HPLC-MS separation and detection of dialkyl phosphonates and trialkyl phosphites in reaction mixtures of 1-alkanols with phosphorous trichloride

The interaction of aliphatic alcohols ROH with phosphorus trichloride in the absence of bases leads to the formation of exclusively dialkyl phosphonates (RO)2PHO. To obtain trialkyl phosphites (RO)3P, insufficiently described to date, the pH of the reaction mixtures was adjusted by adding N,N-dimethylaniline (pKa 5.1 ± 0.1). The identification of trialkyl phosphites directly in reaction mixtures without preparative isolation implies a joint consideration of their mass spectra and gas chromatographic retention indices, provided that all other components of such mixtures are identified. The need to comply with this condition has led to the identification of several homologues of a previously uncharacterized series of 2-alkoxy-1,3,2-dioxaphospholanes, formed from a random admixture of ethylene glycol. The use of such combined mass spectrometric parameters as homologous increments of retention indices helps to distinguish trialkyl phosphites from isobaric dialkyl phosphonates; the compounds of both series belong to the homologous group of y = 12, y ≡ M(mod14). Despite the absence of molecular ion signals in the electron ionization (EI) mass spectra, the molecular weights of trialkyl phosphites can be estimated. In addition, a comparison of the homologous increments of retention indices shows that, compared with dialkyl phosphonates, trialkyl phosphites are significantly less polar.

A heuristic approach to optimization of complex physicochemical processes in the form of a genetic algorithm for solving problems is presented. In comparison with other evolutionary methods, the genetic algorithm allows working with large search spaces and complex evaluation functions, which is especially important in the study of multifactor physicochemical systems. Due to the relatively high need for computing resources when working with large and complex search spaces, optimization of existing calculation organization schemes has a positive effect on the accuracy of the calculated results. The paper presents a modified genetic algorithm that minimizes the number of iterations to achieve a given accuracy when solving the problem of finding the optimal composition of the initial reaction mixture. For a complex physicochemical process, an optimization problem is formulated which consists in finding the composition of the initial reaction mixture that promotes maximization (or minimization) of a given target parameter. The optimality criterion is determined by the type of the problem being solved and, when organizing calculations, is focused on the maximum yield of the target product. The main steps of implementing the genetic algorithm include creating an initial set of solutions and subsequent iterative evaluation of their quality for subsequent combination and modification until optimal values are achieved using mechanisms similar to biological evolution. To improve the efficiency of the method and reduce the number of iterations, a modification of the genetic algorithm is proposed which boils down to a dynamic estimate of the “mutation” parameter, depending on the diversity of individuals in the formed population of solutions. In a series of computational experiments, an analysis was made of the influence of the genetic algorithm parameters on the accuracy and efficiency of solving the problem using the example of studying the kinetics of the Michaelis-Menten enzymatic reaction. The results of calculations to determine the optimal composition of the reaction mixture showed that the dynamic determination of the “mutation” parameter contributes to an increase in the accuracy of the problem solution and a multiple decrease in the relative error value reaching 0.77 % when performing 200 iterations and 0.21 % when performing 400 iterations. The presented modified approach to solving the optimization problem is not limited by the type and content of the studied physicochemical process. The calculations performed showed a high degree of influence of the “mutation” parameter on the accuracy and efficiency of the problem solution, and dynamic control of the value of this parameter allowed increasing the speed of the genetic algorithm and reduce the number of iterations to achieve an optimal solution of a given accuracy. This is especially relevant in the study of multifactorial systems when the influence of parameters is non-trivial.

Various literature studies show that increasing the concentration of free acid in the hot injection synthesis of colloidal nanocrystals raises the diameter of the resulting nanocrystals. We analyze this reaction chemistry/nanocrystal property relation by combining reaction simulations with an experimental study on a particular CdSe nanocrystal synthesis. We find that increasing the free acid concentration has the same effect on a real synthesis as raising the solute solubility in the simulations. Both lead to larger sizes and a deterioration of the size dispersion at constant reaction rate. Since free acids are used to coordinate the cation precursors in these syntheses, this leads to a meaningful link between a parameter in reaction simulations and the composition of an experimental reaction mixture. We thus explain the increase of the nanocrystal size with the acid concentration as resulting from an enhanced consumption of the solute by nanocrystal growth, which reduces the number of nanocrystals formed. This link between a simulation parameter and the composition of the reaction mixture provides a rational basis to further explore and understand reaction chemistry/nanocrystal property relations in the hot injection synthesis.

Homologous series of alkyl esters of phosphorus-containing acids, namely, dialkyl phosphonates (synonym: dialkyl phosphites) and trialkyl phosphites, were characterized by electron ionization (EI) mass spectra and gas chromatographic retention indices (GC RI) on semi-standard non-polar stationary phases. It is confirmed that GC/MS characterization of any sets of homologues should not be restricted to independent registration of these analytical parameters. Their joint processing in the form of so-called homologous increments of GC RIs means, firstly, a novel way of evaluating the chromatographic polarity of analytes and, secondly, a new algorithm for the prediction of their molecular weights if no peaks of molecular ions are registered in the mass spectra. A standard GC/MS technique, including determination of GC RIs on semi-standard nonpolar stationary phases in temperature-programmed regime and electron ionization (EI) mass spectra was used. A total of 31 dialkyl phosphonate homologues and 37 trialkyl phosphite homologues were characterized by EI mass spectra and GC RIs for the first time. Joint processing of these analytical parameters allows the calculation of so-called homologous increments of GC RIs, which are useful parameters for evaluating the chromatographic polarity of analytes and predicting their molecular weights. GC/MS characterization of any sets of homologues should not be restricted to independent registration of the mass spectra and chromatographic retention parameters. Their joint processing in the form of so-called homologous increments of GC retention indices means, firstly, a novel way of evaluating the chromatographic polarity of analytes and, secondly, a new algorithm for predicting their molecular weights.

The treatment of a 2′,3′-O-isopropylidene nucleoside with phosphorus trichloride in the presence of trimethyl phosphate and subsequent hydrolysis gave a nucleoside 5′-phosphite in an excellent yield. Triaryl phosphates, trialkylphosphine oxides, and trialkyl phosphites were also found to be potential accelerators of the reaction. Their effects were comparable to that of trimethyl phosphate. The similar treatment of a 5′-O-acetyl nucleoside gave mixed nucleoside 2′- and 3′-phosphites in an approximate mole ratio of 4 : 6. The reaction intermediate, nucleoside phosphorodichloridite, was subjected to partial hydrolysis, giving the corresponding monochloridite. The product was readily oxidized to give the 5′-nucleotide. Chlorine was generally suitable as an oxidizing agent, but iodine gave better results for guanosine derivatives than did chlorine or bromine. The treatment of an unprotected nucleoside with phosphorus oxychloride in a trialkyl phosphate, prior to the reaction with phosphorus trichloride, could be used for the synthesis of a nucleoside 2′(or 3′),5′-diphosphate in a good yield.

Factors influencing production of cationic starches

The various esters of the phosphoric and phosphorous acids have been obtained directly from white phosphorus and aliphatic (or aromatic) alcohols under aerobic atmosphere in the presence of the CuX2 or FeX3 (X = Cl, NO3, C3H7CO2) salts. Irrespective of the variable nature of the used alcohols and catalysts, trialkyl(aryl) phosphates and dialkyl phosphites are a major products, whereas trialkyl(aryl) phosphites and dialkyl phosphates are a minor products of the phosphorylation process. Thanks to the presence of catalysts, the possible side reaction route of the radical chain oxidation of white phosphorus by oxygen to phosphorus oxides has been precluded. A comparison between the catalytic properties of CuX2 and FeX3 has been done. Although both of them have been found an efficient catalysts for the syntheses, the Cu(II) salts are active at 50-65 °C, whereas the Fe(III) based catalytic systems become competitive in terms of catalytic efficiency when reaction is carried out at 70-90 °C. Aromatic alcohols are characterised by less reactivity in this catalytic reaction as compared with an aliphatic ones. The same coordinative redox mechanism of the oxidative P-O coupling of P4 to ROH in the presence of both Cu(II) and Fe(III) catalysts has been proposed. Relevant steps of the catalytic cycle including the complexation of both white phosphorus and alcohol molecules to metal ion, the reduction of catalyst by white phosphorus, and the oxidation of reduced form of catalyst by oxygen have been also considered.

A general outline is given of a kinetic model of oxidation of a hydrocarbon under the conditions of coexistence on the catalyst surface of sections of different oxidation levels. An analytical dependence has been obtained of the selectivity of the process and conversion on the composition of the reaction mixture. A qualitative agreement has been established between the theoretical and experimental dependences of selectivity and conversion on the ratio of the benzene and oxygen concentrations in the reaction mixture.

Selective hydrogenation of acetylene in ethylene feedstocks on Pd catalysts

Reaction of 3-bromobenzyl and 3-bromoacetyl coumarin with phosphites. Synthesis of some new phosphonates and phosphates in the coumarin series

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

BioTechniquesVol. 40, No. 2 Techniques EssayOpen AccessProtein Folding Mechanisms Studied by Time-Resolved Electrospray Mass SpectrometryLars Konermann, Jingxi Pan & Derek J. WilsonLars KonermannDepartment of Chemistry, The University of Western Ontario, London, ON, Canada, Jingxi PanDepartment of Chemistry, The University of Western Ontario, London, ON, Canada & Derek J. WilsonDepartment of Chemistry, The University of Western Ontario, London, ON, CanadaPublished Online:21 May 2018https://doi.org/10.2144/06402TE01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionMass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two "soft" ionization techniques: matrix-assisted laser desorption/ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fission, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).Due to the seamless coupling to the liquid phase, ESI-MS is a powerful tool for studying chemical and biochemical processes in online experiments. The composition of a reaction mixture can be monitored continuously by direct infusion into the mass spectrometer, while the process of interest occurs in solution. The high selectivity of ESI-MS offers the unique opportunity of tracking a multitude of reactive species simultaneously. The result is a global view that provides both accurate kinetic parameters and detailed insights into reaction mechanisms (5).The ESI charge state distribution is a highly sensitive probe for detecting changes in the solution-phase conformation of proteins. In the commonly used positive ion mode, unfolded polypeptide chains acquire a larger number of protons and will thus appear at lower mass-to-charge ratios (m/z) than their folded counterparts. Presumably, this effect is related to the changes in overall compactness upon unfolding, the larger surface area of an unfolded protein, and the fact that in the native state some protonation sites may be buried inside the interior of the protein (6). Also, possible charge compensation mechanisms are being discussed (7). In addition to providing information on the protein conformation, ESI-MS allows tracking the occurrence of protein-ligand and protein-protein interactions. This is due to the gentle nature of the ionization process that often preserves the composition of noncovalent complexes, such that the ligand binding state of a protein can be deduced from the mass of the corresponding ions (Figure 1).Figure 1. Detecting changes in protein conformation and ligand binding state by electrospray ionization mass spectrometry (ESI-MS), using myoglobin as example.(A) Native myoglobin is a tightly folded heme-protein complex, exhibiting relatively low ESI charge states. The degree of protonation for the various peaks is indicated [e.g., 9+ represents (myoglobin + 9H)9+]. (B) Acid-unfolded myoglobin exhibits much higher charge states and a significantly broadened distribution. The mass of the protein ions generated under these conditions is lower, due to the loss of heme upon acid denaturation (16952 versus 17568 Da). The resulting shift in mass-to-charge ratio (m/z) is highlighted for the 9+ charge state (vertical dashed line). Free heme in panel B is denoted as H.For many proteins, folding and unfolding are reversible processes that occur spontaneously upon exposure to a suitable solvent environment, typically within a time range of milliseconds to seconds. The mechanisms of these conformational transitions continue to be a hotly debated research topic (8). Many proteins have been characterized as apparent two-state folders, while others fold through transient intermediates. Studies on folding intermediates are complicated by their short lifetimes and by the fact that they often do not accumulate to a significant extent. Nonetheless, the detection and structural characterization of these elusive species represents a very important approach for deciphering the mechanisms of folding and unfolding. While deviations from two-state behavior can sometimes be inferred from equilibrium experiments, direct information on transient folding intermediates can be gained only through kinetic investigations. Optical stopped-flow spectroscopy is one of the classical approaches for studies of this kind. Additionally, pulsed hydrogen exchange quench-flow methods, combined with off-line analysis by nuclear magnetic resonance (NMR) or MS have proven to be extremely useful (9). Limitations of these well-established methods include the poor selectivity of optical detection and the very laborious nature of quench-flow experiments, where kinetic data must be pieced together from numerous single time point measurements. Online ESI-MS methods represent an interesting alternative for mechanistic investigations on protein folding and unfolding, especially in cases where structural transitions involve changes in the ligand binding state or quaternary structure. Following a brief summary of some technical aspects, we will highlight two examples that demonstrate the use of ESI-MS for kinetic and mechanistic studies on protein conformational changes.Time-Resolved ESI-MSThe continuous-flow rapid-mixing setup depicted in Figure 2 is capable of performing kinetic experiments with ESI-MS detection on a time scale of milliseconds to minutes (10). This system represents a concentric capillary mixer with adjustable reaction chamber volume. Reactant solutions are continuously expelled from both syringes. Syringe 1 is connected to the inner capillary, whereas the solution delivered by syringe 2 flows through the outer capillary. Diffusive mixing occurs at the end of the inner capillary, thereby initiating the reaction of interest. The reaction proceeds while the mixture flows towards the outlet of the apparatus, where ESI takes place. For an average solution flow velocity va, the reaction time t is given by t = I va-1, where I is the distance between mixing point and outer capillary outlet. A detailed analysis of the measured kinetics has to take into account laminar flow effects in the reaction zone. A discussion of this subject is given in Reference 10.Figure 2. Depiction of a capillary mixer for time-resolved studies by electrospray ionization mass spectrometry (ESI-MS).Syringes 1 and 2 deliver a continuous flow of reactants; mixing of the two solutions initiates the reaction of interest. The inner capillary can be withdrawn together with syringe 1 (as indicated by the dashed arrow), thereby providing a means to control the reaction time. Small arrows indicate the direction of liquid flow. Adapted with permission from Reference 10.Kinetic experiments are performed by initially positioning the mixer directly at the ESI source. This represents the earliest time point that can be monitored (t ≈ 5 ms for a total flow rate of 100 µL/min). The assembly consisting of syringe 1, inner capillary, and mixer is then slowly pulled back by a stepper motor-driven mechanism, while solutions from the two syringes are continuously passed through the system. High voltage is applied directly to the stainless steel outer capillary, and ESI occurs at the outlet of the device, which is mounted in a custom-designed ion source. The mass spectrometer monitors the ions emitted from the source, thereby providing data at successively later time points. These experiments generate data in three dimensions: (i) the time axis is determined by the mixer position; (ii) the m/z axis provides information on the identity of the species in the reaction mixture; and (iii) the intensity axis is related to the concentration of each of the various solution-phase species. The setup depicted in Figure 2 can be interfaced with any type of ESI mass spectrometer. For the examples discussed in the following sections, a time-of-flight (TOF) instrument was used. The software routines required to operate the mass spectrometer during these kinetic experiments are analogous to those used during the acquisition of liquid chromatography-MS data. Mass spectra for specific reaction times and intensity-time profiles for selected ionic species can be extracted from the measured total ion current (TIC).Unfolding Kinetics of Large Noncovalent Protein ComplexesThe core oxygenase domain of inducible nitric oxide synthase (iNOSCOD) is a homodimeric protein complex of 103 kDa. Each of the two subunits binds a heme cofactor. In addition, two tetrahydrobiopterin (H4B) moieties are sequestered at the dimer interface. Time-resolved ESI-MS was used to study the acid-induced unfolding of iNOSCOD (11). Following a pH jump from 7.5 to 2.8, the ionic signals corresponding to the intact protein dimer show a rapid decay that is characterized by a relaxation time of 360 ms (Figure 3A). This process occurs concomitantly with the formation of monomeric heme-bound proteins that appear in a wide range of different charge states, representing solution-phase structures from tightly folded conformers all the way to significantly unfolded species (Figure 3B). The disruption of the protein-protein interactions is accompanied by loss of H4B. The heme-bound monomers represent short-lived intermediates that are depleted with a relaxation time of 620 ms. The main products of this process are highly unfolded monomeric proteins in their heme-free (apo) form (Figure 3C). In simple terms, the denaturation of iNOSCOD can be described as a sequential process. Disruption of the native protein dimer generates monomeric species that subsequently undergo unfolding with concomitant loss of heme. A detailed global analysis of the kinetic data reveals a number of additional steps and parallel processes that cannot be discussed here due to space constraints (11). When considered in the context of other recent studies (12), the iNOSCOD kinetics shown here suggest that the occurrence of complex reaction mechanisms involving short-lived intermediates is a common feature for the denaturation of large noncovalent protein complexes.Figure 3. Unfolding kinetics of the core oxygenase domain of inducible nitric oxide synthase (iNOSCOD) homodimer monitored by time-resolved electrospray ionization mass spectrometry (ESI-MS).Shown in this figure are intensity-time profiles obtained for ionic signals representing (A) the native protein dimer, (B) the monomeric heme-containing (holo) reaction intermediates, and (C) the unfolded heme-free (apo) monomers. Red lines represent global fits to the experimental data. cps, counts per second; m/z, mass-to-charge ratio. Adapted with permission from Reference 11.Detection of Hidden Folding IntermediatesAn extension of the experimental approach described above is the use of time-resolved ESI-MS in conjunction with online hydrogen/deuterium exchange (HDX). For this type of experiment, a second mixer is added to the outlet of the device depicted in Figure 2, which allows an approximately 25-ms pulsed HDX labeling step to take place immediately prior to ESI (13). This continuous-flow double-mixing approach represents a powerful tool for the detection of short-lived intermediates during folding (9). In a typical experiment, the first mixing step exposes an initially denatured protein to refolding conditions for a variable amount of time. Subsequently, HDX is initiated by mixing the protein with deuterium oxide (D2O) under rapid exchange conditions (i.e., at slightly basic pD). During this step, labile hydrogens in amide groups and amino acid side chains undergo isotope exchange. Every single HDX event increases the protein mass by 1 Da, such that the extent of labeling can be determined directly from the mass shifts of the protein ions in the spectrum. Importantly, this isotope labeling occurs in a structurally sensitive manner. Largely unfolded proteins have a considerable number of exchangeable hydrogen atoms exposed to the solvent and will therefore show high exchange levels. In contrast, more tightly folded protein structures experience a lower number of exchange events, resulting in a smaller mass shift. The HDX level and ESI charge state distribution represent complementary structural probes; the former reports on the intactness of the hydrogen bonding network and the accessibility of exchangeable sites, whereas the latter monitors the overall compactness of the protein.The question whether the folding kinetics of ubiquitin (molecular weight 8.5 kDa, 144 exchangeable hydrogen atoms) follow a simple two-state mechanism or whether there is evidence for the occurrence of a folding intermediate is currently being debated in the literature (13). Our group studied the refolding of this protein (initially denatured in methanol/water at pH 2.0) using time-resolved ESI-MS with online pulsed HDX. Prior to refolding, the protein shows an ESI mass spectrum exhibiting a broad charge state distribution with a maximum around 11+ (Figure 4A). The transition to the native state was triggered by a change in solution conditions to pH 10.0. Mass spectra acquired during refolding have a bimodal appearance. They exhibit a relatively broad charge state distribution centered at 9+ and a more narrow one encompassing the 6+ and 5+ charge states. Refolding of the protein is reflected in a gradual intensity decrease of the protein ions in charge states around 9+ and a concomitant increase of the 6+ and 5+ ions (Figure 4, B and C).Figure 4. Ubiquitin folding monitored by time-resolved electrospray ionization mass spectrometry (ESI-MS) with online pulsed hydrogen/deuterium exchange (HDX).(A) Spectrum of the unfolded protein prior to triggering the conformational transition. Spectra for reaction times of 40 ms and 3.3 s are shown in panels B and C, respectively. (D and E) Mass shift distributions resulting from HDX for the 9+ (blue dashed line) and 5+ (red solid line) charge states for reaction times of 40 ms and 3.3 s. (F) Proposed three-state folding mechanism of ubiquitin. D, denatured ubiquitin; D*, folding intermediate; F folded state. The charge state distributions (CSDs) of D* and Fare indistinguishable; D and D* exhibit the same HDX behavior. Only the combination of HDX and CSD as nonredundant structural probes allows the folding intermediate D* to be detected. m/z, mass-to-charge ratio; t, time. Adapted with permission from Reference 13.After pulsed HDX, the mass distributions observed for charge states around 9+ exhibit a large shift of 83 (±4) Da relative to unlabeled ubiquitin (Figure 4, D and E, blue dashed curves). For t = 3.3 s, the 6+ and 5+ ions exhibit a much smaller shift of 58 (±2) Da (Figure 4E, red curve). Interestingly, however, for t = 40 ms the HDX behavior observed for the 6+/5+ charge states coincides with that observed for the 13+ to 7+ signals (Figure 4D). It is concluded that the refolding of ubiquitin under the conditions studied here involves three kinetic species: (i) the denatured state D (charge states around 9+, large mass shift); (ii) a folding intermediate D* (charge states 6+/5+, large mass shift); and (iii) the folded state F (charge states 6+/5+, small mass shift). The fact that D and D* show indistinguishable HDX characteristics suggests that these two species undergo rapid interconversion during the labeling pulse. This implies that D and D* are separated by a relatively low energy barrier. In contrast, the transition to F occurs on a slower time scale, which is indicative of a major barrier. A schematic depiction of a possible reaction mechanism is given in Figure 4F.In summary, the refolding of ubiquitin under the conditions used here is not a simple two-state (D → F) process. Time-resolved ESI-MS with online HDX reveals the presence of an intermediate species. This result is in line with the notion that the occurrence of folding intermediates is more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior (13).ConclusionsThe examples discussed above highlight the versatility of time-resolved ESI-MS as a tool for studying protein folding and unfolding on the time scale of milliseconds to seconds. The high selectivity of ESI-MS allows the kinetic behavior of multiple conformations and ligand binding states to be monitored simultaneously, thereby providing detailed insights into reaction mechanisms that may involve short-lived intermediates and parallel path-ways. The inclusion of an online pulsed HDX step adds a further dimension to the experiment that greatly facilitates the detection of transiently populated species. Time-resolved ESI-MS is not restricted to studies on protein folding and unfolding; other interesting applications include the exploration of enzymatic and bio-organic reaction mechanisms (14). One current limitation of all online ESI-MS techniques is their relatively low tolerance for nonvolatile salts and some other solvent additives. However, the use of ultrarapid microfluidic desalting devices and other recently developed strategies will help to resolve this issue (15). In any case, it seems certain that MS-based techniques are about to become an indispensable tool for a wide range of kinetic applications in biological chemistry.AcknowledgmentsThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), the Province of Ontario, and the Canada Research Chairs Program.

Transesterification does not allow to make full conversion of oil to biodiesel because the by-product glycerol cannot be included in the composition of biofuel. Interesterification constitutes a full conversion process with production of triacetin (TA) instead of glycerol, which can be included in the composition of biofuel and allows to increase its yield. Both interesterification and transesterification effectively occur only in presence of catalysts. Results of the investigation of heterogeneous and homogeneous catalysts indicate the superior importance of catalyst solubility in starting reaction mixture. Partial solubility can remarkably lower the activity of homogeneous catalyst and extremely increase that of formally heterogeneous one. The reaction mixture of interesterification reaction is less polar than that of transesterification, and potassium tert-butoxide (t-BuOK) should be more appropriate catalyst for interesterification than sodium methoxide which is used in most cases. The catalytic system t-BuOK/t-BuOH substantially increases the yield of TA and changes the properties of obtained biofuel. Whereas the content of the TA in the interesterification reaction mixture does not achieve the same level from the theoretically predicted as the FAME, the occurrence of side reaction between t-BuOH and TA cannot be excluded. This paper presents a study of the interesterification of rapeseed oil in presence of catalytic system t-BuOK/THF (catalytic system without alcohols) with the aim of establishing the influence of aprotic tetrahydrofuran to the proceeding the reaction, composition of reaction mixtures and their fuel properties. Obtained results show that the absence of alcoholic hydroxyl groups in the catalytic system insufficiently increases the activity of catalytic system but fails to increase the yield of FAME.

Reversed‐phase high performance liquid chromatography (RP‐HPLC) was employed to develop predictive models for fish bioconcentration factors (BCF) of organic compounds. Estimation of BCF from RP‐HPLC retention parameters on octadecyl‐bonded silica gel (ODS), cyanopropyl‐bonded silica gel (CN), and phenyl‐bonded silica gel (Ph) columns were investigated. The results show that, for a set of compounds belonging to different chemical classes, the CN stationary phase is the best one among the three columns and better than n‐octanol/water model for BCF estimation. A multi‐column RP‐HPLC model, using the retention parameters on the CN and Ph columns as the variables of multiple linear regression equations, was further evaluated to estimate BCF of organic compounds belonging to different chemical classes, and the results show that the multi‐column RP‐HPLC model is better than that of any single RP‐HPLC column for BCF estimation.

Several monolithic macroporous polymer sorbents (pore size 1–2 µm) based on alkyl methacrylates and ethylene glycol dimethacrylate as a cross-linking agent were prepared by free radical copolymerization in columns 3×150 mm. The influence of compositions of the reaction mixture and porogens and the nature of the alkyl radical in a mixture of monomers on the hydrodynamic and chromatographic characteristics of the monoliths was studied. The monoliths based on n-butyl methacrylate have rigid macroporous morphology and excellent hydrodynamic characteristics (flow rate up to 5 mL min−1). The efficiency of separation of a mixture of benzene and its derivatives in the version of reversed-phase HPLC was shown to increase with an increase in the fraction of a lauryl methacrylate additive (LMA) in the reaction mixture. The maximum separation efficiency (number of theoretical plates (tp)) was 35 000 tp m−1 for the monolith based on n-butyl methacrylate with 7% LMA in the reaction mixture.