Capillary electrophoretic analyzer for multidimensional separation and detection
The aim of the project is to construct a lab-made capillary electrophoretic (CE) analyzer of a new generation for multidimensional separation and detection of biomolecules. Multidimensional separation will be realized by on-line combination of different separation principles performed in two in-series connected capillaries or in a single capillary with different selectivity sections. Enlarged detectability and increased detection sensitivity will be achieved by a multidimensional detection system. The detection system will be composed of universal, double contactless conductivity detector, UV-photometric absorption detector operating at 206 nm and laser induced fluorescence (LIF) detector with excitation wavelength 266 nm. The universal contactless conductivity detector is capable to detect all types of analytes, including those possessing no chromophores and fluorophores. The double in-series arrangement of the conductivity detector allows precise measurement of the electrophoretic mobilities. The UV-absorption photometric detector will be used for detection of molecules absorbing light in the low UV region. LIF detector with excitation wavelength 266 nm will make possible highly sensitive detection of peptides and proteins containing aromatic amino acid residues of tryptophan and tyrosine on the basis of their native fluorescence, i.e. without the need of their derivatization.
New electrolyte systems and (pseudo)stationary phases for capillary electromigration methods
Within this project the following new electrolyte systems and (pseudo)stationary phases will be developed:
- Highly acidic or highly alkaline background electrolytes (BGEs) for separation and characterization of analytes with low and/or high pKa values.
- BGEs with stereoselective additives (cyclodextrins, glycopeptide macrocyclic antibiotics) for separation of enantiomers and diastereomers of amino acids, peptides nucleosides and nucleotides.
- Isoelectric BGEs composed of single- or multiple-component amphoteric compounds with low conductivity allowing application of high intensity electric field resulting in fast and high-efficient CE separations.
- BGEs with replaceable sieving matrices (liquid solutions of entangled polymer networks) for separation of peptides and proteins according to their size (relative molecular mass).
- Wall-immobilized or in solution dissolved oligopyrrolic macrocycles, porphyrins and their derivatives for combined electrokinetic and chromatographic techniques, electrokinetic chromatography and open-tubular electrochromatography in order to increase the selectivity of capillary electroseparations of both ionogenic and nonionogenic biomolecules (amino acids, peptides, nucleosides, nucleotides and saccharides).
Multidimensional capillary electroseparations
The aim of this project is to develop multidimensional separation systems for separation of complex protein and peptide mixtures and for analysis of particular bioanalytes in complex biomatrices. Multidimensional separation systems will be based on the combination of different modes of high-performance capillary electrophoresis (HPCE), separating the analytes according to different separation principles, such as e.g. separation according to charge distribution (isoelectric point), electrophoretic mobility, size (relative molecular mass), hydrophobicity and specific binding capabilities. This combination will be realized by on-line connected HPCE modes of isoelectric focusing, zone electrophoresis in non-sieving or sieving media, electrokinetic chromatography, affinity electrophoresis and open-tubular electrochromatography, which will be performed in different sections of a single capillary or in on-line and in-series connected capillaries.
Conversion of analytical capillary electromigration separations to preparative free-flow electroseparations
Conversion of analytical capillary electromigration separations to preparative free-flow electroseparations will be based on the correlation between capillary zone electrophoresis (CZE) and free-flow zone electrophoresis (FFZE). Correlation between CZE and FFZE results from the fact that both these techniques employ the same separation principle (zone electrophoresis) and both are performed in the carrierless medium (free solution) using the same composition of the background electrolyte. This correlation will be described by a more precise mathematical model, in which the role of electroosmotic flow in FFZE will be included in a more general way than in our earlier developed model. Based on this correlation the procedure for conversion of analytical CZE separations to preparative FFZE separations will be developed.
Physico-chemical characterization of biomolecules by capillary electromigration methods
Elaboration of more precise procedures for determination of physico-chemical characteristics of biomolecules (peptides, proteins, nucleosides, nucleotides) such as their effective electrophoretic mobilities, absolute (limiting) ionic mobilities, effective charges, isoelectric points, relative molecular masses, Stokes radii, diffusion coefficients, acid-base dissociation constants of their ionogenic groups and association constants of their complexes from the experimental data obtained by CE separations and analyses of these biomolecules.
Development of new procedures for interaction studies of biomolecules (amino acids, peptides, nucleotides, saccharides) with new affinity ligands (oligopyrrole macrocycles, boron clusters) and other low- and high-molecular mass ligands (aptamers, antibodies) forming (bio)specific complexes, by the methods of capillary affinity electrophoresis and capillary affinity open-tubular electrochromatography.