Optical Flow Cell Research Articles

Determination of phosphate using a highly sensitive paired emitter–detector diode photometric flow detector

The use of a novel inexpensive photometric device, a paired emitter–detector diode (PEDD) has been applied to the colorimetric determination of phosphate using the malachite green spectrophotometric method. The novel miniaturized flow detector applied within this manifold is a highly sensitive, low cost, miniaturized light emitting diode (LED) based detector. The optical flow cell was constructed from two LEDs, whereby one is the light source and the second is the light detector, with the LED light source forward biased and the LED detector reversed biased. The photocurrent generated by the LED light source discharges the junction capacitance of the detector diode from 5 V (logic 1) to 1.7 V (logic 0) and the time taken for this process to occur is measured using a simple timer circuit. The malachite green (MG) method employed for phosphate determination is based on the formation of a green molybdophosphoric acid complex, the intensity of which is directly related to phosphate concentration. Optimum analytical parameters such as reaction kinetics, reagent to sample concentration ratio and emitter wavelength intensity were investigated for the spectrophotometric method. Linear calibration plots that obeyed the Beer–Lambert law were obtained for phosphate in the range of 0.02–2 μM. The dynamic range, sensitivity and limits of detection are reported.

An improved flow system for chloride determination in natural waters exploiting solid-phase reactor and long pathlength spectrophotometry

A single and sensitive spectrophotometric method for chloride ions determination based on a commuted flow system with a 100 cm optical path flow cell and a solid-phase reactor containing immobilized silver chloranilate was proposed. This procedure exploited the AgCl formation in the solid-phase reactor leading the chloranilate ions, monitored spectrophotometrically at 530 nm. The analytical signals were 75-fold higher and the sensitivity was 12-fold than that achieved with a 1 cm flow cell, allowed a chloride determination in the 0.5–100 mg l −1 range. The R.S.D. was 1.1% ( n = 20) with a sample throughput of 80 h −1 and a waste generated of ca. 100 ng of chloranilate ions per determination. Four samples of natural waters from São Carlos and Araraquara cities were evaluated using the proposed method. Results agreed with the obtained by a reference method at the 95% confidence level.

Spherical vs. Granular Immobilization Support Selection and Performance on an Optical Flow Cell Immunosensor Based on the Fluorescence of Cyanine‐5

A spherical porous glass support Trisoperl® (TRISO) with four pore diameters (ø 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (‐NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(‐NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(‐NCS) and TRISO(‐NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti‐Goat IgG conjugated with Cyanine‐5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(‐NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(‐NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.

Direct determination of tagitinin C in Tithonia diversifolia leaves by on-line coupling of supercritical carbon dioxide extraction to FT-IR spectroscopy by means of optical fibres

Supercritical fluid extraction (SFE) with carbon dioxide as extraction medium was on-line coupled to a FT-IR spectrometer equipped with a Mercury Cadmium Telluride (MCT) detector using a tailor-made high-pressure fibre optic flow cell. This method was optimised and developed for the monitoring in real time and the quantification of dynamic extractions of tagitinin C from Tithonia diversifolia leaves. In order to demonstrate the method ability to allow the direct quantification of tagitinin C in the extract medium the standard addition method was used. The area integration of curves obtained by plotting the absorbance of the highly specific C O stretching vibration at 1668 cm −1 versus time (i.e. extractograms) was used as instrumental response. The SFE/FT-IR process was successfully validated using the accuracy profile as decision tool. On this basis, a linear regression model was chosen for the calibration curve. The relative standard deviation for repeatability and intermediate precision were between 0.8 and 3.1 %, respectively. Moreover, the method was found to be accurate as the two-sided 95% beta-expectation tolerance interval did not exceed the acceptance limits of 85 and 115% on the analytical range investigated (500–2500 μg of added amount of tagitinin C). The proposed method allowed the non-destructive extraction of tagitinin C and its on-line quantitative determination in less than 25 min thus facilitating the subsequent experiments or the pharmacological studies performed on this compound.

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy.

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.

Photometric detection in flow analysis systems using integrated PEDDs

A novel inexpensive optical-sensing technique has been developed for colorimetric flow analysis. This sensing system employs two LEDs whereby one is used as the light source and the other as a light detector. The LED used as light detector is reverse biased with a 5-V supply so that the photocurrent generated by the incident light discharges the capacitance. Direct digital output is provided by a simple timer circuit that measures the time taken for this discharge process from 5 V (logic 1) to 1.7 V (logic 0). This sensing concept has been applied in flow analysis by constructing an optical flow cell with a pair of LEDs. Calibration of the integrated optical flow cell using a dye resulted in a linear response that obeys the Beer–Lambert law. The flow rate, dynamic range, sensitivity and limits of detection were investigated. The system was also used for pH determination in the range of pH 2.5–6.8 using bromocresol green (BCG). The p K a of BCG was successfully determined by this technique.

Using optical NIR sensor for on-line virgin olive oils characterization

Near-infrared transmittance spectroscopy was applied to on-line control quality and characterization of virgin olive oils. The transmittance spectrum (log 1/ T) was obtained through dispersal equipment, scanning the samples between 750 and 2500. A 1 mm optical path length flow cell and holding 120 μl was employed. Calibration models for: acidity value (AV), bitter taste (k225) and fatty acid composition (FAME), were previously developed in the laboratory using partial least squares (PLS) regression. A total of 190 virgin olive oil samples, gathered during the usual harvesting time of three olive crop seasons, were used. The validation set gave correlation coefficients and standard error of prediction of 0.999 and 0.35, 0.936 and 0.058, 0.998 and 0.604, 0.992 and 0.674% for AV, k225, fatty acid oleic and fatty acid linoleic, respectively. These PLS models, prior to a slope/bias correction, were used to monitor on-line values of these parameters during the processing of virgin olive oils in real olive mills. Samples of oils were obtained for chemical analysis. Application of NIR transmittance spectroscopy on-line to virgin olive oil production line, has allowed to detect, at real-time, the changes produced in the characteristics of the oils during their production. The results indicate similarity between information both NIR and reference laboratory methods.

Novel optical flow cell for measurements of fluid phase behaviour

We describe a novel optical flow cell for rapid automated isobaric measurements of liquid–liquid phase boundaries in binary mixtures. The apparatus was suitable for measurements at temperatures between 298 and 473K at pressures of up to 7MPa. The cell volume was only 35μL but this was later increased to 55μL to accommodate a magnetic stirrer. Measurement of a single point on the temperature–composition phase diagram could be accomplished within about 10min. Results are reported for {(1 −x) H2O + xc-(CH2)4O} at pressures of 3 and 6MPa.

An improved flow system for phenols determination exploiting multicommutation and long pathlength spectrophotometry

A greener and sensitive procedure for spectrophotometric determination of phenols based on a multicommuted flow system with a 100 cm optical path flow cell is presented. The method exploited the oxidative coupling of phenolic compounds with 4-aminoantipyrine in alkaline medium containing potassium hexacyanoferrate(III). Sensitivity was 80-fold higher than that achieved with a 1 cm flow cell, making feasible the determination of phenols in the 10–100 μg l −1 range with a detection limit estimated as 1 μg l −1 phenol. The sampling rate and the coefficient of variation were estimated as 90 determinations per hour and 0.6% ( n=10), respectively. The multicommutation approach allowed a 200-fold reduction of the reagent consumption in comparison with the reference batch method. Moreover, the chloroform extraction for analyte concentration is unnecessary in view of the increase in sensitivity. Recoveries within 93.3 and 106% were achieved for determination of phenol in natural and wastewater samples. Results agreed with the obtained by a reference method at the 95% confidence level.

Kinetic studies of iron deposition in horse spleen ferritin using O 2 as oxidant

An optical flow cell provided a means to conveniently measure the rate of successive Fe 2+ oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe 2+ oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe 2+/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH) 3 formation and the presence (>95%) of Fe(OH) 3 deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5–50 Fe 2+/HoSF established that the reaction is overall first order in Fe 2+ concentration. However, when full progress curves were analyzed at a variety of Fe 2+/HoSF ratios, two first-order reactions ( k 1∼0.035 s −1 and k 2∼0.007 s −1) were found to contribute to the overall Fe 2+ oxidation reaction. The proportion of the fast reaction increased with increasing Fe 2+/HoSF ratio until at ∼400, it was the dominant reaction. For the Fe 2+/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe 2+ oxidation. Comparison of successive additions of 1.0 Fe 2+/HoSF showed that oxidation of the first 8–10 Fe 2+ produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H +/Fe 2+ ratio confirmed this difference in behavior by giving an H +/Fe 2+ ratio of ∼1.0 below and 2.0 for ratios >30 Fe 2+/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.

Determination of total petroleum hydrocarbons in soil by dynamic on-line supercritical fluid extraction with infrared photometric detection

Total petroleum hydrocarbons (TPHs) in soil are determined by on-line dynamic supercritical fluid extraction (SFE) using infrared filter photometry detection. The filter photometer was constructed in the laboratory using a tungsten lamp, an optical notch filter that selects the C–H stretching vibration of the extracted organics, an optical chopper with demodulation electronics, and a PbSe detector. A modified high-pressure fiber optic flow cell was used to couple the SFE system to the photometer. Quantitation of TPHs was accomplished through the construction of calibration curves of integrated absorbance of C–H stretching (over time) versus concentration. Our studies show that the sensitivity of this system is affected by both the optical path length in the high-pressure cell and the SFE fluid flow-rate, and detection limits for TPHs are in the mid part-per-million range. The results of the application of this on-line SFE–IR instrument to the determination of TPHs in real-world samples show good agreement with those obtained from standard Soxhlet extraction–IR methods.

Influence of weak elasticity of dispersed phase on droplet behavior in sheared polybutadiene/poly(dimethyl siloxane) blends

The contribution of weak droplet-phase elasticity is investigated for blends of polybutadiene in poly(dimethyl siloxane) in a simple shearing flow with droplet-phase Weissenberg number, Wid, up to around unity. The elasticity of the polybutadiene-dispersed phase is varied by adding various amounts of high-molecular-weight polybutadiene into low-molecular-weight polybutadiene Newtonian fluid. To isolate the contribution of elasticity, the experiments are conducted at fixed viscosity ratio by varying the experimental temperature to counteract the small effect of high-molecular-weight polymer on droplet viscosity. Droplet deformation and relaxation are measured using an optical flow cell mounted on an optical microscope. As the droplet-phase elasticity increases, the steady-state shape deformation at fixed capillary number, Ca, decreases and the critical capillary number for droplet breakup increases. For a 20% dispersed phase blend, the steady-state capillary number calculated from the volume-averaged dropl...

Optical flow cell and apparatus for solubility, salt deposition and Raman spectroscopic studies in aqueous solutions near the water critical point

A flow cell fitted with large diameter optical ports suitable for visual observation and Raman spectroscopic studies of aqueous solutions to temperatures of 500 °C and pressures to 25 MPa is described. The cell uses commercially available compression fittings that have proven to be reliable and robust to cycles of assembly, temperature exposure, and disassembly. A unique design is given for construction of the optical ports. The design of a heated cylindrical insert for salt solubility, salt deposition and salt nucleation studies in near-critical aqueous solutions is described. A method for in situ detection of the crystalline phase of salt deposited from aqueous solution at near-critical conditions and then surrounded by pure supercritical water is demonstrated for Na 2SO 4, using Raman spectroscopic observation.

The application of the multi-pass rheometer for precise rheo-optic characterisation of polyethylene melts

This paper presents an overview of the application of the Multi-Pass Rheometer (MPR) for the study of polymer processing with particular reference to flow birefringence measurements. The MPR is a two-piston capillary rheometer recently developed at the University of Cambridge. We review previous experiments that have been carried out on molten polymers using the MPR and we report on recent results of flow birefringence experiments for a polyethylene (PE) melt. Using a specially designed optic flow cell, molten PE was passed within a Double Cavity Die. A field-wise flow-induced birefringence technique was used to obtain the stress field in a complex flow of a flowing molten polymer. In general, for the PE tested, the entry stress field in the second cavity was found to be different from that in the first. The differences observed in the overall entry stress fields in the two slit configuration could be explained as resulting from the extensional and shear strain history experienced by the polymer. We have carried out matching K-B.K.Z.–Wagner numerical simulations and found that reasonable consistency can be achieved for certain polymers. As a consequence of these experiments, we have found that the MPR is a very good platform for experimental flow birefringence studies that can be matched with numerical simulation and in addition experiments can be carried out with small 20 g quantities of material.

Short-pathlength, high-pressure flow cell for static and time-resolved infrared spectroscopy suitable for supercritical fluid solutions including hydrothermal systems

An optical flow cell for high pressures and temperatures is described. The use of a novel window design allows for a precise, fixed optical pathlength that can be varied by use of spacers that range from a few micrometers to several millimeters. The cell pathlength is not affected by changes in pressure or temperature. The novel window design may be applicable to other high-pressure spectroscopic cells. The flow-cell design has a minimal sample dead volume, which is important for kinetic studies. The design eliminates the need for brazing or for a soft-sealing material for the optical windows, thereby minimizing the number of materials in contact with the sample. Using only diamond and platinum or platinum alloys as the corrosion resistant materials, the design is optimized for the study of aqueous solutions at high temperatures. Infrared spectra of an aqueous sodium tungstate solution up to 400 °C and 380 bar pressure are presented. Time-resolved infrared data are also presented for the ultraviolet photolysis reaction of β-naphthoyl azide in supercritical carbon dioxide.

Optical flow-cell multichannel immunosensor for the detection of biological warfare agents

An automated optical flow cell multichannel immunosensor for the detection and identification of toxins, viruses and bacterial particles is presented. A solid phase ELISA, based on a peroxidase label for signal generation and on fused silica capillaries as a support for immobilized antibodies, has been employed for analyte detection and identification. The sensing and signal transducing component of the sensor consists of a light-emitting diode and a photodetector. The device is fitted with three channels allowing the simultaneous detection of three agents. An integrated flow injection analysis system ensures automation of the assay cycles. Data on the detection of the bacterial toxin staphylococcal enterotoxin B (SEB), the bacteriophage M13 as a viral agent, and Escherichia coli as a bacterial agent are presented.

A flow-through solid phase UV spectrophotometric biparameter sensor for the sequential determination of ascorbic acid and paracetamol

For the first time, a continuous flow system with solid phase UV spectrophotometric detection (an optosensor) is described for the sequential determination of two analytes based on the alternate use of two carrier/self-eluting agents. The selective and sequential sorption of both on an active solid support (an anion exchanger gel placed in the detection zone into an appropriate quartz flow cell) is performed and their respective UV intrinsic absorbances monitored. Each carrier itself elutes the respective analyte from the solid support, so regenerating the sensing zone. Ascorbic acid and paracetamol in concentrations ranging from 0.3 to 20 μg ml −1 and from 0.4 to 25 μg ml −1, respectively, could be determined with this UV flow-through optosensor using sodium acetate/acetic acid (pH 5.6) and 0.05 M NaCl (pH 12.5), respectively as carrier/self-eluting solutions and Sephadex QAE A-25 anion exchanger gel as solid phase placed in the inner of an 1 mm optical path length quartz flow cell. The RSDs % ( n = 10) were lower than 1.3 (for ascorbic acid) and than 1.5 (for paracetamol). Detection limits (criterion 3σ) as low as 0.02 μg ml −1 were achieved in both cases. Application to the analysis of pharmaceutical samples (in addition to synthetic ones) testifies the utility of this sequential sensor, which tolerates amounts of the species usually accompanying the analytes much higher than those ones found in these samples.

Hydroxynitrile lyase at the diisopropyl ether/water interface: Evidence for interfacial enzyme activity

A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, we establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg(-1). For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis-Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase.

Hydroxynitrile lyase at the diisopropyl ether/water interface: Evidence for interfacial enzyme activity

A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, we establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg−1. For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis–Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 425–436, 1999.