Retention behavior of basic compounds on alkylphosphonate-modified magnesia-zirconia composite stationary phase in RPHPLC

Summary The chromatographic properties of an alkylphosphonate-modified magnesia-zirconia composite stationary phase have been investigated by reversed-phase high-performance liquid chromatography with basic compounds as probes. The influence of organic modifier composition and mobile phase pH was studied. The new stationary phase, similar to a silica-based reversed-phase stationary phase, has hydrophobic properties, but greater pH stability. Use of the phase results in more symmetric peaks for basic compounds. A possible mechanism of retention of basic solutes on the new stationary phase is discussed. The chromatographic behavior of the basic solutes depends mainly on hydrophobic interactions between the solutes and the hydrophobic moiety of the stationary phase. Br?nsted acidic and basic sites on the surface of the new stationary phase play an important role in the retention of ionized solutes by ion-exchange interaction. Promising separations of some basic compounds have been achieved by use of methanolic TRIS buffer, pH 10.0, as the mobile phase.     

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

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

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

以稠环芳烃为探针 ,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系 ,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶 Zorbax ODS对稠环芳烃异构体的选择性 ,并对可能的保留机理进行了讨论。以甲醇 -水 (体积比为 75∶ 2 5)为流动相 ,在烷基膦酸改性锆镁复合氧化物固定相上分离了 8种稠环芳烃类化合物     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

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.     

混合固定相电中溶质的输运特征

 以离子交换和反相固定相构成的混合固定相电中 ,溶质迁移在受到疏水、离子交换作用的同时 ,对于带电溶质而言 ,还受到电泳迁移的影响。根据离子独立迁移原理 ,结合过程中的多种相互作用 ,得到了描述溶质表观迁移速率与其各形态迁移速率、各种相互作用之间相互关系的理论表达式 ;讨论了混合模式电中流动相的 pH及其中的有机调节剂浓度、混合固定相配比等对电渗流的影响及不同形态溶质在柱内的输运特征。结果表明 ,在电中采用混合固定相可以在较大的 pH和有机调节剂浓度范围内得到较强且稳定的电渗流。     

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。     

Counter-ionic effect on the separation of water-soluble compounds applying a hydrophilic stationary phase bonded with a zwitter-ionic polymer

A novel hydrophilic stationary phase bonded with a zwitter-ionic polymer for HPLC was synthesized. The stationary phase, in combination with a mobile phase containing various salts, was evaluated for its ability to separate water-soluble compounds, such as nucleobases, nucleosides and glycosides. The retention of a large majority of the solutes, except for cytosine, was increased by adding anti-chaotropic ions to the mobile phase. These results suggested that the retention of solutes depended on the thickness of the hydration layer on the stationary phase. In the zwitter-ionic polymer adsorbent, the formation of the hydration layer and the ionicity of the zwitter-ionic group on the stationary phase will be controlled by the properties of the ions added to the mobile phase.     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

Using the liquid nature of the stationary phase in counter-current chromatography V. The back-extrusion method

The main feature of counter-current chromatography (CCC) is that the stationary phase is a liquid as well as the mobile phase. The retention volumes of solutes are directly proportional to their distribution coefficients K(D) in the biphasic liquid system used in the CCC column. Solutes with high K(D) coefficients are highly retained in the column. The back-extrusion method (BECCC) uses the fact that the liquid stationary phase, that contains the retained solutes, can be easily moved. Switching the column inlet and outlet ports without changing the liquid phase used as the mobile phase causes the rapid collapse of the two immiscible liquid phases inside the column, the previously stationary phase being gathered at the new column outlet. Then this previously stationary liquid phase is extruded outside the CCC column carrying the retained solutes. The back-extrusion method is tested with a standard mixture of five compounds and compared with the recently described elution-extrusion method. It is shown that the chromatographic resolution obtained during the back-extrusion step is good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. However, a major drawback of the BECCC method is that all solutes are split between the liquid phases according to their distribution ratios when the CCC column equilibrium is broken. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode, eluting solutes with small and medium distribution ratios. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column and produce a broad peak showing after the stationary phase extrusion.     

Modelling of elution-extrusion counter-current chromatography using perfect replacement approach

Basing on the perfect replacement approach the equilibrium cell model is developed to describe the separation process in elution-extrusion counter-current chromatography (EECCC). As is known, EECCC consists of three steps: classical elution, sweeping elution, and extrusion. The perfect replacement approach means that during sweeping elution step, the mobile phase contained in the column moves and interacts with the "old" stationary phase in the same mode as during the classical elution step; the "new" and "old" stationary phases do not mix; and after the contacting with the mobile phase the concentration of solutes in the "old" stationary phase remains constant and this stationary phase volume is pushed ahead to the exit of the column. Equations are presented allowing the simulation of the chromatogram of solutes eluted from the column with the mobile phase during the elution period and the chromatogram of solutes pushed out of the column with the stationary phase during the extrusion period of EECCC. These equations can help to choose the optimal conditions for conducting elution-extrusion counter-current chromatography.     

Retention characteristics of a new butylimidazolium-based stationary phase

A new HPLC stationary phase has been synthesized based on the ionic liquid n-butylimidazolium bromide. Imidazolium was covalently immobilized on a silica substrate through an n-alkyl tether and the retention characteristics of the resulting stationary phase were evaluated systematically. Using 28 small aromatic test solutes and reversed phase conditions, the linear solvation energy relationship approach was successfully used to characterize this new phase. The retention characteristics of the test solutes show remarkable similarity with phenyl stationary phases, despite the presence of a positive charge on the new imidazolium phase. Operated in the reversed phase mode, this new stationary phase shows considerable promise for the separation of neutral solutes and points to the potential for a truly multi-modal stationary phase.     

Preparation and characterization of phosphatidylcholine‐coated zirconia–magnesia stationary phase for artificial membrane chromatography

Immobilized artificial membrane chromatography stationary phase was prepared by coating soybean phosphatidylcholine (PC) on zirconia–magnesia micro‐particles. The stability and chromatographic properties were investigated and compared with the PC‐coated silica chromatography stationary phase prepared by the same method. PC‐coated zirconia–magnesia chromatography stationary phase was more stable than the silica especially on resisting organic solvents. Hydrophobic action was the main factor for the retention of analyte on the new artificial membrane chromatography stationary phase, and electrostatic interaction had some contribution to retention. In addition, the special interaction between analyte and matrix affected retention greatly. Basic solutes were appropriate to be analyzed on PC‐coated zirconia–magnesia stationary phase and acidic solutes were appropriate to be done on the silica one. Hence, the two different matrices artificial membrane stationary phases were perfectly complementary.     

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.     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

Pressurized CEC of neutral and charged solutes using silica monolithic stationary phases functionalized with 3-(2-aminoethylamino)propyl ligands

A novel silica monolithic stationary phase functionalized with 3-(2-aminoethylamino)propyl ligands for pressurized CEC has been presented. The monolithic capillary columns were prepared by a sol-gel process in 75 microm id fused-silica capillaries and followed by a chemical modification. The diamino groups on the surface of the stationary phase are meant to generate the chromatographic surface and a substantial anodic EOF as well as to provide electrostatic interaction sites for charged solutes. The electrochromatographic characterization and column performance were evaluated by a variety of neutral and charged solutes. It was observed that the anodic EOF for the diamine-bonded monolith was greatly affected by the reaction time with 3-(2-aminoethylamino)propyltrimethoxysilane and the PEG amount in the sol-gel reaction mixture in addition to the mobile phase conditions. The monolithic stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. Good separations of various solutes including phenols, nucleic acid bases, nucleosides and nucleotides were achieved under different experimental conditions. Fast and efficient separations were obtained with high plate counts reaching more than 130,000 plates/m.     

Capillary electrochromatography with physically and dynamically absorbed stationary phases

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