Flow-through Sampling Research Articles

Variability of bio-optical properties at sampling stations and implications for remote sensing: a case study in the north-east Gulf of Mexico

Variations of bio-optical properties at oceanographic sampling stations, although important for satellite data validation and algorithm development, have rarely been documented or studied. Using flow-through data and water samples collected from the flow-through system and Niskin bottles at ∼260 stations between summer 1998 and spring 1999 in the north-east Gulf of Mexico (27.5° to 30.4° N, 90° to 80° W), we study the variability of several properties, including chlorophyll-a concentration and Gelbstoff absorption, at the sampling stations. It is found that the standard deviations for both Gelbstoff and chlorophyll are less than 10% of the mean values for more than 90% of the stations, including the coastal stations where water is turbid or Case II. High variations are found in the frontal regions near river plumes. At several stations chlorophyll-a and Gelbstoff vary by nearly two-fold due to spatial and/or temporal variations of the properties near the plume waters. This suggests that for water samples collected from moderately coloured waters (chlorophyll-a >0.25 mg m−3) or coastal river plume waters, special care should be taken to validate the sample data by using multiple samples, a continuous flow-through system, or a concurrent satellite data product map. Otherwise large uncertainties are likely to occur when these data are used to validate satellite estimates.

Poly(dimethylsiloxane)‐based microfluidic device with electrospray ionization‐mass spectrometry interface for protein identification

An easy method to fabricate poly(dimethylsiloxane) (PDMS)-based microfluidic chips for protein identification by tandem mass spectrometry is presented. This microchip has typical electrophoretic microchannels, a flow-through sampling inlet, and a sheathless nanoelectrospray ionization (ESI) interface. The surface of the microchannel was modified with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and the generated electroosmotic flow under acidic buffer condition used for the separation was found to be more stable compared to that generated by the microchannel without modification. The feasibility of the device for flow-through sampling, separation, and ESI-MS/MS analysis was demonstrated by the analysis of a standard mixture composed of three tryptic peptides. Results show that four peaks corresponding to three peptide standards and acetylated products of the standard peptide were well resolved and the deduced sequences were consistent with those expected. Furthermore, the compatibility of this device with other miniaturized devices to integrate the whole process was also explored by connecting a miniaturized enzymatic digestion cartridge and a desalting cartridge in series to the sampling inlet of the microchip for the identification of a model protein, beta-casein.

On-line detection of low naphthalene concentrations with a bioluminescent sensor

On-line tests with Pseudomonas fluorescens HK44 were carried out using a biosensor system with a flow-through cell and sample injections to detect low concentrations of naphthalene. Kinetic experiments permitted a 19 min response time and definition of a 50 min induction period for bioluminescence assays. A mathematical model showed that 0.2 g/l is the best cell concentration to be used in the detection of low naphthalene concentrations, with the highest acceleration rate of bioluminescence sensitivity increase (0.46 nA l/(g min 2)). Calibration curves with different concentrations of naphthalene showed a linear correlation up to 0.4 mg/l (3 μM) corresponding to 211 nA output signal. The lower limit of naphthalene detection by strain HK44 was in the region of 0.02 mg/l (0.16 μM), below the naphthalene health advisory limit for drinking water consumed over a lifetime suggested by EPA.

Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use

A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers of chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, small-scale protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6 M urea, 6 M guanidine, 100 m M NaOH or 500 m M NaOH). Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG. Under all cleaning conditions tested, the log 10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles. In the two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by antibody (Ab) step yield and breakthrough. In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100 m M NaOH) or cycle 50 (500 m M NaOH). For media cycled 300+ times using 6 M urea or 6 M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident. In summary, our studies identify Ab step yield and breakthrough as performance attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled. Thus, we propose that virus removal validation studies should be performed on new media only and these attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media.

Automation for continuous analysis on microchip electrophoresis using flow-through sampling.

Automation of electrophoretic microchips for sequential analysis of different samples is demonstrated. This system used an autosampler, which was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamin B at different concentrations was first loaded into a hydrodynamic flow stream by an autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannel for analysis and detection. Automation was achieved by running two independent programs, one for sample loading by an autosampler and the other one for electrophoretic injection by voltage switching, on the same computer. Using this sampling chip, each loaded volume (0.2-1 microL) can be injected for dozens of electrophoretic analyses (1-10 nL for each injection). The variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Results indicate that the dead volume (approximately 300 nL) due to the connection fitting on the chip could lead to dilution of the loaded sample by a factor of one when 0.2 microL of sample was loaded. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing between two loaded samples to minimize cross contamination without human intervention. Using this sampling chip, the required sample amount and handling time can be greatly reduced compared to the manual method.

Flow-through sampling for electrophoresis-based microchips and their applications for protein analysis.

This work presents a model behind the operation of a flow-through sampling chip and its application for immunoseparation, as well as its integration with a wash/elution bed for protein purification, concentration, and detection. This device used hydrodynamic pressure to drive the sample flow, and a gating voltage was applied to the electrophoretic channel on the microchip to control the sample loading for the separation and to inhibit sample leakage. The deduced model indicates that the critical gating voltage (VC) that is defined as the minimum gating voltage applied to the microchip for sampling is a function of the pump flow rate, the configuration of the microchannel on the chip, and the electroosmosis of the buffer solution. It was found that the theoretical V(C) values calculated from the measured electroosmotic mobilities and flow split ratios were comparable to those experimentally obtained from two microchips with different sampling channel sizes. This had an error percentage ranging from 1 to 20%. Because the hydrodynamic flow is insensitive to electrophoretic mobility, this electrophoresis-based microchip device was free of injection bias due to different ionic strength and electrophoretic mobility in the sample. Additionally, the usefulness of this device was demonstrated for the study of affinity interactions. Mixtures of Cy5-labeled bovine serum albumin (Cy5-BSA) and anti-BSA in various proportions were introduced into the microchip via a syringe pump, and the immunocomplex was electrophoretically separated from the free Cy5-BSA on the microchip. Based on the relative intensity of the free and complex BSA, the binding constant of BSA and anti-BSA was estimated as 3.3 x 10(7) M(-1). Furthermore, a C18 microcartridge (20 microL) was connected to the hydrodynamic inlet of the microchip. Using this device, the wash/elution step can be integrated on-line with the electrophoretic separation and detection on the microchip. Results show that the calibration curve of Cy5-BSA obtained from this integrated device has an R2 value greater than 0.99 and a minimum of quantitation at approximately 10 ng. This direct sampling method is another means of subfractionation, resulting in a relatively greater concentration factor than the average concentration of the whole fraction. Moreover, the electrical field-free bed ensures that the protein interaction will not be affected by the electric field during the wash/elution step.

A High-Throughput Continuous Sample Introduction Interface for Microfluidic Chip-based Capillary Electrophoresis Systems

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with <4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.

Flow-through sampling for electrophoresis-based microfluidic chips using hydrodynamic pumping

This work presents a novel electrophoretic microchip design which is capable of directly coupling with flow-through analyzers for uninterrupted sampling. In this device, a 3 mm wide sampling channel (SC) was etched on quartz substrate to create the sample inlet and outlet and the 75 μm wide electrophoretic channels were also fabricated on the same substrate. Pressure was used to drive the sample flow through the external tube into the SC and the flow was then split into outlet and electrophoretic channels. A gating voltage was applied to the electrophoretic channel to control the sample loading for subsequent separations and inhibit the sample leakage. The minimum gating voltage required to inhibit the sample leakage depended on the solution buffer and increased with the hydrodynamic flow-rate. A fluorescent dye mixture containing Rhodamine B and Cy3 was introduced into the sample stream at either a continuous or discrete mode via an on-line injection valve and then separated and detected on the microchip using laser-induced fluorescence. For both modes, the relative standard deviation of migration time and peak intensity for consecutive injections was determined to be below 0.6 and 8%, respectively. Because the SC was kept floating, the external sampling equipment requires no electric connection. Therefore, such an electrophoresis-based microchip can be directly coupled with any pressure-driven flow analyzers without hardware modifications. To our best knowledge, this is something currently impossible for reported electrophoretic microchip designs.

Rapid and Automated Cartridge-based Extraction of Leukocytes from Whole Blood for Microsatellite DNA Analysis by Capillary Electrophoresis

The human genome contains microsatellite sequences, consisting of 2- to 5-bp repeats, randomly distributed among the chromosomes. These sequences, also called short tandem repeats (STRs), have been demonstrated as important markers for disease diagnostics, genetic mapping, and human identification. The list of human hereditary diseases associated with the unusual expansion or deletion of specific microsatellite loci continues to increase. The two most understood diseases of this type are the CGG repeats in fragile-X syndrome (1) and the CAG repeats in Huntington disease (2). However, other diseases, such as cancer of the colon, head, neck, gastrointestinal track, urinary bladder, liver, lung, breast, and leukocytes [white blood cells (WBCs)], have been shown to be linked to microsatellite instability (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The growing importance of microsatellite markers in diagnostics has motivated us to develop rapid, automated devices to integrate sample preparation, PCR, and capillary electrophoresis (CE) analysis. The work presented here demonstrates an efficient filtration cartridge (15) to automatically process WBCs in a sample of blood for PCR amplification and CE analysis. A flow-through sample preparation procedure is used that does not require vortex-mixing, precipitation, and centrifugation, and is amenable to integration into a microfluidic circuit. TH01 , a well-characterized microsatellite locus, served as the model for this study. TH01 , a gene for human tyrosine hydroxylase, is a …

Scanning probe microscopy and enhanced data analysis on lignin and elemental-chlorine-free or oxygen-delignified pine kraft pulp

Fibres from conventional pine kraft pulp (KP), oxygen-delignified KP (KPO) and elemental-chlorine-free (ECF) bleached KP were characterized by scanning probe microscopy (SPM). The surface morphology of lignin isolated from the respective pine KP was studied. The data analysis of the phase-shift data measured by using different tapping mode imaging parameters enabled the study of both the surface amphiphility and stiffness as a function of delignification. The average phase-shift values were observed to correlate to the respective kappa values, when imaging with small tapping amplitude (30 nm), referred to the capability of SPM to follow local amphipilic (adhesion) differences on a wood fibre surface. The roughness (standard deviation) of the average phase-shift values was taken as a measure of the sample homogeneity. The mean morphological roughness was observed to increase with decreasing bulk lignin concentration. The surface of lignin appeared mainly granular but occasionally some linear structures were observed. The surface roughness of different fractions of flow-through residual lignin samples correlated quite well to the molar mass of the respective fraction. Each grain contained hundreds of lignin molecules. Most of the lignin samples were homogeneous as regards friction and phase-contrast images. The surface of the nondelignified pine KP was typically granular. ECF delignification was more effective than oxygen delignification in removing this granular phase. The decrease in the relative amount of the granular phase correlated to a decreasing kappa value of the pulp. The granular phase is thus suggested to correspond to lignin, but it cannot be ruled out that small amounts of hemicellulose, extractives or a mixture of these components are embedded in this phase. The surface concentration of the granular phase of the KPO samples was, according to the phase-contrast images, clearly higher than the bulk lignin concentration, as indicated by the corresponding kappa values.

Flow-through coaxial sample holder design for dielectric properties measurements from 1 to 350 MHz

A system for measuring the dielectric properties of cereal grains from 1 to 350 MHz with a coaxial sample holder is presented. A signal-flow graph model was used to determine the permittivity of several polar alcohols from the full two-port S-parameter measurements. At the lowest frequencies 1-25 MHz where the phase measurements are less accurate, a lumped-parameter model was used to predict the dielectric loss factor values. The system was calibrated with measurements on air and decanol and verified with measurements on octanol, hexanol, and pentanol. The standard error for the polar alcohols used for verification was 2.3% for the dielectric constant and 7.6% for the dielectric loss factor. Although measurements were taken on static samples, the sample holder is designed to accommodate flowing grain.

SENSINGWHEAT MOISTURE CONTENT INDEPENDENT OF DENSITY

Radio-frequency measurements were taken on four cultivars of hard red winter wheat at 24C, moisturecontents ranging from 9% to 21%, wet basis, and at bulk densities ranging from 0.66 to 0.83 g/cm3. Moisture content waspredicted with an equation developed from the transmission coefficient S21 at three frequencies between 1 and 350 MHz.Measurements at about 40, 240, and 320 MHz were selected from multiple linear regression analysis of data collected onstatic samples in a two-port coaxial flow-through sample holder. Calibration was performed with 49 samples from thefour wheat cultivars harvested, and validation was performed with 84 different samples of the same cultivars. Thestandard deviation of the differences (SEP) for the validation data, compared to standard air-oven moisture contents, was0.36% moisture content, and the bias was 0.06% moisture content.<br><br>Initial attempts to develop a moisture calibration from the intrinsic dielectric properties were unsuccessful. This wasattributed to larger errors in the model used to calculate the loss factor values at the lower frequencies.

On-Line Radioisotope XRF Analysis of Copper, Arsenic, and Sulfur in Copper Electrolyte Purification Solutions

The U.S. Bureau of Mines (USBM) and Weber State University (WSU) have developed an on-line analysis system for copper electrolytic solutions in a laboratory-scale decopperization electrolytic cell using a portable energy-dispersive X-ray fluorescence (XRF) unit. The portable XRF unit utilizes Fe-55 and Cm-244 radioactive isotope sources for sample analysis. Copper, sulfur, and arsenic were successfully analyzed directly in electrolytic purification solutions without any dilution or sample alteration. Interelement deconvolution was conducted to compensate for interferences resulting from matrix absorption of copper fluorescence by sulfur. The ubiquitous presence of sulfur in copper refining operations necessitates sulfur correction in the analysis of copper by XRF. A unique flow-through sampling cell was engineered and constructed for the portable XRF unit which allowed real-time analysis of a continuously pumped sample stream from the electrolytic cell.

An Instrument System for High-Speed Mapping of Chlorophyll a and Physico-Chemical Variables in Surface Waters

A device incorporating microprocessor control and flow-through sampling permits high-speed measurements of chlorophyll a, and physical and chemical variables in aquatic systems. Continuous sampling of chlorophyll a, conductivity, temperature, salinity, incident photosynthetically active radiation (PAR), underwater PAR, and pH facilitates multiple correlations and mapping of variables at both small and large spatial scales. The instrument is portable and can be installed in a small boat for “rapid response” sampling of large areas. The low-voltage DC power requirement and shallow draft make the device especially suitable for work in shallow coastal areas, tidal creeks, bayous, and physically complex aquatic landscapes where larger vessels cannot be operated. Examples of applications of the instrument are discussed. In Fourleague Bay, Louisiana, a shallow estuary on the Gulf of Mexico, we were able to detect horizontal chlorophyll a structure and transient fronts map spatial variations on the scale of a few meters to several kilometers, and follow movements of chlorophyll features through the estuary. These patterns were often not apparent when sampled at discrete stations.

The effect of rock type, grain size, sorting, permeability, and moisture on measurements of radon in soil gas: A comparison of two measurement techniques

Soil surveys of radon conducted in the Coastal Plain of New Jersey, Alabama and Texas indicate that soil composition and grain size exert the strongest control on the concentration of radon measured. Soil-gas radon was measured in-situ using two techniques; one developed by G. Michael REIMER of the U. S. Geological Survey; the other developed by Rogers and Associates Engineering Corp. for use by the Environmental Protection Agency. The Reimer technique aquires a small-volume, grab sample of soil gas, whereas the Rogers and Associatess technique acquires a large-volume, flow-through sample of soil gas. The two techniques yield similar radon concentrations in well-sorted sands, but do not correlate as well for poorly sorted soils and clays.

CIRCLE CELL® FT-IR Analysis of Chemical Warfare Agents in Aqueous Solutions

The qualitative and quantitative FT-IR analyses of dilute aqueous solutions of the chemical warfare threat agents tabun (GA), sarin (GB), and soman (GD) were evaluated with the use of the CIRCLE CELL®. Infrared spectra were recorded with a Nicolet 60SXB FT-IR spectrometer fitted with a liquid-nitrogen-cooled MCT-A detector. The CIRCLE CELL® used a modified micro, high-pressure, flow-through sampling accessory fitted with a ZnSe internal reflection element. Peak heights were evaluated with the Nicolet SUPER QUANT program after spectral subtraction of the solvent. The P=O stretching absorption band, centered around 1240 cm−1, produced good quantification in the concentration range 0.5 to 2.0 mg/mL. In this concentration range the intra-daily precision had a percent coefficient of variation (% CV) of less than 3.0%, while the inter-daily precision yielded a % CV of less than 6.0%. The detection limit was observed to be 0.3 mg/mL for GA and 0.2 mg/mL for GB and GD.

Raman spectroscopy of the liquid-solid interface: monolayer and bilayer adsorption of pyridine on silica

Abstract : Raman spectra of pyridine adsorbed at a silica-solution interface are reported. A flow-through sample cell is employed to control the illuminated surface area of the particulate sample and to minimize background from solution phase species. The technique is applied to study the adsorption of pyridine onto silica gel from carbon tetrachloride solution. Spectroscopic adsorption isotherms are acquired with sufficient precision to allow monolayer and bilayer forms of adsorbed pyridine to be distinguished and quantified. The monolayer form adsorbs with proton transfer decreases uniformly with increasing surface coverage. A Frumkin model fits the monolayer isotherm data and provides an estimate of the decrease in free energy of adsorption with increasing surface coverage. The bilayer isotherm exhibits a concentration threshold which could only be fit by assuming that its energy of adsorption depends strongly on monolayer coverage. The accumulation of this pyridine bilayer appears to play a role in stabilizing the charge density of ion pairs in the monolayer.

A continuous method for monitoring and controlling fermentations: Using an automated HPLC system

Fermentation processes still lack rapid, reliable, and quantitative methodology for the real time analysis of substrates and products. Real time data on fermentation components lend itself not only to monitoring complex mixtures, but also to simple control on demand features. Some of the latest method employed involve the use of immobilized enzyme probes to monitor a substrate such as glucose, or a product such as ethanol in an ultrafiltered stream. Mass spectrometers have been used to analyze fermentation off-gases for volatile components such as O/sub 2/, CO/sub 2/, and ethanol. Each of the enzyme sensor methods is substrate specific and a complex fermentation requires the monitoring of several soluble components. Recently, Ivie proposed using an automated HPLC system to monitor and control an ethanol fermentation. An HPLC system has the advantage of being able to discriminate between several different components, usually in less than 15 min, depending on proper column selection. However, samples must be periodically removed from the fermenter, filtered and manually placed in the HPLC injector system for analysis. The system described here uses an automated, computer controlled HPLC system. The unit was fitted with a continuous flow-through sample vial for the on-line analysis of fermentation broths. Resultsmore » of monitoring a simple yeast fermentatin of glucose to ethanol are reported.« less

Temporal and spatial resolution of pump sampling systems

A simple empirical method is described for determining the fine-scale resolution of continuous flow-through sampling systems. An input delta function is provided by injection of fluorescent dye into the sampler intake. The output fluorescent signal provides time-domain estimates of delay and signal spread; Fourier transformation of the output provides the frequency domain transfer function of the system. Systems with direct intakes (hose or through-hull fitting) can resolve features of 30-s duration or less; however, sea chest intakes cause substantial blurring of the input signal. Attempts to increase vertical spatial resolution by slow lowering rates can result in profiles characterized by appearent micropatchiness and multiple layering, artifacts of the procedure.

Separation and Detection of Arsenical Pesticide Residues and Some of Their Metabolites by High Pressure Liquid Chromatography-Graphite Furnace Atomic Absorption Spectrometry

Abstract A method was developed for the separation and quantitative determination of arsenite, methanearsonic acid (MAA), dimethylarsinic acid (cacodylic acid or CA), and arsenate by high pressure liquid chromatography (HPLC) coupled to a graphite furnace atomic absorption (GFAA) detector. The compounds are separated on a low capacity anion exchange column with gradient elution from 100+ water-methanol (80 + 20, v/v) to 100% 0.02M (NH4)2CO3-methanol (85 + IS, v/v) at 1.2 mL/min and a pressure of about 300 psi. The HPLC was coupled by an automatic sampling unit to the GFAA through an interface consisting of a Teflon flow-through sampling cup. Limit of detection for the arsenic compounds was about 5 ng As injected in aliquots of 20 μL (0.25 ppm As in solution). All 4 arsenical compounds gave similar calibration curves, which were linear from 0.1 to 2.0 ng for single atomizations in the GFAA and from 5 to 200 ng when multiple, sequential atomizations of HPLC eluant were used to quantitate HPLC peaks. Quenching by soil extracts as well as aqueous HPLC solvents was minimal.