Flowing Sample Stream Research Articles

A Sensor‐Controlled Flow Apparatus for Fast Online Titrations

AbstractA process‐suitable titration apparatus was developed for fast online titrations. It consists of computer‐controlled gradient pumps, a sample dilution setup, mixing devices and a microfabricated flow cell with potentiometric sensor. The flow control of the pumps is done via the electrode response signal and a control program applying the feedback algorithm. The apparatus allows the conduction of titration experiments in time cycles of 30 – 50 s in a continuously flowing sample stream. It is about ten times faster than classical batch titrations while accuracy and precision remain of similar magnitude. The long‐term stability and the applicability to process control are shown in field tests. The presented increase of speed, decrease of size and explosion proof make titrations suitable as online technique in the process industry.

Underway determination of alkalinity in estuarine waters by reagent-injection gas-diffusion flow analysis

The development and application of a portable, hybrid reagent-injection gas-diffusion flow analysis technique is described for the underway measurement of total alkalinity in estuarine waters. Injection of pH 4.5 buffer into a continuously flowing sample stream produced gaseous CO 2 that diffused across a microporous PTFE membrane into a weakly buffered acceptor stream containing bromothymol blue indicator. The resultant change in acceptor stream pH was detected photometrically using a super-bright LED with a multi-reflection flow cell and charge coupled device detector. This method gave a detection limit of 0.5 mg CaCO 3 L −1, with reproducibility of 1.0% R.S.D. at 160 mg CaCO 3 L −1, and a measurement rate of 71 injections h −1. The portable FIA system was used for underway analysis of estuarine waters with salinities ranging from that of freshwater to seawater, and there was close agreement between the results obtained by underway analysis and from a reference titration method.

Multiplexed and microparticle‐based analyses: Quantitative tools for the large‐scale analysis of biological systems

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using microparticles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development.

Dynamic digital image analysis: emerging technology for particle characterization

The feasibility of applying dynamic imaging analysis technology to particle characterization has been evaluated for application in the water sector. A system has been developed which captures in-situ images of suspended particles in a flowing sample stream and analyzes these images in real time to determine particle size and concentration. The technology can measure samples having a wide range of particle sizes (approximately 1.5 to 1,000 microm equivalent circular diameter) and concentrations (<1 to >1 million/ml). The system also provides magnified images of particles for visual analysis of properties such as size, shape and grayscale level. There are no sample preparation requirements and statistically accurate results are produced in less than three minutes per sample. The overall system architecture is described. The major design challenges in developing a practical system include obtaining adequate contrast for the range of particle materials found in typical water samples and achieving this under operating conditions permitting an adequate sample processing rate for real time feedback of results. Performance of the instrument is reported in reference to industry accepted particle standards and applications as an analytical tool for the water industries are considered.

Simultaneous in-line concentration for spectrophotometric determination of cations and anions

A flow system is proposed for simultaneous in-line concentration of cations and anions. A sliding-bar commutator was employed to insert an anion and a cation exchange column into a flowing sample stream for serial retention of the analytes. In the injector alternative position, different solutions flowed through the columns for parallel elution of the species in different analytical paths. Three-way solenoid valves allowed the intermittent reagent introduction in the sample zones. Signals were measured by employing two flow-through LED-based detectors. The simultaneous retention of the sample zones in coiled reactors can be also performed to increase the residence time and the analyte conversion rate. The analytical potentiality was demonstrated by the in-line concentration of ammonium and phosphate followed by spectrophotometric detection. For a 90 s loading time, the sampling rate was estimated as 40 determinations per hour, which is three-fold higher than the obtained without performing the tasks simultaneously. Enrichment factors of 8.0 and 18 were estimated for phosphate and ammonium, respectively, yielding detection limits of 1 mg L-1 PO4(3-) and 1 mg L-1 NH4+ (99.7% confidence level). The reagent consumption was lower than 2 mg per determination. Results for freshwater samples agreed with the obtained by reference APHA procedures at the 95% confidence level.

Efficient Detection of Single DNA Fragments in Flowing Sample Streams by Two-Photon Fluorescence Excitation

This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.

Single-Molecule Identification in Flowing Sample Streams by Fluorescence Burst Size and Intraburst Fluorescence Decay Rate

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.

Single-Molecule Detection in the Near-IR Using Continuous-Wave Diode Laser Excitation with an Avalanche Photon Detector

While single-molecule detection in flowing sample streams has been reported by a number of groups, the instrumentation can be somewhat prohibitive for many applications due to the complexity and extensive expertise required to operate such a device. In this paper we report on the construction of a single-molecule detection device that is rugged, compact, inexpensive, and easily operated by individuals not well trained in optics and laser operations. The single-molecule detection apparatus consists of a semiconductor diode laser operating in a continuous-wave (CW) mode and a single photon avalanche diode transducer for converting the detected photons into transistor–transistor logic (TTL) pulses for displaying the data. In addition, the sampling volume is produced by a single-component lens, to create a volume on the order of 1 pL, allowing the sampling of microliter volumes of material on reasonable time scales. The device is targeted for operation in the near-IR region (700–1000 nm), where matrix interferences are minimal. Our data will demonstrate the detection of single molecules for the near-IR dyes IR-132 and IR-125, in methanol solvents in flowing sample streams at sampling rates of 100–250 samples/s. Detection efficiencies for the investigated near-IR dyes were found to be 98% for IR-132 and 50% for IR-125. Previous attempts in our laboratory to detect single molecules of IR-125 using time-gated detection were unsuccessful because of the short upper-state lifetime of this fluorophore (τf = 472 ps).

Flow-through fiber-optic ammonia sensor for analysis of hippocampus slice perfusates

An absorbance based flow-through fiber-optic probe is developed for measurements of ammonia in neurochemical samples. The probe is constructed by trapping a small volume of an internal indicator solution between two sets of optical fibers. This indicator solution is separated from a flowing sample stream by a microporous gas permeable membrane. The flow-through design permits analysis of small aliquots of sample which are injected and pass across the gas permeable membrane. Ammonia in the sample diffuses across this membrane, enters the internal solution, and alters the distribution of a pH sensitive chromophore. The resulting change in absorbance is measured and related to the sample ammonia concentration. The final ammonia sensor possesses a 0.2–20 μM dynamic range with a detection limit of 0.2 ± 0.1 μM and response time of 8 min. The utility of this ammonia sensor is illustrated by measuring extracellular ammonia levels from perfused rat hippocampal tissue. These measurements indicate extracellular ammonia levels fluctuate during tissue depolarization, which is consistent with our findings with other neurological tissues.

Analysis of fluorescence lifetime data for single Rhodamine molecules in flowing sample streams

Fluorescence events for individual molecules of Rhodamine 110 dye in a stream of methanol occur as bursts of 5-300 photoelectrons when the dye molecules transit the volume interrogated by the pulsed excitation laser. The arrival times for these events, extracted by use of time-correlated single-photon counting techniques, are distributed exponentially over a ∼10-ns time window gated to discriminate against prompt scattered light. Still, residual scattered light and solvent-impurity fluorescence contribute significantly to the recorded time distributions, which therefore must be analyzed as sums of an exponential decay and a background. Results obtained from two-parameter nonlinear least-squares fits are compared with maximum-likelihood estimates which ignore the background

Application of Single Molecule Detection to DNA Sequencing and Sizing

AbstractWe are developing a laser‐based technique for the rapid sequencing of 40‐kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, and detection of the individual, fluorescently labeled bases by laser‐induced fluorescence as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will reduce significantly the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.We are also applying our sensitive fluorescence detection to sizing of DNA fragments. Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused, continuous‐wave laser beam in an ultrasensitive flow cytometer at a rate of ∼60 fragments per second. The size of the fluorescence burst emitted by each stained fragment as it passes through the laser beam is measured in one millisecond. We have demonstrated flow cytometric sizing of DNA fragments in a ∼0.1‐pg sample of a restriction digest of lambda DNA in 164 seconds with sizing accuracy better than 98%.

Detection and lifetime measurement of single molecules in flowing sample streams by laser-induced fluorescence

The detection and measurement of fluorescence lifetimes of single Rhodamine-110 molecules in a flowing, aqueous sample stream is described. Time-correlated single-photon counting, used in combination with mode-locked picosecond pulsed excitation, allows the detection of single fluorescent molecules in the presence of significant solvent Raman and Rayleigh backgrounds. The fluorescence lifetime of a detected molecule is estimated from the record of arrival times (relative to the excitation pulse) of photons detected during the molecule’s passage through the ∼1 pL excitation volume.

Single-molecule detection as an approach to rapid DNA sequencing

Current sequencing technologies are insufficient to cope with large-scale projects such as sequencing the human genome and genomes of model organisms. In addition, as genetic lesions associated with specific human diseases are identified, DNA sequencing will be used increasingly for clinical applications. Thus, new approaches are needed to combine high-throughput with accuracy for both research and diagnostic purposes. A novel technology based on detection of individual fluorescent nucleotides in a flowing sample stream is under development.

Rapid DNA sequencing based upon single molecule detection

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

High‐speed gas chromatography using an on‐column thermal desorption modulator

AbstractA short, narrow‐bore (50 μm) column with an on‐column thermal desorp‐ tion modulator provides a technically simple and reliable method to generate high‐ speed gas chromatograms. A thermal desorption modulator prepared on the column but operated at a lower temperature functions as a sample introduction device. The modulator portion of the column extends outside of the oven and is at room temperature. A sample stream flows continuously into the modulator and through it into the column. Retained sample is released from the modulator by an electricaliy generated temperature pulse and carried into the column as a concentration pulse analogous to a sharp injecfion. Because the modulator is part of the column, no external injector is needed, and the volume of the concentration pulse is automatically appropriate for the column diameter. A series of chromatograms can be generated from a continuously flowing sample stream by applying a series of temperature pulses to the modulator. The narrow‐bore column is highly efficient and generates chromatograms with total retention times as short as 1 s. Chromatographic efficiency reaches the column's the oretical limit at very high carrier gas linear velocity. Measured terminal band lengths are shorter than the length of the modulator because the thermal gradient between modulator and column focuses bands as they enter the column. Band durations are less than the modulator thermal pulse duration because sample substances are released from the modulator stationary phase and carried onto the column during a small part of the temperature ramp.

Determination of tarce water in non-polar organic solvents with a flow-injection system and tin tetrachloride or antimony pentachloride

Low levels of water (limit of detection 2-5 mg kg 1̄)can be determined in a non-polar organic solvents such as benzene, 1,2-dichloroethane (DCE) and n-hexane by utilizing the reaction of water with SnCl 4 or SbCl 5. The reaction results of hydrolysis in halide and is accompanied by a decrease in optical absorption. With SnSl 4, the reaction is monitored near 300 nm and with SbCl 5 it is monitored at lower wavelengths (350-420). Niether reactons proceeds well in media containing only DCE and n-hexane. For this reason, the arrangemens involves a halide reagent dissolved in benzene which is merged with a benzene/n-hexane/DCE carrier stream into which samlpe is injected. A configuration in which only 2μl of a concentrated halide reagent solution is injected into the flowing sample stream is also shown to be viable for the determination of water in benzene. A membrane-permeation-based calibration method for preparing trace water standards is described.

Solvent extraction in continuous flow systems with intelligent zone sampling

A 50 to 100-μl segment of an extractant is injected into a flowing sample stream, to which reagents, as necessary, have been added. Following an extraction conduit typically providing a residence time of 1–3 min, the extractant segment is isolated as it flows through the loop of an injection valve. The actuation of this valve is controlled by sensing its loop-filling status with conductivity probes or by appropriate timing. The isolated extractant containing the analyte is injected into a miscible carrier where further chemistry may be carried out before detection. Experimental systems studied include the extraction of C1–C5 n-mercaptons from gasoline by aqueous sodium hydroxide, with detection of the analytes in the aqueous extract following a chromogenic reaction, extraction of a linear alkylbenzenesulfonate from water into chloroform as its ion pair with methylene blue, and the extraction of Auramine O (C.I. 41000) from water solutions containing a fluorocarbon surfactant into chloroform. The effects of various parameters such as the choice of the wetted conduit material, conduit configuration, diameter, residence time, phase ratio, flow rate, etc., are examined and discussed. Both phenomelogically and behaviorally, the present system displays significant differences from the familier uniformly (small) segment-interspersed solvent extraction systems.

Determination of activity coefficients of binary liquids by capillary gas chromatography with thermal desorption modulation for direct headspace sampling

Activity coefficients of the binary liquid mixtures benzene-toluene and acetonechloroform were determined using thermal desorption modulation for direct headspace sampling into a capillary gas chromatograph. A thermal desorption modulator is a short, heated section at the head of the column. An electrical current pulse applied to a thin conductive film heats the modulator section and the stationary phase within it, releasing any retained substances as a concentration pulse which flows into the column. The modulator acts like an automatic and highly reproducible small-volume injector for a continuously flowing sample stream. Short-term relative standard deviations obtained using this technique are approximately 2%.

Process Control Applications of the Peltier Cooled SI (LI) Detector Based EDXRF Spectrometer

AbstractThe demand for on-line analyzers capable of compositional determinations in petroleum and chemical process streams has increased dramatically in recent years. Total control of production plant processes and resources requires the analysis of feed, intermediate, and product materials. This paper will describe a rugged, on-stream energy dispersive X-ray fluorescence (EDXRF) analyzer configured for the continuous determination of composition in solid and liquid samples. The detector in the X-ray sensor is a lithium-drifted silicon crystal which is thermoelectrically cooled (Peltier effect) to achieve operational temperatures. This approach to detector cooling offers advantages over traditional cryogenic liquid cooling when EDXRF is used in the process control environment.Three applications of the thermoelectrically-cooled detector-based EDXRF spectrometer will be presented here. The first is the analysis of a catalyst solution to monitor depletion of the active species. Second, two components of a plating bath solution will be determined simultaneously in a flowing sample stream. In the third application, the spectrometer will be oriented in a downward looking configuration to monitor inorganic constituents in absorbent samples as they are transported by conveyor belt to the X-ray measurement area.

Potential of modified reverse flow injection analysis for continuous monitoring and process control

Modified reverse flow injection analysis is introduced as a novel means for monitoring purposes and online process control. The technique is based on the injection of standard solutions into the continuously flowing sample stream. The transient detector response occurring shortly after the injection reflects the deviation between the analyte concentration of the sample stream and the standard injected. Interpolative calibration and the check of nominal values are two interesting execution modes presented. The beneficial features of the novel approach are discussed and exemplarily demonstrated for practical problems. Experimental conditions are given for the photometric determination of chloride in tap water, phosphate in surface water and the potentiometric determination of fluoride. The respective advantages over common monitoring systems are outlined.