Potential binding of borate ions to mono- and oligonucleotides: a capillary electrophoresis investigation

The potential binding of borate to oligonucleotides and DNA fragments is here investigated. In case of free nucleotides, such as AMP, there appears to be a weak binding, although no free versus complexed species could ever be separated under any experimental condition. The binding was suggested by the strong peak asymmetry and by the fact that, at progressively lower borate molarities in the background electrolyte, the peak shape suddenly switched from fronting to tailing. This indicated, as also confirmed by theoretical simulations, that the AMP-borate complex was the slow, not the fast moving species. On the contrary, in the case of free adenosine, strong binding ensued, since in Tris-acetate buffer this compound was only eluted with the electroendoosmotic flux, being neutral, whereas in Tris-borate it had a much higher mobility, comparable to, although lower than, that of AMP. When running oligonucleotides, at standard borate molarities (ca. 45 mM), and under strict iso-ionic strength conditions, no binding to borate could be demonstrated, since the free mobility of a 24-mer DNA was identical in TA and TB buffers. However, at very high borate molarities (200 mM) and high pH values (pH 8.92), some binding to oligonucleotides could occur, since in these latter conditions the mobility of a 24-mer was seen to be ca. 20% lower than at pH 7.69, a pH value that should discourage any complex formation.     

The free solution mobility of DNA in Tris-acetate-EDTA buffers of different concentrations,with and without added NaCl

The free solution mobility of a high-molecular-weight DNA, linear pUC19, and a 20-bp oligomer called dsA5 have been studied as a function of Tris-acetate-EDTA (TAE) buffer concentration, with and without added NaCl. The two DNAs migrate as separate peaks during capillary electrophoresis, because the mobility of linear pUC19 is higher than that of the 20-bp oligomer. In TAE buffers ranging from 10-400 mM in concentration, the migration times and peak areas of the two DNAs are independent of whether they are electrophoresed separately or in mixtures, indicating that DNA-DNA and DNA-buffer interactions are absent in these solutions. The migration times of the two DNAs vary and the peak areas are not additive when the TAE buffer concentration is reduced to 5 mM or below, indicating that DNA-DNA and DNA-buffer interactions are occurring at very low TAE buffer concentrations. The mobilities of linear pUC19 and dsA5 decrease slowly with increasing conductivity or ionic strength when the conductivity is increased by increasing the TAE buffer concentration. When the Tris buffer concentration is held constant and the conductivity is increased by adding various concentrations of NaCl to the solution, the mobilities of linear pUC19 and dsA5 first increase slightly, then become independent of solution conductivity (or ionic strength), and finally decrease when the NaCl concentration is increased above approximately 50 mM. The mobility variations observed in the various TAE and TAE-NaCl solutions are described qualitatively by Manning's theory, although quantitative agreement is not achieved. The free solution mobilities of single-stranded pUC19 and two 20-base oligonucleotides have also been measured. The free solution mobility of single-stranded pUC19 is approximately 15% lower than that of native pUC19, in agreement with other results in the literature. Somewhat surprisingly, the mobilities of the single- and double-stranded 20-mers are equal to each other in TAE buffers with and without added NaCl.     

Conformational separation of monosaccharides of glycoproteins labeled with 2-aminoacrydone using microchip electrophoresis

The conformational separation of monosaccharides labeled with fluorescent 2-aminoacrydone (AMAC) was performed by electrophoresis on a plastic microchip with light-emitting diode confocal fluorescence detection. The AMAC-labeled five neutral monosaccharide mixture (D-glucose (Glc), D-mannose, D-galactose, L-fucose, and D-xylose) or two amino monosaccharide mixture (N-acetyl-D-glucosamine and N-acetyl-D-galactosamine) were well separated at pH 8.5 and 0.5% w/v methylcellulose of 200 mM borate buffer conditions using microchip electrophoresis. The separation was successfully performed considering the difference in stability of the complex between the hydroxyl residue of the monosaccharide and borate ions, and we found that 200 mM and pH 8.5 of borate buffer conditions were critical. High-speed separation for the neutral monosaccharides (50 s) and for amino monosaccharides (70 s) was attained at a 400 V/cm of electric field condition, showing all peak resolutions were greater than 0.9% and RSD of mobility were less than 1.9%. The detection limits of 0.86 microM for Glc and <1 microM for all other monosaccharides were enhanced with the addition of 0.5% w/v methylcellulose to the buffer. These attainments are fully compatible with conventional CE. The analysis of the subtle differences in the conformational stability and the value of the hydroxyl residue of the borate complex allowed the development of an efficient prospective tool for attaining high-resolution separation of monosaccharide mixtures having complicated and analogous conformations.     

Comparison of A+T-rich oligonucleotides with and without self-complementary sequence using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Both A+T-rich oligonucleotides with and without self-complementary sequences were analyzed using ion- pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) by tryethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) buffer systems to study the characteristics of their retention behavior and electrospray ionization tandem mass spectrometry (ESI-MS/MS) response. The results indicated that the chain length had the same effect on the retention of A+T-rich oligonucleotides in both of TEAA and HFIP buffer systems but the sequence had a different impact on the retention in the two buffer systems. A+T- rich oligonucleotides with a self-complementary sequence were much shorter than those without in the TEAA buffer system whereas a slight difference was observed in the HFIP buffer system. Similar total ion current (TIC) intensity was observed both in oligonucleotides with or without self-complementary sequence. The opposite trend of a change in the TIC intensities with increasing chain length were observed in both the TEAA and HFIP buffer systems. A lower charge state was predominant in the TEAA buffer system whereas a higher charge state was mainly distributed in the HFIP buffer system. The oligonucleotides without self-complementary sequences had a higher charge state than those with self-complementary sequences. A- and T- are more esily formed at a higher charge state whereas the sequence fragments will be formed more easily at a lower charge state in both A+T-rich oligonucleotides with and without self-complementary sequences.     

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 mM carbonate, pH 9.0; 50 mM phosphate, pH 8.5; 50 mM borate, pH 8.3; 50 mM Tris-borate+1 mM EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris-borate-EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.     

Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography

The partition coefficients for the distribution of bilirubin between aqueous phosphateborate buffer and cholic, taurocholic, taurodeoxycholic, and taurochenodeoxycholic micelles have been measured by micellar electrokinetic chromatography at pH 8.5. Determination of the partition coefficients required that the critical micelle concentration and partial specific volumes be determined for each bile salt. Critical micelle concentrations were slightly higher for the trihydroxy bile salts. Partial specific volumes of the bile salt micelles differed very little from each other, and for each bile salt they were constant over the concentration range studied, which was typically from slightly above the critical micelle concentration to 35 mM. Capacity factors were corrected for the effects of applied voltage by extrapolation of the capacity factor to zero applied volts. The free solution mobility of bilirubin, determined in the absence of bile salt, was also corrected for the effects of applied voltage. Plots of extrapolated capacity factor versus phase ratio yield the partition coefficient as the slope of a linear fit to the data. Partition coefficients for bilirubin were significantly higher for dihydroxy bile salts than for trihydroxy bile salts.     

Optimization of separation and migration behavior of cephalosporins in capillary zone electrophoresis

The influences of buffer pH, buffer concentration and buffer electrolyte on the migration behavior and separation of 12 cephalosporin antibiotics in capillary zone electrophoresis using three different types of buffer electrolyte, including phosphate, citrate, and 2-(N-morpholino)ethanesulfonate (MES), were investigated. The results indicate that, although buffer pH is a crucial parameter, buffer concentration also plays an important role in the separation of cephalosporins, particularly when cefuroxime and cefazolin, cephalexin and cefaclor, or cefotaxime and cephapirin are present as analytes at the same time. The electrophoretic mobility of cephalosporins and electroosmotic mobility measured in citrate and MES buffers are remarkably different from those measured in phosphate buffer. With citrate buffer, optimum buffer concentration is confined to a small range (35-40 mM), whereas buffer concentrations up to 300 mM can be used with MES buffer. Complete separations of 12 cephalosporins could be satisfactorily achieved with these three buffers under various optimum conditions. However, the separability of 12 cephalosporins with citrate or MES buffer is better than that with phosphate buffer. As a consequence of a greater electrophoretic mobility of cephalosporins than the electroosmotic mobility with citrate buffer at pH below about 5, some cephalosporins are not detectable. The cloudiness of the peak identification and of the magnitudes of the electrophoretic mobility of cefotaxime and cefuroxime reported previously are clarified. In addition, the pKa values of cephradine, cephalexin, cefaclor, and cephapirin attributed to the deprotonation of either an amino group or a pyridinium group are reported, and the migration behavior of these cephalosporins in the pH range studied is quantitatively described.     

Study of the diffusion of pyrene in silicone polymer coatings by steady state fluorescence technique: effects of pyrene concentration

The concentration dependence of the diffusion coefficient of pyrene in single component and two-component room temperature curing silicone polymer coatings is investigated by the steady state fluorescence technique by measuring the pyrene excimer fluorescence intensity. At pyrene concentrations lower than 10 mM, the intensity of excimer fluorescence is proportional to the concentration and at higher concentrations it deviates from this trend due to concentration quenching. Thermal aging studies show that this concentration quenching can be removed by thermal annealing and the excimer emission intensity approaches the value expected from the trend at lower concentrations. The diffusion coefficient of pyrene at low concentrations in silicone polymer coatings is obtained using the approximate solution of one-dimensional diffusion equation. A modified approach is employed to estimate the diffusion coefficient at higher pyrene concentrations. In this method, the excimer intensity and time scale are shifted, respectively to I_max the maximum value of excimer intensity attained during annealing and t_max, the time taken to reach this. The estimated diffusion coefficients at different pyrene concentrations show a negligible dependence on pyrene concentration in both types of polymers. These results are attributed to the high structural mobility of silicone polymer chains due to their molecular structure.     

Analysis of protein-protein interactions with a multi-capillary electrophoresis instrument

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Dispersion stability and aggregation behavior of TEMPO-oxidized cellulose nanofibrils in water as a function of salt addition

Dispersion stability of TEMPO-oxidized cellulose nanofibrils (TOCNs) in water was investigated through both experimental and theoretical analyses to elucidate the critical aggregation concentration of different salts. The 0.1 wt% TOCN/water dispersions with various NaCl concentrations were evaluated by measuring light transmittance, viscosity under steady-shear flow, and the weight fraction of TOCN that had aggregated. Homogeneous TOCN/water dispersion turned to gel as the NaCl concentration increased. The TOCN dispersion maintained its homogeneous state up to 50 mM NaCl, but aggregated gel particles were formed at 100 mM NaCl. The mixture became separated into two phases (gel and supernatant) at ≥200 mM NaCl. Theoretical analysis using ζ-potentials of TOCN elements in the dispersions revealed that the aggregation behavior upon NaCl addition could be explained well in terms of the interaction potential energy between two cylindrical rods based on the Derjaguin–Landau–Verwey–Overbeek theory. The experiments were extended to analyze critical aggregation concentrations of MgCl₂ and CaCl₂ for the 0.1 wt% TOCN dispersion. In the case of divalent electrolytes, TOCN elements began to form aggregated gel particles at salt concentrations of 2–4 mM, corresponding to the critical aggregation concentration predicted by the empirical Schultz-Hardy rule.     

Separation of diadenosine polyphosphates by capillary electrophoresis

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.     

Enantioseparations of hydrobenzoin and structurally related compounds in capillary zone electrophoresis using heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin as chiral selector and enantiomer migration reversal of hydrobenzoin with a dual cyclodextrin system in the presence of borate complexation

We investigated the enantioseparations of racemic hydrobenzoin, together with benzoin and benzoin methyl ether, in capillary electrophoresis (CE) using the single-isomer heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin (SI-S-beta-CD) as a chiral selector in the presence and absence of borate complexation and enantiomer migration reversal of hydrobenzoin with a dual CD system consisting of SI-S-beta-CD and beta-CD in the presence of borate complexation at pH 9.0 in a borate buffer. The enantioselectivity of hydrobenzoin increased remarkably with increasing SI-S-beta-CD concentration and the enantioseparation depended on CD complexation between hydrobenzoin-borate and SI-S-beta-CD. The (S,S)-enantiomer of hydrobenzoin-borate complexes interacted more strongly than the (R,R)-enantiomer with SI-S-beta-CD. The enantiomers of hydrobenzoin could be baseline-resolved in the presence of SI-S-beta-CD at a concentration as low as 0.1% w/v, whereas the three test analytes were simultaneously enantioseparated with addition of 0.3% w/v SI-S-beta-CD or at concentrations >2.0% w/v in a borate buffer and 0.5% w/v in a phosphate background electrolyte at pH 9.0. Compared with the results obtained previously using randomly sulfated beta-CD (MI-S-beta-CD) in a borate buffer, enantioseparation of these three benzoin compounds is more advantageously aided by SI-S-beta-CD as the chiral selector. The enantioselectivity of hydrobenzoin depended greatly on the degree of substitution of sulfated beta-CD. Moreover, binding constants of the enantiomers of benzoin compounds to SI-S-beta-CD and those of hydrobenzoin-borate complexes to SI-S-beta-CD were evaluated for a better understanding of the role of CD complexation in the enantioseparation and chiral recognition. Enantiomer migration reversal of hydrobenzoin could be observed by varying the concentration of beta-CD, while keeping SI-S-beta-CD at a relatively low concentration. SI-S-beta-CD and beta-CD showed the same chiral recognition pattern but they exhibited opposite effects on the mobility of the enantiomers.     

Assay of the Related Compounds Thiamphenicol, Florphenicol, and Chloramphenicol by CE

The purpose of this study was to examine the migration behaviour of a mixture of thiamphenicol, its two analogues florphenicol and chloramphenicol, and four impurities found in thiamphenicol. Capillary zone electrophoresis was performed with phosphate–borate or borate running buffers, pH 8.5, with or without addition of sodium dodecylsulphate (SDS). Although the compounds could not be resolved in the absence of SDS, supplementation of both types of buffer, at concentrations of 100 mM and higher, with 50 mM SDS enabled separation. On the basis of migration time and resolution, 100 mM borate buffer containing 50 mM SDS was identified as the best for the study. The accuracy and precision of the method were good under these conditions.     

Fluorimetric assay of dopamine, norepinephrine and their 3-O-methyl metabolites by using fluorescamine.

Fluorescamine was subjected to reaction with dopamine and norepinephrine (catecholamines) and with 3-methoxytyramine and normetanephrine (3-methyl metabolites of catecholamines) in phosphate or borate buffer. Catecholamines gave the highest fluorescent intensity at pH 8.0 in phosphate buffer but lower fluorescence in borate buffer. The fluorophores produced in phosphate or borate buffer were the same but the fluorescence intensities were suppressed in borate buffer. The dopamine and norepinephrine fluorophores were separated by high-pressure liquid chromatography on Hitachi 3011 gel with methanol-0.10 M Tris buffer of pH 8.0 (7:3). They were measurable at the 100-pmole level. The metabolites were also measurable by the same chromatography. By using methanol-0.15 M borate buffer of pH 8.0, cate-chol-O-methyltransferase activity might be assayed.     

Transient state of chiral recognition in a binary mixture of cyclodextrins in capillary electrophoresis

The transient state (as the defined point where no enantioseparation is obtained in a dual chiral selector system) of chiral recognition of aminoglutethimide in a binary mixture of neutral cyclodextrins (CDs) was studied by capillary electrophoresis (CE). The following three dual selector systems were used: alpha-cyclodextrin (alpha-CD) and beta-cyclodextrin (beta-CD); alpha-CD and heptakis(di-O-methyl-beta-cyclodextrin) (DM-beta-CD); alpha-CD and heptakis(tri-O-methyl-beta-cyclodextrin) (TM-beta-CD). The S-(-) enantiomer of the analyte was more strongly retained in the presence of either alpha-CD or TM-beta-CD at pH 2.5, 100 mM phosphate buffer, while the R-(+) enantiomer was more strongly retained in the presence of either beta-CD or DM-beta-CD. In the more simple case, the elution order is invariably kept if the enantiomers have the same elution order in either one of the two hosts of the binary mixture. In contrast, the elution order may be switched by varying the concentration ratio of two hosts that produce opposite elution order for this particular analyte. In such a dual selector system, the enantioselectivity will disappear at the transient state at a certain ratio of host1:host2. Moreover, the migration times of the two enantiomers with host, alone (diluted in buffer) is approximately equal to the migration times at the corresponding concentration of host2 alone (diluted in buffer), where the ratio of concentrations of host1:host2 is the same as in the binary mixture at the transient state. As found by nuclear magnetic resonance experiments, the analyte is forming a 1:1 complex with either one of the CDs applied. From this finding, a theoretical model based on the mobility difference of the two enantiomers was derived that was used to simulate the transient state.     

温度和缓冲溶液对胶束电动毛细管色谱的迁移时间窗口的影响

以电渗淌度、胶束电泳淌度和淌度比这3个参数为考察对象,研究了毛细管温度、缓冲溶液种类和浓度对胶束电动毛细管色谱的迁移时间窗口的影响。电渗淌度和胶束电泳淌度均随毛细管温度的升高线性的增加,粘度是这一影响中的主要因素。理论上证明了管壁表面的局部粘度与主体粘度不同。当温度变化时,电渗淌度和胶束电泳淌度的变化幅度不同。降低温度可以扩展迁移时间窗口,虽然扩展幅度较小,但在商品化仪器上易于实现。推导出能统一描述电渗淌度和胶束电泳淌度与缓冲溶液浓度间的关系式。     

Highly efficient separation of isomeric epoxy fatty acids by micellar electrokinetic chromatography

A capillary electrophoresis (CE) method has been developed for simple and direct separation of cis- and trans-12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid isomers. Separation was performed in micellar electrokinetic capillary chromatography (MEKC) using a buffer consisting of 25 mM borate (pH 9.20), 10 mM sodium dodecyl sulfate (SDS) and 10% v/v acetonitrile. The key variables, concentrations of SDS and organic modifier, were optimized by the application of a factorial experimental design. The use of a low micellar concentration, just above critical micelle concentration (CMC), in a background electrolyte containing an organic modifier not only made it possible to dissolve and separate highly hydrophobic fatty acid isomers, but also resulted in improved separation efficiency and selectivity. Separation efficiency up to 4 x 10(5) theoretical plates/m was achieved under an optimized condition. Also investigated were the influence of temperature on separation and the effect of organic modifier concentration on the dynamic exchange of the analytes between micelles and the bulk of the buffer solution. Direct UV was applied for detection of the fatty acids.     

Capillary zone electrophoresis of aldoses,ketoses and uronic acids derivatized with ethylp-aminobenzoate

Summary A number of mono- and oligosaccharides derivatized with ethylp-aminobenzoate have been separated as their borate complexes by capillary zone electrophoresis, using a fused silica capillary containing 175 mM borate buffer, pH 10.5, as carrier. The derivatized carbohydrates were sufficiently separated within 17 min at an applied voltage of 25 kV. On-column UV monitoring allowed detection of these derivatives at the 10 fmol level, and quantification by the relative peak area method allowed reproducible determination of the saccharides at least in the concentration range of 0.5–20 mM in reaction solutions. This method has been applied to the determination of the monosaccharide compositions of polysaccharides extracted from Flos matricariae and Radix althaeae.     

The effect of electric field strength,buffer type and concentration on separation parameters in capillary zone electrophoresis

Summary Probe solutes were used to investigate the effect of buffer type, concentration and applied voltage on solute mobility, column efficiency and resolution in capillary zone electrophoresis. With low conductivity buffers higher concentrations and/or higher voltages could be used to improve column efficiency and resolution. Doubling the concentration of the buffer doubles the amount of heat generated inside the column while doubling the applied voltage cause a 4-fold increase. Solute migration time is approximately an inverse function of the charge density of the buffer's cation. Analysis time is increased by about 30% if the buffer concentration is doubled while it is cut in half if the applied voltage is doubled. Column efficiency is improved (higher theoretical plate count) with increasing buffer concentration and/or applied voltage as long as the heat generated is efficiently dissipated. The separation factor is directly related to analysis time and, therefore, selectivity improves with increasing buffer concentration but decreases with increasing applied voltage. Hence, resolution is optimized by increasing buffer concentration at a moderate applied voltage.