Catalogo dei Corsi di studio

General expected learning outcomes
This course is mainly designed to provide a deeper insight into advanced chromatographic and spectrometric techniques presented during the course of Physical Methods in Organic Chemistry.
Chromatographic techniques allow the qualitative and quantitative determination of complex mixtures, obtained both from synthetic processes and from extractive processes from vegetable drugs and environmental matrices. For these reasons, such techniques play a fundamental role for a future employment in the field of regulatory affairs and healthcare industries.
The student will experience the most modern techniques for the separation of complex mixtures (HPLC, UHPLC, HILIC) and the basic concepts of the coupling between liquid chromatography and mass spectrometry (LC/MS).

Specific expected learning outcomes
Knowledge and understanding
At the end of the course the student has a thorough and advanced knowledge of the chromatographic and spectroscopic methods already presented in the course of Physical Methods in Organic Chemistry. He knows the most modern techniques for the separation of complex mixtures (HPLC, UHPLC, HILIC) and the basic concepts of the coupling between liquid chromatography and mass spectrometry (LC/MS).

Applying knowledge and understanding
At the end of the course the student knows the most widely used chromatographic materials, the theoretical principles and the most innovative chiral stationary phases for the analysis of chiral drugs; he is familiar with the concept of the inversion of the elution order of enantiomers. He knows some of the most recent applications in the field of proteomics (monolithic capillary column approach) and of stereoselective molecular recognition in the gas phase.

Making judgements
At the end of the course the student is able to decide independently which analytical method is more useful to solve a particular trouble that he could meet in the working field, from the characterization of new active ingredients of therapeutic interest to the production and quality control of the drug.

Communication skills
At the end of the course the student has the ability to communicate outward the knowledge he has learned during the course, both toward the scientific community and the labor market. In particular, he must be able to provide clear and direct information on the most modern chromatographic techniques and their applications in the field of drugs.

Learning skills
The student who has passed the exam is able to continue the study by reading the most recent articles published in the literature that the teacher makes available to him on the e-learning platform.

Programme

Cross-reference to theory and principles and main application fields of chromatography. Chromatographic parameters (capacity factor, selectivity, efficiency, resolution). General separation mechanisms [2 hours].

Chromatographic packing materials: capillary columns, siliceous micro-packings, hybrid organo-siliceous, monolith packings. Geometry of the columns (packed, standard, microbore, micropellicular, preparative) [2 hours].

Stereoselective chromatographic separations. Direct and indirect approach. Innovative chiral stationary phases (CSPs) for HPLC: totally synthetic and glycopeptide-containing CSPs. Concept of the reversal of the elution order of enantiomers. Inverted Chirality Columns Approach (ICCA) [4 x 2 hours].

Hydrophilic Interaction Chromatography (HILIC). Sugar immobilization by the click-chemistry approach. Chromatographic analysis of sugars: the concept of the "on-column interconversion" (anomerization) [3 x 2 hours].

Converting HPLC (High Performance Liquid Chromatography) methods into UHPLC (Ultra High Performance Liquid Chromatography) methods [2 hours].

Capillary HPLC based on organic monoliths and its application in proteomics. Analysis of histones and of their post-translational modifications (PTM): acetylation and enzymes involved (histone acetyltransferase, HAT and histone deacetylase, HDAC) in the screening of new potential anticancer drugs [2 x 2 hours].

Introduction to Mass Spectrometry (MS) and key terms. Ionization sources: electronic impact (EI), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI). Analyzers: quadrupole, time of flight (TOF), ion trap. Atmospheric pressure ionization (API) interfaces [2 hours].

Electrospray Ionization (ESI). Factors affecting electrospray nebulization. Capillary voltage, solvent surface tension. Eluent flow-rate. Conductivity and ionic strength of the eluent. Source design. Capillary, sampling cone, on-axis, off-axis, and z-spray geometries [2 hours].

Interfacing HPLC/ESI-MS: direct and with flow splitting. Miniaturization of the LC system for nano-LC/MS and cap-LC/MS specific applications. Choosing a suitable HPLC system to be coupled to ESI-MS. Solvent and additives compatibility. Analyte structural requirements. Effects of organic modifier, eluent pH, non-volatile buffers. Ion pair reagents in ESI. Electrospray cationization. Post column addition. Trifluoroacetic acid (TFA) effect and neutralization.
APCI: source design. Corona discharge. APCI compatibility and application field. Harmonization with ESI. Choosing a suitable HPLC system to be coupled to APCI-MS. Solvent and additives compatibility. Analyte structural requirements [2 x 2 hours].

Key terms: mass-to-charge ratio; multi-charged ions; molecular ion; resolution; mass range. Mass spectra of biomolecules [2 hours].

Tandem mass spectrometry (MS/MS). Collision Induced Dissociation (CID). MS/MS Scan modes: (1) Product Ion Scan; (2) Precursor Ion Scan; (3) Neutral Loss Scan; (4) Selected Reaction Monitoring (SRM) [2 ore].

Gas-phase stereoselectivity by mass spectrometry. Host-guest complexes and fragmentation studies. Orbitrap. Tetramide host molecules A2B2 and A2C2 and their enantiodiscrimination ability towards amino acidic guests [2 x 2 ore].

Exercises in the classroom and illustration of the ChemDraw software for the representation of organic molecules and the prediction of fragmentation (use of the "mass fragmentation" tool). Presentation and interpretation of mass spectra of model drugs [12 exercises].

Adopted texts

The lectures of the teacher and an accurate bibliography on the topics covered is uploaded on the teacher's web page of the E-learning platform and made available to the students registered.

Prerequisites

For an easy understanding of the topics covered in the course it is recommended to have acquired the basics of Physical Methods in Organic Chemistry.

Frequency modes

Being an optional course, the attendance is optional but strongly recommended.

Exam modes

To get the CFU of the course, the student must pass the final exam, which is scheduled to take place at the end of the course, during the periods established.
To access the exam (oral), students must register on the INFOSTUD platform. The exam consists of 3 questions on the whole program (average exam duration: 30 minutes). To achieve a score of 30/30 cum laude, the student must demonstrate that he has acquired excellent knowledge of all the topics covered during the course, showing a natural ability to quickly link between them.

Exam reservation date start	Exam reservation date end	Exam date
01/02/2022	14/02/2022	15/02/2022
10/06/2022	20/06/2022	21/06/2022
08/07/2022	19/07/2022	20/07/2022
10/09/2022	25/09/2022	29/09/2022
10/10/2022	23/10/2022	24/10/2022
10/11/2022	20/11/2022	21/11/2022
01/01/2023	18/01/2023	19/01/2023