Filip Duša

Monoliths in capillary electrochromatography and capillary liquid chromatography in conjunction with mass spectrometry

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.

New solution IEF device for micropreparative separation of peptides and proteins

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.

Simple automated liquid chromatographic system for splitless nano column gradient separations

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 (RSD<0.3%). 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 (RSD<5%).

Combination of micropreparative solution isoelectric focusing and high-performance liquid chromatography for differentiation of biofilm-positive and biofilm-negative Candida parapsilosis group from vascular catheter.

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.

Simple power supply for power load controlled isoelectric focusing

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.

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides.

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 and Capillary Electrophoresis for Fast Screening of Interactions between Phosphatidylcholine Biomembranes and Surfactants

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 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid 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 nonionic) 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.