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Pressurized water extraction (PWE) is a technique suitable for efficient extraction of almond proteins, some of which are known as potent allergens. In this work, we present a PWE protocol that allows fast and complete isolation of almond proteins achieved within one 5-min cycle at 40°C and 15 MPa. The extracted protein contents obtained using PWE or conventional extraction method with 22 selected solvents were compared based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Individual proteins from separation gels were excised and identified using mass spectrometry with database searching. Our results showed that the PWE of almond proteins can offer similar efficiency as the conventional extraction method while being four times faster and providing water-based extracts without any additives with low environmental impact.

In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.

We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry.

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

We present a set of novel low-molecular-mass (LMM) compounds possessing ampholytic properties. The compounds were designed to perform as markers of isoelectric point (pI) in different isoelectric focusing (IEF) formats and feature direct detectability in UV and visible wavelength regions. Capillary isoelectric focusing (cIEF) was used to determine the purity of the focusing species and the compounds' pI values. Nitrophenol-based pI markers (NPIMs) published previously were used as standards for the pI value calibration. The presented compounds focused very well, but small portion of them contained focusing impurities, thus, we recommend them for use in other IEF formats like gel IEF and preparative IEF. Moreover, multi-wavelength detection enabled determination of individual markers based on their specific spectral profiles and different absorption at selected detection wavelengths in the electropherogram. The presented compounds compose a group of chemicals featuring excellent shelf stability and isoelectric focusing properties, inexpensive synthesis, universal/multimode detectability, and good solubility at pI. The presented results provide a solid ground for their use as reference standards in various isoelectric focusing methods.

In this work, we characterize a previously synthesized multi-cationic aminopyrene-based labeling tag for oligosaccharide analysis by capillary electrophoresis with laser-induced fluorescence detection (CE/LIF). The fluorescent tag, 4,4',4''-(8-aminopyrene-1,3,6-trisulfonyl)tris(1-methylpiperazine) (APTMP), was characterized by reaction with standard maltooligosaccharides and the labeling parameters such as fluorescent tag concentration, labeling temperature, and time as well as influence of a reducing agent and its solvent were investigated in terms of labeling efficiency. The nanomolar limit of detection of CE/LIF analysis of APTMP labeled maltopentaose was determined. However, significant amount of the oligosaccharides was reduced to alditols, which negatively affects the yield and rate of the labeling reaction. Under optimized conditions, a highly reproducible labeling by multi-cationic APTMP was obtained; however, the most commonly used labeling by multi-anionic 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) is superior compared to APTMP labeling. Lower reactivity of APTMP compared to APTS can be explained by the loss of nucleophilicity induced by substitution of the sulfonate groups with more electron-withdrawing aminosulfonyl ones. On contrary, APTMP is still a promising tag for oligosaccharide labeling followed by CE-MS in a positive ion mode, which is considered to be more sensitive than MS detection of APTS in a negative ion mode.

The cell membrane is mainly composed of lipid bilayers with inserted proteins and carbohydrates. Lipid bilayers made of purified or synthetic lipids are widely used for estimating the effect of target compounds on cell membranes. However, the composition of such biomimetic membranes is much simpler than the composition of biological membranes. Interactions between compounds and simple composition biomimetic membranes might not demonstrate the effect of target compounds as precisely as membranes with compositions close to real organisms. Therefore, the aim of our study is to construct biomimetic membrane closely mimicking the state of natural membranes. Liposomes were prepared from lipids extracted from L-α-phosphatidylcholine, Escherichia coli, yeast (Saccharomyces cerevisiae) and bovine liver cells through agitation and sonication. They were immobilized onto silicon dioxide (SiO2) sensor surfaces using N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid buffer with calcium chloride. The biomimetic membranes were successfully immobilized onto the SiO2 sensor surface and detected by nanoplasmonic sensing. The immobilized membranes were exposed to choline carboxylates. The membrane disruption effect was, as expected, more pronounced with increasing carbohydrate chain length of the carboxylates. The results correlated with the toxicity values determined using Vibrio fischeri bacteria. The yeast extracted lipid membranes had the strongest response to introduction of choline laurate while the bovine liver lipid extracted liposomes were the most sensitive towards the shorter choline carboxylates. This implies that the composition of the cell membrane plays a crucial role upon interaction with choline carboxylates, and underlines the necessity of testing membrane systems of different origin to obtain an overall image of such interactions.

We present a novel method for concentration and purification of DNA from biological samples. The method is based on isotachophoretic separation of DNA strands in a separation bed made of a disposable nonwoven fabric strip. Application of oxalate as the leading ion prevented corrosion of the carbon anode and also the leading ion was continually removed from the system due to its decomposition into CO2 at the anode. The fractions were marked by three colored markers of electrophoretic mobility closely surrounding the mobility of DNA. The fraction collection was realized by a centrifugal drain of cut out strip segments. The method was evaluated using two purified salmon sperm DNA fragments of lengths 200 bp and 2000 bp. The results confirmed the high DNA concentrating effect of the method (34-fold increase of the original DNA concentration). The composition of running solutions and voltage program were optimized in order to finish the analysis within 30 min. The optimized method was used to extract, concentrate and purify DNA from a crude yeast cell lysate. The maximum DNA enrichment factor decreased to 12 due to the stretching of DNA zones caused by low-molecular contaminants present in the original lysate. The average recovery determined for yeast DNA was 71 ± 11% (n = 3). The connected elimination of the proteins from DNA zones resulted in the purification factor value of 582 for DNA vs proteins. This demonstrates that the presented method is capable to concentrate DNA from the bulk volume and to further purify it from crude cell lysates using a simple instrumentation and low-cost disposable separation bed.

The effects of ionic liquids on model phospholipid membranes were studied by small-angle X-ray scattering, dynamic light scattering (DLS) and zeta potential measurements. Multilamellar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes and large unilamellar vesicles composed of L-alpha-phosphatidylcholine (eggPC) and L-alpha-phosphatidylglycerol (eggPG) (80:20 mol%) or eggPC, eggPG, and cholesterol (60:20:20 mol%) were used as biomimicking membrane models. The effects of the phosphonium-based ionic liquids: tributylmethylphosphonium acetate, trioctylmethylphosphonium acetate, tributyl(tetradecyl)-phosphonium acetate, and tributyl(tetradecyl)-phosphonium chloride, were compared to those of 1-ethyl-3-methyl-imidazolium acetate. With multilamellar vesicles, the ionic liquids that did not disrupt liposomes decreased the lamellar spacing as a function of concentration. The magnitude of the effect depended on concentration for all studied ionic liquids. Using large unilamellar vesicles, first a slight decrease in the vesicle size, then aggregation of vesicles was observed by DLS for increasing ionic liquid concentrations. At concentrations just below those that caused aggregation of liposomes, large unilamellar vesicles were coated by ionic liquid cations, evidenced by a change in their zeta potential. The ability of phosphonium-based ionic liquids to affect liposomes is related to the length of the hydrocarbon chains in the cation. Generally, the ability of ionic liquids to disrupt liposomes goes hand in hand with inducing disorder in the phospholipid membrane. However, trioctylmethylphosphonium acetate selectively extracted and induced a well-ordered lamellar structure in phospholipids from disrupted cholesterol-containing large unilamellar vesicles. This kind of effect was not seen with any other combination of ionic liquids and liposomes. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Our study demonstrates that nanoplasmonic sensing (NPS) can be utilized for the determination of the phase transition temperature (Tm) of phospholipids. During the phase transition, the lipid bilayer undergoes a conformational change. Therefore, it is presumed that the Tm of phospholipids can be determined by detecting conformational changes in liposomes. The studied lipids included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Liposomes in gel phase are immobilized onto silicon dioxide sensors and the sensor cell temperature is increased until passing the Tm of the lipid. The results show that, when the system temperature approaches the Tm, a drop of the NPS signal is observed. The breakpoints in the temperatures are 22.5 °C, 41.0 °C, and 55.5 °C for DMPC, DPPC, and DSPC, respectively. These values are very close to the theoretical Tm values, i.e., 24 °C, 41.4 °C, and 55 °C for DMPC, DPPC, and DSPC, respectively. Our studies prove that the NPS methodology is a simple and valuable tool for the determination of the Tm of phospholipids.

Here, we have reviewed separation studies utilizing monolithic capillary columns for separation of compounds preceding MS analysis. The review is divided in two parts according to the used separation method, namely CEC and capillary LC (cLC). Based on our overview, monolithic CEC-MS technique have been more focused on the syntheses of highly specialized and selective separation phase materials for fast and efficient separation of specific types of analytes. In contrast, monolithic cLC-MS is more widely used and is often employed, for instance, in the analysis of oligonucleotides, metabolites, and peptides and proteins in proteomic studies. While poly(styrene-divinylbenzene)-based and silica-based monolithic capillaries found their place in proteomic analyses, the other laboratory-synthesized monoliths still wait for their wider utilization in routine analyses. The development of new monolithic materials will most likely continue due to the demand of more efficient and rapid separation of increasingly complex samples.

The worldwide use of ionic liquids (ILs) is steadily increasing, and even though they are often referred to as "green solvents" they have been reported to be toxic, especially toward aquatic organisms. In this work, we thoroughly study two phosphonium ILs; octyltributylphosphonium chloride ([P-8444]CI)and tributyl(tetradecyl)phosphonium chloride ([P-14444]CI). Firstly, the critical micelle concentrations (CMCs) of the ILs were determined with fluorescence spectroscopy and the optical pendant drop method in order to gain an understanding of the aggregation behavior of the ILs. Secondly, a biomimicking system of negatively charged unilamellar liposomes was used in order to study the effect of the ILs on biomembranes. Changes in the mechanical properties of adsorbed liposomes were determined by quartz crystal microbalance (QCM) measurements with silica coated quartz crystal sensors featuring a polycation layer. The results confirmed that both ILs were able to incorporate and alter the biomembrane structure. The membrane disrupting effect was emphasized with an increasing concentration and alkyl chain length of the ILs. In the extreme case, the phospholipid membrane integrity was completely compromised. (C) 2015 Elsevier B.V. All rights reserved.

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3-methyl-1-(4-vinylbenzyl)-imidazolium chloride) (PIL-1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi-permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL-1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log Po/w > 2. The ability to separate cationic drugs was shown with β-blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.

The power supply for IEF based on features of the Cockcroft-Walton voltage multiplier (CW VM) is described in this work. The article describes a design of the IEF power supply, its electric characteristics, and testing by IEF analysis. A circuit diagram of the power supply included two opposite charged branches (each consisting of four voltage doublers). The designed CW VM was powered by 230 V/50 Hz alternate current and it generated up to 5 kV and 90 mW at the output. Voltage and current characteristics of the power supply were measured by known load resistances in the range from 10 kΩ to 1 GΩ, which is a common resistance range for IEF strip geometry. Further, the power supply was tested by a separation of a model mixture of colored pI markers using a 175 × 3 × 0.5 mm focusing bed. Automatically limited power load enabled analysis of samples without previous optimization of the focusing voltage or electric current time courses according to sample composition. Moreover, the developed power supply did not produce any intrinsic heat and was easy to set up with cheap and commonly available parts.

A simple splitless gradient liquid chromatographic system for micro and nano column separations has been assembled and tested. It consists of an OEM programmable syringe pump equipped with a glass microsyringe and ten-port selector valve. Gradient of mobile phase was created in the syringe barrel due to turbulent mixing. Capability of suggested system to create various gradient profiles was verified using 50-μl, 100-μl, and 250-μl glass syringes. Acetone, thiourea, and uracil were tested as gradient markers and finally, uracil was proved to be an excellent way of water-acetonitrile gradient tracing. It was found that up to 80% of the total syringe volume is available as a linear gradient section. In context to micro and nano column chromatography, the best results were obtained using the 100-μl syringe. Separations were performed on the capillary monolithic column Chromolith CapRod RP-18e (150mm×0.1mm) and system performance was evaluated using a test mixture of six alkylphenones as well as tryptic digest of bovine serum albumin. Results proved that suggested system is able to create uniform gradients with high repeatability of retention times of test solutes (RSD0.3%). Repeatability of injection of sample volumes in the range of 0.1-3μl was evaluated using on-column preconcentration technique which means that sample was diluted in mobile phase of low eluting strength. Repeatability of the peak areas was measured and statistically evaluated (RSD5%).

The effect of 11 common amidinium, imidazolium, and phosphonium based ionic liquids (ILs) on zebrafish (Danio rerio) and Chinese hamster ovary cells (CHO) was investigated with specific emphasis on the effect of anion and cation chain length and aggregation of phosphonium based ILs. Viability and behavioral alteration in the locomotor activity and place preference, after IL treatment of 5 days postfertilization larvae, was recorded. Behavior and histological damage evaluation was performed for adult fish in order to get insight into the long-term effects of two potential biomass-dissolving ILs, [DBNH][OAc] and [P4441][OAc]. To get an understanding of how IL aggregation is linked to the toxicity of ILs, median effective concentrations (EC50) and critical micelle concentrations (CMC) were determined. The long-chain ILs were significantly more toxic than the short-chain ones, and the anion chain length was shown to be less significant than the cation chain length when assessing the impact of ILs on the viability of the organisms. Furthermore, most of the ILs were as monomers when the EC50 was reached. In addition, the ILs used in the long-term tests showed no significant effect on the zebrafish behavior, breeding, or histology, within the used concentration range.

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.

Nanoplasmonic sensing (NPS), based on localized surface plasmon resonance, with sensors composed of glass covered with golden nanodisks and overlaid with a SiO2 coating was applied in this study. Egg phosphatidylcholine (eggPC), being an easily accessible membrane-forming lipid, was used for preparation of biomimicking membranes. Small unilamellar vesicles with an approximate hydrodynamic diameter of 30 nm, formed by sonication in HEPES buffer, were adsorbed within 10 min on the sensor surface either as intact vesicles or as a planar bilayer. The adsorbed biomembrane systems were further utilized for interaction studies with four different well-known surfactants (negatively and positively charged, zwitterionic, and non-ionic) and each surfactant was tested at concentrations below and above the critical micelle concentration (CMC). Our results allowed the evaluation of different NPS patterns for every particular supported membrane system, surfactant, and its concentration. The most significant effect on the membrane was achieved upon the introduction of zwitterionic surfactant micelles, which in fact completely solubilized and removed the lipid membranes from the sensor surface. Other surfactant micelles interacted with the membranes and formed mixed structures remaining on the sensor surface. The studies performed at the concentrations below the CMCs of the surfactants showed that different mixed systems were formed. Depending on the supported membrane system and the type of surfactant, the mixed systems indicated different formation kinetics. Additionally, the final water rinse revealed the stability of the formed systems. To investigate the effect of the studied surfactants on the overall surface charge of the biomembrane, capillary electrophoresis (CE) experiments were carried out in parallel with the NPS analysis. The electroosmotic flow mobility of an eggPC-coated fused silica capillary was used to measure the total surface charge of the biomembrane after its treatment with the surfactants. Our results indicated in general good correlation between CE and NPS data. However, some discrepancies were seen while applying either zwitterionic or positively charged surfactants. This confirmed that CE analysis was able to provide additional data about the investigated systems. Taken together, the combination of NPS and CE proved to be an efficient way to describe the nature of interactions between biomimicking membranes and amphiphilic molecules.

Here, we have reviewed separation studies utilizing monolithic capillary columns for separation of compounds preceding MS analysis. The review is divided in two parts according to the used separation method, namely CEC and capillary LC (cLC). Based on our overview, monolithic CEC-MS technique have been more focused on the syntheses of highly specialized and selective separation phase materials for fast and efficient separation of specific types of analytes. In contrast, monolithic cLC-MS is more widely used and is often employed, for instance, in the analysis of oligonucleotides, metabolites, and peptides and proteins in proteomic studies. While poly(styrene-divinylbenzene)-based and silica-based monolithic capillaries found their place in proteomic analyses, the other laboratory-synthesized monoliths still wait for their wider utilization in routine analyses. The development of new monolithic materials will most likely continue due to the demand of more efficient and rapid separation of increasingly complex samples.

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3-methyl-1-(4-vinylbenzyl)-imidazolium chloride) (PIL-1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi-permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL-1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log Po/w2. The ability to separate cationic drugs was shown with β-blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.

The effect of three phosphonium and imidazolium ionic liquids (ILs) on the interaction between liposomes and common pharmaceuticals found in wastewaters was studied. The liposomes comprised zwitterionic phosphatidyl choline and negatively charged phosphatidyl glycerol. A set of common cationic, anionic, and neutral compounds with varying chemical composition and unique structures were included in the study. The electrophoretic mobilities of the analytes were determined using conventional capillary electrophoresis (CE), using CE under reversed electroosmotic flow mobility conditions, and in the presence of ILs in the background electrolyte (BGE) solution by electrokinetic chromatography (EKC). In order to evaluate the impact of ILs on the interaction between the compounds and the liposomes, EKC was performed with liposome dispersions, with and without ILs. The retention factors of the compounds were calculated using BGEs including liposome dispersions with and without ILs. Two phosphonium based ILs, namely tributyl(tetradecyl)phosphonium chloride ([P14444]Cl) and octyltributylphosphonium chloride ([P8444]Cl), were chosen due to their long alkyl chains and their low aggregation concentrations. Another IL, i.e. 1-ethyl-3-methylimidazolium acetate ([emim][OAc]), was chosen based on our previous study, which suggests that it has a minimal or even nonexistent effect on liposomes at the used concentrations.The results indicate that the studied ILs have an effect on the interactions between wastewater compounds and liposomes, but the effect is highly dependent on the concentration of the IL and on the IL alkyl chain lengths. Most of the ILs hindered the interactions between the liposomes and the compounds, indicating strong interaction between ILs and liposomes. In addition, the nature of the studied compounds themselves affected the interactions.

The power supply for IEF based on features of the Cockcroft-Walton voltage multiplier (CW VM) is described in this work. The article describes a design of the IEF power supply, its electric characteristics, and testing by IEF analysis. A circuit diagram of the power supply included two opposite charged branches (each consisting of four voltage doublers). The designed CW VM was powered by 230 V/50 Hz alternate current and it generated up to 5 kV and 90 mW at the output. Voltage and current characteristics of the power supply were measured by known load resistances in the range from 10 kΩ to 1 GΩ, which is a common resistance range for IEF strip geometry. Further, the power supply was tested by a separation of a model mixture of colored pI markers using a 175 × 3 × 0.5 mm focusing bed. Automatically limited power load enabled analysis of samples without previous optimization of the focusing voltage or electric current time courses according to sample composition. Moreover, the developed power supply did not produce any intrinsic heat and was easy to set up with cheap and commonly available parts.

This study utilizes the high-performance liquid chromatography technique in combination with the new micropreparative solution isoelectric focusing fractionation on non-woven fabric strip for the characterization and differentiation of biofilm-positive and biofilm-negative forms of Candida parapsilosis sensu stricto on the basis of the changes in the composition of their cell-surface. Treatment of yeasts by boiling in distilled water relased surface substances from yeasts cells. Consequently, the optimized procedure has been used for fast identification of the highly pathogenic biofilm-positive Candida parapsilosis group in real clinical material – sonicate from vascular catheters. Moreover, the capillary isoelectric focusing was used as supporting and control technique. Obtained results suggest that this new method can be used to distinguish between biofilm-positive and negative forms of Candida parapsilosis sensu stricto.

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.

There have been published several papers on voltammetric ultra-trace determination of some nucleic bases in the presence of Cu(II) using a mercury electrode or solid amalgam electrodes. Carbon electrodes can be used for the same purposes. The aim of this work was to employ the behaviour of purines in the presence of Cu(II) to determine these metal ions. Primarily we aimed on investigation of basic electrochemical behaviour of detection copper at hanging mercury drop and carbon paste electrodes by using of cyclic and differential pulse voltammetry. Thanks to formation of the complex adenine-Cu(II) under the specific conditions the peak of this complex was measured at -0.6 V. The detection limit (3 S/N) for Cu(II) ions was estimated as 5 nM. By using of this approach we were able to determine the content of copper in animal tissues even at tens of nM. Moreover we utilized suggested approach to monitor the level of copper ions in samples of blood serum of patients with metabolic disorder.