Selectivity in reversed-phase separations Influence of the stationary phase

The selectivity difference between 15 different stationary phases was measured using a large number of analytes at 2 or 3 different pH values (3, 7 and 10) with acetonitrile and methanol as the mobile phase modifiers. The packings discussed include standard C(8) and C(18) packings, packings with embedded polar groups, a phenyl packing, a pentafluoro-phenyl packing, an adamantylethyl packing and others. The major selectivity differences observed are discussed in detail. Specific effects such as pi-pi interactions on phenyl packings or hydrogen-bond interactions on phases with embedded polar groups are confirmed.     

Separation properties of novel and commercial polar stationary phases in hydrophilic interaction and reversed-phase liquid chromatography mode

Four novel nonionic polar stationary phases were synthesised by anchoring first 2-mercaptoethanol and 1-thioglycerol, respectively, onto vinylised silica (ME and TG packings) followed by an on-phase oxidation with excess hydrogen peroxide in aqueous medium which yielded sulphoxide analogues of the embedded sulphide groups, i. e. oxidised 2-mercaptoethanol (MEO) and oxidised 1-thioglycerol (TGO) packings. Chromatographic characteristics of these stationary phases were evaluated comparatively to three commercial so-called 'diol' columns. U-shaped response curves of retention factors of adenosine and guanosine with hydro-organic eluents containing 5-95% v/v ACN as well as noticeable CH(2)-increment selectivity demonstrated multimodal separation capabilities of the developed amphiphilic materials, i. e. columns can be operated both in hydrophilic interaction chromatography (HILIC) and in RP mode. Although the selector ligands were physico-chemically related, considerably differing retention and selectivity patterns were observed in the HILIC mode. Thereby the introduction of additional hydroxyl groups in the chromatographic ligand resulted in selectivity increments that were different from those obtained by sulphur oxidation. For example, a set of five vitamins delivered five different elution orders with the overall seven columns. A close examination of HILIC separations of nucleobases and nucleosides on the developed packings revealed that (i) the amount of ACN in the eluent adopts a pivotal role in adjusting retention, (ii) the linearity of the relationship log (retention factor) versus log (volume fraction of water in the eluent) increases with phase polarity in the range of 5-40% v/v water, (iii) the slopes are higher with solutes having more polar interactive sites, (iv) the van't Hoff plots are linear (range 15-45 degrees C) with negative retention enthalpy values DeltaH (-4.5 to -14.5 kJ/mol) and (v) the -DeltaH values tend to be higher with more polar phases and more polar analytes. Based on these data the HILIC retention mechanism is described to be composed of both partitioning and adsorption processes. Distinct types of polar interactive sites in the chromatographic ligands may generate mixed-mode HILIC separation conditions that may additionally be superimposed by surface silanol contributions.     

Solute-solvent interactions between a range of solutes and trifluoropropyl siloxane stationary phases in terms of gas-liquid chromatography activity coefficients

Summary Several poly(3,3,3-methyltrifluoropropyl siloxane) stationary phases with a low percentage of trifluoropropyl have been recharacterised by means of activity coefficients at temperatures in the range 60–140°C. The temperature effect of activity coefficients was studied. Thermodynamic magnitudes: excess Gibbs energy, excess enthalpy and excess entropy for 44 solutes on these polymers were calculated, and their relationships with solutes’ molecular connectivity indexes were tested. Solute-polymer interactions were calculated at 120°C according to the solvation parameter model, and several correlations for selected solutes and polymers were investigated, mainly the effect of solutes’ structure on the non-polar interactions and the effect of the solute dipole moment on the polar interactions. In addition, the influence of polymer polarity on the different polar and non-polar interactions was investigated.     

Prediction of theoretical plate number in isothermal gas chromatographic analysis on capillary columns

A method for the prediction of the efficiency of gas chromatographic analysis in isothermal conditions by using experimental data of 1-alcohols and n-alkanes measured on capillary columns filled with polar and non-polar stationary phases in isothermal and isobaric conditions is described. The theoretical plate height trend indicates the change of separation efficiency as a function of inlet pressure and column temperature. By evaluating the variation of the diffusion coefficients of the analysed compounds into the mobile and stationary phase it is possible to predict the column efficiency and the number of theoretical plates at any temperature.     

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 mechanisms in super/subcritical fluid chromatography on packed columns

Whereas the retention rules of achiral compounds are well defined in high-performance liquid chromatography, on the basis of the nature of the stationary phase, some difficulties appear in super/subcritical fluid chromatography on packed columns. This is mainly due to the supposed effect of volatility on retention behaviours in supercritical fluid chromatography (SFC) and to the nature of carbon dioxide, which is not polar, thus SFC is classified as a normal-phase separation technique. Moreover, additional effects are not well known and described. They are mainly related to density changes of the mobile phase or to adsorption of fluid on the stationary phase causing a modification of its surface. It is admitted that pressure or temperature modifications induce variation in the eluotropic strength of the mobile phase, but effects of flow rate or column length on retention factor changes are more surprising. Nevertheless, the retention behaviour in SFC first depends on the stationary phase nature. Working with polar stationary phases induces normal-phase retention behaviour, whereas using non-polar bonded phases induces reversed-phase retention behaviour. These rules are verified for most carbon dioxide-based mobile phases in common use (CO(2)/MeOH, CO(2)/acetonitrile or CO(2)/EtOH). Moreover, the absence of water in the mobile phase favours the interactions between the compounds and the stationary phase, compared to what occurs in hydro-organic liquids. Other stationary phases such as aromatic phases and polymers display intermediate behaviours. In this paper, all these behaviours are discussed, mainly by using log k-log k plots, which allow a simple comparison of stationary phase properties. Some examples are presented to illustrate these retention properties.     

Packed column supercritical fluid chromatography with light-scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile phases

Summary Evaporative light scattering detectors can be used to detect organic substances without chromophoric groups in packed column supercritical fluid chromatography (SFC). A detector of this type has been used to detect squalane and glucose after SFC with various packed columns and binary mobile phases. In this study, the amount of organic modifier in carbon dioxide/modifier mixtures was varied. The results give further insight into the mechanisms that influence retention behaviour in packed column separations with super- and subcritical mobile phases. Squalane is an ideal non-polar test solute which shows long retention times on non-polar columns while its elution can be accelerated by non-polar modifiers in carbon dioxide. Glucose is an extremely polar solute containing hydroxyl groups. Elution of this sugar can be improved with polar modifiers. Column packings with polar end groups lead to high capacity ratios and long retention times for glucose. Most columns used in this study contained silica-based packing materials. For purposes of comparison, a polymeric packing (HEMA RP-18) was also employed.     

木糖醇和麦芽糖醇型亲水作用色谱固定相的制备及其分离性能的评价

利用-NCO和-OH的加成反应,通过简单的两步反应将木糖醇和麦芽糖醇成功地键合于硅胶表面,制备了两种新型糖醇类亲水作用色谱固定相。流动相中乙腈含量对保留的影响曲线表明,这两种糖醇固定相具有典型的亲水作用色谱固定相性质,对极性和亲水性化合物有很强的保留作用。利用这两种固定相成功分离了水溶性维生素、水杨酸及其类似物、碱基及其相应的核苷和淫羊藿苷类似物等模型混合物,同时糖醇固定相展现了新颖的选择性,特别是相对于线形的木糖醇键合固定相,非线形的麦芽糖醇键合固定相表现出了对糖基的独特保留能力。此外,缓冲盐的pH和浓度对保留的影响表明静电作用在这两种糖醇固定相的保留机理中也发挥着一定的作用。本文所发展的糖醇类固定相具有良好的分离性能,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

Temperature dependence of equivalent chain length values on capillary columns of different polarity

The influence of temperature and capillary column stationary phase polarity on the equivalent chain length (ECL) values of unsaturated fatty acid methyl esters (FAMEs) is discussed. Comparisons are made of a bonded, nonpolar methyl silicone, bonded and nonbonded polyethylene glycols, and a highly polar, stabilized cyanosilicone stationary phase. The change in the ECL values over a 20° temperature range is used to demonstrate selectivity shifts and the influence of temperature on the separation of FAMEs on these phases. The effect of the degree of unsaturation of the FAME components, on the various stationary phases is also investigated.     

Retention and selectivity of stationary phases for hydrophilic interaction chromatography

More and more polar stationary phases have become available for the separation of small polar compounds in the past decade as hydrophilic interaction chromatography (HILIC) continues to find applications in new fields (e.g., metabolomics and proteomics). Bare silica phases remain popular, especially in the bio-analytical area. A wide range of functional groups (e.g., amino, amide, diol, sulfobetaine, and triazole) have been employed as polar stationary phases for HILIC separation. This review provides a survey of the popular stationary phases commercially available and discusses the retention and selectivity characteristics of the polar stationary phases in HILIC. The purpose of the review is not to provide a comprehensive overview of literature reports, but rather focuses on findings that demonstrate retention and selectivity of the polar stationary phases in HILIC.     

Comparison of column properties in reversed-phase chromatography: monolithic, cholesterolic and mixed bonded stationary phases

A commercial Chromolith C18 column and two new stationary phases with mixed ligands bonded on the Kromasil silica gel support, SG-MIX and SG-Chol, were characterized using simple tests based on the retention of non-polar, basic and acidic compounds. Polar and methylene selectivity tests in acetonitrile-water and methanol-water mobile phases revealed lower hydrophobicities of the SG-MIX and SG-Chol columns in comparison to the Chromolith column. The columns were further characterized using new test criteria - gradient oligomer capacity and isomeric selectivity and peak symmetry of naphthalene di-sulphonic acids in aqueous mobile phases. The cholesterolic column shows greater gradient oligomer selectivity for the separation of oligoethylene glycol samples than the SG-MIX and the Chromolith columns. Increased retention and peak tailing, but decreased isomeric selectivity for naphthalene-di-sulphonic acids was observed with the SG-MIX column, because of interactions with various polar bonded groups.     

Influences of support,stationary phase polarity and loading in gas-liquid chromatographic retention

Summary Support effects for a range of diatomite supports have been determined from variations in both retention volume and retention index data for four solute probes as a function of stationary phase loading. The phases examined were Silicone 702, Silicone QF 1 and Carbowax 600. It was found that when polar phases were used, the main cause of retention data variation was gasliquid interfacial adsorption, support activity being eliminated if the surface of the support was effectively covered by such phases. For non-polar phases, support effects may still be apparent with loadings over 5%. With liquid loadings less than this value, unreal retention volumes may result from solute-support adsorption. Retention data, as a function of stationary phase loading, for highly fluorinated and non-fluorinated compounds reveals that the Retention Indices of the former are as significant as the latter class. In both cases high stationary phase loadings are recommended for the reporting of retention data.     

Monte-carlo simulation of gas chromatographic separation for the prediction of retention times and peak half widths

Summary A new method for prediction of gas chromatographic retention times and peak half widths is based on the renewal theory. The only requirements are the heats of vaporization of the compounds to be separated and one calibration measurement. With this data, retention times and peak half widths can be predicted for isothermal as well as temperature-programmed gas chromatography. For the separation of non-polar substances on non-polar stationary phases the prediction error for retention times is approx. 1–2%. First simulations of polar molecules and polar stationary phases indicate that this method is also applicable in these cases but some extension will be required.     

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.     

Selectivity differences between alkyl and polar‐modified alkyl phases in reversed phase high performance liquid chromatography

Compared to conventional C18 phases, polar‐modified phases have distinct differences with regards to chromatographic behavior. In the present study, ODS phases and polar‐modified phases were synthesized. The columns containing these new packings demonstrated satisfactory stability under both acidic (pH 1.5) and basic (pH 10) conditions. We evaluated the selectivity differences between alkyl and polar‐modified alkyl RP columns by using a range of neutral analytes. The polar‐modified alkyl phases showed excellent peak shapes for almost all compounds. We also compared the selectivity differences between them for separating nucleotides by using 100% aqueous mobile phase and tricyclic antidepressants in the intermediate pH mobile phases. The results demonstrated that polar‐modified phases display a significantly reduced hydrophobic nature and a significantly reduced silanol activity compared to the conventional C18 phases.     

Naphthalene sulphonic acids--new test compounds for characterization of the columns for reversed-phase chromatography

Non-substituted naphthalene sulphonic acids are strong acids, which are completely ionised in aqueous and aqueous-organic solutions. Because of repulsive electrostatic interactions, they are more or less excluded from the pores of the column packing materials commonly used in reversed-phase chromatography. The ionic exclusion can be suppressed by increasing the ionic strength of the mobile phase. In aqueous sodium sulphate solutions, very good selectivity was observed for isomeric naphthalene di- and tri-sulphonic acids, allowing reversed-phase separations of these strongly ionic compounds without addition of ion-pairing reagents to the mobile phase. The retention of the isomeric acids increases proportionally to the dipole moment, which can be explained by its effect on increasing exposure of the naphthalene ring to hydrophobic interactions with the non-polar stationary phases. Chromatographic behaviour of isomeric naphthalene di- and trisulphonic acids was investigated on 25 different columns for reversed-phase chromatography. The elution order of the isomers is the same on all the columns, but very strong stationary phase effects were observed on the retention and on the band asymmetry, depending on polar interactions with residual silanol groups and other polar adsorption centres in the stationary phases. These effects are independent of the organic solvents, as the tests are performed in purely aqueous mobile phases and allow classification of the columns into several groups.     

Insights into the Retention Mechanism on a Pentafluorophenylpropylsiloxane-Bonded Silica Stationary Phase (Discovery HS F5) in RP-LC

The solvation parameter model is used to elucidate the retention mechanism of neutral compounds on the pentafluorophenylpropylsiloxane-bonded silica stationary phase (Discovery HS F5) with methanol-water and acetonitrile-water mobile phases containing from 10 to 70% (v/v) organic solvent. The dominant factors that increase retention are solute size and electron lone pair interactions while polar interactions reduce retention. A comparison of the retention mechanism with an octadecylsiloxane-bonded silica stationary phase based on the same silica substrate and with a similar bonding density (Discovery HS C18) provides additional insights into selectivity differences for the two types of stationary phase. The methanol-water solvated pentafluorophenylpropylsiloxane-bonded silica stationary phase is more cohesive and/or has weaker dispersion interactions and is more dipolar/polarizable than the octadecylsiloxane-bonded silica stationary phase. Differences in hydrogen-bonding interactions contribute little to relative retention differences. For mobile phases containing more than 30% (v/v) acetonitrile selectivity differences for the pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica stationary phases are no more than modest with differences in hydrogen-bond acidity of greater importance than observed for methanol-water. Below 30% (v/v) acetonitrile selectivity differences are more marked owing to incomplete wetting of the octadecylsiloxane-bonded silica stationary phase at low volume fractions of acetonitrile that are not apparent for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. Steric repulsion affects a wider range of compounds on the octadecylsiloxane-bonded than pentafluorophenylpropylsiloxane-bonded silica stationary phase with methanol mobile phases resulting in additional selectivity differences than predicted by the solvation parameter model. Electrostatic interactions with weak bases were unimportant for methanol-water mobile phase compositions in contrast to acetonitrile-water where ion-exchange behavior is enhanced, especially for the pentafluorophenylpropylsiloxane-bonded silica stationary phase. The above results are compatible with a phenomenological interpretation of stationary phase conformations using the haystack, surface accessibility, and hydro-linked proton conduit models.     

Retention properties of novel β-CD bonded stationary phases in reversed-phase HPLC mode

With the given special structures, the CD bonded stationary phases are expected to have complementary retention properties with conventional C18 stationary phase, which will be helpful to enhance the polar selectivity in RP mode separation. In this work, two β-cyclodextrin (β-CD) bonded stationary phases for reversed-phase HPLC, including 1, 12-dodecyldiol linked β-CD stationary phase (CD1) and olio (ethylene glycol) (OEG) linked β-CD stationary phase (CD2), have been synthesized via click chemistry. The resulting materials were characterized with FT-IR and elemental analysis, which proved the successful immobilization of ligands. The similarities and differences in retention characteristics between the CD and C18 stationary phases have been elucidated by using comparative linear solvation energy relationships (LSERs). The force related to solute McGowan volume has no significant difference, while the hydrogen bonding and dipolar interactions between solutes and CD stationary phases are stronger than between solutes and C18, which is attributed to the special structures (CD and triazole groups) of CD stationary phases. Chemical origins are interpreted by comparison between CD1 and CD2. Similar dispersive interactions of CD1 and CD2 are attributed to their similar length of spacer arms. CD2 which contains OEG spacer arm has relative weaker HBD acidity but stronger HBA basicity. CD stationary phases display no serious different methylene selectivity and higher polar selectivity than in the case of C18. Higher acid selectivity and lower basic selectivity are observed on CD2 than on CD1. Distinctive retention properties and good complementary separation selectivity to C18 make the novel CD bonded stationary phases available for more application in RPLC.     

“Click chemistry” preparation of WCX packings for protein separation

Click chemistry was applied to immobilize three kinds of alkyne-carboxylic acids onto azide-modified silica gel to prepare three novel stationary phases for weak cation exchange chromatography(WCX).The developed protocol combines the benefits of operational simplicity,exceptionally mild conditions and high surface loadings.Six kinds of standard proteins were separated completely on the novel packings.Compared with commercial WCX columns,the three kinds of novel WCX packings prepared by click chemistry approach have better resolution and selectivity.Lysozyme was purified successfully from egg white with the novel WCX column by one step.The purity was more than 97%and a high specific activity was achieved to be 81,435 U/mg.The results illustrate the potential of click chemistry for preparation of stationary phase for IEC.     

The orthogonal character of stationary phases for gas chromatography

A database of system constants for 32 open-tubular columns at 100 degrees C is used to identify stationary phases for obtaining a wide selectivity space in comprehensive GC. Three parameters based on the Euclidean distance (D-parameter) or vectors (d-parameter and costheta) in hyperspace are used to establish the chemical similarity and retention correlation as an inverse scale of selectivity differences. It is shown that the poly(methyloctylsiloxane) stationary phase is the best candidate for a low-selectivity stationary phase and affords a wider selectivity space when combined with a selective polar stationary phase than poly(dimethylsiloxanes). The most suitable polar stationary phases are poly(ethylene glycols) or bis(cyanopropylsiloxane-co-silarylenes and to a lesser extent poly(methyltrifluoropropylsiloxanes). No systems are truly orthogonal but angles between individual stationary phase vectors of about 75 degrees are possible by choosing the correct combination of stationary phases.