UV Absorption Measurements Research Articles

Complexation of bovine serum albumin with cationic polyelectrolytes at pH 7.40 – Formation of soluble complexes

Complexation of bovine serum albumin (BSA) with the cationic polymer poly([3-(methacryloylamino)propyl]trimethylammonium chloride) (PMAPTAC) and its graft copolymer with poly(N, N-dimethylacrylamide), (PMAPTAC-g-PDMAM75), was investigated at pH=7.40, in water, and in 0.15M NaCl, close to physiological conditions, as a function of the Polycation/BSA charge ratio, r. Turbidity, and UV absorption measurements, indicated the formation of insoluble stoichiometric complexes between BSA and PMAPTAC in water, but not in 0.15M NaCl. A potentiometric study, in agreement with this behavior, showed proton release for the BSA/PMAPTAC mixtures in water, but not in 0.15M NaCl. In the case of the mixtures of BSA with the graft copolymer PMAPTAC-g-PDMAM75, the solutions obtained even in water, were clear. Nevertheless, soluble complex formation in water was confirmed by potentiometry, ζ-potential and static light scattering measurements, while in 0.15M NaCl not any complex formation was detected.

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2

Porcine pancreatic phospholipase A2, a small and disulfide rich protein, is extremely resistant against chemically or thermally induced unfolding. Despite this marked resistance, the protein displays broad unfolding transitions resulting in comparatively low apparent thermodynamic stability. Broad unfolding transitions may result from undetected folding intermediates, residual structures in the unfolded state or an inhomogeneity of the native state. Using circular dichroism, fluorescence, and NMR spectroscopy, we ruled out the existence of stably populated folding intermediates, whereas UV absorbance measurements hinted at stable residual structures in the unfolded state. These residual structures proved, however, to have no impact on the folding parameters. Studies by limited proteolysis, CD, and NMR spectroscopy under non-denaturing conditions suggested pronounced dynamics of the protein in the native state, which as long as unrestrained by acidic pH or bound Ca2+ ions exert considerable influence on the unfolding transition.

Lean body mass assessment based on UV absorbance in spent dialysate and dual-energy x-ray absorptiometry.

The aim of the study was to explore the possibility of assessing lean body mass (LBM) based on UV absorbance measurements in spent dialysate. 9 patients on chronic three-times-a-week HD (4 female, 5 male, mean age 58.8 ± 8.6 years) were studied. Blood and spent dialysate samples were collected for 3 consecutive hemodialysis (HD) sessions from every patient. A double-beam spectrophotometer was used for the determination of UV absorbance in the collected spent dialysate samples. Dual-energy x-ray absorptiometry (DXA) scans were performed on an interdialytic day. LBM was calculated based on creatinine concentration in blood (LBMblood) and UV absorbance in spent dialysate (LBMa) and assessed by DXA (LBMDXA). Also, in vitro experiments were carried out to investigate the effect of tissue hydration on DXA measurements. Although LBMa was slightly lower compared to LBMblood, the estimates based on UV absorbance in spent dialysate presented greater accuracy and precision compared to LBMDXA. The significant difference between LBMblood and LBMDXA was with high probability caused by the altered tissue hydration of HD patents. In summary, the results show that it is possible to asses LBM based on UV absorbance in spent dialysate.

Chain dimensions and intermolecular interactions of polysilanes bearing alkyl side groups over the UV thermochromic temperature

To elucidate temperature change of intermolecular interactions and chain dimensions for poly(dialkylsilane)s with or without thermochromism, synchrotron-radiation small-angle X-ray scattering and UV-absorption measurements were carried out for relatively low-molar mass poly(n-hexylmethyl)silane (PSi-1), poly(n-decylmethyl)silane (PSi-2), and poly(n-hexylpropyl)silane (PSi-3) samples in isooctane over a wide temperature range down to −94 °C. While the dimensional properties for the three polysilanes were almost irrespective of temperature, the second virial coefficient for PSi-2 and PSi-3 significantly decreased and became negative below the thermochromic temperature at −60 °C and −50 °C, respectively. Analyses in terms of the quasi-two-parameter theory for the wormlike chain showed that conformational change detected by UV-absorption behavior does not cause appreciably the unperturbed chain dimensions of the polysilane samples. Furthermore, aggregates were detected by ultra-small-angle X-ray scattering measurements for high molar mass PSi-3 samples at low temperatures. These results indicate that intermolecular interactions between poly(dialkylsilane) molecules become attractive around the thermochromic temperature.

UV absorption‐based inverse modeling of protein chromatography

UV absorbance measurements play an important role in bioprocess development. Yield and purity are often evaluated in terms of peak percentages in analytical SEC or ion‐exchange chromatography. Also, industrial chromatography steps are usually controlled based on UV data with pooling decisions according to absorbance thresholds. Model‐based process development would make elaborate screening experiments redundant, once the model has been calibrated to the specific process step. So far, absorbance measurements could not be used directly for modeling chromatography steps as the commonly applied models rely on mass or molar concentration. This study presents mechanistic modeling of an industrially relevant chromatography setting without any knowledge of the feed composition. The model equations were rewritten to employ boundary conditions in UV absorbance units, the absorption coefficients were shifted into the isotherm, and standard parameter estimation procedures could be applied. An anion‐exchange chromatography case study of a target protein expressed in Escherichia coli and 11 lumped impurity peaks demonstrated practical applicability. The target protein concentration in the feed material was estimated from chromatograms. Using this method, initially unknown feed concentrations can be determined a posteriori for ion‐exchange and multimodal chromatography from single‐component absorbance curves.

Advances in inline quantification of co-eluting proteins in chromatography: Process-data-based model calibration and application towards real-life separation issues.

Pooling decisions in preparative liquid chromatography for protein purification are usually based on univariate UV absorption measurements that are not able to differentiate between product and co-eluting contaminants. This can result in inconsistent pool purities or yields, if there is a batch-to-batch variability of the feedstock. To overcome this analytical bottleneck, a tool for selective inline quantification of co-eluting model proteins using mid-UV absorption spectra and Partial Least Squares Regression (PLS) was presented in a previous study and applied for real-time pooling decisions. In this paper, a process-data-based method for the PLS model calibration will be introduced that allows the application of the tool towards chromatography steps of real-life processes. The process-data-based calibration method uses recorded inline mid-UV absorption spectra that are correlated with offline fraction analytics to calibrate PLS models. In order to generate average spectra from the inline data, a Visual Basic for Application macro was successfully developed. The process-data-based model calibration was established using a ternary model protein system. Afterwards, it was successfully demonstrated in two case studies that the calibration method is applicable towards real-life separation issues. The calibrated PLS models allowed a successful quantification of the co-eluting species in a cation-exchange-based aggregate and fraction removal during the purification of monoclonal antibodies and of co-eluting serum proteins in an anion-exchange-based purification of Cohn supernatant I. Consequently, the presented process-data-based PLS model calibration in combination with the tool for selective inline quantification has a great potential for the monitoring of future chromatography steps and may contribute to manage batch-to-batch variability by real-time pooling decisions.

Photoexcited triplet states of UV-B absorbers: ethylhexyl triazone and diethylhexylbutamido triazone.

The excited states of UV-B absorbers, ethylhexyl triazone (EHT) and diethylhexylbutamido triazone (DBT), have been studied through measurements of UV absorption, fluorescence, phosphorescence, triplet-triplet absorption and electron paramagnetic resonance spectra in ethanol. The energy levels of the lowest excited singlet (S1) and triplet (T1) states and quantum yields of fluorescence and phosphorescence of EHT and DBT were determined. In ethanol at 77 K, the deactivation process of EHT and DBT is predominantly fluorescence, however, a significant portion of the S1 molecules undergoes intersystem crossing to the T1 state. The observed phosphorescence spectra, T1 lifetimes and zero-field splitting parameters suggest that the T1 state of EHT can be assigned to a locally excited (3)ππ* state within p-(N-methylamino)benzoic acid, while the T1 state of DBT can be assigned to a locally excited (3)ππ* state within p-(N-methylamino)benzoic acid or p-amino-N-methylbenzamide. The quantum yields of singlet oxygen generation by EHT and DBT were determined by time-resolved near-IR phosphorescence measurements in ethanol at room temperature. EHT and DBT did not exhibit significantly antioxidative properties by quenching singlet oxygen, in contrast to the study by Lhiaubet-Vallet et al.

Factors influencing natural organic matter sorption onto commercial graphene oxides

Nanosized graphene materials are being considered as a class of new, high surface area sorbents suitable for water treatment applications. This study explored commercially available graphene powders of differing sizes, surface areas, and surface compositions for their ability to sorb dissolved natural organic matter (NOM) from water under varying solution conditions within batch reactors. The sorption kinetics of NOM on graphene powders were rapid and reached equilibrium within hours. Sorption isotherms for all graphenes and all NOM types were all best described with the Freundlich model. Sorption affinity improved with increasing graphene specific surface area, more graphene carbon content, greater NOM aromatic content, and lower solution pH. Graphene sorption behavior is compared to carbon nanotubes and granular activated carbon, and high surface area graphene may possess superior sorption rates and capacities, whereas low surface area graphene may be entirely ineffective. The high surface area graphene examined here also showed selectivity for the aromatic and high molecular weight NOM fractions within measurements of specific UV absorbance and size exclusion chromatography. The results suggest that aromatic interactions significantly participate in NOM binding, but that electrostatic interactions may also influence sorption capacity depending on solution pH and graphene surface charge.

Stabilization of a Bimolecular Triplex by 3'-S-Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation.

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3'-S-phosphorothiolate (3'-SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. (1) H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3'-SP modifications is more compact. The 3'-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures.

Biodegradability of anthropogenic organic matter in polluted rivers using fluorescence, UV, and BDOC measurements.

The presence of highly urbanized and polluted areas affects both the quantity and the composition of organic matter in rivers through effluent loads and urban runoff discharges in watersheds. In such context, this paper aims to evaluate the biodegradability of anthropogenic organic matter in polluted rivers. Stream water samples were collected in three different sites considering a non-impacted area, a highly urbanized site located after a sewage treatment plant, and a site downstream of the watershed. For the biodegradation experiment, two adaptations of biodegradable dissolved organic carbon (BDOC) essay were evaluated to assess the decomposition rates between 10days, with added nutrients, in the dark at 20°C. The organic matter biodegradation was monitored by distinct parameters such as dissolved organic carbon (DOC), total organic carbon (TOC), particulate organic carbon (POC), fluorescence excitation-emission matrix (EEM), and UV absorbance measurements. The measured BDOC ranged from 0.8mg/L at site IG01 (low anthropogenic occupation) to 4.2mg/L at site IG02 (high impacted area), with averaged percentage of initial DOC ranging from 20 to 56%, while an average of 28% up to 95% of POC can be considered as biodegradable. This pattern of biodegradation of fluorescent components was also observed through a decrease of tryptophan-like and tyrosine-like fluorescence peak intensity during the incubation time. The results also showed a higher decrease of humic-like fluorescence peak intensity at polluted sites (IG02 and IG05). Our experimental approach and monitoring strategy suggests that the evaluation of the organic matter biodegradability is essential to understand the fate and transformation mechanism of organic matter in urbanized and polluted rivers. And, considering a water quality planning and management perspective, this approach is important to identify the presence and location of organic compounds potentially important for dissolved oxygen depletion in stream waters.

Characterisation of algogenic organic matter during an algal bloom and its implications for trihalomethane formation

Abstract It is predicated that algal blooms will become an increasing problem under changing climatic conditions. This is particularly concerning for the potable water treatment industry since algogenic organic matter (AOM) in surface waters supplying water treatment works (WTWs) can cause a number of treatment issues. However, whilst previous studies have shown that AOM is distinct from terrigenous, humic-dominated organic matter, limited information exists relating to changes in the character of AOM during different algal growth phases. In this study, reservoir water containing dissolved organic carbon (DOC) dominated by humic material was enriched with nutrient medium to create an algal bloom. Over the course of the algal bloom, DOC was characterised using XAD-fractionation and UV absorbance measurements. In addition, the reactivity of DOC with chlorine both before and after XAD-fractionation was assessed using trihalomethane formation potential (THMFP) and bromine incorporation measurements to monitor whether THM yield and speciation varied between different growth phases. Characterisation of DOC during the algal bloom indicated a shift towards more hydrophilic, aliphatic (low specific UV absorbance; SUVA) DOC with the release of extracellular organic matter (EOM) and later intracellular organic matter (IOM) during cell lysis. XAD-fractionation results suggest that algae produce predominantly hydrophilic neutral (HPIN) DOC. In contrast to some existing research, our study shows a marked change in DOC reactivity over time with a reduction in standardised THMFP (STHMFP) and the initial rate of THM formation observed as the algal bloom progressed. However, bromine incorporation increased with culture age.

Interaction of Ketoconazole with Bovine Serum Albumin: Electrochemical, Spectroscopic and Molecular Modeling Studies

Formulation and Evaluation of Ketoconazole Polymeric Films for Topical ApplicationMohamed Salama, Mahmoud A. Mahdy, Amina Mohamed, Aref T. Mohamed, Eseldin I. Keleb, Assad A. Omar, Nagib A. Elmarzugi

Temporally and spectrally resolved UV absorption and laser-induced fluorescence measurements during the pyrolysis of toluene behind reflected shock waves

Toluene is frequently used as fluorescence tracer in high-temperature combustion applications. A quantitative analysis of laser-induced fluorescence (LIF) signals requires the knowledge of photophysical properties and decomposition kinetics. Using spectrally and temporally resolved ultraviolet absorption and LIF measurements, we studied the spectral properties of toluene and its pyrolysis products behind shock waves between 810 and 1,755 K. Transient absorption spectra were acquired between 220 and 300 nm. The temporal behavior of the absorption at 266 nm was compared to simulations based on literature kinetics models of toluene pyrolysis and available high-temperature absorption cross-sections of toluene, benzyl radicals, and C7H6 as a product from benzyl decomposition. Experiment and simulation agree well at the beginning of the pyrolysis process, whereas for longer reaction times deviations occur presumably due to the build-up of high molecular weight species, which contribute to the observed absorption but have unknown spectral properties. Additionally, LIF emission spectra were recorded following 266-nm excitation at selected reaction times. From measurements up to 1,220 K, the relative fluorescence quantum yield of toluene was derived, extending existing data to higher temperatures. Products from toluene pyrolysis were found to be the major contributors to the LIF signal at higher temperatures.

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry, Circular Dichroism, and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

Distibylation of Acetylenes with Ph2Sb–SbPh2: Synthesis, Crystal Structures and Phosphorescence Properties of Bis(diphenylstibyl)ethenes

Abstract Treatment of tetraphenyldistibine (1) with terminal arylacetylenes, XC6H5C≡CH (2a-c, X=H, F, OMe), gave the corresponding anti-addition products (E)-aryl-1,2-bis(diphenylstibyl)ethenes 3a-c in 11 - 17% yields. A similar reaction of 1 with 1,4-diphenylbutadiyne provided (E)-1,4-diphenyl- 1,2-bis(diphenylstibyl)but-1-en-3-yne (3d) in 28% yield. X-Ray diffraction studies on crystals of 3a, 3b and 3d revealed a congested configuration around the ethylene core. The optical properties of the adducts 3a-d were examined by measuring UV absorption and photoluminescence spectra. Interestingly, 3d showed phosphorescence in the solid state, which was enhanced at low temperature.

Evaluating fluorescence spectroscopy as a tool to characterize cyanobacteria intracellular organic matter upon simulated release and oxidation in natural water

Intracellular organic matter (IOM) from cyanobacteria may be released into natural waters following cell death in aquatic ecosystems and during oxidation processes in drinking water treatment plants. Fluorescence spectroscopy was evaluated to identify the presence of IOM from three cyanobacteria species during simulated release into natural water and following oxidation processes (i.e. ozone, free chlorine, chloramine, chlorine dioxide). Peak picking and the fluorescence index (FI) were explored to determine which IOM components (e.g., pigments) provide unique and persistent fluorescence signatures with minimal interferences from the background dissolved organic matter (DOM) found in Colorado River water (CRW). When IOM was added to ultrapure water, the fluorescence signature of the three cyanobacteria species showed similarities to each other. Each IOM exhibited a strong protein-like fluorescence and fluorescence at Ex 370 nm and Em 460 nm (FDOM), where commercial fluorescence sensors monitor. All species also had strong phycobiliprotein fluorescence (i.e. phycocyanin or phycoerythrin) in the higher excitation range (500–650 nm). All three IOM isolates had FI values greater than 2. When IOM was added to CRW, phycobiliprotein fluorescence was quenched through interactions between IOM and CRW-DOM. Mixing IOM and CRW demonstrated that protein-like and FDOM intensity responses were not a simple superposition of the starting material intensities, indicating that interactions between IOM and CRW-DOM fluorescing moieties were important. Fluorescence intensity in all regions decreased with exposure to ozone, free chlorine, and chlorine dioxide, but the FI still indicated compositional differences compared to CRW-DOM. The phycobiliproteins in IOM are not promising as a surrogate for IOM release, because their fluorescence intensity is quenched by interactions with DOM and decreased during oxidation processes. Increases in both FDOM intensity and FI are viable qualitative indicators of IOM release in natural waters and following oxidation and may provide a more robust real-time indication of the presence of IOM than conventional dissolved organic carbon or UV absorbance measurements.

In vitro anthelmintic activity of Millettia auriculata leaves and stems

ObjectiveTo study the anthelmintic activity of phytochemically charaterized chloroform extracts of Millettia auriculata leaves and stems. MethodsChloroform extracts of leaves and stems were prepared. Phytochemical characterisation involved preliminary screening, optimisation of solvent system in thin layer chromatography (TLC) and UV absorption measurement of each bright yellow (in view of a common physical property of flavonoids) TLC fraction for each extract. Anthelmintic activity of each extract was carried out for different concentrations (10, 20 and 40 mg/mL) using Pheretima posthuma. ResultsPhytochemical characterisation of the extracts revealed the presence of steroids/triterpenes, phenolics/flavonoids and carbohydrates with optimum TLC-separation in chloroform-hexane, 19:1. All the investigated extracts possessed significant anthelmintic activity (P<0.001) at a minimal dose of 10 mg/mL when compared with the corresponding concentration of reference drug albendazole. Leaf extract was found to have more efficacy and potency than that of stem and albendazole. ConclusionsThe results of this study indicated that Millettia auriculata can be used as anthelmintic drug. It would be also interesting to find out any novel or existing chemical entities showing anthelmintic activity with mechanism of action.

Dual-wavelength recording, a simple algorithm to eliminate interferences due to UV-absorbing substances in capillary electrophoresis.

Analytical interferences have been described due to the presence of various exogenous UV-absorbing substances in serum. Iodine-based X-ray contrast agents and various antibiotics have been reported to interfere with interpretation of serum protein pherograms, resulting in false diagnosis of paraproteinemia. In the present study, we have explored the possibility of measuring UV absorbance at two distinct wavelengths (210 and 246 nm) to distinguish between true and false paraproteins on a Helena V8 clinical electrophoresis instrument. This study demonstrates that most substances potentially interfering with serum protein electrophoresis show UV-absorption spectra that are distinct from those of serum proteins. Scanning at 246 nm allows detection of all described interfering agents. Comparing pherograms recorded at both wavelengths (210 and 246 nm) enables to distinguish paraproteins from UV-absorbing substances. In case of a true paraprotein, the peak with an electrophoretic mobility in the gamma-region decreases, whereas the X-ray contrast media and antibiotics show an increased absorption when compared to the basic setting (210 nm). The finding of iodine-containing contrast media interfering with serum protein electrophoresis is not uncommon. In a clinical series, interference induced by contrast media was reported in 54 cases (of 13 237 analyses), corresponding with a prevalence of 0.4%. In the same series, 1631 true paraproteins (12.3%) were detected. Implementation of the proposed algorithm may significantly improve the interpretation of routine electrophoresis results. However, attention should still be paid to possible interference due to presence of atypical proteins fractions (e.g., tumor markers, C3).

Determination and Validation of Henry’s Constant for Ozone in Phosphate Buffers Using Different Analytical Methodologies

Different procedures for measuring the concentration of ozone in aqueous buffered solutions have been reviewed for the determination of Henry’s constant at different temperatures and pH. Iodometry, indigo trisulfonate and iron (II) chemical methods were compared with the direct UV absorbance measurement of ozone at 258 nm. From these data a new value for the molar extinction coefficient of ozone is proposed as 3840 ± 109 M−1 cm−1. After the verification of the ozone analysis methods, the solubility of ozone in phosphate buffer medium was studied with in-situ absorbance direct measurements in a device with high gas-liquid transference rate, minimizing the gas desorption process on sample manipulation. The decomposition of ozone was not detected at the experimental conditions tested and, consequently, the observed solubility of ozone is constant, with the pH ranging from 2 to 7.6. The solubility of ozone was measured between 5 and 35 °C, resulting in a dimensionless solubility ratio of HB0 = 1.797 exp[0.0277·t(°C)].

Nanostructured biocompatible thermal/electrical stimuli-responsive biopolymer-doped polypyrrole for controlled release of chlorpromazine: Kinetics studies

Biocompatible nanostructured conductive heparin-doped polypyrrole film was fabricated and employed as a high-capacity cation exchanger for programmable release of neuroleptic drug, chlorpromazine (CPZ) with thermally and electrical dual-stimulation. Releasing behavior were studied at different applied potentials and temperatures by in-situ monitoring of UV absorbance measurements. Three mathematical models (Higuchi, Power, and Avrami equation) were employed to investigate kinetics of the release. Based on the obtained results, the Avrami model found to be more comprehensive than two other ones for mathematical description of electro-stimulated release of CPZ. A quantitative relationship between activation energy parameters (Ea, ΔG≠, ΔH≠, and ΔS≠) and release conditions (applied potential and temperature) has been developed and established to predict release rate constants at various applied conditions.