Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase

Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (d-LH₂) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A—incubation time (t_inc = 15 min), inactivation time of the enzyme (t_inac = 2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP] = 40 μM) and luciferase ([Luc] = 0.1 μM); method B—concentrations of L-AMP ([L-AMP] = 2 μM), luciferase ([Luc] = 50 nM) and luciferin ([LH₂] = 30 μM). Method A has a linear response over the range of 0.1-20 μM of PPi, with a limit of detection (LOD) of 0.5 μM and a limit of quantitation (LOQ) of 1.8 μM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 μM PPi and 5.9% at 8 μM PPi. Method B has a linear response over the range of 0.75-6.0 μM of PPi, with LOD and LOQ of 0.624 and 2.23 μM, respectively, and a R.S.D. of 5.1% at 2.5 μM PPi and 4.9% at 5 μM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P₃).     

自掺杂磺酸化聚苯胺之最优化实验设计(英文)

应用实验设计法包括部分析因设计与中心组合设计,优化自掺杂磺酸化聚苯胺的导电性.部分析因设计发现,需用低温(4℃)、短反应时间(1 h)和少量发烟硫酸(10 mL)等实验条件进行磺酸化反应才能得到好的导电性(ca.127 mS.cm-1);中心组合设计与确认实验表明,其最佳导电性(ca.141 mS/cm)的磺酸化条件是4℃、75 m in反应时间以及8 mL 30%的发烟硫酸.应用傅立叶变换红外光谱(FTIR)、四探针与X光光电子能谱等分析制备的材料.此外,发现磺酸化速率与程度与聚合反应时所使用的酸有关.     

Application of a Factorial Design Method to Determine the Accuracy of a Solution to an Inverse Heat Conduction Problem

A numerical/experimental inverse procedure was employed to estimate the temperature-dependent thermal conductivity of a solid body in which 1D heat conduction in a top heated cylindrical sample is assumed. The emphasis is focused on the issue of sensitivity of results to selected assumptions made in inverse calculations. It has been found that the accuracy of heat capacity evaluation brings the largest contribution to final errors (up to 74%). Density accounts for one-fourth to one-third of the total error of determination. The failure to ensure unidirectional heat conduction in a sample during an experiment is important only at elevated temperatures.     

Investigation of the limitations of saturated fractional factorial experimental designs,with confounding effects for an HPLC ruggedness test

Summary Recently much attention has been directed to the validation of analytical methods. The level of validation will depend on the method application. If a method is developed for general use, for instance by different analysts, instruments or laboratories, then the effect of these changes need evaluation. This is normally referred to as ruggedness testing, where the limitations of the method to changes in the specified conditions are examined.This paper examines the use of Plackett-Burman fractional factorial experiment designs for ruggedness testing in high performance liquid chromatography (HPLC). The ability of these designs to identify confounding effects is applied to the stability indicating analysis of Salbutamol and its major degradation product, AH4045. The results show that confounding effects, proving extremely pertinent to HPLC, can be identified.The assay proved rugged to most tested changes although not to the use of column packing material from different manufacturers.     

Highly sensitive ion preconcentration method based on flow sorbent extraction using multiwall carbon nanotubes

In this work a solid phase on-line uranium ion preconcentration system coupled with spectrophotometry has been developed. The method is based on uranyl ion preconcentration at pH 3.75 onto multiwall carbon nanotubes treated with HNO₃. After preconcentration, the uranyl ions are eluted with 0.32?mol?L^?1HCl followed by reaction with 3,6-bis[(2-arsonophenyl)-azo]-4,5-dihydroxy-2,7-naphthalendisulfonic acid 0.08%[w/v] (Arsenazo III), which had maximum monitored absorbance of 650?nm. Effects of the pertinent experimental parameters on the system were investigated by means of 2^6?2 fractional factorial design, while optimization was carried out using the Doehlert matrix. Under optimized conditions, detection and quantification limits were found to be 0.21 and 0.7?µg?L^?1, respectively. The analytical curve ranged from 5 to 150?µg?L^?1 (r?=?0.998), while the relative standard deviations (RSD) were 3.27 and 2.56% for the respective uranium concentrations of 10 and 100?µg?L^?1 (n?=?10). The features obtained for the on-line preconcentration system were: preconcentration factor of 228, concentration efficiency of 57?min^?1, consumption index of 0.13?mL and sample throughput of 15?h^?1. In order to assess the accuracy of the proposed method, addition and recovery studies were carried out on spring water samples from different sources and synthetic seawater with satisfactory results ranging from 94.85 up to 103.65%.     

Optimization strategies for radiation induced grafting of 4-vinylpyridine onto poly(ethylene-co-tetraflouroethene) film using Box-Behnken design

The radiation induced grafting of 4-vinylpyridine (4-VP) onto poly(ethylene-co-tetrafluoroethene) (ETFE) was optimized using the Box-Behnken factorial design available in the response surface method (RSM). The optimized grafting parameters; absorbed dose, monomer concentration, grafting time and reaction temperature were varied in four levels to quantify their effect on the grafting yield (GY). The validity of the statistical model was supported by the small deviation between the predicted (GY=61%) and experimental (GY=57%) values. The optimum conditions for enhancing GY were determined at the following values: monomer concentration of 48 vol%, absorbed dose of 64 kGy, reaction time of 4 h and temperature of 68 °C. A comparison was made between the optimization model developed for the present grafting system and that for grafting of 1-vinylimidazole (1-VIm) onto ETFE to confirm the validly and reliability of the Box-Behnken for the optimization of various radiation induced grafting reactions. Fourier transform infrared (FTIR), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) were used to investigate the properties of the obtained films and provide evidence for grafting.     

Evidence of Ternary Inclusion Complexes Formation Using Factorial Design and Determination of Their Formation Constant

Evidence of the interaction among the [PbAc]⁺ and[PbEDTA]^-2 with the -cyclodextrin (-CD)is shown, using a 2⁴ factorial design. The factors used werethe [Pb(II)], [-CD], [Ac^-] and [EDTA] concentrationsat pH = 6.2. The statistical results indicate that the changes inchemical response for these species are associated to theinteraction among these factors. Spectrophotometric andelectrochemical studies were undertaken to assess the interactions.The equilibrium constants for inclusion complexes were determinedFor -CD-[PbAc]⁺ and -CD-[PbEDTA]^-2.     

Relationship between coffee husk caffeine degradation and respiration of Aspergillus sp. LPBx in solid-state fermentation

Studies were carried out in a packed-bed column fermentor using coffee husk as substrate in order to verify a relationship between caffeine degradation and the respiration of Aspergillus sp. LPBx. Fermentation conditions were optimized by using factorial design experiments. The kinetic study showed that the caffeine degradation was related to the development of mold and its respiration and also with the consumption of reducing sugars present in coffee husk. From the values obtained experimentally for oxygen uptake rate and CO₂ evolved, we determined a biomass yield of 3.811 g of biomass/g of consumed O₂ and a maintenance coefficient of 0.0031 g of consumed O₂/(g of biomass·h). The maximum caffeine degradation achieved was 90%.     

Asymptotically minimum variance unbiased estimation for a class of power series distributions

Summary The problem of finding an asymptotically minimum variance unbiased estimator (A.M.V.U.E.) for the parameter of certain truncated power series distributions, is discussed, when the generating function of their coefficients are i) polynomials of binomial type ii) generalized ascending factorials iii) polynomials with coefficients the well known Eulerian numbers.     

400GeV/cpp碰撞多粒子产生的自仿射分形

利用４００ＧｅＶ／ｃｐｐ碰撞多粒子产生的实验数据进行了自仿射分形分析，并与自相似分析相比较，结果表明在实验分辨能力范围内，自仿射分形分析具有嵔虾玫谋甓刃形