溶质在动态改性氧化锆液相色谱柱上的保留行为

分别采用硬脂酸、环糊精和十二烷基磺酸钠动态改性自制的ZrO2微球,研究了流动相中甲醇和改性剂浓度对苯酚及苯甲酸的衍生物、苯胺衍生物及芳香烃类化合物的色谱保留行为的影响。中性及碱性化合物的保留时间较短,色谱峰对称;酸性化合物保留时间较长,色谱峰拖尾较严重。改性氧化锆表现出反相色谱性能。     

Mixed-mode reversed-phase and ion-exchange separations of cationic analytes on polybutadiene-coated zirconia

The retention and selectivity of the chromatographic separation of basic (cationic) analytes on a polybutadiene-coated zirconia (PBD-ZrO2) stationary phase have been studied in greater detail than in previous studies. These separations are strongly influenced by the chemistry of the accessible surface of zirconia. In the presence of buffers which contain hard Lewis bases (e.g., phosphate, fluoride, carboxylic acids) zirconia's surface becomes negatively charged due to adsorption of the buffer anion at the hard Lewis acid sites. Consequently, under most conditions (e.g., neutral pH), cationic analytes undergo both hydrophobic and cation-exchange interactions. This mixed-mode retention process generally leads to greater retention factors for cations relative to those on silica-based reversed phases despite the lower surface areas of the zirconia phase, but, more importantly, adsorption of hard Lewis bases can be used to control the chromatographic selectivity for cationic analytes on these zirconia-based stationary phases. In contrast to our prior work, here we show that when mixed-mode retention takes place, both retention and selectivity are easily adjusted by changing the type of hard Lewis base buffer anion, the type of buffer counter-ion (e.g., sodium, potassium, ammonium), the pH, and the ionic strength of the eluent as well as the type and amount of organic modifier.     

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.     

Superheated water as eluent in high-temperature high-performance liquid chromatographic separations of steroids on a polymer-coated zirconia column

High-temperature liquid chromatography (HTLC), with a superheated water mobile phase, has been shown to be a feasible replacement for medium-polarity acetonitrile-water mixtures as an eluent in reversed-phase HPLC. Instrumental parameters of flow-rate, injection volume and mobile phase preheating were shown to have significant effects on the quality of the chromatographic peaks. The selectivity and retention patterns of testosterone and several related compounds were investigated on a porous zirconia, polybutadiene-coated column at temperatures up to 200 degrees C and compared with that of a porous silica, octadecylsilane-coated column and the zirconia column under traditional reversed-phase conditions of an acetonitrile-water mobile phase at 40 degrees C. The selectivity differences observed for testosterone and related compounds show that the separation mechanisms are complementary and unique selectivity is obtained with the zirconia column under HTLC conditions.     

The retention behaviour of polar compounds on zirconia based stationary phases under hydrophilic interaction liquid chromatography conditions

The most separations in HILIC mode are performed on silica-based supports. Nevertheless, recently published results have indicated that the metal oxides stationary phases also possess the ability to interact with hydrophilic compounds under HILIC conditions. This paper primarily describes the retention behaviour of model hydrophilic analytes (4-aminobenzene sulfonic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 3,4-diaminobenzoic acid, 3-aminophenol and 3-nitrophenol) on the polybutadine modified zirconia in HILIC. The results were simultaneously compared with a bare zirconia and a silica-based HILIC phase. The mobile phase strength, pH and the column temperature were systematically modified to assess their impact on the retention of model compounds. It was found that the retention of our model hydrophilic analytes on both zirconia phases was mainly governed by adsorption while on the silica-based HILIC phase partitioning was primarily involved. The ability of ligand-exchange interactions of zirconia surface with a carboxylic moiety influenced substantially the response of carboxylic acids on the elevated temperature as well as to the change of the mobile phase pH in contrast to the silica phase. However, no or negligible ligand-exchange interactions were observed for sulfanilic acid. The results of this study clearly demonstrated the ability of modified zirconia phase to retain polar acidic compounds under HILIC conditions, which might substantially enlarge the application area of the zirconia-based stationary phases.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Ion‐exchange vs reversed‐phase chromatography for separation and determination of basic psychotropic drugs

Ion exchange chromatography, an alternative to reversed‐phase (RP) chromatography, is described in this paper. We aimed to obtain optimal conditions for the separation of basic drugs because silica‐based RP stationary phases show silanol effect and make the analysis of basic analytes hardly possible. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types of buffers at acidic pH and with the addition of organic modifiers: methanol and acetonitrile. The obtained results reveal a large influence of the salt cation used for buffer preparation and the type of organic modifier on the retention behavior of the analytes. These results were also compared with those obtained on an XBridge C₁₈ column. The obtained results demonstrated that SCX stationary phases can be successfully used as alternatives to C₁₈ stationary phases in the separation of basic compounds. The most selective and efficient chromatographic systems were applied for the quantification of some psychotropic drugs in fortified human serum samples. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis

Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100‐5 C18 and LiChrosorb RP‐select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers – acetonitrile or methanol – in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?     

Cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia for chiral separation in high performance liquid chromatography.

Porous zirconia particles are very robust material and have received considerable attention as a stationary phase support for HPLC. We prepared cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia (CDMPCCZ) as a chiral stationary phase (CSP) for separation of enantiomers of a set of 14 racemic compounds in normal phase (NP) and reversed-phase (RP) liquid chromatography. Retention and enantioselectivity on CDMPCCZ were compared to those on CDMPC-coated zirconia (CDMPCZ) to see how the change in immobilization method of the chiral selector affects the retention and chiral selectivity. In NPLC, retention was longer and the number of resolved racemates was smaller on CDMPCCZ than on CDMPCZ. However, chiral selectivity factors for some resolved racemates were better on CDMPCCZ than on CDMPCZ. The longer retention on CDMPCCZ is likely due to strong, non-chiral discriminating interactions with the carbon layer on CDMPCZ. In RPLC only two racemates were resolved on CDMPCCZ, but retention times were shorter than, and resolutions were comparable to, those in NPLC, indicating a potential for improving chromatographic performance of the CDMPCCZ column in RPLC with optimized column preparation and separation conditions.     

A humic acid stationary phase for the high performance liquid chromatography separation of buckminsterfullerenes: theoretical and practical aspects

The influence of the mobile phase composition and column temperature on the chromatographic separation of five buckminsterfullerenes (C60, C70, C76, C78, C84) on a stationary phase based on silica gel with chemically bonded humic acid (Bonded humic acid column (BHAC)) was studied. The retention behavior of the fullerenes was measured under isocratic conditions with different mobile phase compositions, ranging from 0.05-0.70 (v/v) of toluene in cyclohexane. The column temperature was analysed in the range 35-75 °C. The retention factors of the five fullerenes do not depend linearly on the toluene fraction but follow a quadratic relationship. The best chromatographic conditions for baseline separation of the five fullerenes were selected. The retention of the fullerenes on the HA stationary phase was strongly affected by temperature. Positive values of thermodynamic parameters (changes of enthalpy and entropy) were due to the abnormal solubility behaviour of fullerenes in toluene in the temperature range 35-75 °C. The information obtained in this work makes this BHAC very simple to prepare and low cost, useful for fullerene research applications.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia-zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia-zirconia reversed stationary phases (C₁₈PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia-zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9-12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C₁₈PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the pK_a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.     

Application of silica-based hyper-crosslinked sulfonate-modified reversed stationary phases for separating highly hydrophilic basic compounds

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, low selectivity and irreproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as (-)SO(3)-HC-C(8)-L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the (-)SO(3)-HC-C(8)-L phase showed significantly improved retention and separation selectivity for hydrophilic amines. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hyper-crosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparing the (-)SO(3)-HC-C(8)-L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.     

Separation of 9,10-anthraquinone derivatives: evaluation of C18 stationary phases

Chromatographic study of natural products helps to determine their molecular composition and to identify their sources (biological, geographical, etc.). However, identifying anthraquinoids is still a challenge because this chemical family is composed of more than half a thousand molecules. In the present work, a series of C18 stationary phases were systematically studied to evaluate their separation potential for these compounds in high performance reversed phase liquid chromatography (RPLC). The stationary phases with various physicochemical properties were evaluated with respect to the separation of 30 standards. Selectivity of the studied stationary phases and symmetry of the obtained peaks were chosen as the evaluation criteria. Calculations of symmetry and separation capacity parameters have been proposed. These parameters are not dependent on the dimensions of the columns and were used for the classifications of phases. In conclusion, this study is intended to be a guide as to which stationary phases analysts can use for resolving separation of a complex mixture of anthraquinoids.     

Polybutadiene-coated zirconia packing materials in solvating gas chromatography using carbon dioxide as mobile phase

Summary In this paper, practical considerations of column efficiency, separation speed, thermal stability, and column polarity of capillary columns packed with polybutadiene-coated zirconia were investigated under solvating gas chromatography (SGC) conditions using carbon dioxide as mobile phase. When compared with results obtained from conventional porous octadecyl obtained from conventional porous octadecyl bonded silica (ODS) particles, PBD-zirconia particles produced greater change in mobile phase linear velocity with pressure than conventional ODS particles under the same conditions. The maximum plate number per second (N_t) obtained with a 30 cm PBD-zirconia column was approximately 1.5 times higher than that obtained with an ODS column at 100 °C. Therefore, the PBD-zirconia phase is more suitable for fast separations than conventional ODS particles in SGC. Maximum plate numbers per meter of 76,900 and 63,300 were obtained using a 57 cm×250 μm i.d. fused silica capillary column packed with 3 μm PBD-zirconia at 50 °C and 100 °C, respectively. The PBD-zirconia phase was stable at temperatures up to 320 °C under SGC conditions using carbon dioxide as mobile phase. Polarizable aromatic compounds and low molecular weight ketones and aldehydes were eluted with symmetrical peaks from a 10 cm column packed with 3 μm PBD-zirconia. Zirconia phases with greater inertness are required for the analysis of more polar compounds by SGC.     

Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.     

正反相模式下键合纤维素手性固定相对手性化合物的拆分

在自行合成的键合型纤维素-(3,5-二甲基苯基氨基甲酸酯)(CDMPC-CSP)手性固定相上,在正相模式下对苯偶姻和甲霜灵外消旋体进行了拆分,考察了流动相中醇以及温度在手性识别中对手性分离的影响。同时考察了反相模式下外消旋体的拆分。     

Polyethyleneimine Immobilized on Silica Endcapped with Octadecyl Groups as a Stationary Phase for RP-LC

A new reversed stationary phase was prepared, based on thermal immobilization of trimethoxysilylpropyl modified polyethyleneimine onto silica particles endcapped with octadecyl molecules. The physicochemical and morphological properties of the stationary phase were characterized by solid state cross-polarization and magic angle spinning ²⁹Si nuclear magnetic resonance, infrared spectroscopy, porosimetry, and elemental analysis. For the studies on reversed phase high-performance liquid chromatography (HPLC) retention, separation of the established Tanaka and Engelhardt test mixtures was performed. The stationary phase showed a typical partition mechanism for the reversed phase; however, the low hydrophobicity required a low organic content solvent in the mobile phase for chromatographic separation of more hydrophobic compounds. The stationary phase also showed low residual silanol activity for the elution of basic compounds due to the protection offered by octadecyl endcapped molecules and the competition provided by the imine groups of the polymeric layer. The proposed stationary phase possesses interesting selectivity and is convenient for applications requiring the separation of more retentive compounds in conventional HPLC columns using more aqueous mobile phases.     

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.