Capillary electrochromatography with novel stationary phases: II. Studies of the retention behavior of nucleosides and bases on capillaries packed with octadecyl-sulfonated-silica microparticles

An octadecyl-sulfonated silica (ODSS) stationary phase specially designed for performing capillary electrochromatography (CEC) at relatively strong electroosmotic flow (EOF) proved useful for the separations of some nucleosides and bases. The ODSS stationary phase is composed of a hydrophilic, negatively charged sublayer to which a nonpolar top layer containing octadecyl ligands is covalently attached. The charged sublayer contains sulfonic acid groups which ensure a relatively strong EOF. Due to the presence of permanently charged sulfonic acid groups in the sublayer, the hydrophilic nature of the sublayer and the hydrophobic character of the top octadecyl layer, retention and selectivity of charged and relatively polar nucleosides and bases on the ODSS stationary phase are based on electrostatic interaction, hydrophilic interaction, and reversed-phase mechanisms. This yielded for the ODSS stationary phase a unique selectivity towards the nucleosides and bases, thus allowing their rapid separation. To gain insight into the chromatographic behavior of nucleosides and bases on the ODSS stationary phase, the results were compared to those obtained on an octadecyl-silica (ODS) capillary under otherwise the same elution conditions. Due to the difference in the nature of the organic layers on the surface of the ODSS and ODS stationary phases, the elution order on both stationary phases differed significantly, and the ODSS capillary proved more suitable for the separation of the nucleosides and bases than the ODS capillary.     

Dual Retention Mechanism in Two-Dimensional LC Separations of Barbiturates,Sulfonamides, Nucleic Bases and Nucleosides on Polymethacrylate Zwitterionic Monolithic Micro-Columns

In-house prepared zwitterionic polymethacrylate micro-columns using in situ polymerization of N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MEDSA) functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) cross-linker provided excellent stability and reproducibility of preparation and separation efficiency of 60,000–70,000 theoretical plates m⁻¹ for small molecules under isocratic conditions. The column showed a dual retention mechanism, reversed-phase (RP) in highly aqueous mobile phases and aqueous normal-phase (HILIC) in acetonitrile-rich mobile phases. This property can be used to obtain complementary separation and combined information on the sample from repeated injections of a sample on a single column, in different mobile phases characteristic for the HILIC and for the RP modes, which is in fact a form of offline two-dimensional chromatography on a single column. The dual retention mechanism has been observed with a variety of columns, however, often with impractically narrow retention range in one of the two modes. To take full advantage from the combined single-column RP–HILIC experiments, the column should provide a sufficiently broad mobile phase interval both in the RP and in the HILIC mode. The BIGDMA-MEDSA micro-columns proved suitable earlier for the combined RP–HILIC separations of some phenolic compounds and flavonoids. In the present work, we investigated the effects of the mobile phase composition on the retention of a variety of polar compounds over full retention range of buffered aqueous acetonitrile mobile phases, to find potentially useful HILIC and RP retention ranges for barbiturates, sulfonamides, nucleosides and nucleic bases. In the HILIC mode, proton donor–acceptor interactions show a major effect on retention and selectivity of separation, whereas the size of the non-polar hydrocarbon part of the sample molecule is the most important factor in the water-rich mobile phases. The sample structure strongly affects the composition of aqueous–organic mobile phases at which the transition between the two retention modes occurs. Of the investigated sample types, barbiturates show better separation under reversed-phase conditions, whereas nucleosides and nucleic bases in the HILIC mode. Aromatic carboxylic acids and sulfonamides can be separated either in the reversed phase or under HILIC conditions, the two separation modes showing complementary selectivity of separation.

     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. I. Preparation and characterization of sulfonated naphthalimido-modified silyl silica gel

A novel packing material, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), was prepared for the use as a stationary phase of capillary electrochromatography (CEC). The sulfonic acid groups on SNAIP stationary phase contributed to the generation of electroosmotic flow (EOF) at low pH and served as a strong cation-exchanger. In CEC with SNAIP, a mixed-mode separation was predicted, comprising hydrophobic and electrostatic interactions as well as electrophoretic migration process. In order to understand the retention mechanism on SNAIP, effects of buffer pH, concentration, and mobile phase composition on EOF mobility and the retention factors of barbiturates and benzodiazepines were systematically investigated. Moreover, the retention behavior of barbiturates on SNAIP was investigated and compared with those on octadecyl silica (ODS), phenyl-bonded silica, and 3-(1,8-naphthalimido)propyl-modified silyl silica gel to confirm the presence of pi-pi interaction on its retention mechanism. It was observed that a column efficiency was more than 85,000 N/m for retained compounds and the relative standard deviations for the retention times of EOF marker, thiourea, and five barbiturates were below 2.5% (n = 4). Under an applied voltage of 20 kV and a mobile phase consisted of 5 mM phosphate (pH 3.8) and 40% methanol, the baseline separation of five barbiturates was achieved within 3 min.     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Capillary electrochromatography with monolithic silica columns III. Preparation of hydrophilic silica monoliths having surface-bound cyano groups: chromatographic characterization and application to the separation of carbohydrates, nucleosides, nucleic acid bases and other neutral polar species

Two synthetic routes have been introduced and evaluated for the preparation of hydrophilic silica-based monoliths possessing surface-bound cyano functions. In one synthetic scheme, the silica monolith was reacted in a single step with 3-cyanopropyldimethylchlorosilane to yield a cyano phase referred to as CN-monolith. In a second synthetic route, the silica monolith was first reacted with gamma-glycidoxypropyltrimethoxysilane (gamma-GPTS), followed by a reaction with 3-hydroxypropionitrile (3-HPN) to give a stationary phase denoted CN-OH-monolith. Although the gamma-GPTS was intended to play the role of a spacer arm to link the 3-HPN to the silica surface, this spacer arm became an integral part of the hydrophilic stationary phase. Thus, the CN-OH-monolith can be viewed as a double-layered stationary phase (i.e., stratified phase) with a hydroxy sub-layer and a cyano top layer. Due to its stronger hydrophilic character, the CN-OH-monolith yielded higher retention and better selectivity than the CN-monolith. The CN-OH-monolith was demonstrated in the normal-phase capillary electrochromatography (CEC) of various polar compounds including phenols and chloro-substituted phenols, nucleic acid bases, nucleosides, and nitrophenyl derivatives of mono- and oligosaccharides. The CN-OH-monolith yielded a relatively strong electroosmotic flow over a wide range of mobile phase composition, thus allowing rapid separation of the polar compounds studied.     

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.     

Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography

A stationary phase composed of silica-bonded sulfonated cyclofructan 6 (SCF6) was synthesized and evaluated for hydrophilic interaction chromatography (HILIC). The separation of a large variety of polar compounds was evaluated on different versions of the stationary phase and compared with the same separations obtained with commercially available HILIC columns. The new columns successfully separate polar and hydrophilic compounds including β blockers, xanthines, salicylic acid related compounds, nucleic acid bases, nucleosides, maltooligosaccharides, water soluble vitamins and amino acids. The separation conditions were optimized by changing the composition and the pH of the mobile phase. The dependence of analyte retention on temperature was studied using van't Hoff plots. The newly synthesized stationary phase showed broad applicability for HILIC mode separations.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

High-Performance Liquid Chromatography of some Bases and Nucleosides on Alkylphosphonate-Modified Magnesia-Zirconia Column

ABSTRACT

The high-performance liquid chromatographic behavior of some bases and nucleosides was studied on a new reversed-phase stationary phase, alkylphosphonate-modified magnesia-zirconia. The effect of mobile phase variable such as methanol content, ionic strength and pH on their behavior was investigated. It was found that the retention behavior of the bases and nucleosides on the new stationary phase is similar to that on ODS stationary phase. The retention mechanism on the new stationary phase was also discussed. The separation of some bases and nucleosides was accomplished on the new stationary phase.     

Preparation of phosphorylcholine‐based hydrophilic monolithic column and application for analysis of drug‐related impurities with capillary electrochromatography

A hydrophilic monolithic CEC column was prepared by thermal copolymerization of zwitterionic monomer 2‐methacryloyloxyethyl phosphorylcholine (MPC), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2‐methylpropene‐1‐sulfonate (MPS) in a polar binary porogen consisting of methanol and THF. A typical hydrophilic interaction LC retention mechanism was observed for low‐molecular weight polar compounds including amides, nucleotides, and nucleosides in the separation mode of hydrophilic interaction CEC, when high content of ACN (>60%) was used as the mobile phase. The effect of the electrostatic interaction between the analytes and the stationary phase was found to be negligible. The poly(MPC‐co‐PETA‐co‐META or MPS) monolithic columns have an average column efficiency of 40 000 plates/m and displayed with a satisfactory repeatability in terms of migration time and peak areas. Finally, the column was successfully applied to determine the impurities of a positively charged drug pramipexole which are often separated by ion pair RP chromatography due to their high hydrophilicity. All four components can be baseline separated within 5 min with BGE consisting of ACN/20 mM ammonium formate buffer (pH 3.0; 80/20).     

The analysis of pharmaceutical bases on a silica stationary phase by capillary electrochromatography using aqueous mobile phases

Summary The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3. We have previously demonstrated that the addition of mobile phase additives such as TEA-phosphate at low pH values has resulted in excellent CEC analysis of bases on reversed-phase packing materials. The same approach was applied to the analysis of bases on the silica phase in order to improve peak shape. Excellent chromatography was obtained for the analysis of strong pharmaceutical bases such as benzylamine, nortriptyline and diphenhydramine. The experimental investigations have shown that the CEC separation of a range of pharmaceutical bases can routinely be achieved with excellent peak shapes and peak efficiencies as high as 320,000 plates m⁻¹.     

Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases

The development of new RP stationary phases containing polar groups has provided the chromatographer with a variety of stationary phase choices with differing selectivities. Polar endcapped and polar embedded group stationary phases have found use in solving a wide variety of separation problems, especially for the efficient separation of organic bases as well as separations necessitating the use of highly aqueous mobile phases. In this report, the retention thermodynamics of small, nonpolar solutes on traditional alkyl, polar endcapped, and polar embedded group stationary phases are compared. It is found that the nonpolar (methylene) transfer enthalpy is less favorable when polar embedded group phases are used, when compared to traditional or polar endcapped phases. In contrast, the transfer enthalpy of a phenyl group is found to be more favorable when a polar endcapped phase is used. In addition, the retention characteristics of these phases are compared using a set of solutes with differing solvatochromic parameters. Hydrogen-bond acids appear to have enhanced retention on polar embedded group phases, while hydrogen-bond bases have enhanced retention on polar endcapped phases.     

新型咪唑嵌合氨基亲水作用色谱固定相的制备及其色谱性能

发展新型高效的亲水作用色谱分离材料对于极性化合物的分离分析具有重要的意义。本文设计合成了一种新型咪唑嵌合的氨基亲水作用色谱固定相（Sil-IEASP）,并分别采用傅里叶变换红外光谱仪、热重分析仪和元素分析仪对该固定相进行了表征,结果表明该固定相制备成功。以核苷和核酸碱基为样品,分别考察了流动相中的水含量、盐浓度和pH对其保留的影响,结果表明所发展的固定相具有良好的亲水作用特性;此外,缓冲盐浓度和pH几乎不影响上述物质的保留。进一步将该固定相应用于分离尿嘧啶、腺嘌呤、胞嘧啶、尿苷和3种位置异构体（邻三联苯、间三联苯和苯并菲）,与常用的氨基固定相相比,本文所发展的固定相具有更好的分离效果,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

亚微米中孔SBA-15 棒状固定相在毛细管电色谱分离中的应用(英文)

采用中孔SBA-15棒状硅胶颗粒填充毛细管柱用于毛细管电色谱(CEC)分离。这一亚微米材料直径为400 nm并具有沿相同方向伸展的高度有序、均一的圆柱形中孔。棒状的特殊形态使得填充柱的通透性良好,简化了尺寸微小的CEC柱的填充过程。修饰后的棒状SBA-15填充毛细管柱成功应用于反相和离子交换电色谱分离非极性和极性样品,获得了较高柱效(140000理论塔板/m)。流速3.2cm/min时获得最低理论塔板高度为7.1 mm。范迪米特曲线说明了SBA-15孔结构的传质阻力特征。分别以芳香酸、人参、天麻提取物为样品,对亚微米固定相毛细管电色谱柱加以评价。该固定相显示出了较高的分离能力,为纳米材料在色谱固定相中的应用提供了一个新的思路。     

核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离

建立苯胺甲基键合硅胶固定相(PAMS)高效液相色谱分离核苷与碱基的方法；研究流动相有机溶剂浓度、磷酸缓冲液pH值、离子强度对核苷和碱基在该键合固定相上的色谱保留及分离选择性的影响，用磷酸缓冲液(pH＝4)为流动相快速分离了部分核苷与碱基。     

Preparation and evaluation of a lysine-bonded silica monolith as polar stationary phase for hydrophilic interaction pressurized capillary electrochromatography

A silica-based monolith as polar stationary phase was described for hydrophilic interaction pressurized capillary electrochromatography (HI-pCEC). The polar monolithic column was prepared by on-column reaction of lysine with epoxy groups on a gamma-glycidoxypropyltrimethosysilane-modified silica monolith. The stationary phase yielded strong hydrophilic interaction due to the slightly polar hydroxyl groups, and the strong polar lysine ligand with amino groups and carboxylic groups contained on the surface of the monolith. In order to evaluate the hydrophilic character of lysine ligand, the chromatographic behaviors of epoxy monolith (before lysine bonded) and diol monolith (hydroxyl groups contained) were also investigated. Two groups of comparative experiment were developed in terms of the separation of typical neutral non-polar and polar compounds performed in a mobile phase of aqueous-acetonitrile solution. Results showed that the lysine monolith was much more hydrophilic than the diol monolith, which presented less hydrophobic than the epoxy monolith. For further study on its hydrophilic character, the lysine monolith was demonstrated in the HI-pCEC mode for the separations of various polar compounds such as phenols, nucleic acid bases and nucleosides.     

The use of derivatized cyclodextrins as solubilizing agents in the preparation of macroporous polymers employed as amphiphilic continuous beds in capillary electrochromatography

Employing solubilization by complexation with CDs, new mixed-mode monolithic stationary phases for CEC and micro-LC were synthesized. Free radical copolymerization was performed in aqueous solution with a CD-solubilized hydrophobic monomer, a water-soluble crosslinker (piperazinediacrylamide), and a charged monomer (vinylsulfonic acid). Different hydrophobic methacrylate monomers (isobornyl, adamantyl, cyclohexyl, and phenyl methacrylate) were investigated. Chromatographic properties of the synthesized monoliths were studied with aqueous and nonaqueous mobile phases with hydrophobic and polar analytes. Due to the amphiphilic nature of the polymers synthesized, the elution orders obtained correspond to the RP mode and to the normal-phase mode dependent on the polarity of the mobile phase. However, observations made with polar solutes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties and on the specific permeability was elucidated. Run-to-run, day-to-day, and capillary-to-capillary reproducibility of electroosmotic mobility and retention factors were determined. Comparison of retention data with those of a commercial octadecyl silica gel HPLC column reveals that the methylene selectivity of the monolithic capillaries prepared in this study is very similar to that of routinely used octadecyl silica gels.     

大黄酸键合硅胶固定相的简便制备及色谱性能评价

以大黄酸为原料,γ-氨丙基三乙氧基硅烷(KH-550)为偶联剂,简便制备了一种新型羧基键合硅胶固定相(RBSP),并用红外光谱、热重分析及元素分析对其结构进行表征.考察了流动相中甲醇含量对键合固定相色谱性能的影响,并以含酸性、中性和碱性化合物的混合物为溶质,评价了RBSP的色谱性能.以甲醇-水为流动相,用C18柱作参比,研究了该键合硅胶作为HPLC固定相对两种大豆异黄酮化合物和几种生物碱基的分离,并对其色谱分离机理进行了初步探讨.实验结果表明,该固定相(RBSP)具有较好的反相色谱性能,同时由于键合相中含有酚羟基及酰胺基团,能为多种溶质提供作用位点,对极性化合物的分离具有明显优势,且分离速度快,可有效用于极性化合物的分离分析.     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.