Determination of the rotational barriers of atropisomeric polychlorinated biphenyls (PCBs) by a novel stopped-flow multidimensional gas chromatographic technique

The rotational barriers ΔG^‡ (T) of the four atropisomeric polychlorinated biphenyls (PCBs) 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95), 2,2′3,3′,4,6′-hexachlorobiphenyl (PCB 132), 2,2′,3,3′,6,6′-hexachlorobiphenyl (PCB 136), and 2,2′,3,4′,5′,6-hexachlorobiphenyl (PCB 149) were determined via on-line enantiomerization kinetics by a new stopped-flow multidimensional gas chromatographic technique (stopped-flow MDGC) employing Chirasil-Dex as chiral stationary phase for enantiomer separation. The calculated rotational barriers ΔG^‡ (T) of the trichloro-ortho-substituted atropisomers are 184 ± 2 kJ/mol for PCB 95, 189 ± 4 kJ/mol for PCB 132, and 184 ± 1 kJ/mol for PCB 149 at 300°C. The rotational barrier ΔG^‡ (T) of tetrachloro-ortho-substituted PCB 136 is at least (or higher than) 210 kJ/mol at 320°C. Chirality 10:316–320, 1998. © 1998 Wiley-Liss, Inc.     

Novel direct access to enantiomerization barriers from peak profiles in enantioselective dynamic chromatography: enantiomerization of dialkyl-1,3-allenedicarboxylates

The axially chiral allenes dimethyl-1,3-allenedicarboxylate 1 and diethyl-1,3-allenedicarboxylate 2 show characteristic plateau formation during enantioselective GC separation on the chiral stationary liquid phase Chirasil-beta-Dex. The elution profiles, obtained from temperature-dependent dynamic GC (DGC) experiments (1: 100-140 degrees C; 2: 110-150 degrees C) were evaluated with the recently derived approximation function (AF) k1(approx) = f(t(R)(A),t(R)(B),w(h)(A),h(plateau), N) to yield the enantiomerization rate constant directly k(1). These values were compared with those obtained by computer-aided simulation with ChromWin. The Eyring activation parameters of the experimental interconversion profiles were determined to be: DeltaG(#)(298.15 K) = 103.6 +/- 0.9 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.4 kJ mol(-1), DeltaS(#) = -198 +/- 7 J K(1) mol(-1) for dimethyl-1,3-allenedicarboxylate 1, and DeltaG(#)(298.15 K) = 103.5 +/- 1.1 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.5 kJ mol(-1), DeltaS(#) = -197 +/- 9 J K(-1) mol(-1) for diethyl-1,3-allenedicarboxylate 2. The approximation function (AF) presented here allows the fast determination of rate constants k(1) and activation barriers of enantiomerization DeltaG(#) from chromatographic parameters without extensive computer simulation.     

Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on‐column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3‐phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N² position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N² position on enantiomerization kinetics. However, methylation on N⁴ position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Determination of polyhydroxyalkanoates in activated sludge by ion chromatographic and enzymatic methods

Two new detection methods for the determination of poly-beta-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete recoveries of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.     

Determination of enantiomerization barriers of hypericin and pseudohypericin by dynamic high‐performance liquid chromatography on immobilized polysaccharide‐type chiral stationary phases and off‐column racemization experiments

Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high‐performance liquid chromatography (HPLC) using immobilized polysaccharide‐type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar‐organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5‐dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5‐dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on‐line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (ΔG) of 97.6–99.6 kJ/mol for both compounds (investigated temperature range 25–45°C). Complementary variable temperature off‐column (i.e., in solution) racemization experiments delivered ΔG = 97.1–98.0 kJ/mol (27–45°C) for hypericin and ΔG = 98.9–101.4 kJ/mol (25–55°C) for pseudohypericin. An activation enthalpy of ΔH^# = 86.0 kJ/mol and an activation entropy of ΔS^# = ?37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and ?82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions—at least as long as stereoselective intermolecular interactions with other chiral entities are absent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Fast and precise access to enantiomerization rate constants in dynamic chromatography

An analytical solution of the unified equation to evaluate elution profiles of interconverting enantiomers in dynamic chromatography is presented. Rate constants k1 and k(-1) and Gibbs activation energies are directly obtained from the chromatographic parameters (retention times tR A and tR A of the interconverting enantiomers, the peak widths at half height wA and wB, and the relative plateau height hp), and the initial amounts A0 and B0 of the enantiomers without any iterative and time consuming computational step. Therefore, this equation is no longer limited to racemic analytes. The analytical solution presented here was validated by comparison with a dataset of 125,000 simulated elution profiles of enantiomerizations. Furthermore, it was found that the recovery rate from a defined dataset is on average 40% higher using the unified equation compared to evaluation methods based on iterative computer simulation. The new equation was applied to determine the enantiomerization rate constant of 1-n-butyl-2-tert-butyldiaziridine by enantioselective gas chromatography. The activation parameters (DeltaH(double dagger) = 112.6 +/- 2.5 kJ/mol and DeltaS(double dagger) = -27 +/- 2 J/(K mol) were obtained from temperature-dependent measurements between 100 degrees C and 140 degrees C in 10K steps.     

Determination of 3,4-dioxyphenyl-L-alanine by spectrophotometric and chromatographic methods in enzymatic synthesis using microbial cells

A possibility of using the ninhydrin reaction for 3,4-dihydroxyphenyl-L-alanine (DOPA) determining during the synthesis from pyrocatechol and ammonium pyruvate was verified by using free and immobilized cells of Citrobacter freundii, st. 62. Spectrophotometric assay was performed at the adsorption maximum for the DOPA-ninhydrin complex at 390 nm. DOPA can be reliably quantified in the presence of all components at 20-fold and greater dilution of the reaction mixture. A high-sensitive quantitative assay of the reaction mixture was developed based on phase-reversed HPLC. A quantitative correlation was observed between spectrophotometric and chromatographic assays. The assays were employed to study in detail the initial period and equilibrium of DOPA synthesis and its characteristic features, which made it possible to construct a kinetic model of the process.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.     

Purification of plasmid DNA by chromatographic methods

Chromatographic methods have been used to purify the DNA of plasmid RP1. DNA was purified in two stages. DNA was precipitated by ethanol and separated from RNA and proteins in Sepharose 4B column after lysis of plasmid containing cells by alkaline solution of sodium dodecylsulphate. Separation of the total DNA preparation and isolation of plasmid DNA was achieved at the second stage by chromatography on the hydroxyapatite column. The resulting purified plasmid DNA was free of RNA, protein and linear fragments of chromosomal DNA. The plasmid DNA kept intact native structure and possessed the transforming activity. The DNA of RP1 yield after purification by the described technique presented 70-80 micrograms per g of wet biomass.     

Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods

The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 × 10² s^?1, (4.5–9.3) S^?1, and (2–5) × 10^?3 s^?1. In the fastest phase, a substantial amount of secondary structure (40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group. © 1994 Wiley-Liss, Inc.     

The separation of enantiomeric sugars by chromatographic methods

This paper has reviewed the number of chromatographic methods by which one may determine the absolute configuration of sugars. Both indirect methods (converting the enantiomeric pair into diastereomers) and direct methods (using chiral stationary phases) have been discussed. Resolving reagents for the indirect methods include chiral hydroxy compounds, chiral amines, and chiral thiols; with subsequent separation of the diastereomers either by gas-liquid chromatography or by high pressure liquid chromatography. Direct methods discussed have exclusively utilized chiral substitution of organopolysiloxane phases for the separation of enantiomeric sugars as volatile derivatives by gas-liquid chromatography.     

Determination of the dead time of a stopped-flow fluorometer

This investigation was carried out to develop a convenient alternative method for examining the performance and determining the dead time of a stopped-flow fluorometer. We examined the kinetics for the formation of the fluorescent Mg2+-8-hydroxyquinoline chelate in aqueous solutions. The reversible association of the Mg2+ ion with 8-hydroxyquinoline is a second-order process whose on and off rate constants are dependent on pH. We estimated that the Mg2+ ion chelate has a fluorescence quantum yield of 0.02 in aqueous solutions. Using this reaction we measured the dead time of a stopped-flow fluorometer at different pH values. Measurements of the dead time were found to be reproducible and accurate. The Mg2+-8-hydroxyquinoline reaction fulfills the requirements for a convenient test reaction for dead time measurement of stopped-flow fluorometers. Although the usefulness of the reaction is primarily to determine the dead times of stopped-flow instruments operating in the fluorescence mode, the reaction can also be used for testing an instrument operating in the absorbance mode.     

Characterization of ozonated vegetable oils by spectroscopic and chromatographic methods

In this work the effect of ozonation on olive oil, soybean oil, oleic-, linoleic- and linolenic acid was studied. The effects of ozonation time on the oils and acids were analyzed by 1H, 13C NMR. Further, the peroxide- and acid values, the viscosity and the molar mass were determined for pure and ozonated oils. The fatty chains in both ozonated oils showed a gradual decrease of unsaturation with the gradual increase of ozonation time. Reaction products were identified according to Criegee mechanism. The major product in the early stage of the reaction was ozonide. The disappearance of unsaturation and formation of ozonide was almost equal. Ozonation increased the peroxide and acid values for both oils, the increase being higher for soybean oil. After long ozonation times higher molar mass species, as well as low molar mass species were observed. These are interpreted as oligomeric ozonides and cross-ozonides, respectively.     

Determination of the enantiomerization energy barrier of some 3-hydroxy-1,4-benzodiazepine drugs by supercritical fluid chromatography

In this study, nucleotide adsorption-desorption behaviour of boronic acid-carrying uniform, porous particles was investigated. The particles were produced by a "multi-step microsuspension polymerization" in the form of poly(styrene-vinylphenyl boronic acid-divinylbenzene) terpolymer. In the first step of the production method, uniform polystyrene latex particles (6.2 microm in size) were obtained by dispersion polymerization. These particles were first swollen by a low molecular mass organic agent (i.e. dibutylphthalate, DBP) and then by a monomer mixture including styrene (S), 4-vinylphenyl boronic acid (VPBA) and divinylbenzene (DVB). The particle uniformity was protected in both swelling stages by adjusting DBP/polystyrene latex and monomer mixture/polystyrene latex ratios. Polymerization of the monomer mixture in the swollen seed particles provided boronic acid-carrying uniform, porous particles 11-12 microm in size. To have uniform particles with different porosities and boronic acid contents, the feed concentration of boronic acid-carrying monomer and the monomer/seed latex ratio were changed. The particles were tried as sorbent for the adsorption of a model nucleotide (i.e., beta-nicotinamide adenine dinucleotide, beta-NAD). In the beta-NAD adsorption experiments, the maximum equilibrium adsorption was obtained at pH 8.5 which was very close to pKa of boronic acid. The incorporation of boronic acid functionality provided a significant increase in the beta-NAD adsorption. In contrast to plain poly(styrene-co-divinylbenzene) particles, four-fold higher beta-NAD adsorption was obtained with the boronic acid functionalized particles. Beta-NAD was desorbed from the particles with the yields higher than 90% by weight.     

Determination of adenosine 5'-triphosphate by the luciferase system with a stopped-flow spectrometer.

A stopped-flow spectrometer is used for ATP assay by firefly luciferase-luciferin method. It allows one to record initial rise of the light intensity and to differentiate the light produced due to the conversion of ADP to ATP by nucleoside diphosphokinase in the firefly lantern when other nucleoside triphosphates are present. Addition of luciferin (0.27 mm) to luciferase extract increases the light intensity by a factor of 50–100. This method can be used to measure ATP in the picomole range.     

Direct calculation and computer simulation of the enantiomerization barrier of oxazepam in dynamic HPLC experiments--a comparative study

Dynamic chromatographic methods constitute a versatile approach to the rapid and precise determination of enantiomerization barriers of stereolabile drugs. In the present study enantioselective dynamic high-performance liquid chromatography (DHPLC) was employed to determine the enantiomerization barrier of oxazepam. Dynamic elution profiles, exhibiting plateau formation and/or peak broadening between 20 and 60 degrees C at pH 2.6 and pH 8 were obtained in the presence of the chiral stationary phase (CSP) Nucleodex-beta-PM (permethylated beta-cyclodextrin chemically bonded to silica) using a 6:4 mixture of phosphate buffer and methanol as mobile phase. Evaluation of the experimental chromatograms was performed by the novel approximation function (AF) (without computer simulation), and by the stochastic model implemented in the ChromWin simulation software (with computer simulation) furnishing the respective apparent forward rate constants, k(1)(app)(T). From the rate constants, k(1)(app)(T), measured at variable temperatures, the kinetic Eyring activation parameters, deltaG(T)(#), deltaH(#) and deltaS(#), of the enantiomerization of oxazepam were obtained. By variation of the flow rate of the mobile phase, the expected independence of the enantiomerization barrier from the chromatographic time scale was demonstrated for the first time.     

Measurement of DNA oxidation in human cells by chromatographic and enzymic methods

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG.     

Determination of phenylpropionic acid in fermentation medium by chromatographic techniques

Phenylpropionic acid was determined in the fermentation medium by gas chromatography using an internal standard and by reversed phase high-performance liquid chromatography. The two methods were evaluated by using the standard quantitative criteria employed in analytical chemistry.