Piperazine quaternary diammonium salts as additives to background electrolytes in capillary zone electrophoresis

The differential behavior of five different quaternary mono- and diammonium salts, among the 18 investigated, in modulating the electroendoosmotic flow (EOF) and analyte separations in capillary zone electrophoresis is evaluated. It is found that quaternary diammonium salts with positive charges separated by more than four carbon atoms, while exhibiting a very strong affinity for chromatographic silica beads, to the point of exhibiting Rf values close to zero, display, on the contrary, a very poor affinity for the silica wall of capillaries. Compounds separated only by a C2 unit (i.e., 1,4-dialkyl-1,4-diazoniabicyclo[2,2,2,]octane, salts 17 and 18) show high Rf values due to strong ion pair association. The unique behavior of quaternary monoammonium salts possessing an iodinated alkyl (butyl or octyl) tail (i.e., 1, 6, and 7) is attributed to their ability to be covalently affixed to the silica wall via alkylation of ionized silanols at alkaline pH values. They thus strongly modulate and typically invert the EOF, even when not present in the background electrolyte. On the contrary, all diammonium salts, devoid of such alkyl tails, are unable to modulate the EOF and to prevent analyte binding to the silica wall, since they are rapidly removed from the wall by the voltage gradient. However, if added in small amount to the background electrolyte, they offer excellent separations of mixtures of very similar organic acids and prevent any interaction with the capillary wall.     

Mechanism of action of quaternary diamino quenchers in capillary zone electrophoresis

The synthesis of two novel amino compounds, able to quench and reverse the electroosmotic low (EOF) in capillary electrophoresis is here reported. These chemicals are derivatives of two previously described quaternarized piperazines, 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4I) and 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2.2.2] octane iodide (M7C4I), believed to bind covalently to the silica surface via alkylation of ionized silanols through their terminal, reactive iodine. The novel compounds, although unable to form a covalent bond, due to lack of the o-butyl iodine, are found to be very efficient in suppressing protein interaction with the wall and reversing the EOF. On the basis of their behavior in solution, the minimal structural motifs for strong binding of amino compounds to the silica wall have been derived and are thought to be: (i) the presence of two quaternary nitrogens in the molecule; (ii) a correct distance between said charged nitrogens, represented by a butyl chain (C(4)); (iii) an hydrophobic "decoration" of the molecules, consisting on a high CH(2)/N ratio (in the present case 8:1).     

Omega-iodoalkylammonium salts as permanent capillary silica wall modifiers. Comparative analysis of their structural parameters and substituent effects

Following previous work on the modification and inversion of electroendoosmotic flow (EOF) of naked silica by a cyclic diamine [1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide] [J. Chromatogr. A 894 (2000) 53], the present report considerably expands previous data by describing additional compounds of the same series of omega-iodoalkylammonium salts. Four of them are able to instantaneously reverse the EOF, thus producing a cationic surface with a highly stable reverse EOF. All these compounds are believed to become covalently attached to the silica surface via alkylation occurring by nucleophilic substitution of ionized silanols on the silica wall by the omega-iodo functionality in the modifier. The unique advantage of such compounds, as compared to adsorbed polymers or oligoamine EOF quenchers, is that they are not needed any longer in the background electrolyte, after the initial conditioning step inducing the covalent bond. It is additionally demonstrated, by running a mixture of cinnamic acid compounds, that some of the omega-iodoalkylammonium salts can act as modulators of analyte migration, thus inducing separations of otherwise identical compounds, such as isomeric species. Such interactions can only occur when the analytes drift close to the silica wall, and must be rapidly reversible, since no peak tailing or broadening is experienced.     

Stationary phase structures enhancing electroosmotic flow in capillary electrochromatography

Several chemically bonded silicas with C18 groups were examined with respect to electroosmotic flow (EOF) velocities under CEC conditions. Stationary phases with low hydrophobic selectivity generally provided high EOFs. The stationary phases prepared by using octadecyltrichlorosilane showed greater EOF than those from octadecyldimethylchlorosilane. Restricted-access reversed-phase (RARP) packing materials having C18 groups inside the pores and silanols on the external surfaces showed higher EOF than monomeric C18 phases with similarly high hydrophobic selectivity. The RARP-type structure having silanols at the external surface seems to be effective for increasing EOF while maintaining the hydrophobic character of the solute binding sites.     

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.     

Novel, trifunctional diamine for silica coating in capillary zone electrophoresis

A novel compound ?quaternarized piperazine [(N-methyl,N-4-iodobutyl)-N'-methylpiperazine] (QPzI)? for the coating of a silica capillary able to reduce or invert the electroosmotic flow (EOF) in capillary zone electrophoresis is reported. Unlike standard oligoamines (like spermine and tetraethylene pentamine) which are very efficient in quenching macromolecule interaction with the silica wall, but only in acidic pH ranges, QPzI acts all along the pH scale, including alkaline pH ranges. It is believed that QPzI behaves like a trifunctional derivative: it forms ionic bonds with dissociated silanols via its quaternary nitrogen, hydrogen bonds via its tertiary nitrogen and, most importantly, a covalent bond via alkylation of ionized silanols through the terminal iodine atom in the butyl chain. Excellent separations are obtained with a variety of organic compounds, such as aromatic carboxylic acids, tryptophan metabolites and arylalkanoic acids. Such separations could not be obtained in naked capillaries in the presence of oligoamines and on some occasions not even with capillaries coated with a covalent layer of neutral polymers. In separations taking place in alkaline media, QPzI is not added to the background electrolyte, but is used simply in the capillary pre-conditioning step, a unique feature strongly supporting the hypothesis of its covalent binding to the silica surface. In difficult separations, such as in the case of o-/p-OMe-phenylacetic acids or nicotinic/picolinic acid, which would not normally occur under standard conditions, it is believed that QPzI acts as a discriminator, thus playing an active role in the separation process, rather than simply modulating the EOF.     

Tuning Cooperativity by Controlling the Linker Length of Silica-Supported Amines in Catalysis and CO(2) Capture

Cooperative interactions between aminoalkylsilanes and silanols on a silica surface can be controlled by varying the length of the alkyl linker attaching the amine to the silica surface from C1 (methyl) to C5 (pentyl). The linker length strongly affects the catalytic cooperativity of amines and silanols in aldol condensations as well as the adsorptive cooperativity for CO(2) capture. The catalytic cooperativity increases with the linker length up to propyl (C3), with longer, more flexible linkers (up to C5) providing no additional benefit or hindrance. Short linkers (C1 and C2) limit the beneficial amine-silanol cooperativity in aldol condensations, resulting in lower catalytic rates than with the C3+ linkers. For the same materials, the adsorptive cooperativity exhibits similar trends for CO(2) capture efficiency.     

Reversed Phase Chromatography – the Mystery of Surface Silanols

The nature of surface silanols is reviewed and their influence on retention of basic solutes in reversed phase chromatography is demonstrated and evaluated. The influence of the type of organic modifier and/or pH of buffer on retention of basic analytes with silica based RP columns is evaluated, and means for characterization of RP columns are discussed. Attempts are described to determine the surface silanol concentration of RP columns by chromatographic methods. Surprisingly the experimentally measured silanol concentrations with bare silica and with RP are by one order of magnitude lower than discussed in literature. At pH 7.6 values of 0.12 µmol m^?2 have been measured for benzylamine with RP columns, corresponding to about 43% of the measurable silanols of plain silica. The break-through curves of amines let suggest that even at low pH values unprotonated species are present within the pores of the RP stationary phases and their interaction with the bonded alkyl groups contribute to retention.     

Fluorescent probe as reporter on the local structure and dynamics in hydrolysis-condensation process of organotrialkoxysilanes

To get some information on the aggregation behaviors of the products derived from different organotrialkoxysilanes, the hydrolysis-condensation processes of some organotrialkoxysilanes have been examined by means of pyrene as fluorescent probe. The organotrialkoxysilanes used in the research were n-octadecyltri-methoxysilane (ODTMS), n-octyltrimethoxysilane (OTMS), 3-glycidoxypropyltrimethoxysilane (GTMS), 3-methacryloxypropyltrimethoxysilane (MAPTMS), and propyltrimethoxy-silane (PTMS). The results show that pyrene as fluorescence probe can respond sensitively not only to the organization state of the hydrolysates but also to the change in the organization state during the condensation process. The organization states during the hydrolysis and condensation can be explained in terms of structures of the products. In the initial stage, the silanols with long organic chains are amphiphilic molecules, and such nature of the silanols can be compared to that of a surfactant. Therefore, the excimer emission of pyrene is extremely obvious because of such silanols being prone to form aggregates. In the case of silanols having short alkyl groups or epoxy groups, these silanols homogenously disperse in solution, which results in the appearance of an only monomer emission of pyrene. In the late stage, the fluorescence behavior of pyrene is also sensitive to structural evolution of the silicates. The fluorescence spectra of pyrene during the condensation of the silanols with short alkyl groups or epoxy groups are almost in silence, indicating that the condensation products, with a low condensation degree, homogeneously disperse in solution. For the silanols with long hydrophobic substituents in different lengths, the changes in fluorescence spectra of pyrene during the condensation are varied. Commonly, the excimer emission is noticeable, implying that the condensation products with high condensation degree inhomogenously disperse in solution. However, the relative excimer/monomer fluorescence intensity is alkyl chain-length dependent. The longer alkyl chains in the condensation products result in the appearance of the obvious excimer emission. These phenomena imply that the condensation degree of the products increases with the length of the alkyl chains. Additionally, the distorted spectrum of pyrene appears in the case of the organotrialkoxysilanes with side chain substituent, illustrating that the steric hindrance between the substituents can be monitored by fluorescence of pyrene. All these results are verified by the fluorescence-quenching measurements. The approach in the present study gives new insights into the local structure and dynamics in hydrolysis-condensation process of organotrialkoxysilanes and emphasizes the influence of the self-assembling behavior.     

Preparation and Evaluation of an End-Capped p-tert-Butyl-Calix[4]arene-Bonded-Silica Stationary Phase for Reversed-Phase High-Performance Liquid Chromatography

The preparation, characterization, and potential liquid chromatographic applications of an end-capped p-tert-butyl-calix[4]arene-bonded-silica phase are presented. The phase was prepared by covalently bonding p-tert-butyl-calix[4]arene-derivatized aminosilane to silica gel then end-capping with trimethylchlorosilane. Use of the new phase enabled good separations not only of polar and non-polar aromatic compounds, basic aromatic compounds, and polycyclic aromatic hydrocarbons but also of some isomers. It is more selective toward alkylbenzenes than Kromasil-C₁₈-SiO₂. The retention mechanisms involve strong hydrophobic interaction, strong π–π interaction, and host–guest interaction. Elemental analysis calculations confirm that the large molecules bonded to the silica act as shield against residual surface silanols. Assay of simvastatin and lovastatin on this column is also reported.     

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

正A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.     

Study of cooperative effects of silanols on modified silica by dielectric relaxation method

Two types of chemically modified silica gels with covalently bonded hydrophobic (alkyl, SiO₂-C₈) and hydrophilic (aminoalkyl, SiO₂-NH₂) groups were studied by dielectric relaxation method. For SiO₂-C₈ two relaxation maxima were detected at 140 and 238K. From comparison with bulk silica the lower temperature maximum was assigned to cooperative re-orientation of residual silanols and higher one to melting of physically adsorbed water. Both maxima are much weaker for SiO₂-C₈ then for bulk silica. In Arrhenius coordinates relaxation at 238K exhibits similar behavior as OH groups in polyvinyl alcohol and so it was assigned to mobility of orientation defects in mono-dimensional chain of physically adsorbed water. For SiO₂-NH₂ no low temperature relaxation effect was observed. Intensive relaxation was measured at 248 K only. Strong interaction between supramolecules of silanol groups, bonded aminogroups and adsorbed water was assumed.     

Dynamic capillary coatings for electroosmotic flow control in capillary electrophoresis

Control of the electroosmotic flow (EOF) is critical for achieving optimal separations by capillary electrophoresis. For instance, manipulation of the EOF can yield either high resolution separations or rapid analyses. Dynamic capillary coatings are a simple and cost-effective approach to altering the EOF. The normal EOF can be slowed using buffer additives such as Mg²⁺ and hexamethonium which ion exchange onto the surface silanols to lower the effective wall charge. Alternatively, cationic polyelectrolytes or cationic surfactants can be used to establish a cationic coating on the capillary wall, which results in a reversed EOF. Practical considerations such as pH stability and reproducibility obtainable with an EOF modifier will be discussed.     

Residual silanols at reversed-phase silica in HPLC--a contribution for a better understanding

As porous silica gel is the most common adsorbent and support for bonded stationary phase synthesis, residual silanol groups are a recurring problem in the field of liquid chromatography and other separation techniques. Residual silanols most often have a negative effect on the separation process by causing peak tailing. Therefore, there was an attempt to remove residual silanols during stationary-bonded phase synthesis. The type and surface concentration of residual silanols were measured using different instrumental techniques such as NMR and infrared spectroscopy, calorimetry, and various chromatographic methods. Residual silanols exhibit acidic characteristic and they can ionize depending on the environment. Thus, they posses cation-exchange properties and cause the zeta potential of silica particle in liquid environment. Presented review discusses the influence of the residual silanol groups on the solvation process and retention of polar compounds. The novel methodology of residual silanols determination is presented as well as the influence of the silanols on the zeta potential of the stationary-bonded phases in chromatographic conditions.     

Control of electroosmotic flow in nonaqueous capillary electrophoresis by polymer capillary coatings

In aqueous capillary electrophoresis the electroosmotic flow (EOF) can be strongly suppressed or eliminated by coating the capillary surface silanols either by buffer additive adsorption or chemical modification. Hydrophilic coatings, e.g., polyvinyl alcohol (PVA) proved to be most efficient for EOF control in applications like DNA analysis. In nonaqueous capillary electrophoresis (NACE), however, the EOF cannot be totally suppressed with these capillaries and coating efficiency turned out to be solvent-depending. In this paper, fused-silica capillaries with monomeric and polymeric coatings differing in hydrophobicity and chemical properties (vinyl, vinyl acetate, vinyl alcohol and acrylates with different alkyl chain length) were investigated. Besides studying the EOF characteristics with different organic solvents and water, gas chromatography (GC) measurements were carried out to probe the silanol reduction via ether retention and the surface hydrophobicity by retention of nonane. Good correlations between GC results and EOF magnitude could be found. It could be demonstrated that the polymeric coating has to be solvatized by the buffer solvent to reduce the EOF. The PVA coating was optimal for aqueous systems but not effective for some nonaqueous buffers. On the other hand, polyvinyl acetate and polyethyl acrylate as polymeric coatings proved to be optimal to reduce the EOF in NACE.     

高效液相色谱槲皮素键合硅胶固定相分离极性化合物

槲皮素是一种植物体中含量丰富、价格较便宜的黄酮类化合物,本研究以γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)为偶联剂,将其化学键合到硅胶上,得到一种含天然配体的槲皮素键合硅胶固定相(QUSP)。 采用红外光谱、热重分析、元素分析及固体核磁对其结构进行表征,测得硅胶表面槲皮素的键合量为0.139 mmol/g。 采用不同结构的溶质作探针,在评价固定相反相液相色谱疏水作用性能的基础上,侧重研究新固定相对极性芳香族化合物的分离能力,探讨了新固定相的色谱分离机理。 研究表明,仅采用甲醇或乙腈-水简单流动相,无需用缓冲液精确控制pH值,QUSP就能分别实现吡啶类、芳胺类、苯酚类、苯甲酸类和黄酮类等极性化合物的快速基线分离。 QUSP键合的槲皮素除含疏水性的C6-C3-C6骨架外,黄酮环还能为溶质提供氢键、偶极、π-π、电荷转移等多种作用位点,各种协同作用有利于提高色谱分离选择性,尤其对极性的可离子化的酸性和碱性化合物。     

Branched polyethyleneimine-bonded tentacle-type polymer stationary phase for peptides and proteins separations by open-tubular capillary electrochromatography

A novel tentacle-type polymer stationary phase covalently modified with branched polyethyleneimine (PEI) was developed for peptides and proteins separations by open-tubular CEC (OT-CEC). The preparation procedure included the silanization of capillary inner wall, in situ graft polymerization and PEI functionalization. A wrinkly polymer surface of multitudinous steric amine groups was evenly formed on the capillary inner wall, and anodic EOF could be gained within a wide pH range of 2.5-7.5. The electroosmotic mobility was examined for its dependence on pH as well as PEI concentrations. Good repeatability was gained with RSD for the migration time of EOF marker within 4.8% and satisfactory chemical stability was validated. Due to the existence of amine groups on the surface of tentacle-type polymer stationary phase, the silanol effect that occurs between the positively charged biomolecules and the silanols of the capillary column was greatly suppressed. Compared with a monolayer-coating capillary, seven enkephalin-related peptides were well resolved on the PEI-bonded column with high efficiencies. Favorable separations of peptides and proteins with high column efficiencies were obtained in 144,000-189,000 and 97,000-170,000 plates/m. Branched PEI-bonded tentacle-type polymer stationary phase has been proven to afford satisfactory retention and resolution of peptides and proteins.     

A series of long-chain lamellar hydrated copper(II) alkylsulfonates with different chain molecular assemblies

Reaction of copper(II) salts with n-alkylsulfonate anions yields light blue lamellar Cu(C(n)H(2n + 1)SO3)2 x zH2O displaying distinct (mono/bi-layer) chain packing with increasing alkyl chain lengths. This may be attributed to the amphiphilic nature of the surfactants, i.e., the hydrophilic sulfonate head groups, mediating the coordination, and H-bonding interactions, and the hydrophobic alkyl chains.     

A new easy-to-prepare homogeneous continuous electrochromatographic bed for enantiomer recognition

Completely homogeneous polyacrylamide-based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) were prepared. The latter monomer was synthesized from beta-CD and allylglycidyl ether by a very simple one-step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the "pores" became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by "molecular-sieving", but rather by some type of adsorption ("aromatic adsorption"?).     

Migration behavior of alkylphenols, bisphenol A and bisphenol S studied by capillary electrophoresis using sulfated beta-cyclodextrin.

An application of capillary electrophoresis (CE) using sulfated beta-cyclodextrin (SCD) has been investigated for separating alkylphenols with different chain lengths, as well as bisphenol A and bisphenol S. In the absence of SCD in running buffer, all the phenols migrated at the same velocity as the electroosmotic flow (EOF), whereas the addition of SCD effectively led to the baseline separation of alkylphenols on the basis of the difference in the abilities to bind into the hydrophobic cavity of CD. The host-guest binding constants between analyte phenols and SCD were evaluated from Benesi-Hildebrand plots of the data obtained by two independent methods, CE and UV-visible measurements, demonstrating that the greater the hydrophobicity of the phenols, the larger the binding constants. The effects of organic solvents on the resolution for alkylphenols and bisphenols were also examined. This system using SCD was effective for the separation of 4-octylphenol and 4-nonylphenol isomers having longer alkyl chains.