Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/μL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.     

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral–chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.     

Evaluation and comparison of two selective extraction procedures for heavy metal speciation in sediments.

Two sequential extraction procedures (the former proposed by the Standard, Measurements and Testing-SM&T-program of European Union and the latter derived from those of Tessier and Forstner with various modifications) have been compared, by analyzing a reference material (CRM 601) certified for the content of heavy metals in the three solutions resulting from the SM&T procedure. Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) was used to evaluate the composition of the residues after various extractions. Reliable results, both in terms of precision and accuracy, were obtained for the first two steps of the SM&T scheme. As regards the third step, because it was modified during this work, a direct comparison of our results with those certified is not possible. SEM analysis confirmed that the scheme previously used in the laboratory was more aggressive compared to the SM&T procedure, which has also the advantage that the only CRM available for metal partitioning in sediments is certified using this method.     

An alternating direction scheme on a nonuniform mesh for reaction-diffusion parabolic problems

In this paper we develop a numerical method for two-dimensionaltime-dependent reaction-diffusion problems. This method, whichcan immediately be generalized to higher dimensions, is shownto be uniformly convergent with respect to the diffusion problems.This method, which can immediately be generalized to higherdimensions, is shown to be uniformly convergent with respectto the diffusion parameter.     

$$L^{\infty }$$-norm and energy quantization for the planar Lane–Emden problem with large exponent

For any smooth bounded domain \(\Omega \subset {\mathbb {R}}^2\), we consider positive solutions to

$$\begin{aligned} \left\{ \begin{array}{lr}-\Delta u= u^p &{} \text{ in } \Omega \\ u=0 &{} \text{ on } \partial \Omega \end{array}\right. \end{aligned}$$

which satisfy the uniform energy bound

$$\begin{aligned}p\Vert \nabla u\Vert _{\infty }\le C\end{aligned}$$

for \(p>1\). We prove convergence to \(\sqrt{e}\) as \(p\rightarrow +\infty \) of the \(L^{\infty }\)-norm of any solution. We further deduce quantization of the energy to multiples of \(8\pi e\), thus completing the analysis performed in De Marchis et al. (J Fixed Point Theory Appl 19:889–916, 2017).