Peter C. Stark

Pre-PCR DNA quantitation of soil and sediment samples: method development and instrument design

A simple and straightforward method for the quantitation of dsDNA in soil and sediment matrices has been developed to support rapid, in-the-field PCR analysis of environmental samples. This method uses PicoGreen nucleic acid stain, and a combination of UV/Vis and fluorescence spectroscopy, to quantitate dsDNA in the presence of interfering humic materials. The practical utility of this approach is that it compliments a seven-step DNA extraction procedure for environmental samples. The DNA quantitation method is utilized twice during the extraction procedure. Once, prior to a micro-spin column procedure to maximize the amount of DNA extracted, and a second time, just prior to PCR to optimize the PCR reaction conditions. A field-portable, assay-specific instrument has been developed based on this methodology. Software for this instrument steps the analyst through the experimental procedure, and has been designed such that a minimum of technical expertise is required to perform the assay. Initial data obtained from the prototype unit indicates that this instrument compares with commercial instrumentation in terms of detection limit and sensitivity.

Application of wavelet transforms and an approximate deconvolution method for the resolution of noisy overlapped peaks in DNA capillary electrophoresis

A new procedure for resolving noisy overlapped peaks in DNA separations by capillary electrophoresis (CE) is developed. The procedure combines both a wavelet-based denoising method that effectively denoises the signal and a novel approximate deconvolution technique that resolves the fragment peaks and improves the ability to separate highly overlapped peaks early in the electrophoresis process. Different kinds of overlapped peaks with and without noise simulated by computer as well as some DNA experimental electropherograms were submitted to the new procedure. A second order differential operator with variable coefficients is applied to the entire electrophoresis signal at any given time and approximate deconvolutions of the individual Gaussian peaks are performed. The operator incorporates the effect of the superposition and gives exact annihilation in the neighborhood of each peak. Overlapped peaks with a resolution higher than 0.46 can be resolved directly. Also, the method can determine the peak components of signals with a signal to noise ratio higher than 1.4

Two-Color Capillary Electrophoresis with On-Column Excitation and Wave-Guide Based Fluorescent Detection

An optical wave-guide based two-color capillary electrophoresis laser induced fluorescence (CE-LIF) instrument is described. The wave-guide based approach allows for on column excitation and detect...

AMERICIUM SEPARATIONS FROM HIGH-SALT SOLUTIONS USING ANION EXCHANGE

The aging of the US nuclear stockpile presents a number of challenges, including the increasing radioactivity of plutonium residues due to the ingrowth of 241Am from the β-decay of 241Pu. We investigated parameters that affect the sorption of Am onto anion-exchange resins from concentrated effluents derived from nitric acid processing of plutonium residues. These postevaporator wastes are nearly saturated solutions of acidic nitrate salts, and americium removal is complicated by physical factors, such as solution viscosity and particulates, as well as by the presence of large quantities of competing metals and acid. Single- and double-contact batch distribution coefficients for americium and neodymium from simple and complex surrogate solutions are presented. Varied parameters include the nitrate salt concentration and composition and the nitric acid concentration. We find that under these extremely concentrated conditions, Am(III) removal efficiencies can surpass 50% per contact. Distribution coefficients for both neodymium and americium are insensitive to solution acidity and appear to be driven primarily by low water activities of the solutions.

A new spectroscopy method for in situ rapid detection and classification of micro-organisms

Recent developments in the characterization of particle dispersions have demonstrated that complementary information on the joint particle property distribution (size-shape-chemical composition) of micron and sub-micron particles is available from multiwavelength spectrophotometric measurements. The UV-VIS transmission spectra of the microorganism suspensions reported herein were recorded using a Hewlett-Packard 8453 diode array spectrometer with an acceptance angle smaller than 2 degrees. To eliminate concentration and particle number effects, the transmission spectra were normalized with the average optical density between 230-900 nm. Experimental results demonstrate that microorganisms at various states of growth give rise to spectral differences that can be used for their identification and classification and that this technology can be used for the characterization of the joint particle property distribution for a large variety of continuous, on-line, and in-situ particle characterization applications. An interpretation model has been developed for the quantitative interpretation of spectral patterns resulting from transmission measurements of microorganism suspensions. The interpretation model is based on light scattering theory and spectral deconvolution techniques and yields the quantitative information necessary to define the probability of the detection and identification of microorganisms. A data base of 54 pathogens has been created and demonstrates that the technology can be used in the field for real-time in-situ monitoring applications.

A Full Scanning System and a New Processing Method for Capillary Electrophoresis

A system using a liquid-core waveguide for the full scanning of capillary electrophoresis processes is presented. The system utilizes the liquid-core waveguide as an efficient window for the excitation of separated samples and the collection of light through total internal reflectance, with zeptomolar detection limits. Scanning the excitation laser along the length of the electrophoresis capillary excites individually separated analyte bands, while the fluorescence is collected end-on by an optical fiber coupled to a photomultiplier. A new procedure for denoising and deconvolution was applied to the experimental electropherograms, removing the noise and resolving the highly overlapped peaks observed in early stages of the separation.

Market Research Survey of Commercial Off-the-Shelf Mass Spectrometers for In-Field Analysis. FY 15 Update

This report is an update of the 2013 Market Research Survey1-3 of field-deployable commercial off-the-shelf (COTS) mass spectrometry instruments for safeguards application.

4-tert-Butylcalix[4]arene tetrahydroxamate chelators for theselective extraction of actinide ions: synthesis and preliminary metalion extraction studies1

Organic extractants capable of selective and efficient removal of actinides such as plutonium and americium from aqueous process waste streams are potentially useful in radioactive waste remediation. In this context, with the help of molecular modelling, the 4-tert-butylcalix[4]arene tetrahydroxamate chelators 1 and 2 have been identified as structures capable of achieving the selective solvent extraction of actinide(IV) ions from aqueous solutions. The syntheses of 1 and 2 from 4-tert-butylcalix[4]arene have been accomplished in moderate yields using short synthetic sequences. Some extraction studies have been performed using Th4+ (as a surrogate for Pu4+), Fe3+, UO22+ and Cu2+ to estimate the metal ion selectivity and extraction efficacy of these chelators. Our preliminary results show these ligands are capable of extracting thorium(IV) efficiently but that they may not be selective for its extraction in the presence of iron(III).