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Wyniki wyszukiwania dla: PCR (POLYMERASE CHAIN REACTION)
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Detection of Blastocystis hominis in unpreserved stool specimens byusing polymerase chain reaction
PublikacjaBlastocystis hominis jest pasożytem występującym na całym świecie. Patogenność tego pasożyta nie jest jeszcze w pełni udowodniona, aczkolwiek opisanych jest wiele przypadków, sugerujących, że B. hominis może wywoływać objawy żołądkowo-jelitowe. Wykrywanie tego pasożyta opiera się na bezpośredniej obserwacji mikroskopowej rozmazów i zagęszczonych próbek kału lub rozmazów utrwalonych, jednakże w pewnych przypadkach nie jest to możliwe.Celem...
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A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction
PublikacjaDNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference.We determined DNA methylation levels in several...
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The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis
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Detection ofHelicobacterspecies in liver and stomach tissues of patients with chronic liver diseases using polymerase chain reaction-denaturing gradient gel electrophoresis and immunohistochemistry
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Detection of Helicobacter rodentium-like DNA in the liver tissue of patients with chronic liver diseases by polymerase chain reaction–denaturing gradient gel electrophoresis and DNA sequence analysis
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PCR-ELISA: inexpensive alternative to quantitative PCR
PublikacjaPCR-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay), a combination of PCR and ELISA methods, has been used since late 1980s. The technique is based on specially labelled DNA fragments which are captured by specific DNA sequences and detected by antibodies. The whole procedure of PCR-ELISA is divided into three steps: DNA extraction, PCR reaction and detection by ELISA. The method has been found as very specific...
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Increased concentration of Taq DNA polymerase as a solution for GC-rich templates from clinical and environmental samples
PublikacjaDNA polymerase is an enzyme which plays crucial role in replication and DNA repair. It found application in PCR (polymerase chain reaction) where catalyses process of in vitro DNA synthesis. To meet the demands posed by mod- ern diagnostic, molecular biology or genetic engineering it is necessary to improve DNA polymerases to obtain new or better features useful in these fields. So far implemented modifications in majority are...
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Techniki biologii molekularnej w analityce produktów mięsnych
PublikacjaW pracy przedstawiono możliwości wykorzystania reakcji PCR (Polymerase Chain Reaction) do identyfikacji patogennych drobnoustrojów a także do badania składu surowcowego w mięsnych produktach żywnościowych.
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Fuzyjne polimerazy DNA – otrzymywanie, charakterystyka i zastosowanie
PublikacjaObecnie reakcje PCR (ang. Polymerase Chain Reaction) wykazują bardzo szerokie zastosowanie w diagnostyce medycznej, biologii molekularnej czy inżynierii genetycznej. Efektywność tych reakcji rozumiana jako wydajność i wierność przeprowadzonej amplifikacji jest nieodłącznie związana ze stosowaną polimerazą DNA i warunkami prowadzenia reakcji PCR. Aby sprostać wymaganiom stawianym przez nowoczesne metody diagnostyczne oraz współczesną...
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A simple modification of PCR thermal profile applied to evade persisting contamination
PublikacjaThe polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganisms Based on the Use of a Class IIS Restriction Enzyme and Ligation Mediated PCR
PublikacjaThis study details and examines a novel Ligation-Mediated - Polymerase Chain Reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s)...
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PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera
PublikacjaFungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based...
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5-Bromo-2′-deoxycytidine—a potential DNA photosensitizer
PublikacjaA double-stranded oligonucleotide, 80 base pairs in length, was multiply labeled with 5-bromo-2′-deoxycytidine (BrdC) using polymerase chain reaction (PCR). The modified oligonucleotide was irradiated with 300 nm photons and its damage was assayed by employing DHPLC, LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). Two types of damage were demonstrated, namely, single strand breaks (SSBs) and intrastrand cross-links...
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Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.
Dane BadawczeEnzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes...
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Clinical studies of detecting COVID-19 from exhaled breath with electronic nose
PublikacjaThe COVID‑19 pandemic has attracted numerous research studies because of its impact on society and the economy. The pandemic has led to progress in the development of diagnostic methods, utilizing the polymerase chain reaction (PCR) as the gold standard for coronavirus SARS‑CoV‑2 detection. Numerous tests can be used at home within 15 min or so but of with lower accuracy than PCR. There is still a need for point‑of‑care tests available...
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Phylogenetic trees of genus Oncidium Sw. based on analysis of DNA sequences
Dane BadawczeGenus Oncidium Sw. is widely regarded as a polyphiletic, and the taxonomic boundaries between him and such genera as Odontoglossum Kunth. or Miltonia Lindley remain blurred. The goal of the study was to determine the phylogenetic relationships within the genus Oncidium s.lato based on the DNA sequences analysis. The correlation between molecular data...
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Resistance of Escherichia coli and Enterococcus spp. to selected antimicrobial agents present in municipal
PublikacjaIn this study, the susceptibility to erythromycin (E) and to trimethoprim/sulfamethoxazole (SXT) among isolates of Enterococcus spp. and Escherichia coli was tested, respectively. Both fecal indicators were detected and isolated from raw(RW) and treated wastewater (TW) as well as from samples of activated sludge (AS) collected in a local wastewater treatment plant (WWTP). Biodiversity of bacterial community in AS was also monitored...
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New Approaches for Escherichia coli Genotyping
PublikacjaEasy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing...
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Taq DNA polymerase fused with DNA binding protein with increased resistance to inhibitors from clinical samples
PublikacjaNowadays PCR method is commonly used in molecular diagnostic. However, in many cases PCR is limited, by the presence of inhibitory substances in biological, soil or food samples Efficiency and fidelity of amplification is strongly connected with DNA polymerase and reaction conditions. To meet the requirements of modern diagnostic methods it is essential to seeking for new DNA polymerases with better properties useful in these field....
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Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets
PublikacjaThe DNA coding sequence of TaqStoffel polymer- ase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the Taq DNA polymerase from a relatively low processive...
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Real-time isothermal DNA amplification monitoring in picoliter volumes using an optical fiber sensor
PublikacjaRolling circle amplification (RCA) of DNA can be considered as a great alternative to the gold standard polymerase chain reaction (PCR), especially during this pandemic period, where rapid, sensitive, and reliable test results for hundreds of thousands of samples are required daily. This work presents the first research to date on direct, real-time and label-free isothermal DNA amplification monitoring using a microcavity in-line...
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Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana
PublikacjaBACKGROUND: In recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.RESULTS: We report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (TmaSSB) and Thermotoga neapolitana (TneSSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino...
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Modified DNA polymerases for PCR troubleshooting
PublikacjaPCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase...
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B-GALACTOSIDASE ARTHROBACTER SP. 32cB - OBTAINING THE GENE SEQUENCE, CONSTRUCTION OF THE EXPRESSION SYSTEM, BIOSYNTHESIS AND BIOCHEMICAL CHARACTERIZATION OF THE ENZYME
PublikacjaINTRODUCTION: β-Galactosidase is an enzyme which catalyzes the hydrolysis of O glycosidic bond in β-galactosides. Another activity of β galactosidase is a transglycosylation activity. The main industrial use of this protein is the hydrolysis of lactose in milk in a cooling conditions. Synthesis of galactooligosaccharides, which are mostly used as a prebiotics added to some foods or available as dietary supplements, is only one...
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Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization
PublikacjaDNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the...
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ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping
PublikacjaA DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide....
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Real-Time PCR: molecular technique of many applications
PublikacjaReal-Time PCR is a sensitive DNA amplification technique initially applied in genetics and molecular biology. It enables in vivo copying of the selected DNA fragment (flanked by two primers) by the thermostable polymerase (in the presence of magnesium ions and deoxynucleotide triphosphates) and simultaneous measurement of the fluorescence. For one or more specific sequences in a DNA sample, real-time PCR enables both detection...
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Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors
PublikacjaIn this study, denitrification of ammonium- reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory- scale ‘‘fill-and-draw’’ reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using...
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Molecular identification and genotyping of Staphylococci: genus, species, strains, colnes, lineages, and interspecies exchanges
PublikacjaStaphylococci are increasingly recognized as etiological agents of many opportunistic human and animal infections, emphasizing the need for a rapid and accurate identification, even to a genotypical level of these bacteria. In the recent years, there has been a significant progress in typing and phylogenetic study of Staphylococcus species. Here, we describe molecular methods used in taxonomy as well as staphylococci characterization....
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Mechanism of reaction of RNA-dependent RNA polymerase from SARS-CoV-2
PublikacjaWe combine molecular dynamics, statistical mechanics, and hybrid quantum mechanics/molecular mechanics simulations to describe mechanistically the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp). Our study analyzes the binding mode of both natural triphosphate substrates as well as remdesivir triphosphate (the active form of drug), which is bound preferentially over ATP by RdRp while...
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Insights into the microbial community of treated wastewater, its year-round variability and impact on the receiver, using cultivation, microscopy and amplicon-based methods
PublikacjaApart from chemical constituents, wastewater treatment plant (WWTP) effluents also release microorganisms that can be important to the receiving water bodies either from a sanitary point of view, or taking to the account the biogeochemical potential of the recipients. However, little is known about the treated wastewater microbial community, its composition, seasonal changes, functions and fate in the waters of the receiver. Thus,...
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D-loop sequences retrieved from Canis lupus familiaris mitochondrial genome
Dane BadawczeCanine mitochondrial genome is built of 16727 bp. Non-coding control region (mtCR), called also D-loop, begins with 15458 nucleotide and ends with 16727 nucleotide. The length of this fragment is 1270 bp (Kim et al., 1998). D-loop region is responsible for replication and transcription of mitochondrial DNA. Mutations that occur within it may cause irregularity...
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Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation
PublikacjaThe 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel...
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Synthesis of oxathiolone fused chalkones bearing O-aminoalkyl side chain. Comparison of stability of isomeric benzoxathiolones under alkylation reaction conditions
PublikacjaIzomeryczne oksatiolonowe sprzężone chalkony zawierające O-alkilowe łańcuchy boczne otrzymano w wyniku kondensacji odpowiednich benzoksatiolonów z podstawionymi benzaldehydami. Wyjściowe benzoksatiolony otrzymano w wyniku alkilowania odpowiednich fenoli chlorkami aninoalkilowymi, jednakże reakcja była często komplikowana w wyniku otwarcia pierścienia oksatiolonowego poprzez grupę aminową. Określono aktywność cytotoksyczną i przeciwdrobnoustrojową...
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A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model
PublikacjaWe have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the...
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An investigation on the role of GMA grafting degree on the efficiency of PET/PP-g-GMA reactive blending: morphology and mechanical properties
PublikacjaGlycidyl methacrylate (GMA) has been grafted on polypropylene (PP) with the aid of styrene (St) comonomer, by changing dicumyl peroxide initiator content, GMA level, and St concentration. The performance of the resulting PP-g-GMA reactive material towards static and dynamic mechanical properties of poly (ethylene terephthalate) (PET) was monitored in terms of grafting reaction variables and compatibilizer content. Fourier transform...
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Evaluation of possibilities of genotyping of Candida glabrata clinical isolates with RAPD-PCR method and microsatellite analysis
PublikacjaThe aim of the study was to compare the discriminatory power of RAPD-PCR method using RSD10 primer and microsatellite (GACA)4 analysis for genotyping clinical isolates of C. glabrata. Isolates were received from patients of two Polish hospitals: Children's Memorial Health Institute in Warsaw (n=17) and Medical University of Gdansk (n=37). Species identification was confirmed by two phenotypic methods: growth on chromagar plates...
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Effect of hydroxylated soybean oil and bio-based propanediol on the structure and thermal properties of synthesized bio-polyurethanes
PublikacjaThe aim of this work was to obtain bio-polyurethanes using synthetic compounds and bio-components, i.e. bio-glycols. Bio-polyurethanes were prepared by means of the prepolymer method. Prepolymers were synthesized from 4,4′-diphenylmethane diisocyanate (MDI) and a polyol mixture containing 75% by weight of commercial polyether and 25% by weight of hydroxylated soybean oils (H2 or H3), the latter being obtained in the reaction with...
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Loop-mediated isothermal amplification (LAMP) as a diagnostic tool in detection of infectious diseases
PublikacjaLoop-mediated isothermal amplification (LAMP) is a gene amplification method which amplifies DNA with high specificity and efficiency under isothermal conditions. Because of its rapidity and simplicity, it is a valuable diagnostic tool in the early detection and identification of infectious diseases. LAMP method is based on the use of a set of four to six specially designed primers spanning six to eight distinct sequences on the...
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PCR detection of Scopulariopsis brevicaulis
PublikacjaScopulariopsis brevicaulis is known as a most common etiological factor of the mould toenail infections. There are also reports indicating that S. brevicaulis could cause organ and disseminated infections. Nowadays microscopic observations from the direct sample and culture are crucial for the appropriate recognition of the infection. In this paper is presented a PCR-based method for S. brevicaulis detection. The specificity of...
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Molecular detection of Candida krusei
PublikacjaThe species identification of fungi belonging to genus Candida is an important issue as this genus becomes the emerging problem of nosocomial infections. As Candida krusei presents intrinsicresistance to the fluconazole that is the drug of the first choice in case of invasive candidiasis the PCR identifying the DNA of C. krusei was elaborated. The analytical sensitivity of the assay on spiked blood samples was estimated at 3-5...
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Novel application of terminal restriction fragments length polymorphism method (t-RFLP) for identification of six clinically important Candida sp.
PublikacjaTerminal restriction fragments length polymorphism method (t-RFLP) is a modified classical RFLP method. The main difference is based on carrying out PCR using a fluorescently labeled primer or primers. The result of such modification is that after digestion of amplicons only terminal fragments of PCR product (containing fluorescent dye) are visualized. T-RFLP method has found application in environmental microbiology and epidemiology....
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Synthesis, structure and properties of poly(ether-urethane)s synthesized using a tri-functional oxypropylated glycerol as a polyol
PublikacjaThe main aim of this work was to obtain poly(ether-urethane)s using tri-functional polyoxyalkylene polyol (Rokopol G1000), which introducing the chemical cross-links into the structure of polyurethanes. Poly(ether-urethane)s were prepared using two-step method, called prepolymer method, which involves in the first step the reaction of 4,4'-diphenylmethane diisocyanate (MDI) and tri-functional polypropylene glycol glycerol triether...
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Solid-State Modification of Poly(butylene terephthalate) with a Bio-Based Fatty Acid Dimer Diol Furnishing Copolyesters with Unique Morphologies
PublikacjaCopolyesters based on poly(butylene terephthalate) (PBT) and a bio-based fatty acid dimer diol (FADD) were prepared by solid-state modification (SSM). The chemical incorporation of the FADD into the backbone of the PBT was proven using various techniques. It was clear that the incorporation rate was diffusion-limited rather than reaction-limited. From quantitative 13C NMR a nonrandom chemical microstructure was obtained at all...
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Emulsifi ers from renewable materials: an eco-friendly synthesis and properties
PublikacjaThe focus of this study was the preparation of novel bio based polyglycerol emulsifiers characterized by a one pot synthesis, thus by modified properties with respect to interfacial activity and effectiveness as emulsion stabilizers. The final products of the esterification process, carried out in the presence of carboxylates were used directly as emulsifiers (without purification or fractionation). Polyglycerol emulsifiers obtained...
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Catena-Poly[[(tetrahydrofuran-[kappa]O)lithium(I)]-bis([mu]-trimethylsilanolato-[kappa]2O:O)-gallium(III)-bis([mu]-trimethylsilanolato-[kappa]2O:O)-[(tetrahydrofuran-[kappa]O)lithium(I)]-[mu]-bromido]
PublikacjaThe title chain polymer compound, [GaLi2Br(C3H9OSi)4(C4H8O)2]n, was obtained in the reaction of GaBr3 with Me3SiOLi in toluene/tetrahydrofuran. The GaIII atom, located on a twofold rotation axis, is coordinated by four trimethylsilanolate ligands and has a distorted tetrahedral geometry. The LiI atom is four coordinated by one bridging Br atom located on an inversion centre, two trimethylsilanolate ligands and one tetrahydrofurane...
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Effect of modified soybeen oil amount on rheological characteriza-tion of polyurethane pre-polymers
PublikacjaPolyurethanes (Pu’s) are the polymeric materials which have got urethane groups in the structure. The properties of Pu’s depend both on the method of preparation and monomers used. Polyurethanes are produced by two methods known as one step or two step method called as “pre-polymers method”, especially for the case of segmented polyurethanes (SPU’s). These materials are thermoplastic block copolymers of the (AB)n type consisting...
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Synthesis, structure and properties of poly(ester-urethane)s obtained using bio-based and petrochemical1,3-propanediol and 1,4-butanediol
PublikacjaIn this paper, the poly(ester-urethane)s obtained using petrochemical and bio-based chain extenders were prepared and characterized. The influence of glycols’ origin on the chemical structure, mechanical and thermal properties of the prepared polyurethanes was studied. The materials were synthesised by prepolymer method. The first step involved the reaction of α,ω-dihydroxy(ethylene-butylene adipate (POLIOS 55/20) with 4,4’-diphenylmethane...
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Application of surface active amino acid ionic liquids as phase-transfer catalyst
PublikacjaFive structurally related morpholinium derived ionic liquids containing N-acetyl-glycinate anion were synthesized and their thermal stability, surface properties and activity as phase transfer catalysts investigated. The thermal properties were studied by differential scanning calorimetry, while the adsorption at the air/water interface and micellization behavior was analyzed by surface tension measurements, conductometry and isothermal...
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Synteza, struktura chemiczna i właściwości bezizocyjanianowych poliuretanów otrzymywanych z wykorzystaniem CO2 oraz surowców pochodzenia naturalnego.
PublikacjaZ uwagi na wciąż rosnącą świadomość proekologiczną, politykę zrównoważonego rozwoju, a także nurt zielonej chemii wiodącym trendem w przemyśle tworzyw sztucznych jest poszukiwanie alternatywnych metod otrzymywania materiałów polimerowych. Komercyjnie poliuretany otrzymywane są w wyniku reakcji poliaddycji di- lub poliizocyjanianów z poliolami i małocząsteczkowymi przedłużaczami łańcucha. Z uwagi na niekorzystne właściwości diizocyjanianów,...