Syllabus
1. Theory of electromigration - forces affecting electromigration, electrophoretic mobility, the effect of ionic strength, the effect of pH, relaxation effect, electrophoretic effect, electroosmotic flow.
2. Separation efficiency - practical consequences of low/high efficiency in analysis, the theoretical plates model, calculation of efficiency, contributions to band broadening (diffusion, sample zone length, Joule heating, adsorption to capillary walls, detection cell, electromigration dispersion).
3. Instrumentation - basic components of the instrument, separation capillaries, current and its stability, sample injection, field-amplified sample stacking techniques, detection (direct and indirect UV/VIS, fluorescence, contactless conductivity detection, system peaks), short-end separations, data sampling frequency.
4. Separation modes ICapillary zone electrophoresis (CZE) - separation mechanism, choice of background electrolyte, its pH and ionic strength, manipulation of the electroosmotic flow direction and velocity, dynamic and permanent capillary coatings. Micellar electrokinetic chromatography (MEKC) - separation mechanism, micelles as a pseudostationary phase, elution window, surfactants used in MEKC. Capillary electrochromatography (CEC) - separation mechanism, stationary phases, electroosmotic flow, comparison with liquid chromatography.
5. Separation modes IICapillary gel electrophoresis (CGE) - separation mechanism, physical and chemical gels for CGE, DNA sequencing, Sanger method. Capillary isoelectric focusing (CIEF) - separation mechanism, development of pH gradient, mobilization of the system. Capillary isotachophoresis (CITP) - separation mechanism, leading and terminating electrolyte, autofocusing effect, specifics of the ITP experimental records, application of transient isotachophoretic effect for sample stacking in CZE. Capillary as a microreactor - on-capillary enzyme reactions and derivatization of analytes, electrophoretic and diffusion-driven mixing of zones.
Annotation
The lecture gives an overview of electromigration analytical methods from theoretical and practical points of view and is taught using the flipped classroom method. The learning is organized in two phases. 1) One week is always dedicated to self-study. Students themselves go through materials that are available in Moodle. Then they submit homework (also through Moodle). The homework helps the teacher to see what needs further explanation. Homework is not a part of the final classification, it serves only to check the understanding of the topics from self-study. Nevertheless, if the teacher decides that the student did not pay proper attention to the homework or if there is a critical misunderstanding he can ask the student to rework the homework. 2) The following week, a presence lesson is held during which problematic parts are clarified and the students use and fix the knowledge acquired during the self-study in solving various problems.
After completing the given section the student will be able to: 1 Theory
Describes forces acting on the ion in the electric field.
Defines and explains the following terms: migration time, electrophoretic mobility (apparent, effective), and electroosmotic flow.
Interprets an electropherogram.
Based on an electropherogram, calculates the apparent and effective mobility of an analyte.
Describes the effect of experimental conditions (background electrolyte composition, pH, ionic strenght, voltage, capillary dimensions, temperature) and properties of analytes (charge, size) on their electrophoretic mobility and velocity of electroosmotic flow.
In Peakmaster program, calculates the pH and ionic strength of a background electrolyte based on its composition.
In Peakmaster program, chooses background electrolyte composition to match a given pH and ionic strength.
In Peakmaster program, performs a simulation of electrophoretic separation. 2 Separation efficiency
Defines separation efficiency.
Explains the advantages of high separation efficiency.
Names factors influencing separation efficiency and describes their effect.
Names measures to suppress overheating of the solution in the capillary.
Defines electromigration dispersion and explains the mechanism of its occurrence.
Identifies peaks deformed by electromigration dispersion. 3 Instrumentation
Names and describes basic parts of an electrophoretic instrument.
Explains the difference between hydrodynamic and electrokinetic sample injection. Describes advantages and disadvantages of both techniques.
Describes the principle of pre-concentration techniques field-amplified sample stacking, field-amplified sample injection, large-volume sample stacking, pH-mediated sample stacking, and transient isotachophoresis.
Explains the principle of detection techniques and describes their advantages and disadvantages: direct UV, indirect UV, fluorescence, and contactless conductivity detection.
Estimates if, for a given background electrolyte and analytes, the detection technique provides a signal and if the peak will be positive or negative.
Chooses detection technique suitable for given analytes.
Explains "short-end separation" term.
Calculates the approximate migration time of the analyte for a given total and effective capillary length from migration time in a capillary of different total and effective length.
Identifies compounds in an electropherogram based on electropherograms of standards and UV spectra of peaks. 4 Separation modes I
Describes separation principle in the following separation modes: Capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Names parameters necessary to be considered while optimizing a capillary zone electrophoresis method and describes their effect on the separation.
Suggests a reasonable background electrolyte for separation of given analytes in capillary zone electrophoresis.
Using the Peakmaster program optimizes conditions of an electrophoretic separation (to reach given resolutiuon of separation efficiency).
Indicates the direction of electroosmotic flow based on experimental conditions.
Describes ways how to influence velocity and direction of electroosmotic flow. 5 Separation modes II
Describes separation principles in the following separation modes: Capillary gel electrophoresis, capillary isoelectric focusing, and capillary isotachophoresis.
Describes principle of the Sanger DNA seqencing method.
Identifies whether an isotachophoretic system is aimed for the separation of cations or anions, based on leading and terminating electrolytes.
Explains how electrophoretic capillary can be used as a microreactor.
Suggests injection sequence for reactants to perform a chemical/biochemical reaction inside the capillary.
Chooses appropriate separation mode for given analytes.
General/valid for multiple sections
Estimates order of analytes in electropherogram based on experimental conditions in different separation modes.
Identifies a mistake in experimental conditions of an electromigration separation and suggests how to correct it.