A Multi-Pumping Gradient Calibration Module for Potentiometric Determination of Nitrate in Surface Water.

The novel, automated, multi-pumping flow system (MPFS) for online calibration and determination of nitrate in surface water is presented for the first time. The system was equipped with micropumps of three different nominal volumes (10, 20, and 50 µL). As a result, it was possible to prepare from one standard, directly in a flow system, up to seven standard solutions. Determination of nitrate was conducted in stop-flow conditions and is based on a commercially available ion selective electrode (ISE) application. It was found that the linearity and slope of the calibration graphs depend mainly on the characteristics of the ISE. The obtained results were very repeatable, owing to the high precision of the micro-pumps used. The R.S.D. for the stroke volume of each micro-pump was below 1%. The accuracy of the method was checked through determination of nitrate in surface water samples. The obtained results were compared with those of the reference method (photometric Hach cuvette tests). It was found that, at a 96% confidence level, the difference between the results obtained by the proposed method and the reference method was statistically insignificant. The accuracy of the method was confirmed through the determination of nitrate in Certified Reference Material. The relative deviation (R.D.) of the measured and the certified concentrations was 5%.

Novel Integrated Flow-Based Steam Distillation and Titration System for Determination of Volatile Acidity in Wines.

Novel integrated flow-based steam distillation and titration system with spectrophotometric detection was developed for determination of volatile acidity in wines. Using the system, the distillation procedure was carried out in an automatic manner, starting with introducing into a heated steam distillation module a sample and subjecting it to steam distillation. Under selected conditions, all the analyte was transferred to the distillate; therefore, the system did not require calibration. The collected distillate and titrant were introduced into the next monosegments in varying proportions, in accordance with the developed titration procedure, and directed to the detection system to record the titration curve. The titration was stopped after reaching the end point of titration. Procedures for distillation and titration were developed and verified separately by distillation of acetic acid, acetic acid in the presence of tartaric acid as well as acetic acid, tartaric acid, and titratable acidity, with precision (relative standard deviation) and accuracy (relative error) for both procedures lower than 6.9 and 5.6%, respectively. The developed steam distillation and titration systems were used to determine volatile acidity in samples of white and rosé wines separately and as the integrated steam distillation and titration system, both with precision lower than 9.4% and accuracy better than 6.7%.

Fuzzy Clustering Methods to Identify the Epidemiological Situation and Its Changes in European Countries during COVID-19.

The main research question concerned the identification of changes in the COVID-19 epidemiological situation using fuzzy clustering methods. This research used cross-sectional time series data obtained from the European Centre for Disease Prevention and Control. The identification of country types in terms of epidemiological risk was carried out using the fuzzy c-means clustering method. We also used the entropy index to measure the degree of fuzziness in the classification and evaluate the uncertainty of epidemiological states. The proposed approach allowed us to identify countries' epidemic states. Moreover, it also made it possible to determine the time of transition from one state to another, as well as to observe fluctuations during changes of state. Three COVID-19 epidemic states were identified in Europe, i.e., stabilisation, destabilisation, and expansion. The methodology is universal and can also be useful for other countries, as well as the research results being important for governments, politicians and other policy-makers working to mitigate the effects of the COVID-19 pandemic.

Assessing the level of the material deprivation of European Union countries.

The purpose of this paper is to assess the level of material deprivation in European Union countries in 2016 from both a local and a global perspective. The Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) was used in the study. Based on research, five main types of the level of the material deprivation of European Union countries were identified. Research findings suggest that the population of old EU countries is less severely affected by material deprivation than people living in new member states. Also, the level of global material deprivation was assessed. The study was based on 2016 statistical data delivered by Eurostat.

Novel Approach to Sample Preconcentration by Solvent Evaporation in Flow Analysis.

A preconcentration module operated in flow mode and integrated with a sequential injection system with spectrophotometric detection was developed. Using the system, preconcentration was performed in continuous mode and was based on a membraneless evaporation process under diminished pressure. The parameters of the proposed system were optimized and the system was tested on the example of the spectrophotometric determination of Cr(III). The preconcentration effectiveness was determined using the signal enhancement factor. In the optimized conditions for Cr(III), it was possible to obtain the signal enhancement factors of around 10 (SD: 0.9, n = 4) and determine Cr(III) with precision and intermediate precision of 8.4 and 5.1% (CV), respectively. Depending on the initial sample volume, signal enhancement factor values of about 20 were achieved. Applicability of the developed preconcentration system was verified in combination with the capillary electrophoresis method with spectrophotometric detection on the example of determination of Zn in certified reference materials of drinking water and wastewater. Taking into account the enhancement factor of 10, a detection limit of 0.025 mg L-1 was obtained for Zn determination. Zn was determined with precision less than 6% (CV) and the results were consistent with the certified values.

A chemiluminescence method for screening of fluoroquinolones in milk samples based on a multi-pumping flow system.

An automated and miniaturized chemiluminescence method for screening of fluoroquinolones in milk samples was proposed. The method was based on magnetic dispersive micro-solid phase extraction of analytes followed by the chemiluminescence determination of the total fluoroquinolones content using a multi-pumping flow system. In the developed method, Zr-Fe-C magnetic nanoparticles were used as an efficient sorbent for separation of fluoroquinolones from sample matrix. The chemiluminescence intensity obtained for Ce4+-SO32--Tb3+ chemiluminescence system was greatly increased in the presence of the analytes. Under the optimal conditions, the detector response for fluoroquinolones was linear in the concentration ranges from 5·10-9 to 1·10-6 mol L-1 with respect to fleroxacin. The limit of detection, calculated from a blank test based on 3σ, was 2·10-9 mol L-1 with respect to fleroxacin. The presented method demonstrated to be a good tool for available and cost-effective point-of-need screening fluoroquinolones in milk samples.

A novel automatic flow method with direct-injection photometric detector for determination of dissolved reactive phosphorus in wastewater and freshwater samples.

The novel automatic flow system, direct-injection detector (DID) integrated with multi-pumping flow system (MPFS), dedicated for the photometric determination of orthophosphates in wastewater and freshwater samples is for the first time described. All reagents and the sample were injected simultaneously, in counter-current into the reaction-detection chamber by the system of specially selected for this purpose solenoid micro-pumps. The micro-pumps provided good precision and accuracy of the injected volumes. For the determination of orthophosphates, the molybdenum blue method was employed. The developed method can be used to detect orthophosphate in the range 0.1-12 mg L-1, with the repeatability (RSD) about 2.2% at 4 mg L-1 and a very high injection throughput of 120 injections h-1. It was possible to achieve a very small consumption of reagents (10 µL of ammonium molybdate and 10 µL of ascorbic acid) and sample (20 µL). The volume of generated waste was only 440 µL per analysis. The method has been successfully applied, giving a good accuracy, to determination of orthophosphates in complex matrix samples: treated wastewater, lake water and reference sample of groundwater. The developed system is compact, small in both size and weight, requires 12 V in supply voltage, which are desirable for truly portable equipment used in routine analysis. The simplicity of the system should result in its greater long-time reliability comparing to other flow methods previously described.

An automatic chemiluminescence method based on the multi-pumping flow system coupled with the fluidized reactor and direct-injection detector: Determination of uric acid in saliva samples.

A novel approach for the automatic chemiluminescence (CL) analysis of the complex samples is proposed. A multi-pumping flow system was successfully combined with fluidized reactor and direct-injection CL detector. The possibility of the approach was demonstrated on the determination of uric acid in saliva samples. Uric acid is clinically important analyte and its determination in biological fluids is related to human organism dysfunctions, such as gout. For the first time, the fast luminol - N-bromosuccinimide (NBS) reaction in an alkaline medium was used for the CL determination of uric acid in saliva samples. The CL intensity is greatly quenched in the presence of the analyte. The method includes on-line separation of uric acid from the saliva samples based on fluidized beds strategy using anion-exchange resin Dowex® 2×8 followed by the elution and CL determination using a direct-injection CL detector. The stroke pulsations of the solenoid micro-pumps provided the floating of the anion exchange resin in a sample phase and uric acid separation from the sample matrix into a sample pre-treatment block of flow system. To obtain efficient elution of analyte an eluent circulation was applied. Under the optimal conditions, the detector response for uric acid was linear in the logarithmic concentration ranges from 6·10-6 to 1·10-3molL-1. The limit of detection, calculated from a blank test based on 3σ, was 2·10-6molL-1. Fluidized bed strategy allows us to exclude saliva matrices influence on the luminol-NBS CL reaction, which results in improved selectivity. The applicability of the method developed is demonstrated with the help of real sample analysis. The obtained results are confirmed by reference HPLC-UV method.

Application of direct-injection detector integrated with the multi-pumping flow system to chemiluminescence determination of the total polyphenol index.

In this work, we present a novel chemiluminescence (CL) method based on direct-injection detector (DID) integrated with the multi-pumping flow system (MPFS) to chemiluminescence determination of the total polyphenol index. In this flow system, the sample and the reagents are injected directly into the cone-shaped detection cell placed in front of the photomultiplier window. Such construction of the detection chamber allows for fast measurement of the CL signal in stopped-flow conditions immediately after mixing the reagents. The proposed DID-CL-MPFS method is based on the chemiluminescence of nanocolloidal manganese(IV)-hexametaphosphate-ethanol system. The application of ethanol as a sensitizer, eliminated the use of carcinogenic formaldehyde. Under the optimized experimental conditions, the chemiluminescence intensities are proportional to the concentration of gallic acid in the range from 5 to 350 ng mL(-1). The DID-CL-MPFS method offers a number of advantages, including low limit of detection (0.80 ng mL(-1)), high precision (RSD = 3.3%) and high sample throughput (144 samples h(-1)) as well as low consumption of reagents, energy and low waste generation. The proposed method has been successfully applied to determine the total polyphenol index (expressed as gallic acid equivalent) in a variety of plant-derived food samples (wine, tea, coffee, fruit and vegetable juices, herbs, spices).

Modelling the structure of sludge aggregates.

The structure of sludge is closely associated with the process of wastewater treatment. Synthetic dyestuff wastewater and sewage were coagulated using the PAX and PIX methods, and electro-coagulated on aluminium electrodes. The processes of wastewater treatment were supported with an organic polymer. The images of surface structures of the investigated sludge were obtained using scanning electron microscopy (SEM). The software image analysis permitted obtaining plots log A vs. log P, wherein A is the surface area and P is the perimeter of the object, for individual objects comprised in the structure of the sludge. The resulting database confirmed the 'self-similarity' of the structural objects in the studied groups of sludge, which enabled calculating their fractal dimension and proposing models for these objects. A quantitative description of the sludge aggregates permitted proposing a mechanism of the processes responsible for their formation. In the paper, also, the impact of the structure of the investigated sludge on the process of sedimentation, and dehydration of the thickened sludge after sedimentation, was discussed.

Direct-injection chemiluminescence detector. Properties and potential applications in flow analysis.

We present a novel chemiluminescence detector, with a cone-shaped detection chamber where the analytical reaction takes place. The sample and appropriate reagents are injected directly into the chamber in countercurrent using solenoid-operated pulse micro-pumps. The proposed detector allows for fast measurement of the chemiluminescence signal in stop-flow conditions from the moment of reagents mixing. To evaluate potential applications of the detector the Fenton-like reaction with a luminol-H2O2 system and several transition metal ions (Co(2+), Cu(2+), Cr(3+), Fe(3+)) as a catalyst were investigated. The results demonstrate suitability of the proposed detector for quantitative analysis and for investigations of reaction kinetics, particularly rapid reactions. A multi-pumping flow system was designed and optimized. The developed methodology demonstrated that the shape of the analytical signals strongly depends on the type and concentration of the metal ions. The application of the detector in quantitative analysis was assessed for determination of Fe(III). The direct-injection chemiluminescence detector allows for a sensitive and repeatable (R.S.D. 2%) determination. The intensity of chemiluminescence increased linearly in the range from about 0.5 to 10 mg L(-1) Fe(III) with the detection limit of 0.025 mg L(-1). The time of analysis depended mainly on reaction kinetics. It is possible to achieve the high sampling rate of 144 samples per hour.

Application of direct-injection detector integrated with the multi-pumping flow system to photometric stop-flow determination of total iron.

A novel direct-injection detector (DID) integrated with multi-pumping flow system (MPFS) for the photometric determination of iron is proposed. Paired emitter-detector diodes have been used as a photometric detection system. The sample and reagent were injected using appropriate solenoid pulse micro-pumps directly into the detection chamber where effective mixing occured. The use of proposed stop-flow detector considerably simplified the analytical procedure. The potassium thiocyanate has been chosen as a chromogenic reagent for photometric Fe(III) detection. The total volume of reagent and sample/standard solutions involved in the detection process was adjusted to the volume of the reaction-detection chamber. Calibration graph was found to be linear in the range up to 10mgL(-1). The detection limit (3s(b)/S) was 0.15mgL(-1). The repeatability (R.S.D.), calculated from 10 analyses of sample containing 5mgL(-1) Fe(III), was 1.5% and the sample throughput 180 determinations per hour. The consumption of sample and reagent was 20µL each with the waste generation at the level of 0.24mL. The applicability of the proposed method to the determination of total iron in groundwater samples has been proved. The analytical parameters are compared to those obtained exploiting the MPFS system with typical configuration containing a confluence point and reaction coil.

A novel direct-injection photometric detector integrated with solenoid pulse-pump flow system.

A novel flow photometric detector based on paired emitter-detector diodes (PEDD), coupled with solenoid pulse micro-pumps is presented. The photometric detection chamber also plays a role of the reaction chamber. Both solutions, a sample and a reagent, are injected by solenoid micro-pumps directly and simultaneously into the chamber in countercurrent. This method ensures fast and effective mixing of the injected solutions. A good accuracy and precision of the injected volumes assure the pulse micro-pumps. This method, in comparison with other flow methods, does not require the application of the reaction coil. Thanks to this, the dispersion of the sample is minimized. The presented flow network is easy to control, miniaturize and exhibits a very low consumption of reagents and the sample. Two chemical systems were chosen and presented as models of photometric reactions: the first - Fe(III) with thiocyanate, and the second - Cr(VI) with 1,5-diphenylcarbazide. To obtain the highest repeatability, the total volume of the solutions should be smaller than the volume of the reaction-detection chamber. In this case, the whole coloured product remains in the chamber. The use of the proposed direct-injection PEDD detector considerably simplifies analytical procedures. The shape of analytical signals and their potential applications have been discussed.

Ascorbic Acid determination in commercial fruit juice samples by cyclic voltammetry.

A method was developed for assessing ascorbic acid concentration in commercial fruit juice by cyclic voltammetry. The anodic oxidation peak for ascorbic acid occurs at about 490 mV on a Pt disc working electrode (versus SCE). The influence of the potential sweep speed on the peak height was studied. The obtained calibration graph shows a linear dependence between peak height and ascorbic acid concentration in the domain (0.1-10 mmol.L(-1)). The equation of the calibration graph was y = 6.391x + 0.1903 (where y represents the value of intensity measured for the anodic peak height, expressed as muA and x the analyte concentration, as mmol.L(-1), r(2) = 0.9995, r.s.d. = 1.14%, n = 10, C(ascorbic acid) = 2 mmol.L(-1)). The developed method was applied to ascorbic acid assessment in fruit juice. The ascorbic acid content determined ranged from 0.83 to 1.67 mmol.L(-1) for orange juice, from 0.58 to 1.93 mmol.L(-1) for lemon juice, and from 0.46 to 1.84 mmol.L(-1) for grapefruit juice. Different ascorbic acid concentrations (from standard solutions) were added to the analysed samples, the degree of recovery being comprised between 94.35% and 104%. Ascorbic acid determination results obtained by cyclic voltammetry were compared with those obtained by the volumetric method with dichlorophenol indophenol. The results obtained by the two methods were in good agreement.

Modeling the induction of lipid membrane electropermeabilization.

Experiments show significant effects of an electric field on lipid membrane, leading to a pore formation when a high intensity field is applied. The phenomenon of electroporation is preceded by the induction and expansion of defects, responsible for the pre-pore excitation. We examine the mechanism of the induction of the field-driven defects by Monte Carlo simulations. The study is based on the improved Pink's model, which includes explicit interactions between the polar heads and energy of interactions between the heads and the field. No anomalous deformation of the molecules is considered. The study, provided for bilayer dipalmitoyl-phosphatidylcholine (DPPC) membrane in the gel (300 K) and fluid (330 K) phases, shows dependence of the membrane conformational and energetical state on the value of the electric field. We observe that the electric field affects the number of molecules in the gel and in the fluid states. In the layer at the negative potential, when the transmembrane voltage is above U(c) approximately 280 mV, lipid heads abruptly reorient and the number of local spots with fluid conformation increases. The other layer slightly tends to tighten its structure, producing additional mechanical stress between layers. Lipids showed complete insensitivity to the electric field within physiological limits, U<70 mV.

Simulation of electroporated cell by chronopotentiometry.

Chronopotentiometry on planar lipid bilayer (BLM) is proposed as a method for modeling the electrical phenomena in electroporated cell. Two techniques are discussed: constant-current and linear-current chronopotentiometry. It is proposed that the constant-current chronopotentiometry may provide basis for modeling the electroporated cell shortly after the removal of the electric field, when activity of cellular pumps counteracts ionic fluxes through the electropore and ionic channels. The linear-current method can be considered for modeling the cell in the later stage after electroporation, when energetical resources of the cell are gradually getting exhausted and the activity of pumps decreases. Based on this idea, it may be postulated that the electropore in the cell has fluctuating dynamics whose stochastic characteristics, similarly as biological channels, shows 1/f noise. The model implies that the fluctuations would disappear leaving the electropore with a constant resistance when efficiency of the pumps becomes very small. The results of chronopotentiometry also may suggest that opening time, conductivity and selectivity of the electropore can be controlled by the cell environment or membrane composition.

Self-similar processes and flicker noise from a fluctuating nanopore in a lipid membrane.

Stochastic properties of a fluctuating nanopore generated and sustained by an electric field in a lipid bilayer membrane are studied. It is shown that the process of voltage fluctuations, in the current clamp experiment, is a stochastic fractal with long memory, which is the main reason for its nonstationarity. The aging process contributes to the nonstationarity if molecular interactions in the membrane are weak. An attempt to classify the process reveals a non-Gaussian distribution with long tails, which contradicts the hypothesis of fractional Brownian motion, showing that stable motion may be possible. The self-similarity index, estimated by three different methods, depends on current value and membrane sensitivity to electric field in a well defined and explicable manner. The stochastic analysis provided for calculated conductance of nanopore revealed the process close to 1/f noise, the result observed only for the pores not exceeding 1 nm in diameter, induced in membranes with strong molecular interactions. Our results show that such a pore is the simplest biological system needed for flicker noise to occur, and the complexity of highly regulated protein channel is not a necessary factor. A case of noise 1/f(2), observed for a pore with impeded dynamics, suggests a process without memory in such a situation. A physical interpretation is presented for some of the results.

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies.

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.

Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes.

This paper presents the application of chronopotentiometry in the study of membrane electroporation. Chronopotentiometry with a programmable current intensity was used. The experiments were performed on planar bilayer phosphatidylcholine and cholesterol membranes formed by the Mueller-Rudin method. It was demonstrated that a constant-intensity current flow through the bilayer membranes generated voltage fluctuations during electroporation. These fluctuations (following an increase and decrease in membrane conductance) were interpreted as a result of the opening and closing of pores in membrane structures. The decrease in membrane potential to zero did not cause the pore to close immediately. The pore was maintained for about 200 s. The closing of the pore and recovery of the continuous structure of the membrane proceeded not only when the membrane potential equalled zero, but also at membrane potentials up to several tens of millivolts. The fluctuations of the pore were possible at values of membrane potential in the order of at least 100 mV. The size of the pore changed slightly and it closed after some time below this potential value.