Analysis of acrylamido-buffers for isoelectric focusing by capillary zone electrophoresis

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n-mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1% Ficoll-400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).     

部分水解法制备高分子量水溶性(丙烯酰胺/丙烯酸/2-丙烯酰胺-2-甲基丙磺酸)三元共聚物

系列的高分子量水溶性丙烯酰胺 /丙烯酸 /2 丙烯酰胺 2 甲基丙磺酸 (AM/AA/AMPS)三元共聚物(P3A)由相应的 (AM/AMPS)二元共聚物通过部分水解方法制得 .聚合物的结构和组成使用电位滴定和13 C NMR谱测定 ,得到的结果指出 ,在设定的试验条件下 ,水解过程中 ,高分子链上AMPS单元具有充分的稳定性 ,而丙烯酰胺基平稳地转变为丙烯酸 .在所有不同聚合物 (P2A)情况下 ,由于阴离子基团和OH-离子的静电相斥作用 ,酰胺基的水解反应均遵循自动减缓动力学的模式 ,同时 ,最后反应转化率趋向极限 ,AM剩余值位于 3 0mol%左右 ,另外对各种三元共聚物 (P3A)的溶液特性粘数和组成的关系亦作了详细的研究 .     

Chiral 2,2-disubstituted oxirane ethers and carboxylic esters. New ferroelectric liquid crystals and dopants for induced ferroelectric phases

Abstract

The synthesis, phase behaviour and spontaneous polarization of a new class of chiral LCs and chiral dopants for induced ferroelectric phases of general structure A, possessing a (2S)-2-hydroxymethyloxirane unit, and B, possessing a (2R)-2-oxirane carboxylic acid unit connected to mesogenic building blocks are described. One of these new compounds exhibits a S*_c phase. A carbonyl group adjacent to the oxirane ring does not increase the spontaneous polarization which is in contrast to the results obtained for 2,3-disubstituted oxiranes. A comparison with analogous species containing a (2S)-2-hydroxymethyloxetane, a (2R)-2-hydroxymethylthiirane or a (2S,3R)-2-hydroxymethyloxirane unit is given.     

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.     

Kinetic and mechanistic studies of the reaction of a range of bases and metal-hydroxo complexes with the phosphonate ester 2,4-dinitrophenyl ethyl methylphosphonate in aqueous solution

The base hydrolysis and spontaneous water hydrolysis of the phosphonate ester 2,4-dinitrophenyl ethyl methylphosphonate (DNPEMP) has been studied in detail and the activation parameters for these processes determined. The catalytic effect of a series of 19 oxygen and nitrogen bases and metal hydroxo complexes has been studied. The Brønsted plot of pK versus logkN has a slope (β) of 0.47, which is fairly typical for phosphate and phosphonate esters. alpha-Effect nucleophiles such as 2-iodosobenzoate, hypochlorite and the hydroperoxide ion (HO-2) exhibit enhanced reactivity, as does fluoride ion and the bifunctional metal complex [Cu(tmen) (OH2)(OH)]+ (tmen = N,N,N',N',-tetramethylethylenediamine). Reactions involving these species appear to take place by a nucleophilic pathway, while the other oxygen and nitrogen bases act as general base catalysts. The solvent deuterium isotope effect for Et3N catalysis, kH2O/kD2O=1.3, is consistent with general base catalysis. The mechanisms of these reactions are discussed in detail.     

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.     

Tetrachlorogallate(III) complexes with basic triphenylmethane and diphenylnaphthylmethane dyestuffs

A general survey is given of the properties of tetrachlorogallate(III) complexes with basic dyestuffs of the triphenylmethane and diphenylnaphthylmethane class; solvents for extraction, the absorption spectra and molar absorptivities, the optimum concentrations of the dyestuffs and of hydrochloric acid, the stability of the compounds and their composition are also described. Analytical methods for the determination of traces of gallium by means of many of these complexes are suggested.     

Multivalent weak electrolytes - risky background electrolytes for capillary zone electrophoresis

Multivalent weak acids and bases are useful components of buffers in electrophoresis. The use of such buffers as background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is, however, risky due to the existence of unsafe regions in the analytical window of the separation. This contribution discusses the problems and shows that multivalent weak species in BGEs bring about the same effects as mixtures of two independent co-ions, i.e., the presence of two centers of symmetry in the electropherograms and the existence of a migrating system zone with a mobility in between these two centers of symmetry. The system zone deteriorates the analytical separation and detection of the analytes in its neighborhood. Illustrative experimental examples for both cationic and anionic CZE are shown and related discussion is given. Finally, some basic rules are formulated to avoid the preparation of risky BGEs.     

Noncovalent inhibition of the serine proteases, alpha-chymotrypsin and trypsin by trifluoro(organo)borates

A series of potassium organotrifluoroborates were synthesized. Their stability to hydrolysis was determined in D2O, TRIS and phosphate buffer. It was found that in both D2O and TRIS buffers, these compounds are quite stable, whereas in phosphate buffer rapid hydrolysis occurs. Based on these results, a study was undertaken to determine whether potassium organotrifluoroborates can serve as protease inhibitors. It was found that potassium organotrifluoroborates increased inhibition by at least an order of magnitude over the corresponding boronates. Dixon plots showed that these compounds are reversible competitive inhibitors of alpha-chymotrypsin and trypsin. Based on 19F NMR, we speculate that they inactivate the enzymes as a result of the formation of hydrogen-bonds between fluorine atoms of the inhibitors and the serine protease.     

Comparaison de l''action catalytique de thiols monofonctionnels et de certains thiols bifonctionnels (les mercaptoalcoyl-2 benzimidazoles) sur l''hydrolyse de l''acetate de p-nitrophenyle

Synthesis of bifunctional thiols (2-alkylmercaptobenzimidazoles) and kinetic studies of the catalysis of the hydrolysis of p-nitrophenylacetate by these compounds, and by seven thiols indicates high activity of the 2-mercaptomethylbenzimidazoles in their neutral form, interpreted as the result of nucleophilic catalysis by the thiol function in its neutral state combined with general basic catalysis by the heterocycle.     

Synthesis of NiGa layered double hydroxides. A combined EXAFS, SAXS, and TEM study. 2. Hydrolysis of a Ni2+/Ga3+ solution

Takovites are nickel-based layered double hydroxides (LDH) with a general formula that can be written as Ni(1-x)Al(x)(OH)2, A(z-)(x/z), yH(2)O, where A is a compensating interlayer anion. As in some other LDH samples, the positive charge of the layer can be adjusted upon synthesis and various anions can be exchanged in the interlayer region. It is then important to understand the synthesis pathway of these materials. We then undertook a study on the hydrolytic behavior of pure Ni salts and mixtures of Ni and Ga salts. This paper focuses on the hydrolysis of Ni(2+) and Ga(3+) ions, together in solution, carried out by base addition. The samples will be defined by their hydrolysis ratio R = [OH(-)]/([Ni(2+)] + [Ga(3+)]). Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) were used to obtain information on the colloidal species size and shape on a large scale. Each hydrolyzed sample was also studied by Ni K-edge and Ga K-edge extended X-ray absorption fine structure (EXAFS) to obtain information on the local structure of the species in suspension. SAXS curves reveal the presence of bidimensional objects whatever the R values. The platelets formed for R values >/=1.1 are slightly thicker and smaller in size, which may be linked to their different natures. Complementary information is provided by TEM analysis: the first colloids formed have a structure very close to that of alpha-GaOOH, as shown by electronic diffraction. Those structures are progressively replaced by Ni-Ga LDH platelets with increasing hydrolysis ratio, which are the only species in suspension for R = 2.0, as shown by XRD. EXAFS results confirm the complete hydrolysis of gallium before the formation of Ni-Ga LDH phases.     

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

In an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7-9; Servalyt, pH 7-9; Bio-Lyte, pH 8-10 and Pharmalyte, pH 8-10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given M(r) value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different M(r) chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are 'poor carrier ampholytes', in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with alpha,beta-unsaturated acids.     

Efficient electrophilic catalysis of 1,5-anhydrocellobiitol hydrolysis by Al(III); implications for the conservation of "rosin-alum" sized paper

Added Al2SO4 greatly accelerates the hydrolysis at 150.0 degrees C of 1,5-anhydrocellobiitol, a model for the beta(1 --> 4) linkages in cellulose, in succinate buffers of room temperature pH 3.05 and 3.35; E(a) values for 20 mM Al(III)- and H+-catalysed hydrolyses are 109 +/- 3 and 140 +/- 1 kJ mol(-1), respectively.     

Activity of a New Metallomicelle Catalytic System on the Hydrolysis of bis(4-nitrophenyl) Phosphate Ester

A new metallomicellar system containing cerium ion (III), a macrocylic polyamine ligand and the hexadecyltrimethyl ammonium bromide (CTAB) was constructed and used as catalyst in the hydrolysis of bis(4-nitrophenyl) phosphate ester (BNPP). The catalytic rate of BNPP hydrolysis was measured kinetically with UV-vis spectrophotometric method. The results indicated that the metallomicellar system exhibited relatively high stability and excellent catalytic function in BNPP hydrolysis, and the reaction rate of the BNPP catalytic hydrolysis, compared with BNPP spontaneous hydrolysis, increased by a factor of ca. 1 × 10⁸ due to the catalytic effect of the active species and the local concentration effect of the micelle in metallomicellar system. The experimental results also showed that the mono-hydroxy complex made of the macrocyclic polyamine ligand and cerium (III) is the real active species as catalyst in BNPP catalytic hydrolysis, and the micelles provide a useful catalytic environment for reaction. On the basis of the research results, the reaction mechanism of BNPP catalytic hydrolysis was proposed in this work.     

Analysis of basic compounds by reversed-phase liquid chromatography-electrospray mass spectrometry in high-pH mobile phases

The separation of basic compounds in high-pH mobile phases results in extended retention, excellent peak shapes and good chromatographic efficiency. A severe decrease in sensitivity with electrospray mass spectrometric detection in positive ion mode (ESI+-MS) is expected under conditions that suppress analyte ionization in solution. We compared the responses of a large number of various basic drugs covering a wide range of hydrophobic (log P 0.09 to 7.6) and basic character (pKa 6.8-10) in LC-ESI+-MS/MS in 0.1% formic acid in water and acetonitrile, to responses in 10mM ammonium hydrogencarbonate buffers of different pH (7.8-11), and acetonitrile. Contrary to common expectations, high-pH mobile phases do not affect negatively the responses of basic compounds in ESI+. Analyte responses and limits of detection are comparable, or most often better in high pH compared to acidic mobile phases.     

Comparison of Octadecyl-Bonded Alumina and Other Stationary Phases for Lipophilicity Estimation by High Performance Liquid Chromatography

Abstract

A recently developed octadecyl-bonded alumina stationary phase (ODA) was evaluated for determining the lipophilicities of organic compounds by high performance liquid chromatography. Using a column packed with this material and mobile phases consisting of methanol and aqueous buffers, the correlation between the octanol-water partition coefficients (log P's) of compounds of various chemical classes and the logarithms of their chromatographic capacity factors (log k's) was found to be superior to that obtained using columns packed with octadecylsilica, poly butadiene-coated alumina or octadecyl-derivanzed polystyrene-divinylbenzene copolymer. In contrast to results obtained with other columns, phenols and other hydrogen-bonding compounds did not need to be treated as a separate data set on the ODA column to obtain good correlations between log k's and log P's. The resistance of ODA to degradation by alkaline solvents allowed the use of a basic mobile phase (pH > 10) for suppressing ionization and determining the lipophilicities of organic bases which could not be evaluated within the stable pH range of ODS (pH 2–8). The log P's of five basic pharmaceutical compounds determined in this manner were found to be significantly higher than previously reported values. Evidence is presented which indicates that the previously reported log P values of these compounds are inaccurate, due to improper correction for ionization.     

Mechanistic studies on the hydrolysis of a dimeric alkylzinc bis(2-pyridylmethyl)amide

Bis(2-pyridylmethyl)amine 7 reacts with selected dialkylzinc compounds to give dimeric alkylzinc bis(2-pyridylmethyl)amides 8. Regardless of the steric bulk of the alkyl substituent, dimers with central Zn₂N₂ rings are formed. Compounds 8 undergo spontaneous hydrolysis reactions upon exposure to air/moisture which can be partially controlled if the alkyl substituent R is bulky enough [R = CH(SiMe₃)₂]. A dimeric compound 9 containing both zinc-alkyl substituents and a μ-OH functionality results. In the course of this reaction, an amide instead of the expected RH is eliminated. Extensive DFT calculations show that the facile formation of a three-centered Zn[μ-(HO?H?NHR)]Zn functionality is a crucial step. Further evidence for the importance of Zn[μ-(X?H?Y)]Zn intermediates (X, Y = O and now N) in the general mechanism of hydrolysis catalyzed by binuclear zinc compounds is thus provided.     

Chiral spiro-connected 2,3,3-trisubstituted oxiranes New dopants for induced ferroelectric phases

Abstract

The synthesis, phase behaviour and spontaneous polarization of a new class of chiral dopants for induced ferroelectric phases of general structure A and B, possessing a 2,3,3-trisubstituted oxirane ring spiro-connected with four-, five- or six-membered carbocycles are described. In a series of these compounds with the same mesogenic building blocks the one with the five-membered carbocycle exhibits the highest induced spontaneous polarization. Introduction of a carbonyl group adjacent to the oxirane ring leads to an increased induced spontaneous polarization. The sign of the helical twisting power is positive for compounds with a cyclobutane ring and negative for those with a cyclopentane or a cyclohexane ring, although the absolute configuration at the chiral carbon is the same for all compounds.     

Improvement of extraction recovery for the monobutyltin species from sediment

An improvement of the extraction recovery of the MBT species from sediment has been achieved by using a more polar solvent, toluene, and tropolone as chelating agent. A recovery of 84.7 ± 6.3% from spiked sediment has been achieved, which is the highest recovery of MBT species from sediment so far reported using solvent extraction techniques. The relative detection limit for organotin compounds in general in sediment (2 g) is 0.5 ng g⁻¹ when gas chromatography with atomic emission spectrometric detection is used for the analysis. Relative standard deviations (%) for recovery of MBT, DBT and TBT in spiked sediment range from 2.5 to 6.3% (at 0.5 μg g⁻¹ level). The extraction solution also recovers satisfactorily 7 other commonly used organotin compounds. Analyses of selected environmental samples and the Reference Sediment PACS-1 are given.     

Pathways of proton transfer in acetal hydrolysis

The process of acetal hydrolysis is analyzed in terms of the competing steps of proton transfer and heavy atom reorganization. The results of this analysis are portrayed in a series of three-dimensional reaction coordinate diagrams. The observed pathway of hydrolysis (A-1, general acid catalyzed, or spontaneous) is shown to depend on the energy of the various possible intermediates in these reactions.