Spectrophotometric determination of Cu(II) with sequential injection analysis

A sequential injection system, based on the reaction of Cu(II) with diethyldithiocarbamate (DDTC), was developed for the determination of Cu(II) in plant food and water samples. The extraction procedure, generally used to extract the Cu(II)–DDTC complex for subsequent analysis was eliminated in this procedure. The complex was detected spectrophotometrically in aqueous solutions at 460 nm. The physical and chemical parameters depicting the system were studied to obtain optimum conditions for sample analysis. The system developed is fully computerized and able to monitor Cu(II) in samples at seven samples per hour with a relative standard deviation of <4.50%. The calibration curve is linear from 0.5–5.0 mg/l with a detection limit of 0.2 mg/l. Interferences were reduced by introducing multiple flow reversals, to increase mixing between the reagent and sample zones, and subsequently enhance working of the masking agents (EDTA/citrate).     

Merging zones in flow injection analysis : Part 4. Simultaneous spectrophotometric determination of total nitrogen and phosphorus in plant material

The merging zones approach is used in a single flow injection system for the simultaneous Spectrophotometric determination of nitrogen and phosphorus in plant material based on the Berthelot and molybdophosphate reactions. A multiple proportional injector is designed to introduce samples and reagents into water carrier streams in such a way that only one analytical path with one detection unit is required. For both processes, catalysis is employed; a detailed study of the reagent composition is reported for the Berthelot reaction. The proposed system is characterized by a high degree of sample and reagent dispersion; 60 samples per hour can be analyzed with a consumption of reagents in the range of microliters per determination. Total recoveries of 98.3% N and 99.1% P were estimated from 20 runs of a typical plant sample containing around 3% N and 0.3% P in dry matter. The results of the present method agree with those obtained by Technicon AutoAnalyzer procedure.     

Simultaneous differential rate determination of iron(II) and iron(III) by flow-injection analysis

Flow-injection procedures for the simultaneous spectrophotometric determination iron(II) and iron(III), relying on the different kinetic-catalytic behaviour of iron(II) and iron(III) in the redox reaction between leucomalachite green and peroxodisulphate with and without the presence of the activator 1,10-phenanthroline, are described. Exploiting the fact that one of the chemical reactions is very rapid whereas the other one is comparatively slower, two experimental procedures are presented. In the first, two individual zones of sample solution are injected simultaneously into separate carrier streams of reagent in a two-line system. Taking advantage of the different residence times of the samples in the manifold lines, the resulting colour formation is measured by a single optical detector with two separate flow cells aligned within the same optical path. The second approach is based on the use of a single-line flow-injection system, exploiting the formation of a double peak as a result of injecting a large sample zone, sandwiched between reagent zones of appropriate composition. In this manner two time-resolved signals for the kinetically governed processes can be obtained and thus used for quantification of the individual species.     

Determination of glucose in soft drink and sugar-cane juice employing a multicommutation approach in flow system and enzymatic reaction

A flow system based on a multicommutation approach was developed for the determination of glucose and sucrose employing enzymatic reactions. The determination was based on the reaction with D-glucose generating hydrogen peroxide catalyzed by glucose-oxidase (GOD). Subsequently, the H₂O₂ generated reacts with 4-aminefenazone plus phenol to form 4-(p-benzoquinone-mono-imine) fenazone detected at 510 nm. This reaction is catalyzed by the peroxidase enzyme (POD). The flow network comprised a set of three-way solenoid valves and was controlled by means of a microcomputer furnished with an electronic interface and running a software written in Quick BASIC 4.5. The flow network and control software were designed to implement the multicommutation approach providing facilities to handle sample and reagent solutions, so that, sample dilution could be easily performed on line. Accuracy was assessed by comparison with results obtained by known procedures and no significant difference at the 95% confidence level was observed. Other advantageous features were a linear response ranging from 0.05 to 0.20% (w/v) glucose without prior dilution, a reagent consumption of 336 μL per determination, an analytical throughput of 30 samples per hour.     

Determination of glucose in soft drink and sugar-cane juice employing a multicommutation approach in flow system and enzymatic reaction

A flow system based on a multicommutation approach was developed for the determination of glucose and sucrose employing enzymatic reactions. The determination was based on the reaction with D-glucose generating hydrogen peroxide catalyzed by glucose-oxidase (GOD). Subsequently, the H₂O₂ generated reacts with 4-aminefenazone plus phenol to form 4-(p-benzoquinone-mono-imine) fenazone detected at 510 nm. This reaction is catalyzed by the peroxidase enzyme (POD). The flow network comprised a set of three-way solenoid valves and was controlled by means of a microcomputer furnished with an electronic interface and running a software written in Quick BASIC 4.5. The flow network and control software were designed to implement the multicommutation approach providing facilities to handle sample and reagent solutions, so that, sample dilution could be easily performed on line. Accuracy was assessed by comparison with results obtained by known procedures and no significant difference at the 95% confidence level was observed. Other advantageous features were a linear response ranging from 0.05 to 0.20% (w/v) glucose without prior dilution, a reagent consumption of 336 μL per determination, an analytical throughput of 30 samples per hour. Received: 24 August 1998 / Revised: 13 November 1998 / Accepted: 19 December 1998     

An Automated Multi-Pumping Pulsed Flow System with Spectrophotometric Detection for the Determination of Phosphate in Natural Waters

In this work, an automated multipumping pulsed flow system was implemented for the determination of phosphate in natural waters. The developed flow approach was based on the spectrophotometric determination of phosphate by using the vanadomolybdate reaction. The exploitation of a very simple manifold configuration relying on the utilization of just two active components, in this case two solenoid actuated micropumps that were accountable for sample and reagent insertion and commutation, reaction zone formation, and solutions propelling, provided a great operational and optimization simplicity, low reagent consumption, and waste minimization.

Linear calibration plots for phosphate concentrations of up to 20 mg L^?1 (R² = 0.999, n = 6) were obtained, with a detection limit of 0.2 mg L^?1. The sampling rate was about 60 samples per hour. The system was applied to the monitoring of phosphate in local streams at specific sampling stations.     

Exploiting kinetic spectrophotometric determination of captopril, an angiotensin-converting enzyme inhibitor, in a multi-pumping flow system

The implementation of a differential kinetic spectrophotometric method for the determination of angiotensin-converting-enzyme inhibitors in pharmaceutical formulations is described. The determination method was based on the monitoring (350 nm) of the reaction between captopril and iodate, in the presence of iodide, versus time and was fully automated by exploiting the multi-pumping flow concept. The developed multi-pumping flow system included four discretely actuated solenoid micro-pumps as unique flow manifold active components. The automatic control of the solenoid micro-pumps, under time-based and pulse-counting routines, allowed the implementation of a reliable and versatile analytical determination, with the additional advantage of permitting a runtime access to important analytical parameters, such as flow rate, sample insertion and reagent addition synchronisation, facilitating this way the establishment of an approach for kinetic measurements, directly due to the efficient solution handling and accurate timing control.

A linear range of determination was verified for captopril concentrations between 10.0 and 60.0 μg mL⁻¹ with a sample throughput of about 100 determinations per hour. The results were in agreement with those obtained by the reference procedure with relative deviations between 1.81 and 4.48%.     

The use of tryptic marker-peptides for the quantitative analysis of cystatin C

The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis of proteins. To that end, cystatin C is chosen as a model protein. It not only functions as a proof of concept protein but the growing interest in cystatin C as a new marker of kidney failure provides a practical application at the same time. The use of trypsin-based proteolysis, to obtain so-called marker-peptides, simplifies the quantification of a protein to the quantification of a single or a number of peptides. Reproducibility of the trypsin proteolysis procedure is vital and has been optimised. A number of the marker-peptides obtained are selected for LC-MS(/MS) analysis. They are completely separated by high-pressure LC allowing maximum selectivity and mass spectrometric multiple reaction monitoring sensitivity. By doing so, linear calibration curves can be obtained for cystatin C over two orders of magnitude. Experiments have been performed on a triple quadrupole mass spectrometer by single ion monitoring (maximum sensitivity) as well as by multiple reaction monitoring (maximum specificity).     

A flow injection analysis technique for the determination of chloride using reflectance detection

A flow injection (FI) determination for chloride has been developed using the light reflectance of the precipitate formed by the reaction of chloride with silver(I) as the method of detection rather than turbidimetry, as in the previous FI method using this reaction. The dynamic range of the analysis is increased to 0-10 mM chloride with a 10 mM silver(I) reagent and to 0-50 mM chloride with a 50 mM silver(I) reagent by using this mode of detection. The ability to select the injected reagent from an option of two concentrations via the control program is incorporated into the FI system, enhancing the versatility of the analysis. The dynamic range is further extended to 100 mM chloride by measuring the signal levels at the trailing portion of the response curve. The consumption of reagent is kept to a minimum by merging injected zones of sample and reagent instead of using a constant reagent stream.     

Isothermal distillation in flow injection analysis: Determination of total nitrogen in plant material

An isothermal distillation unit is incorporated in flow injection systems. The influence of surfactant, flow rates, alkalinity, ionic strength, collector stream pH, reagent concentration and sample volume in ammonia distillation are discussed. A method for the determination of total nitgrogen in plant digests employing Nessler reagent is proposed. This method, based on merging zones approach, is characterized by a sampling rate of 100 samples per hour, a precision better than 3%, a reagent consumption of only 100 μl per sample and virtually no base-line drift. The results agree with those obtained with the indophenol blue colorimetric method. The incorporation of isothermal distillation in flow systems with potentiometric measurement is also described.     

Autoadaptative sequential injection system for nitrite determination in wastewaters

A novel autoadaptative sequential injection system for the analysis of nitrite is described. The automatic determination uses a direct spectrophotometric method, based on the Griess-Ilosvay reaction. In this method the absorbance of the purple azo dye formed is measured at 555 nm. In the sequential injection operation, the sample and the reagent are aspirated and mixed by reverse flow. The sequencing and overlapping of stacked (reagent) zones as well as selection of volumes have been studied in detail. The proposed analytical system is intelligent, simple and robust, allowing for nitrite determination in a double concentration range, by a simple and automatic programmable operation change. These two ranges are 0.0-3.0 and 0.0-20.0 ppm with detection limits being 0.048 and 0.4 ppm, respectively. Next surroundings have been developed allowing autocalibration and independent monitoring of nitrite concentration. The experimental set-up has been evaluated applying it to real samples analysis of very diverse concentration samples coming from a WWTP. The throughput of the method was 12 samples per hour.     

Stopped-in-loop flow analysis of trace vanadium in water.

The new concept of stopped-in-loop flow analysis (SIL-FA) is proposed, and an SIL-FA method for the catalytic determination of vanadium is demonstrated. In an SIL format, a sample solution merges with reagent(s), and the well-mixed solution is loaded into a loop. The solution in the loop is separated by a six-way switching valve from the main stream. While the reaction proceeds in the stationary loop, the SIL-FA system does not need to establish a baseline continuously. This leads to a reduction in reagent consumption and waste generation compared with traditional flow injection analysis.     

Computer simulation of affinity capillary electrophoresis

A computer-simulated model of affinity capillary electrophoresis is developed. Unlike existing models, it is able to describe the situation where the concentrations of sample molecules and ligand molecules are commensurable, or even the situation where the zones occupied by these molecules are not mixed initially. The model permits to study the dependence of the spatial and temporal distributions of sample molecules on various parameters such as reaction rate constants, concentrations of sample and reagent, electromigration velocities of sample and reagent and sample injection volume. A collection of peak shapes for different values of parameters is presented. The dependence of peak variance on the ratio of the time of analysis to the characteristic time of reaction is studied.     

Sampling strategies exploiting multi-pumping flow systems

In this work new strategies were exploited to implement multi-pumping flow systems relying on the utilisation of multiple devices that act simultaneously as sample-insertion, reagent-introduction, and solution-propelling units. The solenoid micro-pumps that were initially used as the only active elements of multi-pumping systems, and which were able to produce pulses of 3 to 25 microL, were replaced by syringe pumps with the aim of producing pulses between 1 and 4 microL. The performance of the developed flow system was assessed by using distinct sample-insertion strategies like single sample volume, merging zones, and binary sampling in the spectrophotometric determination of isoniazid in pharmaceutical formulations upon reaction with 1,2-naphthoquinone-4-sulfonate, in alkaline medium. The results obtained showed that enhanced sample/reagent mixing could be obtained with binary sampling and by using a 1 microL per step pump, even in limited dispersion conditions. Moreover, syringe pumps produce very reproducible flowing streams and are easily manipulated and controlled by a computer program, which is greatly simplified since they are the only active manifold component. Linear calibration plots up to 18.0 microg mL(-1), with a relative standard deviation of less than 1.48% (n=10) and a throughput of about 20 samples per hour, were obtained.     

Sequential injection analyzer for glycerol monitoring in yeast cultivation medium

A smart and versatile flow system for the at-line monitoring of glycerol based on sequential injection analysis is proposed. Formaldehyde, generated by oxidation of glycerol with sodium periodate, is transformed into 2,4-diacetyl-1,4-dihydrolutidine applying the Hantzsch condensation reaction with acetylacetone and ammonium. Dual-wavelength detection was carried out to minimize the contribution of the schlieren effect using a single blue LED. In-line sample dilution is accomplished applying the concept of zone-penetration and a new concept of sample splitting. Under optimized physical and chemical variables, regression curves over two dynamic working ranges of 0.1-4 and 1-40 g l⁻¹ were attained. The injection throughputs were 14 and 12 h⁻¹, respectively. Applying on-line data evaluation and conditional inquiries, the smart and independent selection of the adequate analytical procedure for the required working range was accomplished. The system was successfully applied to the at-line monitoring of glycerol in a continuous, cell-free medium flow from a yeast cultivation process during batch and fed-batch phase with glycerol as the only carbon source.     

A catalytic multi-pumping flow system for the chemiluminometric determination of metformin

A multi-pumping flow system for the chemiluminometric determination of the hypoglycaemic drug metformin was implemented. The developed methodology was based on the metformin-induced inhibition (metformin acts as a Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between luminol and hydrogen peroxide. The flow manifold configuration was based on the utilisation of multiple solenoid-actuated micro-pumps that were simultaneously accountable for sample/reagent introduction and reaction zone formation/propulsion, thus resulting in a fully automated, simple and highly selective multi-pumping flow system. A versatile sample manipulation allowed the establishment of distinct sampling strategies with low reagent consumption. The characteristic pulsed flow ensured an effective sample/reagent mixing leading to a better and faster reaction zone homogenisation and thus improved analytical signals. Linear calibration plots were obtained for metformin hydrochloride concentrations ranging from 5 to 15 mg L^–1 with a relative standard deviation lower than 2.0% (n=5). Detection limit was 0.94 mg L^–1, and the sampling rate was about 95 determinations per hour. The developed methodology was applied to the analysis of pharmaceutical formulations and the obtained results were in agreement with those furnished by the reference method with relative percentage deviations of lower than 1.5%.     

Investigating the effects of conductivity on zone overlap with EMMA: computer simulation and experiment

In this paper, we demonstrate, using both experiment and simulation, how sample zone conductivity can affect plug-plug mixing in small molecule applications of electrophoretically mediated microanalysis (EMMA). The effectiveness of in-line mixing, which is driven by potential, can vary widely with experimental conditions. Using two small molecule systems, the effects of local conductivity differences between analyte plugs, reagent plugs and the BGE on EMMA analyses are examined. Simul 5.0, a dynamic simulation program for CE systems, is used to understand the ionic boundaries and profiles that give rise to the experimentally obtained data for EMMA analyses for (i) creatinine determination via the Jaffe reaction, a reaction involving a neutral and an anion, and (ii) the redox reaction between gallate and 2,6-dichloroindophenol, two anions. Low sample conductivity, which is widely used in CE analyses, can be detrimental for in-line reactions involving a neutral reactant, as rapid migration of the ionic component across a low conductivity neutral zone results in poor reagent plug overlap and low reaction efficiency. Conversely, with two similarly charged reagents, a low conductivity sample plug is advantageous, as it allows field-amplified stacking of the reagents into a tight reaction zone. In addition, the complexity of simultaneously overlapping three reagent zones is considered, and experimental results validate the predictions made by the simulation. The simulations, however, do not appear to predict all of the observed experimental behavior. Overall, by combining experiment with simulation, an enhanced appreciation for the local field effects in EMMA is realized, and general guidelines for an advantageous sample matrix can be established for categories of EMMA analyses.     

Determination of sucrose in sugar-cane juice and molasses by flow-injection spectrophotometry

An automated procedure is proposed for the spectrophotometric determination of sucrose in sugar-cane juice and nonlasses. The previously diluted and filtered sample is introduced into a flow-injection analyzer designed with two merging streams, producing two samples zones. One zone is transported directly towards the merging-stream confluence; the other zone reaches this site after flowing through a heated coil in which partial and reproducible sucrose inversion is attained under controlled conditions of acidity and temperature. At the confluence point, a buffered periodate stream is added to oxidize the sugar. The consumption of periodate, which mainly reflects the fructose content, is measured spectrophotometrically as a transient lowering of the iodine concentration, produced by reaction of periodate with iodide. The two processed zones proceed sequentially to the flow cell and two peaks are recorded. The sucrose content in the sample is proportional to the difference in peak heights. System dimensioning and the effects of temperature, pH, reagent concentrations, flow rates and the presence of other reducing sugars in the sample are discussed. With the proposed system, about 30 sugar-cane juice samples can be analyzed per hour, and sample clarification is not required. Precise results (r.s.d. <1%), in agreement with those obtained by h.p.l.c., are achieved for sucrose contents of 11–14% (w/v) in cane juice. Modifications of the system for analysis of molasses (16–52% w/w sucrose) are described.     

Increased throughput in quantitative bioanalysis using parallel-column liquid chromatography with mass spectrometric detection

The feasibility of quantitative bioanalysis by parallel-column liquid chromatography in conjunction with a conventional single-source electrospray mass spectrometer has been investigated using plasma samples containing a drug and its three metabolites. Within a single chromatographic run time, sample injections were made alternately onto each of two analytical columns in parallel at specified intervals, with a mass spectrometer data file opened at every injection. Thus, the mass spectrometer collected data from two sample injections into separate data files within a single chromatographic run time. Therefore, without sacrificing the chromatographic separation or the selected reaction monitoring (SRM) dwell time, the sample throughput was increased by a factor of two. Comparing the method validation results obtained using the two-column system with those obtained using the corresponding conventional single-column approach, the methods on the two systems were found to be equivalent in terms of accuracy and precision. The parallel-column system is simple and can be implemented using existing laboratory equipment with no additional capital outlays. A parallel-column system configured in this manner can be used not only for the within-a-run analysis of two samples containing two different sets of chemical entities, but also for the within-a-run analysis of two samples containing the same set of chemical entities.     

Merging zones in flow injection analysis: Part 5. Simultaneous determination of aluminium and iron in plant digests by a zone-sampling approach

A flow injection procedure is proposed for the simultaneous determination of aluminium and iron in plant material. The method is based on a flow configuration involving zone sampling and merging zones. Aluminium is determined spectrophotometrically with eriochrome cyanine R as reagent and iron by atomic absorption spectrometry. The advantages of this method over other procedures already reported are discussed. The effects of reagent composition for the aluminium determination are described in detail. The zone-sampling approach permits an easier pH control in the aluminium determinations so interferences caused by variations in sample acidity are avoided without the need for very concentrated buffers. The merging zones configuration greatly reduces the consumption of reagents. The proposed method permits the analysis of about 120 samples (240 determinations) per hour, with good precision (r.s.d. < 2%) in both the aluminium and iron channels. The results agree with those obtained by inductively-coupled argon plasma spectrometry.