Automated microflow NMR: routine analysis of five-microliter samples

A microflow CapNMR probe double-tuned for 1H and 13C was installed on a 400-MHz NMR spectrometer and interfaced to an automated liquid handler. Individual samples dissolved in DMSO-d6 are submitted for NMR analysis in vials containing as little as 10 microL of sample. Sets of samples are submitted in a low-volume 384-well plate. Of the 10 microL of sample per well, as with vials, 5 microL is injected into the microflow NMR probe for analysis. For quality control of chemical libraries, 1D NMR spectra are acquired under full automation from 384-well plates on as many as 130 compounds within 24 h using 128 scans per spectrum and a sample-to-sample cycle time of approximately 11 min. Because of the low volume requirements and high mass sensitivity of the microflow NMR system, 30 nmol of a typical small molecule is sufficient to obtain high-quality, well-resolved, 1D proton or 2D COSY NMR spectra in approximately 6 or 20 min of data acquisition time per experiment, respectively. Implementation of pulse programs with automated solvent peak identification and suppression allow for reliable data collection, even for samples submitted in fully protonated DMSO. The automated microflow NMR system is controlled and monitored using web-based software.     

Thermophoretic collection and analysis of submicrometer ag particles emitted from a graphite tube-type electrothermal vaporizer

Using thermophoretic collection with a cooled Cu probe, particles generated in an electrothermal vaporizer (ETV) from a AgNO(3) solution sample have been collected and analyzed using transmission electron microscopy and high-energy electron diffraction (HEED). Ag particles are spherical and, interestingly, have diameters falling into one of four size regimes, <5, 15, 25, and >40 nm. An extremely large number (estimated at >10(8)) of particles with diameters of <100 nm are collected. For an aqueous AgNO(3) solution deposited in the ETV, the HEED pattern of the collected aerosol particles exiting the pulse heated ETV matched body centered cubic Ag((s)), and the large number of diffraction spots suggests that particles are composed of microcrystalline domains. The features of the particles confirm earlier predictions of homonucleation as the primary particle formation mechanism. "Groupings" of apparently disconnected particles were a unique feature seen from the pulse-heated vaporization of the dried sample. This morphology is unique and does not appear like any other clustering or aggregation of particles reported elsewhere. It is not clear what causes this particle grouping, although the absence of these groupings when a 700 °C thermal pretreatment step was introduced suggests that the AgNO(3) decomposition plays a role in their formation. It is suggested that the particles formed from homonucleation are created very near the graphite surface and are cooled quite rapidly to form the solid silver particles. A mechanism is presented to explain the appearance of silver in the gas phase at temperatures below the vaporization temperature for silver metal.     

Predicting peak shape in capillary zone electrophoresis: a generic approach to parametrizing peaks using the Haarhoff-Van der Linde (HVL) function.

We have found that the Haarhoff-Van der Linde (HVL) peak function provides excellent fitting to the shapes of CZE peaks. Initially designed for overloaded peaks in gas chromatography, this function describes a Gaussian peak when there is no peak distortion, and a triangular peak when there is no diffusional peak broadening. As such, it is ideal for CZE peaks distorted by electromigration dispersion (EMD). Fitting peaks with this function gives four parameters: three of them can be related to the Gaussian peak that would have been obtained in case of no EMD; the last one is a measure of the peak distortion. Using moving boundary theory, this peak distortion parameter may readily be expressed in terms of analyte and background electrolyte mobilities and concentrations, electric field, and sample injection length. The variance of an HVL peak is shown to be described by a universal function, and a master equation is presented. The region where EMD adds less than 10% to the Gaussian variance is shown to be very narrowly spread around the mobility matching condition. Under typical CZE operating conditions with an analyte at 1% of the BGE concentration, significant peak distortion is always present. Because the total peak variance is not an addition of the Gaussian and triangular contributions, the HVL model and the methodology introduced here should always be used to correctly combine variances.     

Second-order peak detection for multicomponent high-resolution LC/MS data

The first step when analyzing multicomponent LC/MS data from complex samples such as biofluid metabolic profiles is to separate the data into information and noise via, for example, peak detection. Due to the complex nature of this type of data, with problems such as alternating backgrounds and differing peak shapes, this can be a very complex task. This paper presents and evaluates a two-dimensional peak detection algorithm based on raw vector-represented LC/MS data. The algorithm exploits the fact that in high-resolution centroid data chromatographic peaks emerge flanked with data voids in the corresponding mass axis. According to the proposed method, only 4 per thousand of the total amount of data from a urine sample is defined as chromatographic peaks; however, 94% of the raw data variance is captured within these peaks. Compared to bucketed data, results show that essentially the same features that an experienced analyst would define as peaks can automatically be extracted with a minimum of noise and background. The method is simple and requires a priori knowledge of only the minimum chromatographic peak width-a system-dependent parameter that is easily assessed. Additional meta parameters are estimated from the data themselves. The result is well-defined chromatographic peaks that are consistently arranged in a matrix at their corresponding m/z values. In the context of automated analysis, the method thus provides an alternative to the traditional approach of bucketing the data followed by denoising and/or one-dimensional peak detection. The software implementation of the proposed algorithm is available at http://www.anchem.su.se/peakd as compiled code for Matlab.     

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.     

Online standard additions calibration of transient signals for inductively coupled plasma mass spectrometry

An online standard additions calibration method for transient signals in ICPMS is demonstrated in which a small volume of standard is injected as a spike into the sample/carrier stream, overlaying the analyte peak. This technique provides the advantages of conventional standard additions but requires only a single sample run. The method corrects for matrix effects and is suitable for transient signals in which the severity of the matrix effect changes over the analyte peak. The method uses a peak-fitting program to determine the area of the underlying peak and is shown to be effective for the determination of trace metal concentrations in both a high ionic strength matrix and in a biological matrix (urine). Eight analytes with concentrations in the range of 0.82-233.2 mug L-1 in urine were simultaneously determined using a standard spiking solution of 75 mug L-1 injected through a 100-muL loop. The measured concentrations for analytes free of spectral interferences agreed with the certified values, and the precision achieved was comparable to that achieved by the certifying agency. Using a conventional cross-flow nebulizer and Scott-type spray chamber, the accuracy obtained for online standard additions calibration was within 2%, and the precision was within 5%.     

Correction of H3+ contributions in hydrogen isotope ratio monitoring mass spectrometry

Two fundamentally different approaches, termed "pointwise" and "peakwise," are currently used to correct hydrogen isotope ratio monitoring data for the presence of H3+ ion contributions. Consideration of the underlying assumptions shows that the peakwise approach is valid only for peaks with the same functional shape and only when background signals do not vary. The pointwise correction is much more versatile and can be used even when peak shapes and sizes, as well as background signals, vary significantly. It is not exact and is limited in accuracy by (1) the signal-broadening effects of electronic time constants, (2) the analog-to-digital conversion frequency, and (3) the highest frequency of the sample signal. To minimize errors for typical gas chromatographic signals, time constants of <500 ms and analog-to-digital sampling intervals of < or =250 ms are needed. Errors are further minimized by matching sample and standard peaks in both amplitude and D/H ratio. Using the pointwise algorithm, we demonstrate that a series of 14 homologous n-alkanes varying in concentration over a 5-fold range can be analyzed with a mean precision of 2.3 per thousand and no systematic errors.     

爆破条件对爆破震动信号分析中小波包时频特征的影响

非平稳信号的小波包分析是在小波变换基础上发展起来的 ,它对小波分析中没有细分的高频部分进一步分解 ,从而能够对信号局部信息进行更为精细的掌握。爆破条件是影响爆破震动时频特征分布的主要因素之一。本研究中 ,针对在不同段药量、不同微差间隔时间及近似相同的其它条件下产生的爆破震动信号 ,运用小波包分析方法对其进行了时频分析 ,主要探讨了段药量、段微差间隔时间对爆破震动时频分布的影响规律。段药量对爆破震动波形时频特征的影响主要体现在各层小波包主振频带内的细节信号峰值质点振速方面 ,各细节信号的峰值质点振速随段药量增加而增大 ,但主振频带分布保持基本的一致性 ,同一主振频带下小波包细节信号的阻尼比也趋于一致 ;段微差时间间隔对爆破震动时频特征的影响主要表现在 :延长各主振频带小波包细节信号的振动持时 ,不同微差单段波形的叠加增加了主振频带个数 (优势频率个数 )并使各频带内的优势频率值有微弱增大的趋势     

Optimized preprocessing of ultra-performance liquid chromatography/mass spectrometry urinary metabolic profiles for improved information recovery

Ultra-performance liquid chromatography coupled to mass spectrometry (UPLC/MS) has been used increasingly for measuring changes of low molecular weight metabolites in biofluids/tissues in response to biological challenges such as drug toxicity and disease processes. Typically samples show high variability in concentration, and the derived metabolic profiles have a heteroscedastic noise structure characterized by increasing variance as a function of increased signal intensity. These sources of experimental and instrumental noise substantially complicate information recovery when statistical tools are used. We apply and compare several preprocessing procedures and introduce a statistical error model to account for these bioanalytical complexities. In particular, the use of total intensity, median fold change, locally weighted scatter plot smoothing, and quantile normalizations to reduce extraneous variance induced by sample dilution were compared. We demonstrate that the UPLC/MS peak intensities of urine samples should respond linearly to variable sample dilution across the intensity range. While all four studied normalization methods performed reasonably well in reducing dilution-induced variation of urine samples in the absence of biological variation, the median fold change normalization is least compromised by the biologically relevant changes in mixture components and is thus preferable. Additionally, the application of a subsequent log-based transformation was successful in stabilizing the variance with respect to peak intensity, confirming the predominant influence of multiplicative noise in peak intensities from UPLC/MS-derived metabolic profile data sets. We demonstrate that variance-stabilizing transformation and normalization are critical preprocessing steps that can benefit greatly metabolic information recovery from such data sets when widely applied chemometric methods are used.     

A strategy to prevent signal losses, analyte decomposition, and fluctuating carbon contamination bands in surface-enhanced Raman spectroscopy

Signal losses and fluctuating carbon contamination bands are "bottlenecks" in the application of surface-enhanced Raman spectroscopy (SERS) for reliable chemical analysis. They originate mainly from prolonged laser irradiation of the sample during data collection, which causes analyte decomposition and/or loss of the enhancing capabilities of the adsorption site. In this work, a laser illumination/signal collection technique, the "multiple points collection" (MPC) method is introduced to circumvent these problems. The MPC method is based on the use of a pair of galvanic mirrors to scan the laser beam rapidly and steadily across the sample surface. Each position is irradiated for <10 mus, at a rate of approximately 0.5 Hz. The SER spectrum is obtained by summing the signals collected from a large array of non-overlapping sample points. The MPC is compared with the conventional "single point collection" method, in which the laser beam is statically focused onto a particular spot and the scattered signals acquired. The MPC has the following advantages: (1) illumination and collection efficiencies are not compromised, (2) signal losses originating from analyte decomposition and/or alteration of the enhancing capabilities of the adsorption site are avoided, (3) high-quality SER spectra for analytes such as biomolecules and dipicolinic acid (a common marker for bacteria spores) can be easily obtained, and (4) the occurrence of broad amorphous carbon bands and the commonly observed temporal fluctuations in SERS are prevented. The success of the MPC is attributed to the reduction of local sample heating, as the time interval between the laser irradiations of a spot is much longer than the actual irradiation time itself.     

Spectral velocity profiles for detailed ultrasound flow analysis

The operation of a novel ultrasound multigate instrument capable of computing in real-time the fast Fourier transform (FFT) of Doppler signals detected from 64 equally spaced range cells is presented. The new system provides up to 50 velocity profiles per second, which are displayed in such a manner that information about the full spectral content of Doppler signals at all the investigated depths is continuously monitored over a PRF-wide frequency range which can be set arbitrarily between -PRF and +PRF. Experimental results are presented, which demonstrate that the true velocity profile can be accurately detected through the computation of “local” maximum velocities obtained by properly correcting the maximum frequency of each spectrum. There is also a discussion on how the results of multigate analysis are influenced by the sample volume length, a parameter which can be usually set by modifying the duration of the transmitted burst. In particular, it is shown that, in regions close to the vessel walls, the shear rate can be measured with a spatial resolution related to the spacing between subsequent range cells and not to the sample volume length     

EPR properties of intact and deproteinated dentin

The effect of sample preparation on dentin electron paramagnetic resonance (EPR) spectra was investigated. The analysis was performed on dentin samples prepared by pure mechanical treatment or by an alkaline deproteination method. It was observed that (1) mechanical treatment induces both stable and transient signals, depending on the specific mechanical operation applied; and (2) sodium hydroxide deproteination removes the native signal, but at the same time introduces new confounding signals in the EPR spectrum. Also, it increases the radiation sensitivity of dentin. These findings suggest that attention must be paid to dentin sample preparation.     

Dual fluorescence and electrochemical detection on an electrophoresis microchip

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 microM have been achieved for dopamine, catechol, NBD-arginine, and NBD-phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD-arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.     

Hilbert-Huang变换在瞬态信号检测中的应用

空投物体入水时产生具有时域冲击特性的信号,一般称之为瞬态信号。Hilbert-Huang变换可通过经验模态分解和Hilbert谱分析两种方法,从时频角度对瞬态信号进行分析。介绍了Hilbert-Huang变换的基本原理,建立了瞬态信号的数学模型,提出了基于Hilbert-Huang变换的信号重构瞬态信号检测方法,并与传统能量检测器的性能进行了对比分析,最后通过软件仿真和湖试数据处理验证了该方法的可行性和有效性。     

Acoustic emission from stress corrosion cracks in aligned GRP

Acoustic emission (AE) produced by the propagation of stress corrosion cracks in an aligned glass fibre/polyester resin composite material has been recorded. Tests have been carried out over a range of crack growth rates and the variation of AE with crack velocity/applied stress intensity has been examined. The main source of AE is fibre fracture and there is a one-to-one relationship between the number of fibre fractures and the number of high-amplitude AE signals. This enables crack growth to be monitored directly from acoustic emission. The amplitude of AE signals produced by fibre failure appears to be proportional to the fracture stress of the fibres, although further analysis requires a greater understanding of the generation, transmission and detection of AE signals. This work demonstrates that stress corrosion cracking is an ideal source for the study of AE produced by fibre fracture without complications caused by interface effects, such as fibre debonding or pullout.On leave from the Technical University of Wroclaw, Wroclaw, Poland.     

Detection of malaria parasites in blood by laser desorption mass spectrometry

A novel method for the in vitro detection of the protozoan Plasmodium, the causative agent of malaria, has been developed. It comprises a protocol for cleanup of whole blood samples, followed by direct ultraviolet laser desorption (LD) time-of-flight mass spectrometry. Intense ion signals are observed from intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during their growth in human red blood cells. The LD mass spectrum of the heme is structure-specific, and the signal intensities are correlated with the sample parasitemia (number of parasites per unit volume of blood). Parasitemia levels on the order of 10 parasites/microL blood can be unambiguously detected by this method. Consideration of laser beam parameters (spot size, rastering across the sample surface) and actual sample consumption suggests that the detection limits can be further improved by at least an order of magnitude. The influence of experimental factors, such as desorbed ion polarity, laser exposure and fluence, sample size, and parasite growth stage, on the threshold for parasite detection is also addressed.     

Spurenelemente in Halbleitern – eine analytische Herausforderung für die ETV ICP OES

Trace Elements in Semiconductors – A Challenge for the Analysis with ETV ICP OES Direct analysis of solids is characterized by some benefits compared to dissolution‐based methods. Techniques for the direct analysis of semiconductors (Indium phosphide, Silicon carbide, Aluminium nitride, Gallium arsenide) provide an advantageous alternative to methods using wet digestion in sample preparation. The calibration procedure is the major difficulty of all techniques applied for direct solid sample analysis, as there is a lack of suitable reference materials of semiconductors. Calibration was carried out using both dried liquid standards and the analyte‐addition technique. ETV ICP OES can be a powerful technique for the determination of trace elements in semiconductors.     

水声信号多参量估计的PWVD改进方法

Pseudo Winger-Ville分布（PWVD）是处理非平稳信号的有力工具,因其具有较高的时频聚集性更适合于实时处理,且选择合适的窗函数可降低旁瓣大小,提高分辨率。传统的PWVD可以对信号瞬时频率进行无偏估计,但其频率分辨率和时间分辨率不能同时兼顾;提出了一种插值PWVD法可克服此不足,极大地减小了频谱泄漏的负面影响,有效提高了频率估计与时间估计的精度。仿真和实测数据的分析结果验证了插值PWVD法的可行性与有效性。     

A high-capacity LC/MS system for the bioanalysis of samples generated from plate-based metabolic screening

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.     

Supersaturated designs for robustness testing

Supersaturated designs are factorial designs in which the number of factors examined exceeds the number of experiments performed. They do not allow an estimation of individual effects, since even the main effects are confounded. However, the total variance of a response estimated from such a design can in principle be used as a measure for the robustness of the method. A number of case studies were examined to determine whether the variance estimated from a supersaturated design is similar to the one from a Plackett-Burman design. This was found to be the case, which means that the estimated variance describes well the variation in the response caused by the variation in the factors.