反相液相色谱中生物大分子保留自由能及相比的测定

依据液相色谱中溶质的计量置换保留模型（SDM-R）和反相液相色谱（RPLC） 中小分子溶质柱相比的热力学新定义,建立了准确测定生物大分子柱相比的理论模 型和实验方法,准确测定了生物大分子的柱相比。同时依据计量置换参数logI和Z 具有热力学平衡常数的性质,并以logI和对1/T作图呈线性为途径,解决了生物大 分子色谱热力学研究中存在的三个难题,准确测定了七种标准蛋白质在RPLC保留过 程中的自由能变,并发现在RPLC中柱相比对保留过程中溶质总自由能变的贡献值几 乎可与溶质在RPLC保留过程中的净自由能变相当。     

Chiral discrimination of phenoxypropionic acid herbicides on teicoplanin phase: Effect of mobile phase modifier

Summary The chiral recgonition mechanism for a series of phenoxypropionic acid herbicides was investigated in reversed phase liquid chromatography (RPLC) on a teicoplanin stationary phase over a wide range of column temperatures. Thermodynamic constants, of the transfer of an enantiomer from the mobile to the teicoplanin stationary phases were determined. The van't Hoff plots for all solutes had a break at a critical temperature T^* showing a variation in the enantiomer retention mechanism due to a change in the conformational state of the teicoplanin, structure. Additionally, enthalpy-entropy compensation confirmed both the change in enantiomer interaction mechanism observed for regions T<T^* and T>T^* and the independence of this mechanism from (i) herbicide molecular structure,s i.e. the position of the chloro group on the phenol ring and (ii) the carbon absolute configuration. Moreover, the increasing enantioselectivity with increasing methanol fraction in the mobile phase was attributed to restriction of the solute association in the teicoplain, stationary phase, leading to favorable stereoselective interactions. This behavior was used to optimize chromatographic conditions for separation of herbicide enantiomers on teicoplanin.     

Effect of phase ratio on van't Hoff analysis in reversed-phase liquid chromatography,and phase-ratio-independent estimation of transfer enthalpy

In analysis of the thermodynamics of the transfer of a solute from the mobile phase to the stationary phase in reversed-phase liquid chromatography, it is nearly always assumed that the phase ratio is constant. This type of analysis is typically performed by applying a form of the van't Hoff equation, which relates the retention factor to temperature via the enthalpy and entropy of transfer. When non-linear van't Hoff plots are observed, it is often assumed that the enthalpy and entropy of transfer change with temperature. However, when the possibility of a change in the phase ratio is considered, it becomes apparent that non-linear van't Hoff behavior may or may not be due to changes in enthalpy or entropy. In this work, we present mathematical evidence that phase ratio changes, if they occur, can cause deviations from linearity in a van't Hoff plot. We also show that the phase ratio influence can be eliminated by considering the molecular difference between two solutes instead of the solutes themselves. The resulting selectivity van't Hoff plots may be linear, even when the van't Hoff plots of the two solutes are non-linear. In such cases, temperature-dependent phase ratio changes, and not necessarily changes in the transfer enthalpy, may be responsible for the curved van't Hoff plots of the individual solutes. In addition, we present chromatographic evidence that different solutes may "see" different thermodynamic phase ratios. It is clear that the concept of a phase ratio in reversed-phase chromatography is not nearly as well defined as a phase ratio in a bulk system like a liquid-liquid extraction.     

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。     

An Integrated Theory of Adsorption and Partition Mechanism and Eash Contribution to Solute Retention in Reversed Phase Liquid Chromatography

With the combination of the the stoichiometric displacement model for retention (SDM-R) in reversed phase liquid chromatography (RPLC) and the stoichiometric displacement model for adsorption (SDM-A) in physical chemistry,the total number of moles of the re-solvated methanol of stationary phase side.nr,and that of solute side in the mobile phase,q,corresponding the one mole of the desorbing solute,were separately determined and referred as the characterization parameters of the contributions of the adsorption mechanism and partition mechanism to the solute retention,respectively.A chromatographic system of insulin,using mobile phase consisting of the pseudo-homologue of alcohols(methanol,ethanol and 2-propanol)-water and trifluoroacetic acid was employed.The maximum number of the methanol layers on the stationary phase surface was found to be 10.6,only 3 of which being valid in usual RPLC,traditionally referred as a volume process in partition mechanism.However,it still follows the SDM-R.Both of q and nr of insulin were found not to be zero,indicating that the retention mechanism of insulin is a mixed mode of partition mechanism and adsorption mechanism.When methanol is used as the organic modifier,the ratio of q/nr was 1.13,indicating the contribution to insulin retention due to partition mechanism being a bit greater than that due to adsorption mechanism.A linear relationship between q,or nr and the carbon number of the pseudo-homologue in the mobile phase was also found.As a methodology for investigating the retention mechanism retention and behavior of biopolymers.a homologue of organic solvents as the organic modifier in mobile phase has also been explored.     

Phenomenon of dual‐ and single‐retention behaviors of solutes and its validation by computational simulation in linear programmed temperature gas chromatography

The current theory of programmed temperature gas chromatography considers that solutes are focused by the stationary phase at the column head completely and does not explicitly recognize the different effects of initial temperature (T_o) and heating rate (r_T) on the retention time or temperature of a homologue series. In the present study, n‐alkanes, 1‐alkenes, 1‐alkyl alcohols, alkyl benzenes, and fatty acid methyl esters standards were used as model chemicals and were separated on two nonpolar columns, one moderately polar column and one polar column. Effects of T_o and r_T on the retention of nonstationary phase focusing solutes can be explicitly described with isothermal and cubic equation models, respectively. When the solutes were in the stationary phase focusing status, the single‐retention behavior of solutes was observed. It is simple, dependent upon r_T only and can be well described by the cubic equation model that was visualized through four sequential slope analyses. These observed dual‐ and single‐retention behaviors of solutes were validated by various experimental data, physical properties, and computational simulation.     

线性参数logI对反相液相色谱中的非极性小分子溶质体系的表征

从理论上阐明了计量置换平衡常数的对数ｌｏｇＫａ对液相色谱中计量置换保留模型的线性参数ｌｏｇＩ起着主导作用。ｌｏｇＩ表示溶质对固定相的亲合势，且具有热力学平衡常数的性质。     

反相液相色谱中计量置换线性参数logI的热力学特征

 依据液相色谱中溶质计量置换保留模型及线性参数logI(与1mol溶质对固定相的亲和势大小有关的常数),通过作图得知非极性和极性小分子溶质及生物大分子的logI与绝对温度的倒数1/T,以及小分子溶质的logI与其在正辛醇-水中分配系数的对数logPo/w呈线性关系,从两方面进一步证明了logI具有热力学平衡常数的性质。基于小分子溶质、生物大分子的logI和分配系数大小的差别,对两者在反相液相色谱中的保留对柱长的依赖关系给予了定量的说明。     

反相液相色谱中计量置换线性参数logI的热力学特征

依据液相色谱中溶质计量置换保留模型及线性参数 log I(与 1mol溶质对固定相的亲和势大小有关的常数 ) ,通过作图得知非极性和极性小分子溶质及生物大分子的 log I与绝对温度的倒数 1/T,以及小分子溶质的log I与其在正辛醇 -水中分配系数的对数 log Po/ w呈线性关系 ,从两方面进一步证明了 log I具有热力学平衡常数的性质。基于小分子溶质、生物大分子的 log I和分配系数大小的差别 ,对两者在反相液相色谱中的保留对柱长的依赖关系给予了定量的说明     

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse‐phase liquid chromatography at different methanol‐water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol‐water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.     

A comparative study of hydrophobicity of octadecyl and alkylamide bonded phases based on methylene selectivity

The conformational dynamics of home‐made monomeric and polymeric (octadecyl – C18 and alkylamide – AP) stationary phases have been investigated by analyzing the dependence of retention on the reciprocal temperature for different organic compounds. Theoretical considerations were based on the results of methylene selectivity determination. Two selected PAHs – naphthacene and benzo[a]anthracene, whose structures differ in the position of the ring – were chosen as solutes to study the thermodynamics of retention behavior. Standard enthalpies (ΔH ⁰) were obtained from the slope of the plots of the capacity factor logarithm (ln k) against the reciprocal temperature (1/T), i. e. van't Hoff plots, in a temperature range from 285 to 315 K. The conformation of the stationary phase (monomeric and polymeric) structure and the length of bonded ligands with their specific functional (N‐acylamide) groups had a significant effect on retention.     

Determination of liquid-liquid partition coefficients by separation methods

By essence, all kinds of chromatographic methods use the partitioning of solutes between a stationary and a mobile phase to separate them. Not surprisingly, separation methods are useful to determine accurately the liquid-liquid distribution constants, commonly called partition coefficient. After briefly recalling the thermodynamics of the partitioning of solutes between two liquid phases, the review lists the different methods of measurement in which chromatography is involved. The shake-flask method is described. The ease of the HPLC method is pointed out with its drawback: the correlation is very sensitive to congeneric effect. Microemulsion electrokinetic capillary electrophoresis has become a fast and reliable method commonly used in industry. Counter-current chromatography (CCC) is a liquid chromatography method that uses a liquid stationary phase. Since the CCC solute retention volumes are only depending on their partition coefficients, it is the method of choice for partition coefficient determination with any liquid system. It is shown that Ko/w, the octanol-water partition coefficients, are obtained by CCC within the -1 < log Ko/w < 4 range, without any correlation or standardization using octanol as the stationary phase. Examples of applications of the knowledge of liquid-liquid partition coefficient in the vast world of solvent extraction and hydrophobicity estimation are presented.     

Solute Retention in Column Liquid Chromatography. VI. Enthalphy Entropy Compensation: The Column Temperature and Mobile-Phase Composition

Abstract

The concept of enthalpy-entropy compensation in “non-polar” reverse-phase (ODS) column liquid chromatography is examined in terms of the column temperature (10[ddot]-35[ddot]C) as well as mobile-phase composition (acidified water/tetrahydrofuran) for the solutes: phenol, p-nitrophenol, 2,4-dinitrophenol, o-chlorophenol, o-nitrophenol, 2,4-dimethylphenol, 2,4- dinitro-o-cresol, 4-chloro-m-cresol, and 2,4-dichlorophenol. Plots of log (solute capacity factor, k') against reciprocal column temperature, T^?1 exhibit approximate linearity only over the volume-fraction based mobile phase compositional range ?THF = 0 to 0.2. Substantial deviations occur beyond ?THF = 0.3. Plots of In k' against -ΔH[ddot] (compensation plots) yield a random distribution of data, indicating that what enthalpy-entropy compensation may exist for the systems studied is masked both in terms of temperature and mobile-phase composition. It is concluded that interaction(s) of the solutes with the stationary sorbent are more complex than has heretofore been supposed.     

Chromatographic characterization of a bonded liquid crystal stationary phase for HPLC

Summary A chromatographic and thermodynamic study of the compound [4-(allyloxy)benzoyl]-4-methoxyphenyl (ABMP) as a model of a chemically bonded liquid crystal stationary phase for HPLC was undertaken. A number of polycyclic aromatic hydrocarbons (PAHs) and two small solutes, carvone and pulegone, were studied under varying solvent and temperature conditions. Plots of log k vs. % organic in the mobile phase were not completely linear in all cases. The van't Hoff plots revealed at least one phase transition. The enthalpies of solute transfer from the mobile phase to the ABMP phase were determined for several PAHs. All tests indicate that ABMP possess liquid crystal properties when bonded to particulate silica.     

Mixed-mode chromatography and its applications to biopolymers

Mixed-mode chromatography is a type of chromatography in which a chromatographic stationary phase interacts with solutes through more than one interaction mode. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, high selectivity, high sample loading, high speed, and the ability to replace two conventionally corresponding columns in certain circumstances. In this work, some aspects of the development of mixed-mode chromatography are reviewed, such as stationary phase preparation, combinations of various separation modes, separation mechanisms, typical applications to biopolymers and peptides, and future prospects.     

反相高效液相色谱中溶质保留过程的热力学研究 Ⅱ. 焓变及其分量, 吸附及解吸附焓变

依据液相色谱中溶质计量置换保留模型, 从理论上分别推导出了在反相高效液相色谱(RP-HPLC)中lgI(与1mol溶质对固定相的亲合势有关的常数)和Z(对应于1mol溶质被固定相吸附时释放出置换剂的摩尔数)对绝对温度导数的线性关系式。发现非极性与极性小分子溶质的lgI和Z均具有热力学衡常数的特征。如在前报[1]报告的吉布斯自由能变一样, 也推导出了可将溶质保留过程中的总吸附焓变△H(Pa)分成两个独立的分量, 吸附焓变△(1,a)和解吸附焓变△H(Z,D)的表达式。可同时对在流动相中不同置换剂浓度条件下的△H(Pa)和△(Z,D)进行估算,并与实验值进行了比较, 偏差小于±10%。并用△H(1,a)和△H(Z,D)值对溶质在RP-HPLC的保留过程中热变化进行了解释。     

Role of the Dissolution Mechanism in Determining Chromatographic Separation of Biogenic Amines from Their Precursors and Metabolites by HPLC-EC

Abstract

The current study was designed to elucidate the theoretical basis for chromatographic separation of biogenic amines on an octadecyl-silica (C-18) reverse phase column by determining the intermolecular forces between the solute and the stationary phase. The solutes mass transfer diffusion and the heat effect between solutes and stationary phase were assessed by a convenient method. This study demonstrates that the dissolution mechanism plays a major role in the process of chromatographic resolution of biogenic amines and their precursors and metabolites by HPLC-EC.     

Temperature programmed retention indices: Calculation from isothermal data. Part 2: Results with nonpolar columns

The procedure for calculating linear temperature programmed indices as described in part 1 has been evaluated using five different nonpolar columns, with OV-1 as the stationary phase. For fourty-three different solutes covering five different classes of components, including n-alkanes and alkyl-aromatic compounds, both isothermal and temperature programmed indices were determined. The isothermal information was used to calculate temperature programmed indices. For several linear programmed conditions accuracies better than 0.51^T-units were usually obtained. The results are compared with published procedures. It is demonstrated that isothermal retention information obtained on one column can be transferred to another column with the same stationary phase but different column dimensions and/or phase ratio. The temperature programmed indices calculated in this way also have an accuracy better than 0.51^T-u. The temperature accuracy and precision of the GC-instrumentation used was of the order of 0.1°C. All calculations can be run with a Basic-programmed microcomputer.     

Influence of temperature and mobile phase composition on retention properties of oligomeric bonded phases in reversed-phase liquid chromatography (RPLC)

Summary The effect of temperature and mobile phase composition (methanol-water) on the retention behaviour of an oligomeric series of n-octylsilyl bonded phases in reversed-phase liquid chromatography has been investigated. Plots of lnk against 1/T (van't Hoff plot) and the enthalpy of transfer (ΔH^o) yields linear relationships under the conditions studied. The ΔH^o values of the aromatic hydrocarbons and n-alkyl benzoates are higher than those of the polar compounds due to their higher level of interaction with the stationary phase. A linear plot of ΔH^o vs. ΔS^o suggest that the retention process, which is essentially controlled by non-specific (dispersive) interactions between the solutes and the bonded ligands, is identical for all cases evaluated. The existence of similar retention mechanisms is confirmed by the constant value of the enthalpy-entropy compensation temperature of the columns for a given class of componds. As expected, decreasing the methanol content (% v/v) of the mobile phase results in increased eluite retention times. The methylene and phenyl selectivities are found to be independent of the carbon content of the stationary phases and varied only with the eluent composition. In addition to their high stability under aggressive mobile phase conditions as previously reported, the results of this study generally showed that the solute retention process on oligomeric phases are similar to those exhibited by the conventional reversed phases.     

Factors Influencing the Isothermal Retention Indices of 51 Solutes on 12 Stationary Phases of Different Polarity: Applicability of the Solvation Parameter Model

Isothermal retention indices (I) at 333–413 K on 12 stationary phases (SPs) covering a wide polarity range of a variety of volatile solutes belonging to 7 one-heteroatom chemical function series and 10 non-series solutes have been determined. The I values were computed with a method (LQG method) which does not require the determination of holdup times of the chromatographic column. I values of some compounds never before studied are reported. The influence on the retention indices of the column temperature, methylene number, and polarity of both the stationary phase and the solute has been studied. The solvation parameter model (SPM) as a function of I has been used for predicting I values, and for unraveling the influence of the polarity of stationary phase and solute on the retention indices. Seeley et al.’s formulation of the SPM has been used for quantifying the influence of polar and non-polar interactions on the I, and for checking the agreement between calculated and experimental values. According to our results, the I values obtained by the modified SPM can be considered equal to the experimental I values at the 99 % confidence level.