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.     

Borate-containing background electrolytes to improve CE separation in bare capillaries

The paper describes how borate-containing BGEs modify ζ-potential and so EOF in bare fused silica capillaries. This surface modification can be used to suppress EOF and improve the separation performance of CZE including capillary sieving electrophoresis (CSE). Boric acid forms complexes with polysaccharides used as sieving matrices in CSE and other compounds containing hydroxyl groups, including polyol bases such as Tris, triethanolamine, and Bis-Tris propane. High concentration of boric acid in BGEs leads to a strong interaction of boric acid with the silica surface of the capillary wall and this suppresses or even completely eliminates ζ-potential and EOF. Using a polyol base with several charge-carrying amino groups, such as Bis-Tris propane, can actually reverse EOF. We demonstrate the use of various borate-containing BGEs in bare fused silica capillaries for size-separation of DNA fragments, size-separation of proteins by SDS CSE, and also by CZE in the absence of any sieving matrix.     

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).     

Surfing silica surfaces superciliously

The present mini-review summarizes the experience gathered by our group in developing different classes of novel quaternarized heterocyclic compounds able to modulate and reverse the electroendoosmotic flow (EOF) in a most peculiar manner. The first class comprises mono-salt compounds, with the determinant omega-iodoalkyl chains of different lengths (typically C4-C8), able to be adsorbed by silicas, at alkaline pH, and spontaneously alkylate ionised silanols, thus becoming covalently affixed to it. The second class is constituted by di-salt compounds, attached at the termini of an alkyl chain of variable lengths (here too, typically, C4-C8). This second class is unable to bind covalently silica surfaces, although, in thin-layer chromatography, it exhibits an extraordinary affinity for silica beads, contrary to the first one. On the basis of the strikingly different behaviour, structural rules are derived for the minimum requirements for general classes of amines to bind to silica walls and modify EOF. For compounds unable to bind covalently to the wall, the most important structural motif is two quaternary nitrogens spaced apart by a C4 chain: this seems to be the average distance (i.e., 0.8 nm) between two adjacent, ionized silanols for a snug fit. The other structural binding motif is the "hydrophobic decoration", i.e., the ratio of charged groups to alkyl residue in the various amines; amines with high levels of such alkane groups (i.e., with higher hydrophobicity), seem to bind more tenaciously to the wall, probably due to hydrophobic interaction not to the wall but among the amine derivatives themselves, when carpeting the silica.     

Organic and inorganic di-cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis

EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400-2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investigated, Ca++, Mg++, Sr++ and Ba++, the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33-1 mM range) with barium (10-20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and >40 EO units in length. In this last case, best results in terms of reproducibility and separation efficiency of the more heavy homologues were obtained using Li+ salt in small amounts.     

Electroosmotic flow modulation in capillary electrophoresis by organic cations from ionic liquids

This paper describes the ability of several ionic liquids cations for electroosmotic flow modulation in capillary electrophoresis. Organic salts based on phosphonium, sulfonium, cysteinium, ammonium, and guanidinium cations were selected to study this property. In addition, the synergistic effect of these compounds in cyclodextrin chiral separation was also evaluated. In comparison with most studied imidazolium-based ionic liquids, several of the cations studied, are stronger modifiers in terms of electroosmotic flow (EOF) modulation. Phosphonium-based compounds and tri-octyl methylammonium chloride ([Aliquat]Cl) had the strongest ability to reverse EOF both in acidic and in basic conditions and had the lowest EOF reversal concentrations in the presence of hydroxypropyl-β-cyclodextrin. EOF modulation ability of phosphonium cations also contributed to the improvement of chiral separation of DL-propranolol by hydroxypropyl-β-cyclodextrin at lower concentrations in comparison with most commonly used EOF modulators such as tetrabutylammonium phosphate.     

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.     

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.     

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.     

Modification of the electroosmotic flow and separation selectivity of anions in electrochromatography with pseudo-stationary phases of C14-alkyldimethylammoniopropane sulfonate zwitterionic surfactants by addition of salts to the background electrolyte

The effects of salts (NaCl, NaClO4, MgCl2, CeCl3) added to background electrolyte (BGE) solutions (10 mmol L(-1) sodium phosphate, pH 7.2) on electroosmotic flow (EOF) and the separation selectivity of anions (chloride, bromide, iodide, nitrite, nitrate, chlorate, thiocyanate, iodate, chromate, and molybdate ion) by capillary electrochromatography using the zwitterionic surfactant 3-(N,N-dimethylmyristylammonio)propane sulfonate (C14N3S) as a pseudo-stationary phase were investigated. There are two mechanisms affecting the separations: 1. the cations and anions of the added salts interact with the zwitterionic surfactant to varying degrees, thus changing the overall retention of the analytes; and 2. they change the EOF and the resulting apparent mobilities. It was shown that a BGE containing perchlorate and a low concentration of zwitterionic surfactant (2 mmol L(-1)) gave a stable and reproducible EOF and the concentration of perchlorate could be used to manipulate the separation selectivity for polarizable anions, such as iodide and thiocyanate. These effects are discussed in terms of measured association constants describing the interaction of anions and cations with the zwitterion.     

The magnitude and reproducibility of the electroosmotic flow in silica capillary tubes

The influence of a number of factors, such as the nature of the buffer, the presence of additives therein, the manufacturing conditions, and the pretreatment of the capillary, on the magnitude and repeatability of the electroosmotic flow (EOF) in silica capillary tubes has been investigated. It was established that the nature of the buffer plays the most significant role in respect of the stability and magnitude of EOF, whereas the conditions used for manufacture and pretreatment of the tubing are less important. The EOF obtained using buffers consisting of tris(hydroxymethyl)aminomethane and ethylenediamine was more repeatable than that obtained using pure phosphate buffers. Buffer additives such as sodium dodecylsulfate, 1-butanol, sodium salts of 1-pentanesulfonic acid and 1-heptanesulfonic acid, altered the magnitude of the EOF only, not its repeatability.     

Poly(tetrafluoroethylene) separation capillaries for capillary electrophoresis. Properties and applications

Poly(tetrafluoroethylene) (PTFE) is a material widely known for its inertness and excellent electrical properties. It is also transparent in the UV region and has a reasonable thermal conductivity. These properties make PTFE a suitable material for the separation capillary in capillary electrophoresis. Differences in the chemistry of the capillary wall compared to fused silica (FS) can make PTFE an interesting alternative to FS for some special applications. In this work, properties of a commercial PTFE capillary of approx. 100 microm i.d. were investigated, including the dependence of electroosmotic flow (EOF) on pH for unmodified and dynamically modified PTFE, optical properties, and practical aspects of use. The main problems encountered for the particular PTFE capillary used in this study were that it was mechanically too soft for routine usage and the crystallinity of the PTFE caused light scattering, leading to high background absorbance values in the low UV region. The profile of the EOF versus pH for bare PTFE surprisingly showed significantly negative EOF values at pH < 4.2, with an EOF of -30 x 10(-9) m2 V(-1) s(-1) being observed at pH 2.5. This is likely to be caused by either impurities or additives of basic character in the PTFE, so that after their protonation at acidic pH they establish a positive charge on the capillary wall and create a negative EOF. A stable cationic semi-permanent coating of poly(diallyldimethylammonium chloride) (PDDAC) could be established on the PTFE capillary and led to very similar magnitudes of EOF to those observed with FS. A hexadecanesulfonate coating produced a cathodic EOF of extremely high magnitude ranging between +90 and +110 x 10(-9) m2 s(-1) V(-1), which are values high enough to allow counter-EOF separation of high mobility inorganic anions. In addition, pH-independent micellar electrokinetic capillary chromatography (MEKC) separations could be easily realised due to hydrophobic adsorption of sodium dodecylsulfate (used to form the micelles) on the wall of the PTFE capillary. The use of polymers that would be mechanically more robust and optically transparent in the low-UV region should make such CE capillaries an interesting alternative to fused silica.     

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis (CE) were exemplified with p-allylcalix[4]arene (pACX4) which was immobilized to the fused silica surface using gamma-methacryloxypropyl-trimethoxysilane (gamma-MAPS) as linking agent. Successful gamma-ACX4 coating was suggested by the greatly decreased electroosmotic flow (EOF), due to the introduction of phenolic hydroxyl groups on the inner surface of the capillaries. A slight slope of EOF versus pH at pH <8 would help make the separation reproducible. The coated columns also featured a low ultraviolet (UV) absorption background and long lifetime (> 6 months at 4相似文献   

Directional flow induced by synchronized longitudinal and zeta-potential controlling AC-electrical fields

Electroosmotic flow (EOF) in a microchannel can be controlled by electronic control of the surface charge using an electrode embedded in the wall of the channel. By setting a voltage to the electrode, the zeta-potential at the wall can be changed locally. Thus, the electrode acts as a "gate" for liquid flow, in analogy with a gate in a field-effect transistor. In this paper we will show three aspects of a Field Effect Flow Control (FEFC) structure. We demonstrate the induction of directional flow by the synchronized switching of the gate potential with the channel axial potential. The advantage of this procedure is that potential gas formation by electrolysis at the electrodes that provide the axial electric field is suppressed at sufficiently large switching frequencies, while the direction and magnitude of the EOF can be maintained. Furthermore we will give an analysis of the time constants involved in the charging of the insulator, and thus the switching of the zeta potential, in order to predict the maximum operating frequency. For this purpose an equivalent electrical circuit is presented and analyzed. It is shown that in order to accurately describe the charging dynamics and pH dependency the traditionally used three capacitor model should be expanded with an element describing the buffer capacitance of the silica wall surface.     

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.     

毛细管电泳中影响径向电场控制电渗的主要因素

利用自制的二维电场毛细管电泳系统研究了不同因素对径向电场控制电渗能力的影响,发现缓冲液的ｐＨ值、浓度、种类以及管壁表面状态、管径等对电渗的电场调控有关键性的影响。有趣的是,添加剂不影响电场的调控能力,而杯芳烃涂层毛细管却能提高电渗对径向电场的响应能力。利用这种涂层效应有可能实现较高ｐＨ值下电渗的电场调控。     

Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography

Porous silica beads with an average particle diameter between 0.2 and 3 microm have been applied as packing material in capillary electrochromatography (CEC). The experiments were directed to investigate whether it is really feasible and as promising as expected to use such small particles. In CEC, plate heights of H approximately/= 1-2 d(p) can be achieved which is smaller than the plate heights usually attained in high-performance liquid chromatography. Using a capillary packed with 0.5 microm silica beads we achieved a plate height of H = 3 d(p) indicating the presence of dispersive effects like Joule heating. Calculations demonstrate that at a field strength of about 800 V cm(-1) one third of the plate height can be lost by Joule heating effects if the heat is not removed by a cooling system. Additionally, the H(u) curve is still descending at the maximum electroosmotic flow (EOF) velocity we generated with the modified capillary electrophoresis instrument. To fully exploit the potential of submicron size silicas higher field strengths, i.e., higher EOF velocities, must be attained. To study the influence of the kind of packing on the EOF porous as well as nonporous silicas have been applied. The experiments clearly indicate that the EOF of porous and nonporous silicas is the same. Since the EOF is more or less exclusively generated by the packing material the zeta potential of n-octyl bonded 0.5 microm silica has been determined. The dependence of the zeta potential on the pH is identical to the dependence of the EOF on the pH in a packed capillary. The point of zero charge of the silica is at pH 2-3.     

Phenylaminopropyl silica monolithic column for pressure assisted capillary electrochromatography

A novel stationary phase phenylaminopropyl silica (PhA-silica) monolith was successfully prepared for pressure assisted capillary electrochromatography (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by using [3-(phenylamino)propyl]trimethoxysilane as surface modification reagent to produce the phenylaminoporpyl function. The secondary amino groups on the surface of the monolithic stationary phase contributed to the generation of anodic electroosmotic flow (EOF) under acidic conditions. The phenyl group together with the spacer (-(CH(2))(3)-) in PhA-silica provides sufficient hydrophobic properties. To evaluate the column performance, effects of buffer pH and mobile phase composition on the mobile phase linear velocity and the retention factors of alkylbenzenes, phenols and anilines were investigated in pCEC mode. The monolithic stationary phases exhibit typical reversed-phase (RP) electrochromatographic behavior toward neutral solutes. Hydrophobic as well as electrophoretic migration process within the monoliths was observed for the separation of basic solutes such as anilines without peak tailing.