On the mechanism of solute retention in RP-HPLC systems with B+AB1+AB2 type eluents

Summary Intermolecular interactions between the solute and the moieties constituting the mobile phase significantly contribute to the overall retention pattern of a given solute in a given chromatographic system. In this paper retention of solute is discussed in the case of the B+AB₁+AB₂ type mobile phase, which, in a quasithermodynamic way, can be divided into seven individual moieties. One evaluates the influence of each moiety on solute retention, and refers these regularities to the polarity of the solutes.     

梯度液相色谱保留时间公式在梯形梯度洗脱研究中的应用

根据前期得到的梯度液相色谱保留时间计算公式,在不指定溶剂强度模型形式的前提下,探讨了梯形梯度洗脱的一些特点。对于溶质在梯形梯度坡度上流出时的情形,推导得到溶质流出色谱柱所对应的流动相组成(φ_R)随梯度斜率(B)变化的表达式。该公式表明,在该情形中φ_R将会随着B值的增加而增加。对于溶质在梯形梯度最后一个等度区间流出时的情形,如果初始和终止流动相组成保持不变而仅有梯度的斜率发生变化时,从理论上证明了溶质保留时间(t_R)与梯度斜率的倒数(1/B)之间呈线性关系。实验中以C18色谱柱为固定相,甲醇-水为流动相,联苯为样品,测定了不同流动相组成以及梯形梯度条件下的保留时间,所得到的实验值与理论值吻合,从而验证了理论方法的正确性。     

醋酸水流动相体系中硅胶键合相上生物大分子保留行为的研究

 提出了以醋酸 水作为流动相的体系中 ,在ODS柱上分离生物大分子的反相高效液相色谱 (RPLC)方法。实验结果表明 ,醋酸 水的洗脱能力强于甲醇 水 三氟醋酸体系 ,在一定程度上克服了色谱分离中一些蛋白质的不可逆吸附且具有便于冷冻干燥的优点。用参数Z(1mol溶剂化溶质被溶剂化固定相吸附时从两者接触表面释放出置换剂的摩尔总数 ) ,logI(与 1mol溶质对固定相亲和势有关的常数 )和 j(与 1mol溶剂对固定相亲和势有关的常数 )对 9种蛋白质在此流动相体系中的保留进行了表征。     

反相液相色谱中溶剂强度规律的研究

 以溶质计量置换保留模型 (SDM R)为依据 ,通过研究 3种正链醇同系物溶剂置换剂对 14种正链醇同系物溶质色谱保留行为的影响 ,发现计量置换参数Z(对应 1mol溶质被吸附时从溶质与固定相接触处释放出的溶剂的总摩尔数 )、lgI(与 1mol溶质对固定相亲和势有关的常数 )和 j(与 1mol溶剂对固定相亲和势有关的常数 )均随着同系物置换剂相对分子质量的增大而减小 ,并呈现出线性变化 ,表明溶剂强度与溶剂分子的大小有关 ,分子愈大 ,溶剂洗脱能力愈强 ,并遵循同系物规律。     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

A New Equation for Solute Retention in Liquid Chromatography

In consideration of the adsorption of solvent, diluent and solute molecules on the surface of a stationaryphase, a new equation for solute retention in liquid chromatography is presented. This equation includesthree parameters: the displacement equilibrium constant (Ksd) between the solvent and diluent molecules onthe surface of the stationary phase, the total number(N) of the solvent and diluent molecules released fromthe stationary phase after one solute molecule being adsorbed, and the parameter (I) related to the thermody-namic equilibrium constant for the solute adsorption on the stationary phase. Over the whole concentrationrange of the solvent in the mobile phase, the experimental retention data can be well described by this equa-tion, parameters K~, N and I can be obtained by the regression analysis of the experimental retention data,and consequently the number of the solvent and the diluent molecules displaced by one solute molecule fromthe stationary phase can also be derived at different solvent concentrations in the mobile phase,     

在脂肪酸-水流动相中小分子在反相高效液相色谱中的保留表征

在反相液相色谱(RPLC)中用Snyder经验议程和计量置换保留模型(SDM-R)中的参数对深质为脂肪醇同系物,流动相为脂肪酸同系物时深质的保留行为进行了研究,结果表明用SDM-R参数具有明显的优越性,另外,由于用Snyder经验公式中二参数之间的作园无法准确求得斜率,且其不具有明确的物理意义,而由SDM-R二参数作图,不但能准确求得斜率j(与1mol溶剂和固相结合能有关的常数),而且j具有明确的物理意义并符合碳数规律,所以,参数j有可能用于RPLC中表征深剂强度,由此得出,在RPLC中,对同一置换剂面言,随同系物溶质的Z(1mol深剂化深质被深剂化固定相吸附时,从二接解面释放出的置换剂分子数)和logI(与1mol深质和固定相亲和势有关的常数)值增大,它们的保留时间也增大,对同一深质而言,随着在同系物置换剂中碳链的增长,Z和logI值的减小,它的保留时间也缩短,同时还可得出,随着同系物置换剂j值的减小,它们的洗脱能力也增强.     

Correlation between retention and the C18 silica surface coverage in reversed-phase liquid chromatography

Summary Members of three homologous series and five non-homologous solutes with various functional groups were chromatographed on a series of well-characterized C₁₈ reversed stationary phases with a range of methanol-water mobile phases. Measured capacity factors of solutes were correlated with the concentration of C₁₈ ligands on the stationary phase. A linear relationship with the slope depending on a solute molecular structure and the volume fraction of methanol in the mobile phase was obtained. A method for the evaluation of phase ratio is also proposed. Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

The temperature convergence of biopolymers in reversed-phase liquid chromatography

Summary Based on a stoichiometric displacement model for retention (SDM-R) in liquid chromatography, the two linear plots, log/(a constant relating to the affinity of one mole of solute to the stationary phase) andZ (the total moles of the displacing agent released at the interface between stationary phase and solute molecules as one mole of the solute is absorbed by the stationary phase) of small solutes and biopolymers versus the reciprocal of absolute temperature, l/T, in reversed-phase liquid chromatography were theoretically derived and experimentally proved to be linear and to have a common point called the temperature convergent point. The two linear plots could be used instead of the plot of logk to l/T which is normally only valid for small solutes but not for biopolymers for investigations of chromatographic thermodynamics and temperature convergence. The average convergence temperature (T _conv) of biopolymers was theoretically derived to equal the ratio between the slope and the intercept from either one of the two linear plots and to be 130.8±9.3°C for five proteins which are very close to those of biopolymers by calorimetry. A new methodology to investigate chromatographic thermodynamics and possibly for investigating the temperature convergence of biopolymers in the process of protein folding is also presented.     

A STOICHIOMETRIC DISPLACEMENT MODEL FOR RETENTION OF SOLUTE IN LIQUID CHROMATOGRAPHY WITH MULTI-COMPONENT SYSTEM

Considering all the kinds of interactions between solute and solvent, solute and stationary phase, solvent and stationary phase molecules as well as the competitional adsorption among various kinds of solvent molecules on the stationary phase, we present a stoichiometric displacement model of solute retention with four sets of parameters in liquid chromatography. This model was tested with data from both literature and experiments done by ourselves. These results show that this model may fit the experimental data for a liquid chromatography system with various kinds of mobile phases consisting of a complete range of multi-components and with different types of stationary phases.     

Retention mechanism of the multifunctional solute on columns with different coverage densities using highly aqueous reversed‐phase conditions

A series of 11 homemade octadecyl bonded phases with different coverage densities were tested to determine the influence of the stationary phase on the retention in highly aqueous mobile phases. The concentrations of the organic modifiers (methanol and ACN) were in the range of 0–20%_v/v. The coverage density of bonded ligands and the presence of the end‐capping have strong influence on the solute retention. Amoxicillin (AMO) was chosen as the test compound. Dual properties of AMO, which contain hydrophobic skeleton and polar groups (amino, hydroxyl and carbonyl), cause irregular changes of the retention over the stationary phase hydrophobicity and silanol activity at given mobile phase composition. Presented data show that application of non‐standard low coverage density C18 phases allow to determine AMO in the RPLC condition with high retention.     

Factors influencing retention and resolution of substituted alkylbenzenes in reversed-phase liquid chromatography

Summary The retention data of isomeric xylenes, ethyltoluenes and diethylbenzenes, and of mesitylene, benzene, toluene and ethylbenzene were obtained on a reversedphase column using methanol-water and ethanol-water mobile phases at four different temperatures. This database was used to relate the dependence of solute retention and resolution on the polarity of the mobile phase, solute dipole moment, and column temperature. The additivity of the free energy of the transfer of solute molecules or solute segments from the stationary phase to the mobile phase, was examined for the isomeric compounds. For this, the logarithm of the net retention volume was substituted for the free energy. Deviations from the additivity of free energies indicate that the separation of isomeric substituted alkylbenzenes is governed by their differential interactions with both the polar mobile phase and nonpolar stationary phase. Among the disubstituted alkylbenzenes,ortho-isomers favor the mobile phase more andpara-isomers tend to prefer the stationary phase more. Themeta-isomers are found to follow the additivity rule closely. These trends are amplified as the polarity of the mobile phase is increased indicating that these isomers are resolved better in water-rich mobile phases. These findings are substantiated by analogous results from gas-liquid chromatographic retention data, estimation of dipole moment effects, and examination of the entropic and enthalpic contributions to the net retention volume.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

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.     

HPLC separation of fullerenes on two charge-transfer stationary phases

Two charge-transfer stationary phases were prepared by immobilizing p-nitrobenzoic acid and naphthyl acetic acid onto silica. The nitrophenyl moiety and the naphthyl moiety were grafted to silica gel through the spacer of aminoalkyl silanes. The HPLC separation of C60, C70, and higher fullerenes on the new stationary phases was also studied. The influence of mobile phase and column temperature on the separation of C60 and C70 was examined, respectively. The retentions of C60 and C70 on the two stationary phases increased with decreasing toluene content in the mobile phase or with increasing column temperature. Higher fullerenes can be separated well using toluene as the mobile phase on the stationary phase of p-nitrobenzoic acid-bonded silica.     

Retention of C60 and C70 fullerenes on reversed-phase high-performance liquid chromatographic stationary phases.

The separation of C60 and C70 fullerenes on four different polysiloxane stationary phases was examined. It was determined that polar solvents can be used as mobile phases effectively for the separation of fullerene molecules. Unlike previously published work, a polymeric octadecyl siloxane (ODS) stationary phase provided higher separation factors for C70/C60 than did monomeric ODS stationary phases or phenyl substituted stationary phases. For example, for a methanol-diethyl ether (50:50, v/v) mobile phase and C60, k' approximately 5.0 separation factors, alpha = 3.3, were achieved with polymeric ODS compared to alpha = 2.2, with a monomeric ODS stationary phase. A linear solvation energy relationship (LSER) was used to model the importance of solvent interactions and stationary phase interaction to solute retention.     

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.     

Evaluation of ODS-AQ stationary phase for use in capillary electrochromatography

The aim of this study was to evaluate the applicability of ODS-AQ packing material as a stationary phase in capillary electrochromatography (CEC). The electroosmotic flow created on an ODS-AQ stationary phase was measured at different mobile phase compositions and at different column temperatures. It was observed that the electroosmotic flow generated in the column increased by 50% when the temperature of the system was raised from 20 degrees C to 60 degrees C, while all other conditions were kept constant. The electroosmotic flow produced by the ODS-AQ stationary phase was found to be comparable to the flow generated in a column packed with Nucleosil bare-silica material. In addition, a set of polar compounds (D-lysergic acid diethylamide derivatives) was utilized to determine the influence of temperature and mobile phase composition on their chromatographic behavior on an ODS-AQ stationary phase in a CEC mode. A linear relationship between the solute retention factor and column temperatures was seen over the temperature range studied (20 degrees C to 60 degrees C). A quadratic function was used to describe the changes in the solute retention factors with variation of acetonitrile concentration in the mobile phase.