Ewelina Dziubakiewicz

Effect of zeta potential value on bacterial behavior during electrophoretic separation

The aggregation and/or adhesion of bacterial cells is a serious disadvantage of electrophoretic separations. In this study, physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation on the basis of electrophoretic behavior of different clinical strains of Gram‐positive Staphylococcus aureus and Gram‐negative Escherichia coli bacteria. The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in‐line microscope observation using specially designed CE fluorescence stereomicroscope setup. These results suggest that the lower the zeta potential, the higher the number of smaller peaks detected. The direct microscopic observation of electrophoretic movement proved the presence of many small aggregates originating from individual or clustered bacterial cells. On the other hand, lower zeta potential was also observed for dead bacterial cells, which suggested that some of the peaks can be attributed to viable cells while the other to the dead ones.

Zeta potential determination as a new way of stationary phases characterization for liquid chromatography

A set of seven homemade octadecyl silica-based bonded phases was investigated. Their zeta potential data in methanol and ACN as well as in methanol-water and ACN-water solution were obtained using Zetasizer. The influence of both the coverage density of bonded ligands and the end-capping of the modified surface on these data was investigated. Presented results may give useful information about the accessibility of the residual silanols in different mobile phases during the chromatographic analysis. Those measurements may be useful to choose chemically bonded stationary phases for CEC. The results also confirm the phenomena of anion exclusion from the pores of stationary-bonded phase.

Differentiation of Staphylococcus aureus strains by CE, zeta potential and coagulase gene polymorphism

Staphylococcus aureus is a common cause of infection in both hospitals and the community, and it is becoming increasingly virulent and resistant to antibiotics. Possibilities of fast, sensitive and cheap determination of these pathogenic bacteria are extremely important in antimicrobial therapy. In the present study, CE with chemically modified capillary and zeta potential measurements were used for differentiation of three different clinical strains of S. aureus. The data presented in this contribution suggested that electrophoretic behavior and the values of zeta potential should be very useful in distinguishing between closely related strains, which exhibited coagulase gene/protein polymorphism. Understanding the differences between S. aureus strains could help to improve our knowledge about S. aureus pathogenecity and to monitor for and respond to emergence of more virulent strains.

Application of the zeta potential for stationary phase characterization in ion chromatography

Two series of homemade stationary bonded phases for ion chromatography were investigated according to their zeta potential. One set of dendrimer anion exchanger was synthesized on the polymer support whereas the second material was prepared on the silica gel. The zeta potential data in water environment as well as buffered water solution were obtained. The influence of the length of anion-exchanger chains, the type of the support of the modified surface, and charge distribution on these data was investigated. Additionally, the zeta potential was correlated with retention factor of inorganic ions to describe their influence on the retention mechanism in ion chromatography.

Assignment of functional groups in Gram-positive bacteria

The chemical composition, molecular structure and physicochemical properties of five Gram-positive bacterial strain: Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Micococcus luteus were investigated by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), NMR spectroscopy and intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IC MALDI TOF MS). An analysis of FTIR spectra as a function of pH revealed the three major types of cell wall functional groups – carboxyl group, amino group and phosphate group. An analysis of XPS spectra was determinate the major surface components of bacterial cell. 13C NMR and IC MALDI TOF MS spectra of six bacterial species were registered. Our findings indicate that chemical and structural differences in the cell composition of Gram-positive bacteria can be detected. The obtained results also demonstrate that the combination of FTIR, XPS and NMR spectroscopy with IC MALDI TOF MS technique yields useful information and complements other biochemical and physical methods of microbial cells characteristics.

Capillary electrophoresis of microbial aggregates.

Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram‐positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram‐negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca2+). Bound Ca2+ ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high‐intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca2+ between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.

Study of charge distribution on the surface of biocolloids.

Potentiometric titration and zeta potential measurements are crucial techniques for the characterization of the surface properties of bacterial cells. In this study, we investigated the effects of two commonly used electrolytes, NaNO(3) and NaClO(4), on the viability and acid-base properties of Gram-positive bacteria Bacillus subtilis. B. subtilis are non-pathogenic bacteria which are often used to model the surface properties of pathogenic microorganisms of the same genus, including Bacillus anthracis and Bacillus cereus. The survival rates of bacterial cells treated with NaNO(3) were significantly higher in comparison with microorganisms treated with NaClO(4) (5.2-6.8 and 4.1-4.7 log(10) cfu - colony-forming units, respectively). A decrease in the ionic strength (0.1 M, 0.01 M and 0.005 M) of both electrolytes increased viable bacterial cell counts in NaNO(3) treatments and decreased viable bacterial counts in NaClO(4) treatments. Potentiometric titration revealed three dominant types of cell wall functional groups: the carboxyl group (pK(a) values of 4.58-4.89), the amino group (pK(a) values of 9.62-9.89) and the phosphate group (pK(a) values of 7.12-7.49). An increase in the ionic strength of electrolytes led to a decrease in total site concentrations and a drop in buffering capacity at the examined pH values. Based on zeta potential values, measured as a function of pH and ionic strength, the isoelectric point of B. subtilis was determined at pH 2.2 for 0.005 M and 0.01 M NaNO(3). Zeta potential increased with a rise in pH, and it decreased with an increase in ionic strength.

Influence of the charge distribution on the stationary phases zeta potential

A set of seven home-made silica based bonded phases with different functional groups was investigated. Their zeta potential data in methanol and acetonitrile as well as in methanol/water and acetonitrile/water solution were obtained by using a Zetasizer. The influence of polar functional groups on a zeta potential was investigated. The results show that the amines incorporated in the structure of chemically bonded phases of reversed-phase materials are protonated during chromatographic analysis, resulting in changes of the zeta potential from negative to positive values. Acetonitrile causes more negative values and methanol provides positive (or less negative) values of the zeta potential.

Principles of Electromigration Techniques

Electromigration techniques provide the separation of analyzed sample components owing to external voltage generating electrokinetic phenomena—electrophoresis and electroosmosis. Taking into account the relatively large number of parameters dealt with during electrophoretic analyses, it is essential to know their influence on the achieved separation of analytes. In this chapter the theoretical and practical aspects of a resolution optimization, as well as the effect of different separation parameters on the migration behavior are described. These, among others, include migration time, efficiency, selectivity, and resolution. The influence of electrods polarization, applied voltage, temperature, capillary, background electrolyte, and various additives on the separation is also discussed.

Outline of the History

The term ‘electrophoresis,’ that is, the motion of charged particles under the influence of an applied electric field, was formulated as early as the nineteenth century. The first to use electrophoresis as a technique for mixtures separation was Tiselius, who for his accomplishments was awarded the Nobel Prize in Chemistry 11 years later. Others to contribute to the development of capillary electrophoresis were Hjerten, Jorgenson and Lukacs, as well as Terabe. This chapter presents a brief historical outline of electromigration techniques.