Some comments on the use of nitrogen selective detectors

Summary Several detectors which have a selective response toward nitrogen containing compounds are discussed. Data on sensitivities, detection limits and reproducibility of response are given. The application of chemiluminescence to provide a nitrogen selective GC detector is proposed.     

Robust estimates of the theoretical standard deviation to be used in interlaboratory precision experiments

Interlaboratory experiments often contain results that strongly deviate from other results obtained in the same laboratory under repeatability conditions, or laboratory means that strongly deviate from other laboratory means. In ISO 5725-2 [1] and IUPAC [2], the basic standards for the organisation and analysis of interlaboratory experiments for the determination of precision of a measurement method, outlier tests are performed in order to detect such situations and to finally decide whether these values are retained in the analysis or discarded as outliers. This outlier treatment, which always has a subjective aspect, becomes unnecessary by using robust estimates of the repeatability and reproducibility standard deviation. This paper proposes to use Rousseeuw’s Q _n as an extremely robust and efficient estimate of the standard deviation. Two examples show the performance of the new method.     

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10 MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.     

Reproducible method to enrich membrane proteins with high purity and high yield for an LC‐MS/MS approach in quantitative membrane proteomics

The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC‐MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC‐MS/MS label‐free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC‐MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29‐MTX cells. LC‐MS/MS analysis of the membrane‐enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.     

Enhanced Reproducibility of Positive Temperature Coefficient Effect of CB/HDPE/PVDF Composites with the Addition of Ionic Liquid

Developing an effective method for improving the reproducibility of positive temperature coefficient(PTC) effect is of great significance for large-scale application of polymer based PTC composites, owing to its contribution to the security and reliability. Herein, we developed a carbon black(CB)/high density polyethylene(HDPE)/poly(vinylidene fluoride)(PVDF) composite with outstanding PTC reproducibility, by incorporating 1-octyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide([OMIm][NTf_2]) into the composite. After multiple repeated temperature cycles, the PTC performance of as-prepared material keeps almost unchanged and the varition of resistance at room temperature is less than 7%. Our studies revealed that [OMIm][NTf2] contributes to the improvement of PTC reproducibility in two ways:(i)it acts as an efficient plasticizer for refining the co-continuous phase morphology of HDPE/PVDE blends;(ii) it inhibits the crystallization of PVDF through the dilution effect, leading to more overlaps of the volume shrinkage process of HDPE and PVDF melt which results in the decrease of interface gap between HDPE and PVDF. This study demonstrated that ionic liquids as the multifunctional agents have great potential for improving the reproducibility in the application of the binary polymer based PTC composites.     

Microbiosensor based on glucose oxidase and hexokinase co-immobilised on platinum microelectrode for selective ATP detection

ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylindrical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg²⁺ and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosensor can measure ATP concentrations down to a 2.5 μM detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and l-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes.     

Principal eigenvector field segmentation for reproducible diffusion tensor tractography of white matter structures

The study was aimed to test the feasibility of utilizing an algorithmically determinable stable fiber mass (SFM) map obtained by an unsupervised principal eigenvector field segmentation (PEVFS) for automatic delineation of 18 white matter (WM) tracts: (1) corpus callosum (CC), (2) tapetum (TP), (3) inferior longitudinal fasciculus (ILF), (4) uncinate fasciculus (UNC), (5) inferior fronto-occipital fasciculus (IFO), (6) optic pathways (OP), (7) superior longitudinal fasciculus (SLF), (8) arcuate fasciculus (AF), (9) fornix (FX), (10) cingulum (CG), (11) anterior thalamic radiation (ATR), (12) superior thalamic radiation (STR), (13) posterior thalamic radiation (PTR), (14) corticospinal/corticopontine tract (CST/CPT), (15) medial lemniscus (ML), (16) superior cerebellar peduncle (SCP), (17) middle cerebellar peduncle (MCP) and (18) inferior cerebellar peduncle (ICP). Diffusion tensor imaging (DTI)-derived fractional anisotropy (FA) and the principal eigenvector field have been used to create the SFM consisting of a collection of linear voxel structures which are grouped together by color-coding them into seven natural classes to provide PEVFS signature segments which greatly facilitate the selection of regions of interest (ROIs) for fiber tractography using just a single mouse click, as compared with a manual drawing of ROIs in the classical approach. All the 18 fiber bundles have been successfully reconstructed, in all the subjects, using the single ROIs provided by the SFM approach, with their reproducibility characterized by the fact that the ROI selection is user independent. The essentially automatic PEVFS method is robust, efficient and compares favorably with the classical ROI methods for diffusion tensor tractography (DTT).     

Reproducibility evaluation and Al doping of sol-gel-derived single- and multi-layer zinc oxide thin films

Both single- and multi-layer thin films of sol-gel-derived undoped and Al-doped zinc oxide material systems were fabricated for potential use in transparent conducting oxide modules. Functional properties of the resultant films were characterised comparatively. High degree of reproducibility was demonstrated for the characterised properties, with the exception of the electrical conductivity. Influences of Al doping on the characterised properties were also investigated. Al doping reduced the average crystallite size, and led to a denser and less porous morphology, and also caused an increase in transparency in the UV region. Moreover, it was shown that the electrical conductivity of this thin film system could be improved upon either the application of a forming gas process, or Al doping.     

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography

A collaborative study on the robustness and portability of a capillary electrophoresis‐mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis‐mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath‐flow capillary electrophoresis–mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin‐digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3–12 and 9–29% RSD, respectively. These results demonstrate that capillary electrophoresis‐mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis‐mass spectrometry applications in biopharmaceutical analysis and related fields.     

The influence of the memory effect on preparative separations using the amylose tris(3,5-dimethylphenylcarbamate) stationary phase

Acid/base mobile phase modifiers affect enantioseparations in ways that are not yet understood for the lack of systematic studies, which makes the scale-up of preparative separations difficult to predict. Shifts of the selectivity of certain pairs of enantiomers upon exposure of the column to these modifiers is amply documented. Furthermore, once the modifier has been removed from the mobile phase, the improved selectivity remains, this phenomenon has been named the memory effect. We selected four enantiomeric pairs for a systematic study of this memory effect. The selectivity of 4-chlorophenylalanine ethyl ester (4CPEE) improves after a solution of ethanesulfonic acid (ESA) is percolated through the column. The selectivity of propranolol HCl and Tröger's base increases after a solution of diiospropylethylamine is percolated through the column. The selectivity of these three pairs of enantiomers is inversely affected by percolation of the opposite acid/base solution. Each of these four compounds reached an equilibrium concentration that maintained the separation of the enantiomeric pairs. In contrast, the selectivity of trans-stilbene oxide (TSO) is not affected by either acid/base modifier. Preparative separations can be used to detect changes in the active surface of the chiral polymer stationary phase by measuring the change in selectivity and resolution when modifiers are used. Preparative method development was carried out on analytical columns and scale-up to 1 cm ID columns were performed in this study.