Retention characteristics in gas chromatography as function of molecular mass of the stationary phase

The thermodynamic characteristics of the selectivity of stationary phases in gas-liquid chromatography have been used to study the change in selectivity of a stationary phase with its increasing molecular mass within the limits of the same class of compounds. It is shown that the entropy of disssolution is the dominant thermodynamic factor of selectivity in the case of paraffinic stationary phases; a linear relatinship exists between the logarithm of relative retention of different sorbates and the reciprocal molecular mass of the stationary phase. In the case of solutions where hydrogen bonds can be formed, the enthalpy of dissolution serves as the dominant factor; in this case a linear relationship exists between the relative retention of alcohols and the reciprocal molecular mass of the stationary phase (polyethylene glycol). For the other classes of sorbates and stationary phases, the entropy as well as the enthalpy of dissolution contribute to the relative retention when the molecular phase varies within a single class of substances. The theoretical postulations have been confirmed by experimental data for paraffinic stationary phases, polyethylene glycols, and phthalic acid esters.Translated from Teoreticheskaya i éksperimental'naya Khimiya, Vol. 24, No. 1, pp. 81–90, January–February, 1988.     

Efficient preparative separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with a two‐step combined strategy

An efficient two‐step method has been developed for the separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with polysaccharide chiral stationary phases. With respect to retention, selectivity, and resolution of β‐cypermethrin, the effects of chiral stationary phases, cosolvents, mobile phases, and column temperature have been studied in detail. Through a two‐step separation, β‐cypermethrin was firstly separated by using a cellulose‐derived chiral stationary phase to obtain two stereoisomeric pairs, and further resolved on an amylose‐based chiral stationary phase to produce four enantiopure stereoisomers. The electronic circular dichroism patterns of the first‐ and the third‐eluted isomers in methanol solution showed the mirror image of each other in the wavelength range 200∼300 nm, indicating that they were a pair of enantiomers. Moreover, the second‐ and the fourth‐eluted isomers were also enantiomers. This proposed two‐step strategy showed low solvent consumption, fast separation speed, and high‐purity, which may provide an effective approach for preparative separation of compounds with multiple chiral centers and difficult‐to‐separate multicomponent samples.     

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.     

Effect of temperature on the mechanism of retention of fullerenes in liquid chromatography using various alkyl bonded stationary phases

Summary The temperature-dependency of the separation of fullerenes in liquid chromatography (LC) has been examined using various alkyl bonded stationary phases. It has been found that a maximum retention temperature exists with long alkyl bonded stationary phases, whereas there is no similar effect with the newly synthesized alkyl bonded phases which have two phenyl groups at the base of the bonded phase. The interpretation of the retention behavior of fullerenes in the low temperature region on alkyl bonded stationary phases is discussed using information obtained by CP-MAS solid-state NMR spectroscopy and LC.     

Comparison of retention of aromatic hydrocarbons with polar groups in binary reversed-phase high-performance liquid chromatography systems

The retention of aromatic hydrocarbons with polar groups has been correlated as log k1 versus log k2 for reversed-phase high-performance liquid chromatography systems with different binary aqueous mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers. Distinct changes in separation selectivity have been observed between tetrahydrofuran and acetonitrile or methanol systems. Methanol and acetonitrile systems show lower diversity of separation selectivity. The changes in retention and selectivity of aromatic hydrocarbons with various polar groups between any two chromatographic systems with binary aqueous eluents (tetrahydrofuran vs. acetonitrile, tetrahydrofuran vs. methanol and methanol vs. acetonitrile) have been interpreted in terms of molecular interactions of the solute with especially one component of the stationary phase region, i.e. extracted modifier, and stationary phase ordering. The ordering of the stationary phase region caused by modifier type influences the chromatographic selectivity of solutes with different molecular shape.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography part I: Evaluation of silanol terminated dimethyl substituted polymers

The thermal stability of silicones can be improved on replacement of certain of the oxygen atoms in the polymer backbone by phenyl groups. Such a polymer has been synthesized and evaluated for use as stationary phase in fused silica capillary gas chromatography; the polymer was dimethyl substituted and silanol terminated. A selectivity was provided by the phenyl groups in the backbone. For comparative purposes, a silanol-terminated dimethylpolysiloxane has also been evaluated. Both stationary phases gave columns of highest separation efficiency and the supporting fused silica surface was deactivated by the stationary phases on thermal treatment. Further, low column bleeding was observed at the maximum temperature tested, 370°C. The phenyl-containing phase could be immobilized to 60% by heat treatment, but the pure dimethylpolysiloxane was 10% immobilized. The influence on immobilization of factors such as nature of the supporting surface, stationary phase silanol content, reaction temperature and atmosphere in the column during reaction has been studied.     

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.     

Separation of Isomers on Nematic Liquid Crystal Stationary Phases in Gas Chromatography: A Review

Understanding the composition of complex hydrocarbon mixtures is important for environmental studies in diverse fields, but many prevalent compounds cannot be confidently identified using traditional gas chromatography (GC) techniques. Increasing requirements on analyses of isomeric compounds and the problems encountered in their separation demand a study of more efficient systems which exhibit a high selectivity. Kelker and Fresenius first used nematic liquid crystals as stereospecific stationary phases in GC. Nematic liquid crystal has shown this particular selectivity and sensitivity as stationary phases for the separation of isomers having similar volatilities. Because of their unique selectivity towards rigid solute isomers, liquid crystal stationary phases were considered at one time to be a very promising class of materials that give gas chromatographic separations very different from those that can be obtained with any other stationary phase. Since then, a great deal of attention has been paid to the separation properties of this relatively wide group of substances. Liquid crystal can be used to separate a variety of compounds including isomer mixtures which cannot be separated on conventional stationary phases. This paper aims to review all specific experimental results and presents a comparative analytical study of monomeric nematic liquid crystal stationary phases used in GC. A further contribution of this review is in the field of isomeric compounds separation.     

Impact of column temperature and mobile phase components on selectivity of hydrophilic interaction chromatography (HILIC)

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.     

Titania-based stationary phase in separation of ondansetron and its related compounds

Improvements in stationary phase stability have been and remain a great task for research of new stationary phases. Metal oxide-based stationary phases appear as one of perspective alternatives to classical silica based stationary phases regarding to their similar effectiveness, different selectivity, different retention mechanism and mainly better chemical and thermal stability. In this study, the retention behaviour of ondansetron and its five pharmacopoeial impurities on TiO(2)-based reversed phase was investigated. The influence of buffer type, pH and concentration on retention was studied. Different types and amount of organic solvent in mobile phase were tested. The effect of temperature and flow rate on separation was investigated. The separation conditions were optimized and developed method validated. The retention parameters - retention time (t(R)), retention factor (k'), theoretical plate number (N), resolution between peaks due to nearby peaks (R(s)) and symmetry factor (A(s)) have been compared to parameters achieved on polybutadiene-coated zirconia column. The thermodynamic parameters of retention of analysed compounds - enthalpy, entropy and Gibbs free energy - were calculated and compared to those achieved on polybutadiene-coated zirconia column. This work proves similarity of retention behaviour of ondansetron and its five related compounds on zirconia-based and titania-based stationary phases and potential utilisation of polyethylene covered TiO(2)-based reversed stationary phase as an alternative to polybutadiene-coated ZrO(2) stationary phase in pharmaceutical analysis of ondansetron.     

全丁基-β-环糊精与硝酸银的混合液作为毛细管气相色谱固定相的研究

 将合成的全丁基-β-环糊精+硝酸银(聚乙二醇400作溶剂)混合固定液静态涂渍在弹性石英毛细管柱内壁,7组对映异构体、4组芳香族位置异构体化合物等在该柱上得到满意分离。通过测定以上化合物在该柱及全丁基-β-环糊精柱上的热力学参数(焓、熵、自由能及焓变差、熵变差),探讨了固定液对所测化合物的色谱分离机理。结果表明,混合固定液对所测化合物具有协同效应     

Evaluation of different column types for the hydrophilic interaction chromatographic separation of iron-citrate and copper-histidine species from plants

Hydrophilic interaction chromatography (HILIC) has emerged as a very useful separation method for polar analytes, including non-covalent metal species. Several types of stationary phases are available for HILIC applications, differing mainly in their chemical functionalities that supply additional interaction modes and alternative selectivities for the separation of special analytes. With regard to the separation of metal species only few of these stationary phases have been applied to date, and it is not completely clear what are their differences with respect to the chromatographic separation of metal species, but also with respect to species stability during chromatography. Here, a comparison of different column types for the HILIC separation of iron citrate and copper histidine species is presented and the results are discussed with respect to retention mechanisms and chromatographic stability of these metal species. It is shown that different stationary phases display very different separation patterns. In particular, three types of HILIC columns enable successful separation of iron citrates and copper histidine at pH 5.5, namely a crosslinked diol phase, a zwitterionic phase, and an amide phase. Two groups of iron-citrates are separated on all three columns, consisting of a species of 3:3 stoichiometry and another one of mainly 3:4 stoichiometry (plus 1:2 and 2:2 species). For copper-histidine only one stable species is found based on the 1:2 stoichiometry. Detection and unambiguous identification of the different species is possible by employing electrospray mass spectrometry in the negative ionization mode. Species found in standard solutions are consistent with species found in spiked plant samples. Also in unspiked solutions iron citrate of 3:4 stoichiometry (plus 1:2 and 2:2) is detectable, but no species of 3:3 stoichiometry. Significant differences of related species patterns are found in real plant samples.     

High-performance liquid chromatographic separation of rolipram, bupivacaine and omeprazole using a tartardiamide-based stationary phase influence of flow rate and temperature on the enantioseparation

Chromatographic separation of the chiral drugs rolipram, bupivacaine and omeprazole on a tartardiamide-based stationary phase commercially named Kromasil CHI-TBB is shown in this work. The effect of temperature on the chromatographic separation of the chiral drugs using the Kromasil CHI-TBB stationary phase was determined quantitatively so as to contribute toward the design for the racemic mixtures of the named compound by using chiral columns. A decrease in the retention and selectivity factors was observed, when the column temperature increased. Van't Hoff plots provided the thermodynamic data. The variation of the thermodynamic parameters enthalpy and entropy are clearly negative meaning that the separation is enthalpy controlled.     

LC and LC-MS Separation of Peptides on Macrocyclic Glycopeptide Stationary Phases: Diastereomeric Series and Large Peptides

Previous work on the LC separation of peptides had shown that macrocyclic glycopeptide stationary phases to be selective for peptides of five to thirteen amino acids in length. In this work, the selectivity of the teicoplanin stationary phase is compared to that of a C18 stationary phase for seven diastereomeric enkephalin peptides. The teicoplanin stationary phase separated all seven diastereomeric enkephalin peptides in a single chromatographic run. The insertion of d-amino acids into the primary enkephalin sequence produced areas of hydrophobicity that influenced retention order on the C18 stationary phase. However, analogous trends are not observed on the teicoplanin stationary phase, which is more polar and structurally diverse. Optimization of the mobile phase and the use of a step-gradient for the enkephalin separation on the teicoplanin stationary phase is discussed. Also, the selectivity of macrocyclic glycopeptide stationary phases for peptides of 14, 28, 30, and 36 amino acids also is investigated and compared to separation on a C18 stationary phase. A method for eluting peptides with multiple basic amino acids, which tend to be strongly retained on the macrocyclic glycopeptide stationary phases, is presented.     

Determination of water-soluble vitamins in vitamin premixes,bioacitve dietary supplements,and pharmaceutical preparations using high-efficiency liquid chromatography with gradient elution

The influence of the pH and temperature of the mobile phase on the retention and separation of 14 vitamins and vitameric forms has been studied for four different stationary phases. The chromatographic conditions allowing the separation of 12 water-soluble vitamins and vitameric forms have been proposed. It has been established that the best separation of water-soluble vitamins can be achieved by employing gradient elution. The mobile phase A, (0.6% phosphoric acid) pH 1.5–1.8; mobile phase B, acetonitrile. For the separation of nicotinic and ascorbic acid it is preferable to use stationary phases Luna C18(2) and Gemini C18, while for the separation of riboflavin and riboflavin-5-phosphate the Synergy Hydro RP and Zorbaz SB-C18 stationary phases should be used. The selected conditions have been used for the determination of vitamins in commercial samples of vitamin preparations. The results are in good accordance with the producer data.     

Use of a polar-embedded stationary phase for the separation of tocopherols by CEC

A polar-embedded stationary phase (ULTIMA C18) has been investigated for the separation of alpha-, beta-, gamma- and delta-tocopherols by CEC in comparison with commercially available C(18) and C(30) n-alkyl RPs. The behavior of this stationary phase was tested for different mobile phases based on methanol, ACN, or mixtures thereof and different separation parameters such as retention factors and resolution were evaluated. The main feature of this stationary phase is the improved selectivity for the separation of beta- and gamma-tocopherols (positional isomers) when compared with the pure n-alkyl C(18) material, which was unable to resolve these compounds. Additionally, it is possible to observe a reversal in the elution order of the beta- and gamma-tocopherol isomers with respect to that obtained on the C(30) column. The resulting data indicate that the enhanced selectivity obtained with the polar-embedded stationary phase, with respect to the conventional C(18) material, is due to the participation of both hydrophobic and polar interactions: these latter are of the hydrogen bridge type with the amide group of the polar-embedded stationary phase, which increases the retention of the tocopherols and facilitates the discrimination between the beta- and gamma-isomers. Adequate separation of the four tocopherols was obtained by CEC using the polar-embedded stationary phase and 95:5 v/v methanol/water (5 mM Tris, final concentration) as the mobile phase.     

温度对蛋白和β-环糊精手性固定相拆分对映体的影响

 采用三聚氯氰为活化剂分别合成了牛血清白蛋白 (BSA)、人血清白蛋白 (HSA)和 β 环糊精手性固定相 ,研究了温度在色氨酸 ,华法令 ,酮基布洛芬和丹酰化苏氨酸手性拆分中的影响。结果表明 ,在蛋白手性固定相上对映体间的熵变对色氨酸 ,华法令和酮基布洛芬的拆分有很大的影响 ,而丹酰化苏氨酸对映体在 β 环糊精手性固定相上的拆分为典型的焓控过程 ,与蛋白柱有着不同的热力学特性。由于键合方式不同 ,色氨酸在我们合成的BSA手性固定相上的最佳分离温度为 35℃左右 ,而不是文献报道的以戊二醛为活化剂的 2 4℃。     

Joint use of cyclodextrin additives in chiral discrimination by reversed-phase high-performance liquid chromatography: temperature effects

The temperature dependence of chiral separations was investigated in combined system of reversed-phase (RP) liquid chromatography using two chiral additives: single or β native cyclodextrins and their permethylated derivatives. The model tested compounds of pharmaceutical interest were: methylphenobarbital, mephenytoin, morsuximide and camphor. Taking the localization of a complexation process as a criterion – the combined system with two selectors has been rationalized as occurring in three stages. The influence of temperature (in narrow range of 20°C) on retention and enantioselectivity was studied in; System I (complexation occurs in the mobile phase), in System II (complexation on the stationary phase) and in System III (complexation in both phases together). In System III (as for System I) it has been found that the model compounds could be classified into three groups based on their retention dependence on temperature: retention decrease with temperature decrease, retention increase with temperature decrease or no influence of temperature on retention. For all the compounds investigated, decrease in temperature increases the selectivity. Standard enthalpy (ΔH⁰) and entropy (ΔS⁰) changes of solute transfer between the mobile and the stationary phase and standard enthalpy (ΔH⁰_CD) and entropy (ΔS⁰_CD) changes of complex formation were also calculated. In Systems I and III, if the complexation in the mobile phase is favored process compared with interaction with the stationary phases (RP or covered by permethylated cyclodextrin), the shortest retention time and the best selectivity is observed at low temperature.