Programmable flow injection in batch mode: Determination of nutrients in sea water by using a single, salinity independent calibration line, obtained with standards prepared in distilled water.

Interference of the Schlieren effect on sea water analysis by spectrophotometry is caused by the flow of solutions of different ionic strengths through a flow cell. A flow injection assay protocol programmed in a flow-batch format removes this interference and allows the use of a calibration line, obtained in deionized water, for determination of analytes in sea water samples of different salinity. This Single Line Calibration (SLC) technique is validated on the most frequently performed nutrient assays. Automated determinations, performed at rates ranging from 20 to 60 samples/hr, covered seawater sample ranges from nM to mM with limits of detection: 12 nM for nitrite, 94 nM for nitrate, 47 nM for phosphate, and 240 nM for silicate. Reproducibility of the determinations was equal to or better than, 3% r.s.d. and day to day calibration was within 10%. The programmable FI, uses about 1/5th volume of reagents compared to continuous flow techniques.

The performance of a new linear light path flow cell is compared with a liquid core waveguide and the linear cell is used for spectrophotometric determination of nitrite in sea water at nanomolar concentrations.

The sensitivity of a spectrophotometric assay is enhanced either by increasing the concentration of the target molecules within the flow cell or by extending the length of the light path of the flow cell. Determination of nutrients at nanomolar concentrations in sea water has therefore been based either on the preconcentration of the analyte on a microcolumn from a large volume of sample followed by its elution into a conventional 1-2 cm flow cell, or by the use of Liquid Core Waveguide (LCW) with a light path as long as several meters. In order to evaluate the relative improvements of these different approaches to increasing sensitivity we have developed a preconcentration technique for the determination of nitrite in seawater using the Gries reaction and compare its sensitivity and precision to that of non preconcentration techniques using both LCW and Linear Light Path (LLP) cells of different lengths. In this work the performance of the LLP is investigated and compared with the performance of the coiled LCW flow cell. Next, the determination of nitrite, automated by programmable Flow Injection (pFI), was carried out by using LCW and LLP flow cells, as well as by using a 10 cm LLP flow cell together with the preconcentration step of nitrite on a microcolumn. The assay of nitrite in sub micromolar range was most efficiently performed by a combination of pFI with the LLP flow cell without the need for a preconcentration step. The determination was performed at a rate of 40 samples/hour with a Limit of Detection (LOD) = 0.6 nM N using a 50 cm long, and a LOD = 2.5 nM N using a 10 cm long, LLP flow cell. Analysis of sea water samples confirmed that salinity does not affect the sensitivity of the determination. At a much lower cost than LCW, the LLP flow cell can also be easily assembled from components usually at hand in a laboratory.

Flow injection programmed to function in batch mode is used to determine molar absorptivity and to investigate the phosphomolybdenum blue method.

The ultimate goal of flow-based analytical techniques is to automate serial assays of a target analyte. However, when developing any reagent-based assay, the underlying chemistry has to be investigated and understood a step, which is almost always the most challenging component of the optimization effort. The difficulty lies in that almost all reagent-based assays were initially developed and optimized in a batch mode, with the aim to perform assays manually, within a time frame of up to 15 min, while flow injection techniques are designed to monitor concentration gradients at times prior to reaching chemical equilibria and while performing up to two assays per minute. This work resolves this discrepancy by using programming Flow Injection (pFI) that operates in a batch mode within a time frame of 1 min or less, with the aim of optimizing an assay under the same conditions and using the same instrument in which the assay will be performed. This novel concept is verified by determining a molar absorptivity of Fe(II) ferrozine complex and by comparing it with literature data. Next, the pFI-batch technique was used to investigate and optimize the phosphate assay, based on formation of phosphomolybdenum blue, with the aim of maximizing sensitivity and improving the limit of detection of this widely used method.

Determination of traces of phosphate in sea water automated by programmable flow injection: Surfactant enhancement of the phosphomolybdenum blue response.

An assay protocol, based on programmable Flow Injection (pFI), is optimized by tailoring flowrates appropriately to the individual steps of an assay, thus allowing sample and reagent metering, mixing, incubation, monitoring and washout to be carried out more efficiently and in different time frames. This novel approach to flow based methods is applied here to optimize the determination of orthophosphate at nanomolar levels. Programmable Flow Injection was also used to facilitate an investigation of the properties of the phosphomolybdenum blue (PMoB) formed during this assay, by using the stop flow technique - an approach that revealed for the first time the influence of surfactants on the kinetics of formation of PMoB and its spectral characteristics. It was discovered that the two most frequently used surfactants (SDS and Brij) have profound and different influences on the spectra and formation of PMoB and this finding was used to enhance the sensitivity of the phosphate assay at nanomolar levels. The method was applied to the assay of trace levels of phosphate in sea water.

Programmable Fow Injection. Principle, methodology and application for trace analysis of iron in a sea water matrix.

Automation of reagent based assays by Flow Injection is based on sample processing, in which a sample flows continuously towards and through a detector for monitoring of its components. There are three drawbacks to using this approach. The constant continuous forward flow: continually consumes reagents and generates chemical waste and necessitates a compromise when optimizing the performance of the reagent based assay. The reason is that individual steps of an assay protocol, i.e., sample and reagent metering, mixing, incubation, monitoring and efficient washout are carried out most efficiently on different time scales and therefore at different flowrates. Programmable Flow Injection (pFI) eliminates all three drawbacks and permits the execution of optimization of the assay protocol by means of a computer. This paper details this novel approach to method development by optimization of an assay of iron at nanomolar levels and its application to its determination in a sea water matrix. The pFI method was developed in two variants: Stop in Holding Coil (SHC) and Stop in Flow cell (SFC). The SHC method has a Limit of Detection (LOD = 3.1ppb or 55nM Fe, precision of 1.9% r.s.d. at ~ 90nM, and sampling frequency of 90 samples/h. The SFC method had LOD = 0.57ppb or 10nM Fe, precision of 0.8% r.s.d. at ~ 90nM, and sampling frequency of 40 samples/h and its sensitivity is independent of the salinity of the matrix. The SFC method, and its manual equivalent, was used for the determination of dissolved Fe (II) that had been spiked into several samples of seawater that had been diluted with various volumes of deionized water to mimic coastal seawater. The results showed good agreement between both the SFC and the manual methods.

Determination of trace zinc in seawater by coupling solid phase extraction and fluorescence detection in the Lab-On-Valve format.

By virtue of their compactness, long-term stability, minimal reagent consumption and robustness, miniaturized sequential injection instruments are well suited for automation of assays onboard research ships. However, in order to reach the sensitivity and limit of detection required for open-ocean determinations of trace elements, it is necessary to preconcentrate the analyte prior its derivatization and subsequent detection by fluorescence. In this work, a novel method for the determination of dissolved zinc (Zn) at subnanomolar levels in seawater is described. The proposed method combines, for the first time, automated matrix removal, extraction of the target element, and fluorescence detection within a miniaturized flow manifold, based on the Lab-On-Valve (LOV) concept. The key feature of the microfluidic manipulation of the sample is flow programming, designed to pass sample through a mini-column where the target analyte and other complexable cations are retained, while the seawater matrix is washed out. Next, zinc is eluted and merged with a Zn selective fluorescent probe (FluoZin-3) at the confluence point of the LOV central channel using two high-precision stepper motor driven pumps that are operated in concert. Finally, the thus formed Zn complex is transported to the LOV flow cell for selective fluorescence measurement. This work describes the characterization and optimization of the method including Solid Phase Extraction using the Toyopearl AF-Chelate-650M resin, and detailed assay protocol controlled by a commercially available software and instrument. The proposed method features a LOD of 0.02 nM, high precision (<3% at 0.1 and 2 nM Zn levels), an assay cycle of 13 min and a reagent consumption of 150 µL FluoZin-3 per sample, which makes the method highly suitable for oceanographic shipboard analysis. The accuracy of the method has been validated through the analysis of seawater reference standards and comparison with ICP-MS determinations on seawater samples collected in the upper 1300 m of the subtropical south Indian Ocean. This work confirms that integration of sample pretreatment with optical detection in the LOV format offers a widely applicable approach to trace analysis of seawater.

Towards chemiluminescence detection in micro-sequential injection lab-on-valve format: a proof of concept based on the reaction between Fe(II) and luminol in seawater.

Micro-sequential injection lab-on-valve (µSI-LOV) is a well-established analytical platform for absorbance and fluorescence based assays but its applicability to chemiluminescence detection remains largely unexplored. In this work, we describe a novel fluidic protocol and two distinct strategies for photon collection that enable chemiluminescence detection using µSI-LOV for the first time. To illustrate this proof of concept, we selected the reaction between Fe(II) and luminol and developed a preliminary protocol for Fe(II) determinations in acidified seawater. The optimized fluidic strategy consists of holding 100 µL of the luminol reagent in a confined zone of the LOV and then displacing it with 50 µL of sample while monitoring the chemiluminescent product. Detection is achieved using two strategies: one based on a bifurcated optical fiber and the other based on a customized detection window created by mounting a photomultiplier tube atop of the LOV device. We show that detection is possible using both strategies but that the window strategy yields significantly enhanced sensitivity (355×) due to the larger detection area. In our final experimental conditions and using window detection, it was possible to achieve a limit of detection (LOD) of 1 nmol L(-1) and to quantify Fe(II) in acidified seawater samples up to 20.00 nmol L(-1) with high precision (RSD<6%). These analytical features combined with the long-term stability of luminol solution and the full automation and low reagent consumption make this approach a promising analytical tool for shipboard analysis of Fe(II). The intrinsic capacity of the LOV to operate at a low microliter level and to handle solid phases also opens up a new avenue for chemiluminescence applications. Moreover, this contribution shows that LOV can be a universal platform for optical detection, capable of absorbance, fluorescence and luminescence measurements in a single instrument setup.