ACH/SEM2 Separation Methods 2

Lecturer: Karel Lemr
Lecture: 2 hours/week
Credits: 3
Summer semester
Form of course completion: exam

Introduction, liquid chromatography, high performance liquid chromatography (HPLC) – history, basic terms. Principles of separation used in liquid chromatography – thermodynamic aspects of separation processes. Transport of compounds through column – kinetic aspects of separation processes, comparison of HPLC and GC. Extra column volume in HPLC. Separation process in HPLC (resolution, selectivity, capacity, efficiency). Instrumentation in HPLC – solvent delivery systems, injection devices, columns. Detectors, reactive detectors, auxiliary devices. Registration and evaluation of data. Hyphenation of HPLC and spectral techniques (HPLC/MS, HPLC/FTIR, HPLC/NMR). Flow of mobile phase through column, used sorbents and their comparison. Chromatographic systems – description of retention, mechanisms of separation, application. Chromatography with polar stationary phases (normal-phases). Reversed-phase liquid chromatography. Ion-pair chromatography, use of secondary equilibria. HILIC systems. Liquid-liquid chromatography. Separation of chiral compounds. Ion exchagers in analytical chemistry, ion-exchange chromatography. Size-exclusion and affinity chromatography. Selection of chromatographic system, experimental conditions and their optimization. Microcolumn HPLC, preparative liquid chromatography. Further systems in liquid chromatography (low- and medium pressure systems). Paper and thin-layer chromatography – sorbents, selection of experimental conditions, detection, instrumentation, application. 
Migration of ions in electrical field. Ion mobility. Limit, actual and effective mobility of ions. Types of electromigration methods. Electrophoresis and electroosmotic flow. Electrokinetic zeta-potential, electric double-layer. Electrophoretic velocity and its influencing, mobilities and electroomosis. Zonal electrophoresis. Band broadening and suppression of diffusion by stabilization of zone. Analytical and preparative slab gel electrophoresis. Capillary zone electrophoresis. Efficiency and resolution. The influence of Joule heating, electroosmosis, adsorption, electromigration dispersion on efficiency. The influence of chemical modifications of capillary, radial voltage. The choice of running electrolyte, voltage. Instrumentation. Separation of neutral compounds. Micellar electrokinetic chromatography. Special influencing of effective mobilities. Inclusion complexation, crown complexes, afinity complexes, enantiomeric separation. The dynamic changes of composition of running electrolyte during separation processes. Izoelectric focusation, characteristics of focusation. Fucusation in gradient of pH and in gradient of ligand. Self-sharpen effect, the condition of isotachophoresis. Kohlrausch regulation function – the condition of continuity. Leading and terminating electrolytes. The prediction and mathematic optimization of separation conditions. The connection of ITP and CZE. On-line preconcentration techniques. Application of methods on low-molecular compounds and macromolecules.