Ionisation Effects in the Reversed Phase Liquid Chromatographic Separation of Thyromimetic Iodoamino Acids

Abstract

The influence of ionisation equilibria on the retention behaviour of iodoamino acids and related compounds on micro-particulate octadecylsilica supports has been examined. The chromatographic data for these ionogenic solutes have been discussed in terms of current concepts for reversible solvophobic interactions with the hydrocarbonaceous stationary phase. This treatment permits the conditional effects of the mobile phase composition and pH on solute retention to be assessed and the relationship between the molecular surface area of a solute and its retention to a non polar stationary phase evaluated.     

Contribution of Steric Repulsion to Retention on an Octadecylsiloxane-Bonded Silica Stationary Phase in Reversed-Phase Liquid Chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsilioxane-bonded silica stationary phase, Ascentis ^TM C18, with acetonitrile-water and methanol-water mobile phase compositions containing 10–70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (1), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent compositions with a discontinuity at low organic solvent mobile phase compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes excluded from the models. The overwhelming number of residual values, here defined as the difference between experimental and model predicted retention factors for the excluded solutes, could be explained by contributions from steric repulsion. The latter defined as the inability of solutes to fully insert themselves into the solvated stationary phase because of their size or conformation. Steric repulsion resulted in a systematic reduction in retention compared with predicted values for the fully inserted solute. The bonding density of the stationary phase; the type and composition of the mobile phase; and the size, conformation, type and number of functional groups on the solute are shown to affect the contribution of steric repulsion to the retention mechanism.     

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.     

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.     

Reversed-Phase Liquid Chromatography with Mixed Eluents: Partition Model of Retention for Ionogenic Solutes

Abstract

Basing on the pure partition model of solute retention new theoretical equations for ionogenic solutes are discussed. These equations define dependence of the capacity ratio on the mobile phase composition, which is characteristic for the reversed-phase liquid chromatography.     

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

Abstract

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml^?1 in mobile phase containing 5.0 × 10^?2 mol l^?1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml^?1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.     

Solute retention in the stationary phase of a liquid-solid chromatographic system

Summary Utilizing the UNIFAC group model of activity coefficients the retention behaviour of a solute in the stationary phase of a liquid-solid chromatographic system is studied. By comparison of experimentally observed capacity ratios and calculated activity coefficients of solutes in the mobile phase, varying the concentration of a polar moderator, it is shown that the calculated activity coefficients in the stationary phase fit very well the equation formally identical with the Langmuir function. Comparison of activity coefficients in the mobile and the stationary phase proves equivalence between the solvent interaction and the competition theory.     

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.     

Study of the influence of electronic effects on the retention of substitutedN-benzylideneanilines in normal-phase liquid chromatography

Summary A series of 72 substitutedN-benzylideneanilines (NBA) has been studied by normal-phase liquid chromatography by use of an experimental design based on variation of the composition of mobile phases prepared from heptane and three modifiers, tetrahydrofuran, 1-octanol and ethyl acetate, for each of which the specific interactions are different. Seven mobile phases were defined in the experimental design. The chromatographic data obtained are used to discuss the behavior of NBA by application of complementary chemometric methods—hierarchical ascending classification (HAC) and correspondence factor analysis (CFA). Although solute polarity has the greatest effect on retention, construction of HAC and CFA plots shows that solute behavior is also influenced by second-order electronic effects arising as a result of specific interactions between the solutes and the different modifier solvents. A quantitative structure-retention relationship has been established between Hammett's constants and the solutes projection on the first factorial axis of CFA.     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

Characterization of Column Packings in Normal-Phase Liquid Chromatography. I. Retention Behavior of Fat-Soluble Vitamins in Silica Gel-Binary Solvent Systems

Abstract

To characterize packing columns in high performance liquid chromatography, the retention indices of ten fat-soluble vitamins were systematically measured using binary solvents each containing ethyl acetate, tetrahydrofuran and 2-propanol in n-hexane for silica gel chromatography. A linear relationship between the logarithm of the capacity ratio and that of the concentration of the polar solvents was confirmed. The retention sequence of the solutes was determined as follows: retinol > ergocalciferol = cholecalciferol > δ- > γ- > β- > α - tocopherol > menadione > phylloquinone. The retention behavior of retinal was similar to that of tocopherol derivatives, but varied depending on the polar solvent used. Such a retention sequence of fat-soluble vitamins may be explained on the basis of hydrogen bonding interactions between the active functional group on the solute molecules and silanol groups on the silica gel surface. Based on the adsorption selectivity given by the phase systems used, the resolution of each class of vitamins but not that of vitamin D homologues was successfully carried out.     

Relative Strength of Stronger Solvents for Silica,Amino- and Cyano-alkyl Bonded Silica Columns in Normal-Phase Liquid Chromatography

Abstract

The strength and selectivity of solvents such as ethyl acetate, dioxan and ethyl alcohol in n-hexane binaries were determined using steroids as solutes in normal-phase liquid chromatography of silica gel, amino- and cyano-propyl silica columns. Based on the linear relationship between the logarithm of the capacity ratio and logarithm of solvent composition, the relative strength of solvents was determined from the experimental retention data described in our earlier articles. A micro-computer data base was compiled for filing the retention behavior of the steroids. Using this computer data base, the optimization process of binary solvents for a given sample was improved. An example of the phase system design is described.     

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。     

Auswahl von Phasensystemen in der Lösungs-, Adsorptions- und Ionenaustausch-Chromatographie

In contrast to GC selectivity in LC is determined by the composition of both the stationary as well as the mobile phase. Therefore the main problem in LC results in selecting an appropriate phase system for the given separation problem. The selectivity factorα _ijis defined as the ratio of the capacity factors k′ _i k′_jof two solutes, which corresponds to the ratio of their distribution coefficients ^c K _i, ^cK_j. In LLC α _ijis determined by the relative solubility of the solutes in the two immiscible phases, which were prepared from binary or ternary liquid-liquid-systems. Secondary effects on retention are caused by the support. Two variations exist (LLC, Reverse-Phase-LLC) which differ in whether the polar phase is used as stationary or mobile phase, resp. In LSC the same phase variation is possible. Using a polar support and an unpolar solvent α _ijis governed by the relative strength of interactions between the solute molecules and the surface of the support. In Reverse-Phase-LSC, however, using an unpolar support and a polar solvent, these interactions are very weak and α _ijis mainly determined by the solubility of the solutes in the mobile phase. In IEC α _ijdepends on a set of parameters such as the type of ion-exchange matrix, its pore structure and its degree of crosslinking, resp., the type, surface concentration and distribution of functional groups, the type of the eluent ion, its concentration, the ionic strength and pH-value of the eluent, the temperature. Different methods have been developed in order to calculate the distribution coefficients of solutes for a given phase system.     

Insights into the retention mechanism on an octadecylsiloxane-bonded silica stationary phase (HyPURITY C18) in reversed-phase liquid chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsiloxane-bonded silica stationary phase HyPURITY C18 with methanol-water and acetonitrile-water mobile phase compositions containing 10-70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (2), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent composition with a discontinuity at low organic solvent compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 solutes at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes not included in the models. The overwhelming number of residual values [log k (experimental) - log k (model predicted)] were negative and could be explained by contributions from steric repulsion, defined as the inability of the solute to insert itself fully into the stationary phase because of its bulkiness (i.e., volume and/or shape). Steric repulsion is shown to strongly depend on the mobile phase composition and was more significant for mobile phases with a low volume fraction of organic solvent in general and for mobile phases containing methanol rather than acetonitrile. For mobile phases containing less than about 20 % (v/v) organic solvent the mobile phase was unable to completely wet the stationary phase resulting in a significant change in the phase ratio and for acetonitrile (but less so methanol) changes in the solvation environment indicated by a discontinuity in the system maps.     

The Use Of Soft Acid Metal Salts Of Carboxylic Acids For Retention Of Hydrophilic Peptides In Reversed-Phase High Performance Liquid Chromatography

Abstract

The effect of various soft acid cation acetate salts on the retention of the peptides Phe-(Gly)_n, where n=1–4, and on phenylalanine, indicates a correlation between the softness character of the cation of the acetate salt used in the mobile phase and retention in reversed-phase high performance liquid chromatography. Inorganic soft acid cation salts did not exhibit the same increased retention properties. By using silver(I) valerate or silver(I) octanoate as ion-pairing reagents in the mobile phase for RP-HPLC, static retention of peptides was achieved.     

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F13C9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C8) and octadecyl (C18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C18 phase, F13C9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C8, C18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C18 or C8 phases and was even disadvantageous on F13C9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.     

Retention of Ionized Solutes in Reversed-Phase High Performance Liquid Chromatography

Abstract

The mechanism of retention in reversed-phase high performance liquid chromatography is affected by both solute-eluent interactions and the nature of the stationary phase. The hydrophobic expulsion of ionized solutes plays a major role in affecting solute behavior in the water-rich range of hydroorganic eluents. In the water-lean range of eluent composition, there is little hydrophobic expulsion, and specific interactions between the solute and surface can be observed. The nature of the surface affects the retention of a variety of ionized species, both large cations and anions. Octadecylsilane (ODS) bonded phases can exhibit two different binding sites: one exhibiting a weak interaction and the second a strong specific interaction with a solute. Styrenedivinylbenzene polymeric surfaces exhibit the potential for weak dispersion interactions, and in addition pi-bonding interactions with a solute. A variety of solutes have been injected in a water: methanol eluent system in order to assess solute-surface effects on reversed-phase supports.