Separation of structurally related peptides by open-tubular capillary electrochromatography using (metallo)porphyrins as the adsorbed stationary phase

Several (metallo)porphyrins, particularly the porphyrin derivative tetraphenylporphyrin, and complexes of porphyrin derivatives with metal ions (Zn2+, Cu2+, Ni2+, Co2+, Co3+) have been employed as the stationary phase physically adsorbed onto the inner fused-silica capillary surface for open-tubular capillary electrochromatography, and applied for the separation of structurally related peptides. Four octapeptides, derivatives of the B23-B30 fragment of the B-chain of human insulin with minor changes in their sequences (presence of lysine or ornithine in position B-29, presence or absence of phenylacetyl protecting group on the amino group of lysine/ornithine or N-terminal amino group of glycine), were studied as model analytes. Separations were performed both in alkaline (pH 9.0) and in acidic (pH 2.25) background electrolytes, and the changes in the migration/retention behaviour of the model set of peptides were investigated with respect to the porphyrin periphery/central metal atom and the charge of the octapeptides modified. The key moment of successful separation of these peptides seems to be the accessibility of functional groups of the peptides to the interaction with the modifiers tested herein.     

Chemoselectivity and Conformational Analysis of the Reaction of 2‐Acetylcycloalkanones with Benzohydrazide: Synthesis and Reduction of 1‐Aroylcycloalkapyrazole Derivatives

From the reaction of 2‐acetylcyclopentanone and 2‐acetyl‐2‐methylcyclopentanone with benzohydrazide, the 1‐benzoyl‐6a‐hydroxycyclopentapyrazole derivatives 2a and 2b were obtained as the only reaction products, whereas from the reaction of 2‐acetylcyclohexanone an epimeric cis/trans mixture of the 2‐benzoyl‐3‐hydroxy‐2H‐indazole derivative 3c was formed. The dehydration of the isolated compounds 2a and 3c , as well as the NaBH₄ and NaBH₃CN reduction products of 2a were studied. The structural assignments of the compounds derived were established by analysis of their NMR spectra (¹H, ¹³C, DEPT, COSY, NOESY, HETCOR C? H, and COLOC C? H). The chemoselectivity of the reactions of 1a and 1c with benzohydrazide was studied by conformational analysis with MM2 and semiempirical (AM1 and PM3) MO calculations.     

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25 degrees C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.     

Homogeneous nucleation of droplets from supersaturated vapor in a closed system

Kinetic equations describing homogeneous nucleation kinetics within standard model are solved numerically under the condition of a constant number of molecules in the considered system. It has consequences to decrease the supersaturation of the supersaturated vapor during the process of the formation of small droplets of a new phase. The decrease of supersaturation occurs in a short time and reaches some value which remains unchanged for a relatively long time (quasistationary regime), especially at lower initial supersaturations. This time interval decreases with increasing value of the initial supersaturation. In the quasistationary regime the nucleation rate reaches its stationary value. At higher initial supersaturation, the rate of formation of nuclei goes to some maximum value corresponding to the stationary nucleation rate and then decreases with time due to the decrease of supersaturation.     

Nucleation kinetics of folded chain crystals of polyethylene on active centers

Kinetic equations describing formation of nuclei of a new phase on active centers from supercooled melt taking into account exhaustion of active centers are solved numerically. Basic characteristics of nucleation process (total number of nuclei and nucleation rate) of folded chain crystals of polyethylene at low supercooling are determined and compared with measured data. Our model gives good coincidence with experimental measurements of the total number density of nuclei. Nucleation rate reaches some quasistationary limit at sufficiently long time, which is approximately 20% of the stationary nucleation rate determined by the standard way.     

Collisions of slow polyatomic ions with surfaces: The scattering method and results

Surface-induced dissociation (SID) and reactions following impact of well-defined ion beams of polyatomic cations C₂H₅OH⁺, CH₄⁺, and CH₅⁺ (and its deuterated variants) at several incident angles and energies with self-assembled monolayers (SAM), carbon surfaces, and hydrocarbon covered stainless steel were investigated by the scattering method. Energy transfer and partitioning of the incident projectile energy into internal excitation of the projectile, translational energy of products, and energy transferred into the surface were deduced from the mass spectra and the translational energy and angular distributions of the product ions. Conversion of ion impact energy into internal energy of the recoiling ions peaked at about 17% of the incident energy for the perfluoro-hydrocarbon SAM, and at about 6% for the other surfaces investigated. Ion survival probability is about 30–50 times higher for closed-shell ions than for open-shell radical cations (e.g., 12% for CD₅⁺ versus 0.3% for CD₄⁺, at the incident angle of 60° with respect to the surface normal). Contour velocity plots for inelastic scattering of CD₅⁺ from hydrocarbon-coated and hydrocarbon-free highly oriented pyrolytic graphite (HOPG) surfaces gave effective masses of the surface involved in the scattering event, approximately matching that of an ethyl group (or two methyl groups) and four to five carbon atoms, respectively. Internal energy effects in impacting ions on SID were investigated by comparing collision energy resolved mass spectra (CERMS) of methane ions generated in a low pressure Nier-type electron impact source versus those generated in a Colutron source in which ions undergo many collisions prior to extraction and are essentially vibrationally relaxed. This comparison supports the hypothesis that internal energy of incident projectile ions is fully available to drive their dissociation following surface impact.     

Determination of the kinetic parameters of rhodanese by electrophoretically mediated microanalysis in a partially filled capillary

Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (K(m)) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N-(2-hydroxymethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mM beta-alanine-HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of K(m) for thiosulfate of 1.30 x 10(-2) M was consistent with previously published values; the K(m) for cyanide of 7.6 x 10(-3) M was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping-pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese-catalyzed reaction around 20-25 degrees C agreed with literature values.     

Influencing electroosmotic flow and selectivity in open tubular electrochromatography by tetrakis(pentafluorophenyl)porphyrin as capillary wall modifier

A physically adsorbed and covalently bonded porphyrin derivative, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, H₂TPFPP, has been used as a fused-silica capillary wall modifier in open tubular capillary electrochromatography (OT-CEC), and its influence on the electroosmotic flow (EOF) velocity and on the selectivity of OT-CEC separations of a set of model aromatic carboxylic acids has been tested. Whereas most of the coatings of this category bring about an increase in selectivity with a concomitant slow down of the EOF, H₂TPFPP coating, depending on pH of the background electrolyte used, resulted both in decreasing of EOF at pH 8.5 by 5% and in increasing of EOF by 10–43% at pH 6 and 5, respectively. The separation efficiency and the resolution of aromatic carboxylic acids separation in coated capillaries, namely in that one with covalent coating, were better than in the bare fused-silica capillary. The perspectives of H₂TPFPP as capillary wall modifier are visualized in introducing well defined electroosmotic properties of materials used for miniaturized separation channels preparation in chip-based electromigration devices.