Sylabus
Capillary electrophoresis (CE) - sample stacking techniques, enzyme assays
Comprehensive gas chromatography (GCxGC)
Supercritical fluid chromatography (SFC) + chiral separations
Ultrahigh-performance liquid chromatography (UPLC)
Hydrophilic interaction liquid chromatography (HILIC)
Anotace
The course deals with modern separation techniques such as UPLC, SFC, GCxGC and CZE. It will familiarize students with novelties in instrumentation, materials and application approaches.
The course is intended for students who already have basic knowledge of separation methods. The lecture is taught in English for Erasmus students but is open to all students.
The course is organized in so called "flipped class" format in which students are given materials prior to each class to read through them at home. In the class clarifications are provided if necessary and students apply the acquired knowledge to solve problems with teacher's assistance. Active participation of students in the learning process and student-oriented teaching yield better understanding and better fixation of the acquired knowledge.
After passing the individual parts of the course students will be able to: 1 Capillary electrophoresis (CE)
Describe how enzyme assays can be performed in CE.
Compare online enzyme assays in CE with offline enzyme assays using separation techniques - describe advantages and disadvantages.
Describe electrophoretic and diffusion-driven techniques of zone mixing, and discuss their advantages and disadvantages.
Explain why quantification is more precise when separation efficiency is high.
Explain the principle of electromigration dispersion.
Describe the effect of electromigration dispersion on analyte zones.
Identify peaks affected by electromigration dispersion.
Explain the principle of the following techniques: field-amplified sample stacking, field-amplified sample injection, large-volume sample stacking, pH-mediated sample stacking, and transient isotachophoresis.
Discuss the advantages and disadvantages of the abovementioned techniques. 2 Comprehensive gas chromatography (GCxGC)
Classify a gas chromatography method into the following categories: Multicolumn, Two-dimensional, and Comprehensive.
Explain the principle of GCxGC.
Explain the difference between heart-cutting GC and GCxGC.
Describe the function of the modulator.
Explain the conditions needed for a GCxGC system to work correctly.
Describe the raw data obtained, explain how they are transformed, and presented.
Interpret a GCxGC chromatogram.
Define the modulation period and explain the effects of the modulation period being too short or too long.
Explain the difference between thermal and pneumatic modulators.
Explain the difference between single-stage, dual-stage, and twin-stage modulators.
Based on a scheme, classify a modulator and explain its function.
Define the term "orthogonal separation" in GCxGC.
Compare the degree of orthogonality of two GCxGC separations.
Decide for which analytical problems GCxGC is an adequate and appropriate choice. 3 Supercritical fluid chromatography (SFC) + chiral separations
Explain what is a supercritical fluid.
Compare the properties of supercritical fluids with the properties of gases and liquids.
Explain the principle of SFC.
Compare SFC with GC and HPLC in terms of separation selectivity and efficiency.
Explain the importance of mobile phase UV cut-off.
Name the basic components of an SFC instrument.
Explain the differences between SFC and HPLC instruments.
Describe stationary and mobile phases typically used in SFC.
Explain the advantages of using carbon dioxide as a mobile phase in SFC.
Explain the role of cosolvents and mobile phase additives in SFC.
Define chiral separation.
Give examples of practical applications of chiral separations.
Explain the possible approaches to chiral separation in SFC and describe separation mechanisms and interactions taking place.
Define chiral selector.
Recognize chiral stationary phases used in SFC.
Describe how gradient elution is performed in SFC. 4 Ultra-performance liquid chromatography (UHPLC)
Define UHPLC.
Explain the difference between HPLC and UHPLC.
Compare the advantages and disadvantages of HPLC and UHPLC.
Name the basic components of a UHPLC system.
Define the terms separation efficiency, selectivity, retentivity, and resolution.
Explain how separation efficiency, selectivity, and retentivity influence the outcome of separation and resolution.
Name the individual terms of the Van Deemter equation.
Explain how the individual terms of the Van Deemter equation influence separation efficiency.
Relate the individual terms of the Van Deemter equation to the physical properties of separation column and experimental conditions.
Sketch Van Deemter's plot and show how individual terms combine in the resulting Van Deemter plot.
Explain how particle size influences optimum flow rate.
Name factors that can be used to optimize separation in SFC and explain their effects.
Describe core-shell particles.
Explain the advantages and disadvantages of core-shell particles.
Transfer an HPLC method to a UHPLC method using an online method transfer calculator.
Explain in which cases UHPLC is advantageous due to its higher sample throughput and where due to its increased separation efficiency. 5 Hydrophilic interaction liquid chromatography (HILIC)
Explain the separation mechanism in HILIC.
Compare normal-phase, reversed-phase, and HILIC separation modes regarding the polarity of suitable analytes and electrospray ionization-mass spectrometry response.
Describe the properties of analytes suitable for separation by HILIC.
Recognize analytical problems for which HILIC is suitable.
Give examples of typical HILIC stationary and mobile phases.
Describe the effect of acetonitrile content in the mobile phase on the retention of analytes in HILIC.
Explain what kinds of mobile phase additives are suitable for HILIC and their effects on separation.
Explain how gradient elution is performed in HILIC.
Explain the effect of sample solvent on HILIC separation.
Explain the difference between HILIC and electrostatic repulsion hydrophilic interaction chromatography.
Estimate the retention order of analytes in HILIC based on their log D values.
