Characterization of stationary/mobile phase combinations by using markers : Part 1. Preliminary results for the prediction of reversed-phase retention on various stationary phases

The characterization of stationary/mobile phase combinations can be done in a phenomenological way by measuring the k′ values of specific solutes, the markers. These markers can be chosen optimally from a set of test solutes with the use of multivariate techniques. When retention data of solutes on different stationary phases, with varying mobile-phase compositions, are available, a procedure is given to predict the retention of those solutes on new stationary phases. This procedure uses markers to characterize the new stationary/mobile phase combinations.     

A New Application of McReynolds Constants to the Characterization of the Chromatographic Properties of Stationary Phases

An algorithm was proposed for comparing the McReynolds constants of new gas-chromatographic stationary phases with data for not only well-known individual substances but also their binary combinations. It was found that, in most cases, sets of these parameters for the chromatographic properties of new stationary phases are inconsistent with the data for a restricted set of previously characterized phases; however, they are consistent with the properties of phase combinations. This can be interpreted in terms of the absence of specific properties. Exceptions were found only among stationary phases bearing some specific molecular fragments (such as nitro groups) and individual crown ethers.     

Comparison of liquid chromatographic behaviors on N-methylimidazolium functionalized ZrO(2)/SiO(2)-4 and N-methylimidazolium functionalized SiO(2) stationary phases

A new stationary phase N-methylimidazolium functionalized ZrO(2)/SiO(2)-4 (Zr/SilprMim) has been prepared. The chromatographic property of this stationary phase is investigated by ion chromatography (IC) with inorganic and organic anions, and normal phase HPLC with basic compounds and hydroxybenzenes. The effects of pH and the strength of Lewis base of eluent on separation of anions are studied. This new stationary phase is also compared with a N-methylimidazolium functionalized SiO(2) stationary phase (SilprMim). The results show that this new stationary phase can be used in analysis of inorganic anions with great prospects. At the same time, successful separations of some organic anions can be obtained by using phosphate buffer solution as mobile phase. This new stationary phase also has a distinct advantage in the separation of basic compounds in normal phase. But due to the presence of Lewis acid sites on the surface of ZrO(2)/SiO(2)-4, Lewis bases such as hydroxybenzenes adsorb very strongly on this new stationary phase, and Lewis acid sites can be masked or modified by the eluent that contain Lewis base groups.     

Evaluation of polymeric methacrylate-based monoliths in capillary electrochromatography for their potential to separate pharmaceutical compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.     

Analysis of basic compounds at high pH values by reversed-phase liquid chromatography

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.     

Evaluation of differences between Chiralpak IA and Chiralpak AD‐RH amylose‐based chiral stationary phases in reversed‐phase high‐performance liquid chromatography

Polysaccharide‐based chiral stationary phases can be used for the enantioselective separation of a wide range of structurally different compounds. These phases are available with chiral selectors coated or immobilized on silica gel support. The means of attachment of the chiral selector to the carrier can influence the separation performance of these stationary phases. This paper deals with evaluation of differences in the separation abilities of coated Chiralpak AD‐RH versus immobilized Chiralpak IA amylose‐based stationary phases in the reversed–phase mode of high–performance liquid chromatography. A set of chiral analytes was separated under acidic and basic conditions. Differences were observed in the enantioseparation potential of the tested phases. The linear‐free energy relationship and additional evaluation of ionic interactions were used to ascertain whether the interactions that participate in retention and enantioseparation are affected by the means of preparation of these phases. All the interactions covered by the linear‐free energy relationship were significant for the studied phases and their absolute values were almost always higher for the coated phase. Ionic interactions were found to be more important on the immobilized stationary phase but did not contribute to any improvement in the enantioselective separation performance.     

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,     

Synthesis and Evaluation of a Synthetic Polymeric Chiral Stationary Phase for LC Based on the N, N′-[(1R,2R)-1,2-Diphenyl-1,2-Ethanediyl]bis-2-Propenamide Monomer

A synthetic polymeric chiral stationary phase for liquid chromatography based on N, N′-[(1R,2R)-1,2-diphenyl-1,2-ethanediyl]bis-2-propenamide monomer was prepared via a simple solution initiated radical polymerization. This stable chiral stationary phase showed enantioselectivities for a large number of racemates in polar organic and normal phase modes and high sample loading ability. However, none of the generated data has been optimized in terms of column performance. Different enantioselectivities were observed on this new chiral stationary phase compared with the commercial polymeric chiral stationary phase based on N-(2-acryloylamino-(1R,2R)-cyclohexyl)-acrylamine monomer. Consequently, these two chiral stationary phases are considered complementary to one another. Furthermore they utilize the same mobile phase and optimization procedures. This polymeric chiral stationary phase appears to be useful for preparative separations since high amounts of analyte can be injected without loosing enantioselectivity.     

Protein separation using a novel silica-based RPLC/IEC stationary phase modified with N-methylimidazolium ionic liquid

Ionic liquids (ILs) immobilized on silica as novel high performance liquid chromatography (HPLC) stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica (ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.     

Stationary-Phase Optimized Selectivity LC (SOS-LC): Separation Examples and Practical Aspects

Recently the principle of a novel approach was demonstrated:the stationary-phase optimized selectivity liquid chromatography (SOS-LC). In this paper some practical aspects are given and applications are presented to demonstrate the effect and possibilities of this novel procedure. Applying the same mobile phase and constant operating conditions the retention factors (k) of the model compounds have to be determined on three different single stationary phases. The optimal stationary phase combination can be predicted using serially connected columns and the principle of the ''PRISMA'' model, applied so far for mobile phase optimization. Generally the overall selectivity of this separation is better than that of any individual stationary phase. By use of different combinations the selectivity can be finely adjusted and even different elution order might be achieved. As new results of the application of SOS-LC complete separations of 7 flavonoids and 12 pesticides are demonstrated. The conclusions of the experiments dealing with the effect of the physical order of the different stationary phase segments within the serially connected column is also discussed. Finally, the SOS-LC optimization procedure is demonstrated in a form of a flow-chart.     

直链淀粉手性固定相高效液相色谱法拆分比索洛尔对映体

采用直链淀粉手性固定相高效液相色谱法在正相条件下直接拆分了比索洛尔对映异构体。分别以异丙醇、乙醇为有机改性剂,考察了流动相的组成与配比、流速及柱温等因素对比索洛尔对映体分离的影响。确定了比索洛尔对映体的最佳拆分条件:流动相正己烷-乙醇-二乙胺(体积比为88∶12∶0.1),流速0.6 mL/min,检测波长270 nm,柱温20 ℃。该方法可快捷、简便地拆分比索洛尔对映体。     

Sol-gel stationary phases for capillary electrochromatography

A review is presented on the current state of the art and future trends in the development of sol-gel stationary phases for capillary electrochromatography (CEC). The design and synthesis of stationary phases with prescribed chromatographic and surface charge properties represent challenging tasks in contemporary CEC research. Further developments in CEC as a high-efficiency liquid-phase separation technique will greatly depend on new breakthroughs in the area of stationary phase development. The requirements imposed on CEC stationary phase performance are significantly more demanding compared with those for HPLC. The design of CEC stationary phase must take into consideration the structural characteristics that will provide not only the selective solute/stationary phase interactions leading to chromatographic separations but also the surface charge properties that determine the magnitude and direction of the electroosmotic flow responsible for the mobile phase movement through the CEC column. Therefore, the stationary phase technology in CEC presents a more complex problem than in conventional chromatographic techniques. Different approaches to stationary phase development have been reported in contemporary CEC literature. The sol-gel approach represents a promising direction in this important research. It is applicable to the preparation of CEC stationary phases in different formats: surface coatings, micro/submicro particles, and monolithic beds. Besides, in the sol-gel approach, appropriate sol-gel precursors and other building blocks can be selected to create a stationary phase with desired structural and surface properties. One remarkable advantage of the sol-gel approach is the mild thermal conditions under which the stationary phase synthesis can be carried out (typically at room temperature). It also provides an effective pathway to integrating the advantageous properties of organic and inorganic material systems, and thereby enhancing and fine-tuning chromatographic selectivity of the created hybrid organic-inorganic stationary phases. This review focuses on recent developments in the design, synthesis, characterization, properties, and applications of sol-gel stationary phases in CEC.     

Capillary electrochromatography with physically and dynamically absorbed stationary phases

Adsorption is always considered a troublesome effect in capillary electrophoresis (CE) and capillary electrochromatography (CEC). However, the adsorption effect can also be exploited to prepare or optimize the stationary phase in CEC. Compared with the chemical synthesis of new stationary phase materials for CEC, this method is simpler and more convenient. This review is focused on CEC with physically and dynamically adsorbed stationary phases. Separation of some acidic, basic and neutral solutes as well as enantiomers in CEC with dynamically adsorbed stationary phases are presented. The theory for the migration of charged solutes and the stationary phases currently used in CEC are also briefly reviewed.     

Characterization of stationary phases in subcritical fluid chromatography by the solvation parameter model. I. Alkylsiloxane-bonded stationary phases

Varied types of alkylsiloxane-bonded and fluoroalkylsiloxane-bonded stationary phases, all commercially available, were investigated with subcritical fluid mobile phase. The effect of the alkyl chain length (from C4 to C18) and of the nature of the bonding (fluorodecylsiloxane, phenyl-C18 and polar-embedded-C18) on the chromatographic behaviour was investigated by the use of a linear solvation energy relationship (LSER), the solvation parameter model. A large set of test compounds provides precise and reliable information on the intermolecular interactions responsible for retention on these stationary phases used with a subcritical mobile phase. First of all, the results underline the close properties between subcritical fluid and organic liquid. The use of non aqueous mobile phases reduces the cavity energy and the mobile phase acidity generally encountered with aqueous liquid phases, allowing other interactions to take a part in retention. As expected, an increase in the alkyl chain length favours the dispersive interactions between the solutes and the stationary phases. Changes in basicity and acidity of the stationary phases are also related to the chain length, but, in this case, mobile phase adsorption onto the stationary phase is supposed to explain these behaviours. The addition of a phenyl group at the bottom of the C18 chain, near the silica, does not induce great modifications in the retentive properties. The fluorodecylsiloxane and the polar-embedded alkylsiloxane phases display very different properties, and can be complementary to the classical alkylsiloxane-bonded phases. In particular, the fluorinated phase does not favour the dispersive interactions, in comparison to hydrogenated stationary phases, when the basicity of the polar-embedded phase is obviously greater than the one of classical alkylsiloxane-bonded phases, due to the amide function. Finally, logk-logk curves plotted between the different phases illustrate the effect of the interaction properties on the retention of different classes of compounds.     

Enhancement of selectivity in reversed-phase liquid chromatography

In an effort to gain insight into the relationship between stationary phase solvation and selectivity, the use of short- and medium-chained-length alcohols (methanol, n-propanol, n-butanol, and n-pentanol) as mobile phase modifiers in reversed-phase liquid chromatography (RPLC) was investigated to determine their impact on chromatographic selectivity. A wide range of mobile phase compositions was evaluated because of the large effect exerted by solvent strength on selectivity. Employing a set of six vanillin compounds as retention probes, evidence is presented to support the view that an increase in the hydrophobicity of the organic modifier used in RPLC can increase the selectivity of the C18 alkyl bonded phase while simultaneously decreasing the retention time of the eluting solutes. Thus, we are presented with an interesting paradox: higher selectivity and shorter retention times, which can be attributed to changes in either solvent selectivity and/or stationary phase solvation by the organic modifier.     

Application of parallel factor analysis to total synchronous fluorescence spectrum of dilute multifluorophoric solutions: Addressing the issue of lack of trilinearity in total synchronous fluorescence data set

In recent years, total synchronous fluorescence (TSF) spectroscopy has become popular for the analysis of multifluorophoric systems. Application of PARAFAC, a popular deconvolution tool, requires trilinear structure in the three-way data array. The present work shows that TSF based three-way array data set of dimension sample × wavelength × Δλ does not have trilinear structure and hence it should not be subjected to PARAFAC analysis. This work also proposes that a TSF data set can be converted to an excitation–emission matrix fluorescence (EEMF) like data set which has trilinear structure, so that PARAFAC analysis can be performed on it. This also enables the retrieval of PARAFAC-separated component TSF spectra.     

烃类化合物在不同色谱柱上的定量结构-保留相关性研究

运用量子化学中的AMI方法计算烃类化合物的分子结构描述参数,借助逐步回归法建立了烃类化合物在不同极性色谱柱上的QSRR模型。结果表明：烷烃、烯烃、二烯烃类化合物在不同极性的色谱柱上的色谱保留与其分子结构描述参数之间具有较好的线性关系,烃类化合物在不同极性固定相上的保留主要与溶质分子的MR有关,即与溶质分子的色散力有关。随着溶质分子的不饱和度的增加,或固定相极性的增强,溶质分子与固定相之间的电荷传递作用随之增强。而且,烃类化合物在不同极性固定相上的色谱保留的QSRR模型均可用量化参数HOMO、LUMO、EICE以及MR参数来描述。所建立的在不同极性色谱柱上的烃类化合物的色谱保留QSRR模型预测烃类化合物的色谱保留值时具有较好的稳定性和准确性。     

Selectivity optimization in liquid chromatography via stationary phase tuning

In striving for the best possible separation, the selectivity of stationary phases as an optimization parameter is often underestimated although there are many ways to influence this powerful tool. This review serves to provide an insight into the various ways of adapting the selectivity of a separation in liquid chromatography. Approaches via temperature and flow rate tuning are discussed as a basis followed by focusing on the stationary phase as the superior optimization parameter. Highly selective stationary phases hereby provide an advantage for groups of similar analytes. For more complex mixtures, separations can be improved using mixed-mode technologies where different retention mechanisms are combined. Serial coupling, mixed-bed columns, and stationary phase optimized selectivity liquid chromatography provide solutions to various degrees. Finally, the advantages of stationary phase tuning over adaption of mobile phase and/or temperature are presented in terms of optimum application range.     

Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases

The development of new RP stationary phases containing polar groups has provided the chromatographer with a variety of stationary phase choices with differing selectivities. Polar endcapped and polar embedded group stationary phases have found use in solving a wide variety of separation problems, especially for the efficient separation of organic bases as well as separations necessitating the use of highly aqueous mobile phases. In this report, the retention thermodynamics of small, nonpolar solutes on traditional alkyl, polar endcapped, and polar embedded group stationary phases are compared. It is found that the nonpolar (methylene) transfer enthalpy is less favorable when polar embedded group phases are used, when compared to traditional or polar endcapped phases. In contrast, the transfer enthalpy of a phenyl group is found to be more favorable when a polar endcapped phase is used. In addition, the retention characteristics of these phases are compared using a set of solutes with differing solvatochromic parameters. Hydrogen-bond acids appear to have enhanced retention on polar embedded group phases, while hydrogen-bond bases have enhanced retention on polar endcapped phases.