Separation Methods

Chemistry students will become familiar with the information about recent state of separation methods within the system of scientific cognition of material reality and their unavoidable need for generation of interpretable analytical signal. For the purpose of education separation methods we utilize unified approach based on concepts of transport and distribution phenomena that are of use in modern separation methods. Separation methods create frequently basis of chemical analysis methods. They are also base for qualitative evidence and quantitative measurement of characteristics or determination of chemical substances utilized in various human activities and associated control, e.g. analysis of technology products, bioanalysis, analysis of substances in environment, clinical analysis and diagnostics etc. Student will get information related to classification of analytical, preparative and industrial separation processes and methods, respectively. Calculus from separation science beside case study of model situations of daily praxis is the component part of the objective. Theoretical knowledge and calculus basics are taught via lectures and seminars, respectively. Vital part of the objective is also instrumental experimental work in the laboratory.

Sylabus prednášok (Syllabus)

1. Characteristics of separation methods, their function and importance in analytical, preparative and industrial procedures and processes; separation methods based on differences in phase distribution of separated components; separation methods based on differences in velocity of migration of separated components in various fields of force; separation methods based on differences in velocity of separated components permeating through semipermeable membranes.
2. General terms, concepts, terminology characterizing extent of separation; distribution ratio, distribution constant, Nernst distribution law, chemical equilibrium in separation process. Interactions in separation systems. Calculus, thermodynamic and kinetic aspects of separation methods, relation of terms separation and resolution.
3. One-stage separation methods, principle, technique and application; separation by precipitation, co-precipitation. Sublimation, lyophilisation. Electroprecipition, electrolytic deposition. Extraction separation in the system solid phase-liquid, selective leaching, liquid – liquid extraction, cloud-point extraction – micellar systems. Membrane extraction. Salting-out. Simple distillation. Zone melting.
4. Multi–steps separation methods and introduction to chromatographic methods. Multi–stage liquid-liquid extraction, principle of continuous extraction. Multi–stage distillation. Chromatographic separation, classification according to various criteria. Theory of chromatographic phenomena, qualitative and quantitative chromatographic analysis – unified approach.
5. High-resolution gas chromatography (HRGC). Instrumentation in GC. Separation mechanism in GC. Optimization of separation in GC. Advanced techniques. Reaction chromatography, pre-column derivatization, vacant GC. GC-MS. Computing simulations and calculus.
6. High-performance liquid chromatography (HPLC). Instrumentation in HPLC, column vs. planar techniques, computing simulations and calculus. Chromatogram interpretation. Chromatographic phase systems in LC (HPLC, TLC).Advanced techniques. Separation mechanism in HPLC. Pr-column and post-column derivatization (physical, chemical and biological). Typical application of chromatographic methods.
7. Separation of substances by field of force utilization. Electroseparation methods. Principles of action, classification, parameters characterizing electroseparation. Concept of ionic mobility, separation mechanism, column vs. planar techniques, column- coupling technique and detection. Zone electrophoresis. Separations in free solution of carrier electrolyte. Separations in micelle forming solutions, electroosmotic flow, micellar electrokinetic chromatography (MEKC). Basics, instrumentation and practical utilization of computing simulation techniques.
8. Capillary isotachophoresis (cITP) and isoelectric focusing (IEF). Basic principles of ITP, instrumentation and computing simulation technique, analytical signal- isotachopherogram. Basis principles of IEF, instrumentation and computing simulation technique, separation and analysis of proteins.
9. Chip-based electroseparation. Miniaturization of separation columns and channels. Instrumentation and novel approaches to electroseparations. Advance and application of chip-based electroseparations.
10. Mass spectrometry as separation method (MS-MS). Classification of MS methods from point-of view of ions separation in vacuum. Sedimentation separation. Centrifugal separation. Centrifugal field flow fractionation (FFF) – principle and application areas.
11. Membrane separations. Principles, classification. Dialysis and electrodialysis, principle, instrumentation and usage. Ultrafiltration – utilisation in analytical procedures. Gas permeation through membranes. Validation of analytical methods focused to separation methods.
12. Solution of case study examples from various fields of social and productive praxis. Typical utilization of separation methods in analytical procedures, preparative and industrial scale procedures. Application of separations in FIA, SIA an autoanalyzers (clinical diagnostics).
13. Future trends in development of separation methods.

Hodnotenie predmetu (Grading policy)

There will be one running written test for 20 points, during the semester on seminars. Only student obtaining from the test minimum 60% will pass to final exam test. Experimental laboratory work will be graded as follows: continuous compounding will be evaluated by 20 points maximum involving theoretical preparation for laboratory work, activity and quality of results in the experimental work summarized in laboratory protocol. Final test from topics of laboratory will be enabled only to student who submitted protocols from all done laboratory experiments - student can get maximum 20 points, in summary max. 40 points for experimental laboratory work. Only those students will be admitted to final examination who achieve at least 60% of the points from seminar tests and laboratory work evaluation. Final exam related to lecture topics will consist of 40-point test. For clarification of boundary test results it is possible to require additional oral answer to the question for maximum 4 points included. Overall grade will consist of summary evaluation of final exam, seminar tests and experimental work according to formula (0.4 x % final exam) + (0.2 x % seminar tests) + (0.4 x % laboratory work) = resulting %.

Celkové hodnotenie predmetu

Laboratórne cvičenia (Laboratory experiments)

1. Computing simulations of basic separation process in gas chromatography. Simulation of individual contributions of dispersion parameters. Optimisation of separation of components of gas mixture. Van Deemter equation. Capillary gas chromatography. Qualitative and quantitative GC analysis. Calculus of separation parameters of model mixtures (separation of components of hydrocarbon gas). Separation and determination of volatile organic contaminants in water by capillary GC (determination BTEX in water, headspace concentration fast analysis).
2. Computing simulations of basic separation process in liquid chromatography. Simulation of individual contributions of dispersion parameters. Optimisation of HPLC analysis of liquid mixture. Van Deemter equation and H-u curves. Gel chromatography. Column liquid chromatography Qualitative and quantitative LC analysis. Calculus of separation parameters of model mixtures (separation of phenols and phenolic acids, fast analysis). Partial validation of LC methods. Separation and determination of selective medical drugs by HPLC (analysis of pills and determination of paracetamol, guaifenesin, acetylsalicylic acid).
3. Computing simulations of basic separation process in capillary electrophoresis. Simulation of individual contributions of dispersion parameters. Optimisation of operational parameters in capillary electrophoresis. Qualitative and quantitative analysis. Calculus of separation parameters in capillary zone electrophoresis, isotachophoresis and isoelectric focusing in model mixtures. Separation and determination of food additives and contaminants in food by capillary electrophoresis. Centrifugal separation.
4. Electroseparations using chips. Basic differences among separations in macro- and micro-scale. Analysis of body fluids by chip electrophoresis.
5. Liquid-liquid extraction separation, solid-phase extraction (SPE), cloud-point extraction and micelle-forming systems. Ultrafiltration, dialysis, separation of low molecular mass and high molecular mass substances.
6. Mass spectrometry as separation method. FIA mode in MS. Experiment, animation and simulation.

Študijný materiál a Obsluha prístroja

Odporúčaná literatúra (Suggested readings)

• D. A. Skoog, F. J. West, F. J. Holler, S. R. Crouch: Analytical Chemistry. An Introduction. Saunders Coll. Publ.2000
• G. Schwedt:The Essential Guide to Analytical Chemistry, Wiley, New York, 1997
• R. Kellner, J-M. Mermet, M. Otto, Analytical Chemistry, John Wiley & Sons Australia, Ltd,  2013;  E. J. Henley, J. D. Seader, D. K. Roper
• Separation Process Principles, 3rd Ed., Wiley-VCH, 2011; C. E. Meloan, Chemical Separations: Principles, Techniques and Experiments (Techniques in Analytical Chemistry) Wiley-Interscience; 1st Ed., 1999

• Paul Haddad,  Maryam Taraji a Roman Szücs; Prediction of Analyte Retention Time in Liquid Chromatography; Analytical ChemistryVolume 93, Issue 1, Pages 228 – 256; https://doi.org/10.1021/acs.analchem.0c04190