Combined supercritical fluid chromatographic methods for the characterization of octadecylsiloxane-bonded stationary phases

In this paper, we present a combination of a key-solute test based on retention and separation factors of large probe solutes (carotenoid pigments) and a quantitative structure-retention relationship analysis based on the retention factors of small probe solutes (aromatic compounds) to investigate the different chromatographic behavior of octadecylsiloxane-bonded stationary phases of all sorts: classical, protected against silanophilic interactions or not, containing polar groups (endcapping groups or embedded groups). Varied chemometric methods are used to enlighten the differences between the 27 phases tested. The results indicate that the two approaches chosen (carotenoid test and solvation parameter model) are complementary and provide precise information on the chromatographic behavior of ODS phases.     

A simple subcritical chromatographic test for an extended ODS high performance liquid chromatography column classification

This paper describes a new test designed in subcritical fluid chromatography (SFC) to compare the commercial C18 stationary phase properties. This test provides, from a single analysis of carotenoid pigments, the absolute hydrophobicity, the silanol activity and the steric separation factor of the ODS stationary phases. Both the choice of the analytical conditions and the validation of the information obtained from the chromatographic measurements are detailed. Correlations of the carotenoid test results with results obtained from other tests (Tanaka, Engelhard, Sander and Wise) performed both in SFC and HPLC are discussed. Two separation factors, calculated from the retention of carotenoid pigments used as probe, allowed to draw a first classification diagram. Columns, which present identical chromatographic behaviors are located in the same area on this diagram. This location can be related to the stationary phase properties: endcapping treatments, bonding density, linkage functionality, specific area or silica pore diameter. From the first classification, eight groups of columns are distinguished. One group of polymer coated silica, three groups of polymeric octadecyl phases, depending on the pore size and the endcapping treatment, and four groups of monomeric stationary phases. An additional classification of the four monomeric groups allows the comparison of these stationary phases inside each group by using the total hydrophobicity. One hundred and twenty-nine columns were analysed by this simple and rapid test, which allows a comparison of columns with the aim of helping along their choice in HPLC.     

Description of retention characteristics of calixarene-bonded stationary phases in dependence of the methanol content in the mobile phase

Calixarene-bonded stationary phases in HPLC are known to support additional interactions compared to conventional alkyl-bonded phases (π–π interactions, complex-building interactions). Thus it cannot be presumed that the same mechanisms of retention apply and that retention can be predicted in similar ways. Here 31 solutes of highly various molecular structures have been analysed at different mobile phase compositions (0–98% (v/v) methanol) in order to characterise the chromatographic behaviour of the novel stationary phases and to test the applicability of established models predicting retention factors. The influence of a change of the methanol content is discussed for non-polar, polar and ionic solutes and differences of their behaviour on the differing column types are shown. Additionally estimates about underlying retention mechanisms are given.     

Description and comparison of chromatographic tests and chemometric methods for packed column classification

The main tests developed in last 20 years to investigate the chromatographic behaviour and the stationary phase properties are described in this paper. These properties are the hydrophobicity, depending on the surface area and the bonding density, the number of accessible residual silanol groups having sometimes different acidity, which can interact with neutral solutes by hydrogen bonds or with the ionic form of basic compounds and the shape or steric selectivity, depending on both the functionality of the silanising agent and the bonding density. Two types of tests are performed, either based on key solutes having well defined properties such as phenol, caffeine, amitriptyline, benzylamine, acenaphtene, o-terphenyl, triphenylene, p-ethylaniline, carotenoid pigments, or on retention models (solvation parameter, hydrophobic subtraction) obtained from the analyses of numerous and varied compounds. Thus, the chromatographic properties are either related to selectivities or retention factors calculated from key solutes, or they are described by interaction coefficients provided by multilinear regression from retention models. Three types of comparison methods are used based on these data. First, simple plots allow the study of differences between the columns as regards to one or two properties. Columns located in the same area of the plot display close properties. Second, chemometric methods such as principal component analysis (PCA) or hierarchical cluster analysis (HCA) can be performed to compare columns. In this case, all the studied properties are included in the comparison, done either by data projection to reduce the space in which the information is located (PCA) or by distance calculation and comparison for drawing a classification (HCA). Neighbouring columns are expected to provide identical chromatographic performances. These two chemometric methods can be used together, PCA before HCA. The third way is to calculate a discrimination factor from a reference column, through calculation methods based on the Pythagorean Theorem: the lower this factor, the closer the column properties. Following the presentation of the analytical conditions, the compounds and the data treatments used by the teams working in this field, the pertinence of the different selectivities, i.e. of the different probe solute couples or of the different interaction coefficients, are discussed as regards their discrimination capacity. The accuracy of chemometric treatments in the discrimination of stationary phases having different functionalities (octadecylsiloxane (ODS), cyano, fluorinated, phenyl, polar embedded group or "aqua" type) will be discussed, as well as their performances in the finer ODS discrimination. New two-dimensional plots, from data gained by different studies will be suggested, to improve the classification of stationary phases having different nature of bonded chains.     

Cholesteric bonded stationary phases for high-performance liquid chromatography: a comparative study of the chromatographic behavior

The properties of four cholesteric bonded stationary phases differing in the nature of the spacer and the end-capping were assessed using simple chromatographic tests based on the retention of nonpolar compounds and of planar or nonplanar probe solutes. All cholesteric columns showed a hydrophobicity close to that of conventional octadecyldimethylsilyl (ODS) materials. Non-end-capped cholesteric bonded phases showed greater selectivity than ODS ones and both end-capped cholesteric bonded phases exhibit behavior intermediate between that of the non-end-capped original material and that of the ODS bonded phase.     

Preparation and comparison of three zwitterionic stationary phases for hydrophilic interaction liquid chromatography

Surface‐bonded zwitterionic stationary phases have shown highlighted performances in separation of polar and hydrophilic compounds under hydrophilic interaction chromatography mode. So, it would be helpful to evaluate the characteristics of zwitterionic stationary phases with different arranged charged groups. The present work involved the preparation and comparison of three zwitterionic stationary phases. An imidazolium ionic liquid was designed and synthesized, and the cationic and anionic moieties respectively possessed positively charged imidazolium ring and negatively charged sulfonic groups. Then, the prepared ionic liquid, phosphorylcholine and an imidazolium‐based zwitterionic selector were bonded on the surface of silica to obtain three zwitterionic stationary phases. The selectivity properties were characterized and compared through the relative retention of selected solute pairs, and different kinds of hydrophilic solutes mixtures were used to evaluate the chromatographic performances. Moreover, the zwitterionic stationary phases were further characterized by the modified linear solvation energy relationship model to probe the multiple interactions. All the results indicated that the types and arrangement of charged groups in zwitterionic stationary phases mainly affect the retention and separation of ionic or ionizable compounds, and for interaction characteristics the contribution from n and π electrons and electrostatic interactions displayed certain differences.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

A unified classification of stationary phases for packed column supercritical fluid chromatography

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model IV. Aromatic stationary phases

The purpose of the present work was to systematically study the chromatographic behaviour of different aromatic stationary phases in a subcritical fluid mobile phase. We attempted to assess the chemical origin of the differences in retention characteristics between the different columns. Various types of aromatic stationary phases, all commercially available, were investigated. The effect of the nature of the aromatic bonding on interactions between solute and stationary phases and between solute and carbon dioxide-methanol mobile phase was studied by the use of a linear solvation energy relationship (LSER): the solvation parameter model. This study was performed to provide a greater knowledge of the properties of these phases in subcritical fluid chromatography, and to allow a more rapid and efficient choice of aromatic stationary phase in regard of the chemical nature of the solutes to be separated. Charge transfer interactions naturally contribute to the retention on all these stationary phases but are completed by various other types of interactions, depending on the nature of the aromatic group. The solvation vectors were used to compare the different phase properties. In particular, the similarities in the chromatographic behaviour of porous graphitic carbon (PGC), polystyrene-divinylbenzene (PS-DVB) and aromatic-bonded silica stationary phases are evidenced.     

Classification of special octadecyl-bonded phases by the carotenoid test

Amongst the numerous base-deactivated ODS phases obtained by increasing the bonding density or/and by efficient endcapping treatments, some particular stationary phases have been developed, to limit the additional interactions of basic compounds with residual silanols, to work at extreme pH or with rich water mobile phases. Horizontal polymeric phases, sterically protected ones, hybrid silicas, propylene bridge, are particularly used for this purpose. Octadecyl chains with embedded polar groups and hydrophilic endcapping are also used in this goal. The properties of these phases were studied with a simple test consisting in the injection of carotenoid pigments in Subcritical Fluid Chromatography. The molecules used and the nature of the mobile phase allow the determination of hydrophobicity, polar site accessibility and type or/and bonding density of the stationary phases. Whatever the type of the phases, the particular stationary phases do not show any remarkable property, in comparison to other base-deactivated C18-bonded phases. On the other hand, embedded and polar-endcapped phases display a specific behaviour in regard of hydrophilic interactions, which are highlighted by the absence of water in the subcritical fluid. Additional properties of these phases are described, such as steric recognition and retention performances. As expected, polar-embedded phases are less retentive than classical ODS ones, but are sometimes able to provide greater steric recognition. On the other hand, the polar-endcapped phases display greater hydrophobicity than polar-embedded ones. From a simple classification diagram based on chromatographic properties, differences can be noticed between the polar-embedded groups (amide, carbamate, ether, sulfonamide) and between embedded and endcapped phases. Surprising behaviours are also noticed for some on the tested phases.     

Single-step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

Characterization of dynamically prepared phospholipid-modified reversed-phase columns.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.     

Selectivity of some basic solutes on a poly(methyltetradecylsiloxane)-silica stationary phase

Complex analyses of polar compounds, especially basic ones, require more selective stationary phases. The present paper describes a stationary phase prepared by thermal immobilization of poly(methyltetradecylsiloxane) onto chromatographic silica (PMTDS-SiO(2)). This stationary phase presents hydrophobic and ion-exchange interactions that confer both high retention and unique selectivities for basic solutes. The influence of ion-exchange interactions is confirmed by the increase in retention factors of basic solutes when the mobile-phase pH changes from acidic to neutral and by the decrease in retention factors when the mobile-phase pH changes from neutral to alkaline. The ion-exchange properties of the stationary phase are enriched in neutral mobile phase (pH 7-7.5) using soft Lewis bases such as tricine and tris as buffers but are suppressed in both acidic (pH 2.5-6) and highly alkaline mobile phases (pH≤10). Increasing both temperature and flow rate permits more rapid separations while maintaining the selectivity. The stability of the stationary phase is evaluated with acid, neutral and alkaline mobile phases.     

The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs

The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based ILs as modifiers of the chromatographic behaviour of a group of β-blockers is shown. The ILs differed in the adsorption capability of the cation and anion on C18 columns. Mobile phases without additive and containing a cationic (triethylamine, TEA) or anionic (sodium dodecyl sulphate, SDS) additive were used as references for the interpretation of the behaviours. The changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on the changes in retention and peak shape of the β-blockers. The silanol suppressing potency of the additives, and the association constants between the solutes and modified stationary phase or additive in the mobile phase, were estimated. The study revealed that SDS and the ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate are the best enhancers of chromatographic peak shape among those studied.     

Effect of short-chain alcohols on surfactant-mediated reversed-phase liquid chromatographic systems

The behaviour of β-blockers in a reversed-phase liquid chromatographic (RPLC) column with mobile phases containing a short-chain alcohol (methanol, ethanol or 1-propanol), with and without the surfactant sodium dodecyl sulphate (SDS), was explored. Two surfactant-mediated RPLC modes were studied, where the mobile phases contained either micelles or only surfactant monomers at high concentration. Acetonitrile was also considered for comparison purposes. A correlation was found between the effects of the organic solvent on micelle formation (monitored by the drop weight procedure) and on the nature of the chromatographic system (as revealed by the retention, elution strength and peak shape of β-blockers). When SDS is added to the mobile phase, the free surfactant monomers bind the C18 bonded chains on the stationary phase, forming an anionic layer, which attracts strongly the cationic β-blockers. The retention is modified as a consequence of the solving power of the organic solvent, micelles and surfactant monomers. The molecules of organic solvent bind the micelles, modify their shape, and may avoid their formation. They also bind the monomers of surfactant, desorbing them from the stationary phase, which affects the retention. The remaining surfactant covers the free silanols on the siliceous support, avoiding the interaction with the cationic solutes. The retention of β-blockers results from a combination of electrostatic and hydrophobic interactions, the latter being weaker compared to the hydro-organic system. The peak efficiencies and asymmetries are excellent tools to probe the surfactant layer on the stationary phase in an SDS/organic solvent system. The peaks will be nearly symmetrical wherever enough surfactant coats the stationary phase (up to 60% methanol, 40% ethanol, 35% 1-propanol, and 50% acetonitrile).     

咪唑侧基功能化聚离子液体修饰的混合模式色谱固定相的制备及其色谱性能

该文合成了咪唑侧基功能化的离子液体单体1-(4-乙烯基苄基)-3-氰甲基溴化咪唑盐,通过表面引发原子转移自由基聚合将该单体接枝到硅胶表面,制备了一种新型混合模式色谱固定相。采用红外光谱、元素分析及热重分析对其结构进行表征。该色谱固定相具有良好的分离能力。通过研究流动相pH对物质保留的影响,验证了物质在该固定相上存在反相-离子交换保留机理。通过与十八烷基硅烷键合硅胶固定相比较,证实了该聚离子液体固定相对物质保留提供了π-π作用。结果表明,对咪唑侧基功能化是制备新型离子液体固定相的可行方法。     

反气相色谱法测定Kovats及Flory-Huggins保留参数的变化以探究润滑油的氧化机理

 Inverse gas chromatographic technique (IGC) was attempted as a new approach to follow the chemical changes that occur during lubricating base oil oxidation. Three groups of the oxidized base oils were prepared at different oxygen flow rates, periods and temperatures according to IP48 method. The corrected retention volumes (VR) were calculated for a series of selected test solutes possessing different functional groups on the oxidized base oils used as stationary phases. Kovats retention index (I), Flory-Huggins interaction parameter (κ∞1,2), and partial molar free energy of solution (ΔG∞L), were calculated for the given test solutes from their VR. The relationships between the I values and the oxidation variables were plotted and discussed. The obtained results were confirmed by potentiometric titration. The study reveals that the magnitudes of variation of I, κ∞1,2 or ΔG∞L retention parameters depend on the oxidation degree of the base oil. Large differences between the I values permit discrimination between the different oxidation steps.     

Retention characteristics of a new butylimidazolium-based stationary phase. Part II: anion exchange and partitioning

A surface-confined ionic liquid (SCIL) and a commercial quaternary amine silica-based stationary phase were characterized employing the linear solvation energy relationship (LSER) method in binary methanol/water mobile phases. The retention properties of the stationary phases were evaluated in terms of intermolecular interactions between 28 test solutes and the stationary phases. The comparison reveals a difference in the hydrophobic and hydrogen bond acceptance interaction properties between the two phases. The anion exchange retention mechanism of the SCIL phase was demonstrated using nucleotides. The utility of the SCIL phase in predicting logk _IL/water values by chromatographic methods is also discussed.     

Lattice-fluid model for gas-liquid chromatography

Lattice-fluid models describe molecular ensembles in terms of the number of lattice sites occupied by molecular species (r-mers) and the interactions between neighboring molecules. The lattice-fluid model proposed by Sanchez and Lacombe (Macromolecules, 1978;11:1145-1156) was used to model specific retention volume data for a series of n-alkane solutes with n-alkane, polystyrene, and poly(dimethylsiloxane) stationary liquid phases. Theoretical equations were derived for the specific retention volume and also for the temperature dependence and limiting (high temperature) values for the specific retention volume. The model was used to predict retention volumes within 10% for the n-alkanes phases; 22% for polystyrene; and from 20 to 70% for PDMS using no adjustable parameters. The temperature derivative (enthalpy) could be calculated within 5% for all of the solutes in nine stationary liquid phases. The limiting value for the specific retention volume at high temperature (entropy controlled state) could be calculated within 10% for all of the systems. The limiting data also provided a new chromatographic method to measure the size parameter, r, for any chromatographic solute using characteristic and size parameters for the stationary phase only. The calculated size parameters of the solutes were consistent, i.e. independent of the stationary phase and agreed within experimental error with the size parameters previously reported from saturated vapor pressure, latent heat of vaporization or density data.