Anomalous migration of hyaluronic acid oligomers in capillary electrophoresis: correlation to susceptibility to hyaluronidase.

During high-resolution capillary electrophoresis analysis in an electrolyte solution containing a neutral polymer, small oligomers of regularly arranged acidic polysaccharides such as hyaluronic acid and N-acetylneuraminic acid polymers showed reversal of the migration order. This anomalous migration was well correlated with their reported biological activity. In the present study, we analyzed hyaluronidase action on the purified hyaluronic acid oligomers using capillary electrophoresis and found that hydrolytic and transglycosylation actions by hyaluronidase were dependent on the molecular sizes of hyaluronic acid oligomers, and well correlated to their migration profiles. Furthermore, fluorescent polarization technique was employed for understanding the relationship between molecular size of hyaluronic acid oligomers and their electromigrations.     

Separation of hyaluronic acid by ultrahigh-voltage capillary gel electrophoresis

Hyaluronic acid was separated using 95 kV applied potential in a polyacrylamide gel-filled capillary. The results of this separation were compared to those obtained using a capillary electrophoresis instrument operated at a more conventional potential of 15 kV. For lower-molecular-weight oligomers, the separation efficiency was found to improve by about tenfold, and the resolution by about threefold. However, the improvement in resolution declined as the polymer molecular weight increased.     

The aqueous phase nitration of phenol and benzoic acid studied through flow-gated capillary electrophoresis

The aqueous phase nitration of benzoic acid and phenol was investigated via on-line capillary electrophoresis (CE). The presence of nitrated benzoic acid and phenol was supported through appearance of corresponding molecular ion peaks in ESI-MS measurements, and speciation of the nitrated isomers is achieved via the on-line CE method. The nitrated isomers produced in both reactions were successfully separated in <4?min by addition of 15?mM β-cyclodextrin to the electrophoresis buffer. Sequential separations (on-line analysis) allowed the reaction kinetics to be described. For benzoic acid, reaction yields were low (2–3%) however, results suggest both 3- and 2-nitrobenzoic acid form in a 1–1.4 concentration ratio. In addition, 3-hydroxybenzoic acid also forms in significant quantity under our reaction conditions. For the nitration of phenol, the reaction occurred more rapidly with observed yields between ≈10–30% for individual isomers. The yield of 2-nitrophenol was higher than 4-nitrophenol by a ratio of ≈?1.7–2, but 3-nitrophenol was not detected. For both reactions, nitrated and hydroxylated aromatics were the major products and formation of higher molecular weight oligomers was not observed.     

Electrophoretic studies of polygalacturonate oligomers and their interactions with metal ions

Polygalacturonic acid, a linear homopolysaccharide, was investigated by capillary electrophoresis (CE) using linear polyacrylamide-coated capillaries and laser-induced fluorescence (LIF) detection. A successful separation of its fluorescently labeled oligomers was achieved through sieving in polyacrylamide entangled matrices. The reaction conditions for the derivatization of polygalacturonic acid were optimized. In studying the interactions between polygalacturonic acid and various metal ions, the end-label, free-solution electrophoretic (ELFSE) technique, developed earlier in our laboratory (Sudor, J., Novotny, M. V., Anal. Chem. 1995, 67, 4205-4209) was found preferable to the sieving method. ELFSE is fast and convenient in that no polymer solutions are needed for the separation. The investigation showed that for the moderately large oligomers, the strongest binding occurred with calcium and cadmium ions, while the smallest interaction was observed with magnesium ions.     

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.     

High‐Resolution Separation of Oligo(acrylic acid) by Capillary Zone Electrophoresis

Summary: Oligo(acrylic acid)s, produced by RAFT polymerization, have been separated and analyzed for the first time by capillary zone electrophoresis. The resolution obtained by capillary electrophoresis in borate buffers is far higher than that currently achieved using size exclusion chromatography. This work demonstrates that capillary electrophoresis is the technique of choice for the characterization of oligomers of acrylic acid and of other water‐soluble monomers involved in emulsion polymerization processes.

Electropherograms of different acrylic acid (AA) oligomers obtained by CZE.     

Free solution mobility of small single-stranded oligonucleotides with variable charge densities

The free solution mobilities of six single-stranded 16-nucleotide DNA oligomers with the same sequence, containing up to 11 neutral phosphoramidate internucleoside linkages, have been measured by capillary electrophoresis. The mobilities of the partially charged oligomers increase linearly with the logarithm of increasing charge density, as predicted by the Manning theory of electrophoresis (G. S. Manning, J. Phys. Chem. 1981, 85, 1506-1515). For comparison, the mobilities of eight fully charged single-stranded oligomers containing similar numbers of charged phosphate residues have also been measured. The mobilities of the variable length, fully charged oligomers increase more rapidly with the increasing number of charged phosphate residues than the mobilities of the constant size, partially charged phosphoramidate derivatives, because of the larger diffusion coefficients of the modified oligomers.     

高效毛细管电泳在核酸、蛋白质分析中的新进展

高效毛细管电泳以其分离效率高，分析速度快，样品和试剂用量少，易于实现自动化等优点，在核酸、蛋白质等生物样品的分析方面发挥着重要的作用并具有巨大的潜力。本文介绍了近两年来高效毛细管电泳技术的进展，特别是PCR／CE、CE／MS以及电泳芯片技术等方面的新发展，并综述了高效毛细管电泳在核酸、蛋白质分析方面的应用，同时对其前景进行了展望。     

On-column amperometric detection in capillary electrophoresis with an improved high-voltage electric field decoupler

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.     

Synthesis of alpha-(2-->5)Neu5Gc oligomers

A facile synthesis of the sialic acid oligomers alpha-(2-->5)Neu5Gc (1) is presented. Monosaccharides 2-4 with suitable functionality were used as the building blocks. After selective removal of the paired carboxyl and amine protecting groups, the fully protected oligomers were assembled through consecutive coupling of the building blocks by well established peptide coupling techniques. By this approach, fully protected oligomers as large as an octasaccharide were synthesized. Deprotection of these fully protected oligomers was conducted in two steps (LiCl in refluxing pyridine and 0.1 n NaOH) to afford the desired products in high yield. Enzymatic degradation of the octamer with neuraminidase, monitored by capillary electrophoresis (CE), was also accomplished. The stepwise exo-cleavage adducts were all well separated and identified in the CE spectrum. The strategy described here for solution-phase synthesis also provides the basis for future solid-phase synthesis of poly-alpha-(2-->5)Neu5Gc.     

Study of the acid hydrolysis of (3-methacryloxypropyl)trimethoxysilane by capillary electrophoresis-ion-trap mass spectrometry

The sol-gel method is a widely used technique for the synthesis of various functional coating films. Alkoxysilanes such as (3-methacryloxypropyl)trimethoxysilane (MEMO) are largely used as precursors for inorganic-organic hybrid sol-gel materials. Indeed, these compounds can form complex network, through hydrolysis and condensation reactions. The latter have to be perfectly controlled to obtain the required properties. In such a context, we have studied the potentialities of capillary electrophoresis-ion-trap mass spectrometry (CE-MS) coupling to resolve both separation and characterization of the synthesized compounds as a function of the hydrolysis time. The study of acid hydrolysis of MEMO was carried out as an example. After optimization of the running electrolyte in capillary zone electrophoresis (CZE) with UV detection, we characterized the synthesized compounds in CE-MS by using positive detection mode. The obtained resolution in CZE-UV was not entirely satisfactory because of the very closed charge/mass ratio of formed solute but also because of the interaction between the solutes and the capillary walls. Nevertheless, several oligomers were characterized in CE-MS. The absence of detection with regard to oligomers that possess higher molecular masses than octamer is discussed in this work.     

Capillary zone electrophoretic analysis of positively charged poly(ethylene oxide) macromolecules using non-covalent polycation-coated fused-silica capillary and indirect UV detection

Capillary zone electrophoresis was used to show the coupling between NH2-terminated poly(ethylene oxide) and oligomers of lactic acid activated by transforming carboxyl chain ends to acyl chloride ones. The demonstration was based on the use of fused-silica capillary physically modified by pre-adsorption of polycations in the reversed polarity mode. As poly(ethylene oxide) macromolecules are UV transparent, indirect UV detection was used. A creatinine solution at pH 4.8 was selected as background electrolyte. Commercially available polycations with different structures were tested. It was shown that the reversed electroosmosis could be modulated according to the structure of the polycation. The method was then applied to analyse a commercial alpha,omega-diamino poly(ethylene oxide), namely Jeffamine ED 600 characterised by a broad mass dispersion. Data showed that the method can detect and separate amino poly(ethylene oxide) of different structures. When applied to analyse post coupling products, no free NH2-terminated poly(ethylene oxide) segments were detected. Moreover, the method allowed detection of water-soluble oligomers generated by partial degradation of lactic segments during the reaction.     

Off-line and in-line determination of catechol- O-methyltransferase activity in a capillary electrophoretic system

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.     

Capillary electrophoresis--electrospray ionization--mass spectrometry for the characterization of natural organic matter: an evaluation with free flow electrophoresis-off-line flow injection electrospray ionization-mass spectrometry

The separation of Suwannee River natural organic matter (NOM) with capillary zone electrophoresis hyphenated to electrospray ionization-mass spectrometry (CZE-ESI-MS) is presented. The obtained electropherograms and signal distributions are comparable to the mobility distributions obtained with more classical UV detection. A direct comparison of the results was possible with free-flow electrophoresis (FFE), which allows an upscaling of the CZE method and the analysis of the collected fractions in an off-line modus with flow-injection electrospray ionization-mass spectrometry (FI-ESI-MS). The changes of the m/z distributions with mobility are very similar with both methods and show a decrease of the m/z with increasing electrophoretic mobility in the humic hump at alkaline pH; superimposed on this hump a low-molecular-weight fraction migrates at lower mobility. The analysis of benzene carboxylic acids, glycerrhycic acid as well as oligomers of polystyrene sulfonic acid and polyacrylic acid additionally illustrates possible fragmentation, formation of adducts and multiplicity of the charges of the molecules prior to MS detection. These hardly controllable difficulties add a challenge to the interpretation of the obtained m/z distributions of NOM in terms of charge and mass distributions of molecules present in the NOM mixture.     

Confinement effects on DNA hybridization in electrokinetic micro‐ and nanofluidic systems

Spatial confinement, within cells or micro‐ and nanofabricated devices, impacts the conformation and binding kinetics of biomolecules. Understanding the role of spatial confinement on molecular behavior is important for comprehending diverse biological phenomena, as well as for designing biosensors. Specifically, the behavior of molecular binding under an applied electric field is of importance in the development of electrokinetic biosensors. Here, we investigate whether confinement of DNA oligomers in capillary electrophoresis impacts the binding kinetics of the DNA. To infer the role of confinement on hybridization dynamics, we perform capillary electrophoresis measurements on DNA oligomers within micro‐ and nanochannels, then apply first‐order reaction dynamics theory to extract kinetic parameters from electropherogram data. We find that the apparent dissociation constants at the nanoscale (i.e., within a 100 nm channel) are lower than at the microscale (i.e., within a 1 μm channel), indicating stronger binding with increased confinement. This confirms, for the first time, that confinement‐based enhancement of DNA hybridization persists under application of an electric field.     

Capillary electrophoresis of ANTS labelled oligosaccharide ladders and complex carbohydrates

The detection limits of the ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) label and ANTS maltose as a model carbohydrate conjugate were investigated with on-column UV and laser induced fluorescence detection. Under capillary electrophoresis conditions, the concentration and mass detection limits were found to be 5×10^–7 mol/l or 8 femtomole with UV and 5×10^–8 mol/l or 400 attomole with laser induced fluorescence detection, respectively. Including the derivatization reaction, the best concentration detection limit increases to 1×10^–6 mol/l carbohydrate. A model calculation shows that these detection levels are still insufficient to match those of current protein sequencing protocols.Derivatization conditions for dextran and polygalacturonic acid ladders are described with subsequent fast separation in a capillary electrophoresis system under acidic pH buffer conditions. Up to 30 oligomers could be separated in less than 10 min. The application of ANTS labelled carbohydrate analysis in the food industry is demonstrated with the carbohydrate fraction of sweets and the kinetic monitoring of the hydrolysis of polygalacturonic acid.The described ANTS derivitization protocol works with as little as 5 g carbohydrate as demonstrated with a complex oligosaccharide labelled in a reaction volume as little as 2 l. To demonstrate the applicability of this approach to complex carbohydrate analysis, an oligosaccharide mixture derived from human Immunoglobuline G was labelled and separated within 5 min. Separation efficiency and speed are superior to state-of-the-art chromatographic methods. Both electrophoretic and chromatographic methods are complementary because of their different separation mechanism. The implications of using capillary electrophoresis with laser induced fluorescence and appropriate labelling strategies for structural and compositional analysis of complex carbohydrates are discussed.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).     

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

高效毛细管电泳对农药手性拆分的进展

环境中使用的农药绝大多数都具有位置或光学异构体,而且同一农药的不同对映体通常也具有不同的药效和毒性,但是目前有关手性农药的研究还很少,高效毛细管电泳是一门特别适于手性分离研究的技术,综述了近年来利用高效毛细管电泳技术对手性农药进行拆分的研究,最后对该工作的发展前景进行了讨论。