峰识别算法的设计

在变压器油色谱峰识别领域,传统的一阶导数法需要斜率阈值来实现色谱峰识别,因此自动化程度低及容易失真。针对这些缺点,本文在一阶导数法的基础上进行了改进,将迭代移动平均及归一化分析技术应用到色谱峰识别中,通过对信号曲线及方波曲线多次迭代移动平均确定最优的归一化识峰参数,结合色谱峰的绝对保留时间及识峰窗口实现对色谱峰的准确识别。实验结果表明：该算法可以准确识别色谱峰,对噪声、色谱峰的峰宽及峰形变化不敏感,具有很强的自适应性,满足变压器油中气体在线监测装置的现场使用要求。     

Optimization and identification in any kind of multi-step temperature programmed gas chromatography

The theory which predicts the retention time, retention temperature, and peak width for any kind of multi-step TPGC and the principle of optimization has been described. Software for optimization and identification in TPGC has been developed on the basis of this theory. It has also been proven that the relationship between peak width in TPGC and the derived or “invented” retention time is similar to that between peak width and retention time in isothermal processes. The validity of the software has been proved by using it to predict retention temperature, retention time, and peak width for any kind of temperature programming and to predict the optimum temperature program for separation of a multihomolog mixture of industrial alcohols and 15 enantiomeric pairs of amino acids.     

Improvement of an overloaded, multi-component, solvent gradient bioseparation through multiobjective optimization

Solvent gradient chromatography is quite often used in analytical studies for decreasing the analysis time of samples having components with widely different retention behaviour. Several studies, both theoretical and experimental, have been reported on the optimization of gradient profiles in improving analytical separation performance, suggesting various linear and non-linear gradients. In preparative chromatography, on the other hand, though solvent gradient is being increasingly used (especially in bioseparation) to improve the product yield and productivity, there is a dearth of literature and clearer understanding of the effect(s) of modifier gradients on the separation performance. For this, the gradients used in applications are of relatively simple profiles like step or linear gradients, obtained through hand optimization based on experience and intuition. Significant improvements, however, can be expected using the state-of-the art modelling of chromatographic processes and optimization routines running on widely available hi-speed desktop computers. In this work we are reporting such an optimization procedure to improve the purification of an industrial multi-component mixture, containing 65.8% of Calcitonin as the main product, in an overloaded reversed-phase column. The work comprises both theoretical simulations and their experimental validation using multilinear gradients as optimization variable. The study produced interesting insights for modifier gradient design, like using peak deformation of the target peptide to increase yield and productivity, and improved our understanding of the effect of modifier gradients in non-linear separations.     

Reversed-phase high-performance liquid chromatography of ionogenic compounds: comparison of retention models

Two kinds of retention models describing a behaviour of ionogenic substances in reversed-phase chromatographic systems were compared. Model A utilises a concept of limiting retention factors and is especially suitable for the prediction of retention of compounds co-existing in several forms in mobile phase. An effect of the concentration of organic modifier (e.g., methanol) on the magnitudes of the limiting retention factors and equilibrium constants (dissociation constants of the separated substances) can be expressed with the aid of various, more or less sophisticated, relationships. A stoichiometric displacement model (model B) in its original form simply relates the analyte retention to the content of organic modifier in the mobile phase. In this work, it was modified to also express an effect of the mobile phase pH introducing side equilibria (acid-base) into the model. Both models predict a sigmoidal dependence of the analyte retention factor on the mobile phase pH in accordance with experimental data, and allow, among others, to estimate dissociation constants from those data. Experimental dependencies between the analyte retention and the concentration of methanol in the mobile phase comply well with model A, whereas the stoichiometric displacement model could be used only in a limited range of the methanol concentrations.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Computerized separation of chromatographically unresolved peaks

A computerized peak deconvolution software and mass spectra were successfully applied for the deconvolution of overlapped peak cluster in the chromatogram obtained separating the complex mixture of pesticides by retention time locking gas chromatography-mass spectroscopy. The method based on the unique fragment ions in the spectra can be used for deconvolution of peak clusters if mass spectra of overlapped peaks differ. This method allows determining actual retention times of overlapped peaks. Peak areas found by this method however, cannot be used naturally for the quantitative purposes as the abundance of fragment ions used for this deconvolution procedure can dramatically differ. Computer assisted deconvolution of peaks in the peak clusters gives more realistic peak area ratios as at this method it is supposed equal response for all peaks overlapped in a cluster.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Analysis of sample-solvent induced modifier-solute peak interactions in biochromatography using equilibrium theory and detailed simulations

Batch liquid chromatographic columns are often equilibrated with an eluent stream being a mixture of inert compounds and so-called modifiers. The sample injected into the eluent stream usually consists of the solutes to be separated and of a mixture of the same solvents as in the eluent but in general with different concentration values. This results in two groups of peaks moving along the column: the solute peaks and the modifier pertubations. If the adsorptivity of the solute depends strongly on the modifier, as it is often the case in biochromatography, the interference between the two groups of peaks leads to peculiar phenomena like double peaks, split peaks, distorted peaks with anti-Langmurian shape, etc. In this work, these phenomena are analyzed based on an analytical solution of the equilibrium theory model and the results are compared with detailed simulations and experimental data. It is shown that the qualitative behavior is well predicted in the frame of equilibrium theory and general guidelines how to avoid these kinds of interactions are developed.     

原料奶在HPLC-MS负离子模式下的指纹图谱研究

采用高效液相色谱质谱联用技术,在负离子扫描模式下建立了原料奶的乙腈提取成分的指纹图谱。采用乙腈和0.1％的乙酸为流动相进行二元梯度洗脱,柱温30℃,分析时间为85 min。确定了11个共有峰,以7号峰为参照物,通过相对峰面积和相对保留时间进行了方法学考察。结果表明,本方法具有良好的重现性,各指纹峰相对保留时间的RSD<0.79％,相对峰面积的RSD<2.84％。在原料奶指纹图谱基础上,选取有代表性有害物如防腐剂苯甲酸等进行了标准添加,建立了添加图谱,对沈阳地区超市的50个纯牛奶样品进行了筛查,取得初步应用结果。     

Molecular parameters and retention characteristics of unsubstituted polyaromatic hydrocarbons in HPLC

Summary The present research studies the possibility of using the correlation dependence between molecular parameters of unsubstituted polyaromatic hydrocarbons (PAH) and their retention in reversed-phase liquid chromatography to optimize the conditions for the separation and identification of unknown peaks on the chromatograms of multicomponent mixtures. A linear correlation equation, that takes the number and environment of the carbon atom in the PAH molecule into account as well as the differences in the specific interactions of isomeric molecules with polar eluent, has been proposed. The adequacy of the proposed PAH retention model was verified by comparing the calculated retention values with the experimental data. The possibility of identifying unsubstituted PAH according to the number of carbon atoms of various types and according to the values of the molecules lengths (calculated on the basis of the retention of these substances under different eluent compositions) was exemplified by various chromatographic systems (reversed phase-eluent-PAH molecules).