Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.     

Nonaqueous Capillary Electrophoretic Enantioseparation of Water Insoluble Tröger's Base Derivatives Using β‐Cyclodextrin as Chiral Selector

Racemic mixtures of six Tröger's base derivatives were separated by chiral nonaqueous capillary electrophoresis. The separation protocol was optimized first for suitable solvents. Then the applicability of various salts dissolved in organic solvents and their mixtures was evaluated. As chiral selectors β‐cyclodextrin and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin at various concentrations were used. The best enantioselectivity for the studied analytes was obtained utilizing formamide as organic nonaqueous solvent containing a mixture of sodium citrate and tris(hydroxymethyl)aminomethane acetate as electrolytes, and β‐cyclodextrin as chiral additive. The experimental results demonstrated the feasibility of nonaqueous capillary electrophoresis for enantioseparation of Tröger's base derivatives. This technique represents a suitable alternative to more commonly used capillary electrophoresis in aqueous environment. Chirality 25:810–813, 2013. © 2013 Wiley Periodicals, Inc.     

Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p-dioxane, or acetonitrile/water at pH ? pK_a. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π-π* and n-π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one-term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x-ray diffraction studies.     

Separation of four isomeric tropane alkaloids from Schizanthus grahamii by non-aqueous capillary electrophoresis

The potential of non-aqueous capillary electrophoresis was investigated for the separation of four isomeric tropane alkaloids, namely 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-senecioyloxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane and 3alpha-hydroxy-7beta-tigloyloxytropane extracted from Schizanthus grahamii. The composition of the organic solvent and the nature of the electrolyte were of considerable importance with respect to selectivity. Different organic solvents (i.e. methanol, ethanol, acetonitrile, tetrahydrofuran) and mixtures thereof were investigated. Moreover, different electrolytes such as formate, acetate and trifluoroacetate were tested. After optimisation, an electrolyte consisting of 1 M trifluoroacetic acid and 25 mM ammonium trifluoroacetate in methanol:ethanol (40:60, v:v) was selected. It provided an efficient separation of the four positional isomers as well as a good repeatability of migration time (RSD < 0.2%). The method was successfully used with electrospray MS to confirm the molecular mass of the tropane alkaloids.     

Protein design for non-aqueous solvents

Improving protein stability in unnatural and suboptimal environments is a promising application of protein engineering technology. Carefully designed amino acid alterations may lead to dramatic positive effects on the stability of proteins under highly perturbing conditions, such as in non-aqueous solvents. Applications of biocatalysts and proteins with specific binding capabilities in the chemical industry have been severely limited by constraints placed on the solvent environment. With the advent of convenient methods for altering the amino acid composition and even synthesizing entirely new protein molecules, it is worthwhile to consider engineering proteins for stability in non-aqueous solvents. In order to identify the features that a protein would need for stability in organic media, we have been studying the structure and properties of the hydrophobic protein crambin. Crambin is unique in that it is soluble and stable in very high concentrations of polar organic solvents. Crambin and its water-soluble homologs offer a powerful demonstration of protein engineering for non-aqueous solvents. This paper describes the structural features that contribute to crambin's special properties. Based on these observations and consideration of how non-aqueous solvents affect the interactions important in protein folding, a set of rules for designing non-aqueous solvent-stable proteins is proposed.     

Engineering enzymes for non-aqueous solvents.

Enzymes may be redesigned to permit catalysis in non-aqueous solvents by engineering their amino acid sequences, thereby altering their physical and chemical properties to suit the new solvent environment. The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in non-aqueous media. These approaches are illustrated by several examples where protein engineering has resulted in enzymes that are better suited for catalysis in organic solvents.     

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.     

Chiral separations of 1,3,4-thia- and 1,3,4-selenadiazine derivatives by use of non-aqueous capillary electrophoresis

Our aim was to establish suitable conditions for the chiral separation of 12 1,3,4-thia- and 1,3,4-selenadiazine derivatives; some of them were identified in screening tests as potential antituberculotics. To overcome possible problems with the water insolubility of most analytes, we profited by the advantages of non-aqueous capillary electrophoresis. Methanol, formamide, and a mixture of formamide with acetonitrile (1:2, v/v) were used as separation media. Hydroxyethyl-, hydroxypropyl-, and methyl-beta-cyclodextrin were applied as chiral selectors in concentrations of 200 mM. Besides the effect of these different electrophoretic media and selectors, we also investigated the consequences of using different electrolytes (25 mM ammonium acetate/1 M acetic acid and 25 mM citric acid/12.5 mM TRIS). Distinct differences of the separation factors in the different separation media were observed. Depending on structure characteristics of the analytes, we established clear classifications to these cyclodextrins (CD), which were most appropriate for the separation of the enantiomers of the particular analytes.     

Dependence of electroosmotic flow in capillary electrophoresis on Group I and II metal ions

The effect of Group I and II metal ions on electroosmotic flow in capillary electrophoresis in fused-silica capillaries is characterized. The electroosmotic mobility of aqueous mobile phases of lithium, sodium, potassium, calcium and barium acetates in fused-silica capillaries is measured as a function of pH at constant voltage. Cross contamination is avoided by using separate columns for each study and pH control is maintained with the aid of He sparging. The shape of a plot of pH vs. electroosmotic mobility depends on the particular cation used which in turn depends on the surface sorption properties of the ions. Column history is demonstrated to have an effect on electroosmotic flow and therefore retention times. The resolution of a test mixture is optimal in the lithium-based buffer.     

Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Excellent correlation was observed for the electrophoretic mobilities measured by capillary zone electrophoresis versus q/MW2/3, where q is the calculated charge and MW is the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. The correlation suggests that the frictional drag is proportional to the surface area of a sphere that has a volume proportional to the MW. The correlation of electrophoretic mobility with physicochemical properties will facilitate the elucidation of optimum separation strategies for protein and peptide mixtures.     

Effects of non-aqueous solvents on CO2 absorption in monoethanolamine: Ab initio calculations

Monoethanolamine (MEA) is the most typical alkanolamine and its aqueous solutions are widely used for CO₂ absorption with mature technology, but the regeneration process is energy consuming. To reduce the energy demand, non-aqueous solvents, such as methanol and ethanol are proposed to substitute water in amine solutions. To understand the influence of the aqueous and non-aqueous solvents on CO₂ capture process, the chemical reactions of MEA absorbing CO₂ were conducted via ab initio calculations. The non-aqueous solvents discussed in this paper are methanol, ethanol, 1-propanol and 2-propanol. The reaction patterns were investigated and energy barriers were observed. The results show that zwitterion formation and the followed intermolecular hydrogen transfer are proven to be the most possible reaction pattern in both aqueous and non-aqueous solvents. The energy analysis shows that the forward reaction energy barriers increase while the backward barriers decrease as the solvent changes from water to methanol, ethanol, 1-propanol and 2-propanol in turn. The decreases of the energy barriers for backward processes are much higher than the corresponding increases for forward processes. These results indicate that lower energies are required in non-aqueous solvents than in water during the desorption reactions and the non-aqueous solvents are very promising to reduce the regeneration energy consumption in MEA capturing CO₂ process. Moreover, the reaction energy gaps between different solvation effects were found to have linear relationship with the logarithm of the dielectric constant difference, which could provide an easy way to theoretically predict the reaction energies of monoethanolamine absorbing CO₂ in other solvation effect and can be used to screen appropriate CO₂ capture solvent.     

Diffusion and electrophoretic mobility of single-stranded RNA from molecular dynamics simulations

Hydrodynamic properties of small single-stranded RNA homopolymers with three and six nucleotides in free solution are determined from molecular dynamics simulations in explicit solvent. We find that the electrophoretic mobility increases with increasing RNA length, consistent with experiment. Diffusion coefficients of RNA, corrected for finite-size effects and solvent viscosity, agree well with those estimated from experiments and hydrodynamic calculations. The diffusion coefficients and electrophoretic mobilities satisfy a Nernst-Einstein relation in which the effective charge of RNA is reduced by the charge of transiently bound counterions. Fluctuations in the counterion atmosphere are shown to enhance the diffusive spread of RNA molecules drifting along the direction of the external electric field. As a consequence, apparent diffusion coefficients measured by capillary zone electrophoresis can be significantly larger than the actual values at certain experimental conditions.     

Quantification of cholesterol in foods using non-aqueous capillary electrophoresis

A simple method for the rapid quantification of cholesterol in egg yolk and milk by non-aqueous capillary electrophoresis (NACE) is described in this paper. The samples were treated with saponification and then quantified by NACE, in which 100 mM sodium acetate-acetic acid in methanol was employed as the running buffer. The correlation coefficient between the cholesterol concentration and the corresponding peak area was 0.999. The detection limit of cholesterol was 5 microg/ml (twice the signal-to-noise ratio). This method can be used as a routine method for the rapid and sensitive determination of cholesterol in foods.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Effect of water activity on the lipase catalyzed esterification of geraniol in ionic liquid [bmim]PF6

Enzymatic reactions in non-aqueous media have been shown to be effective in carrying out chemical transformation where the reactants are insoluble in water or water is a byproduct limiting conversion. Ionic liquids, liquid organic salts with infinitesimal vapor pressure, are potentially useful alternatives to organic solvents. It is known that the thermodynamic water activity is an important variable affecting the activity of enzymes in non-aqueous solvents. This study investigated the influence of water activity on the esterification of geraniol with acetic acid in ionic liquid [bmim]PF6 catalyzed by immobilized Candida antarctica lipase B. The conversion of geraniol in [bmim]PF6 was significant although the reaction rate was slower than in organic solvents. The profile of initial reaction rate-water activity was determined experimentally, and differed from the data reported for other non-aqueous solvents. A maximum in the initial reaction rate was found at aw = 0.6. The pseudo reaction equilibrium constant, Kx, was measured experimentally for the reaction. The average value of Kx in [bmim]PF6 was 12, 20-fold lower than the value reported for the same system in hexane.     

Effects of organic solvents and substrate binding on trypsin in acetonitrile and hexane media

In this work, we used molecular dynamic (MD) simulation to study trypsin with and without a six-amino-acid peptide bound in three different solvents (water, acetonitrile and hexane) in order to provide molecular information for well understanding the structure and function of enzymes in non-aqueous media. The results show that the enzyme is more compact and less native-like in hexane than in the other two polar solvents. The substrate could stabilize the native protein structure in the two polar media, but not in the non-polar hexane. There are no significant differences in the conformation of the S1 pocket upon the substrate binding in water and acetonitrile media while a reverse behavior is observed in hexane media, implying a possible induced fit binding mechanism in the non-polar media. The substrate binding enhances the stability of catalytic H-bond network since it could expel the solvent molecules from the active site. The enzyme and the substrate appear to be more appropriate to the reactive conformation in the organic solvents compared with aqueous solution. There is much greater substrate binding strength in hexane media than the water and acetonitrile ones since the polar solvent significantly weakens electrostatic interactions, which are observed to be the main driving force to the binding. In addition, some residues of the S1 pocket could remain favorable contribution to the binding despite the solvent change, but with differences in the contribution extent, the number and the type of residues between the three media.

A general approach to desalting oligosaccharides released from glycoproteins

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and -eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.     

Differential Congo Red staining: The effects of pH, non-aqueous solvents and the substrate

Summary Congo Red is an acid-base indicator dye. In free solution the colour and absorption characteristics of Congo Red depend not only on the pH but are also governed by the nature of the solvent environment. In tissue sections stained by Congo Red, alteration of the pH and the use of non-aqueous solvents can effect differential colouring of the tissue components. Stained sections of unmodified and chemically substituted celluloses show that differential red or blue coloration reflects the acidic or basic character of the substrate. In stained tissue sections, secondary protein structure and porosity of the substrate may also influence their colour. The effect of non-aqueous solvents is probably to modify the ionization state of the dye- substrate complex, thus altering the colour of the Congo Red. Such solvents may also change the aggregation or solvation states of the dye, with consequent modification in the colour of tissue components.     

On protein solubility in organic solvent

Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.