SEPARAZIONE E CARATTERIZZAZIONE DI COMPOSTI ORGANICI

12 CFU - 2° Semester

Teaching Staff

ROSARIA SALETTI - Module CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1) - CHIM/06 - 6 CFU
SALVATORE FOTI - Module CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1)
VERA MUCCILLI - Module STRUCTURAL CHARACTERIZATION OF ORGANIC MOLECULES - CHIM/06 - 6 CFU

Learning Objectives

CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1)
The aim of the course is to provide to the students the theoretical principles and the application-related aspects of:

- chomatographic methods for the separation and purification of organic compounds;

- mass spectrometry (MS);

- use of separation techniques (GC, LC) coupled with MS;

- application of the MS to the structural determination of organic compounds.

The BSCM graduate, with the teaching of MASS SPECTROMETRY METHODS AND SEPARATION OF
MOLECOLE ORGANICHE expands and deepens the basic knowledge in the fields of similar and integrative field,
acquired with a first level degree. It also matures an integrated understanding of biological phenomena and an advanced scientific / chemical / biochemical and ecological / environmental preparation.
In particular, the specific training objectives of this course are:
- understand the principles on which the isolation of organic compounds from natural matrices, including biological ones, is based:
- understand the methods and techniques used for this purpose;
- understand the principles underlying the characterization of organic compounds by mass spectrometry;
- understand the techniques used to obtain mass spectra;
- understand how to interpret a mass spectrum;
- understand the principles of chromatography-mass spectrometry coupling.

Furthermore, with reference to the Dublin Descriptors, this course helps to acquire the following transversal skills:

Knowledge and understanding
- inductive and deductive reasoning skills;
- ability to understand the principles and methods on which the separation of organic compounds is based;
- ability to understand the principles and methods on which the identification of organic compounds is based by mass spectrometry.
Ability to apply knowledge
- ability to identify the most appropriate technique for the isolation of organic decomposed;
- ability to identify an organic compound by studying its mass spectrum.
Autonomy of judgment
- critical reasoning skills;
- ability to predict the result of a chromatographic separation;
- ability to predict the result of using a particular mass spectrometry technique.
Communication skills
- ability to describe in oral form, with properties of language and teminological rigor, the result of a chromatographic procedure, mass spectrometry or chromatography / mass spectrometry analysis.
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STRUCTURAL CHARACTERIZATION OF ORGANIC MOLECULES
The course aims to provide students with the basic knowledge and methodology necessary for the interpretation of infrared (IR), ultraviolet-visible (UV-Vis) and nuclear magnetic resonance (NMR) spectra. At the end of the course, the student will be able to carry out a complete structural and stereochemical characterization of organic molecules using in an integrated way spectra (NMR, MS, UV, IR)

Regarding the so-called Dublin Descriptors, the learning outcomes of the course are:

D1 - Knowledge and understanding: The student must demonstrate mastery of the basic knowledge of modern spectroscopic investigation methods as well as the ability to understand the fundamental characteristics of the spectra supplied by each substance (NMR, IR and UV and Massa).

D2 - Ability to apply knowledge and understanding: The student must show the ability to apply their knowledge, and the ability to identify a molecule through the combined analysis of the spectra obtained with the various techniques. He must be able to simulate the spectra of new molecules and to be able to decide which methods are most useful to solve a particular structural problem.
D3 - Independent judgment: students must be able to design and conduct experiments independently. At the end of the course, they will know how to interpret the collected data in a coherent, critical and correct way, correlating them to the appropriate theories. At the end of the course, they will have to know how to formulate hypotheses and discard the incorrect ones.

D4 - Communication skills: The student must be able to communicate his / her knowledge and his / her interpretative ability of the various spectra to specialists and not with an adequate language.

D5 - Learning skills: students will be able to solve a problem of structural identification autonomously, showing the ability to face it through the application of the skills acquired during the course.

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Course Structure

CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1)
Lectures with classroom exercises.
STRUCTURAL CHARACTERIZATION OF ORGANIC MOLECULES
The course is structured to allow the acquisition of the indispensable tools for a straightforward interpretation of spectroscopic data aimed at the structural characterization of compounds of biological, pharmaceutical and nutritional interest.

The course is organised in lectures, classroom exercises and laboratory activities.

During the lectures, in order to stimulate the students' attention, the teacher periodically organizes short tests (5-10 min) that the students will follow anonymously on their electronic devices (smartphones, tablets, etc.).

During the exercises, guided analyzes of IR, MS, UV, mono and bidimensional NMR spectra will be performed to identify an unknown organic molecule. The interpretation of the spectral data is carried out by the teacher interactively with the students.

The laboratory part (2 CFU) aims, instead, to complete the training course provided by the whole class. During the hours of this part of the course, practical exercises will be developed aimed at acquiring the NMR, IR, UV and mass spectra and subsequent interpretation of the relative spectral data.

Detailed Course Content

CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1)
CHROMATOGRAPHY

Theoretical principles of the chromatographic separation.

Retention (retention time and volume). Capacity factor. Selectivity, resolution, peak simmetry. Efficiency and theoretical plate. Differential migration of the analytes and chromatographic band spreading: multiple paths (Eddy diffusion), longitudinal diffusion, mass transfer between mobile, stagnant mobile and stationary phase. Equation of Van Deemter. Band broadening not due to the column.

Classification of the chromatographic techniques:

The adsorption liquid chromatography (liquid/solid, LS). Low pressure liquid chromatography on the column (LPC) and thin layer chromatography (TLC). Description of the atmospheric pressure chromatographic system.

High pressure liquid chromatography on the column (HPLC). Characteristics of the stationary phases used in normal-phase HPLC (liquid/solid) and reversed-phase HPLC (RP-HPLC, liquid/liquid).

Description of the high pressure chromatographic system. The sample valve injector (“loop”). The pumps: syringe pump, single piston and double piston reciprocating pump. Pulse dampers.

Mobile phases for HPLC: physical properties, eluting power and selectivity. Preparation of the mobile phase: dehydration, filtration and degassing. Sample preparation. Stationary and mobile phases. Examples of organic compounds separation by nornal- and reversed-phase HPLC. Optimization of the chromatographic conditions.

Detectors for liquid chromatography: general properties (limit of detection, LOD, and linear dynamic range, LDR). Detectors: UV-Vis, diode array, refractive index, fluorescence. Mass spectrometer.

Size-exclusion chromatography. Principle; stationary and mobile phases. Examples of biopolymers separation.

Ion-exchange chromatography. Princile; stationary and mobile phases. Examples of organic compounds separation: the amino acid analyzer. Post-column derivatization of amino acids by ninhydrin.

Affinity chromatography. Principle; stationary and mobile phases. Dye-protein affinity chromatography for protein purification.

Gas chromatography. Description of a gas chromatograph system. Adsorption gas chromatography (gas solid, GSC) and partition gas chromatography (gas liquid, GLC). Capillary and packed columns. The carrier gas. Solid and liquid stationary phases. Choosing the stationary phase.

Detectors for gas chromatography: thermal conductivity (TCD), flame ionisation (FID), alkaline flame, electron capture (ECD). Mass spectrometer.

MASS SPECTROMETRY

Principles of the method

The Electron Ionization ion source. Construction an operation

Magnetic sector analyzer. Resolution. Double-focusing (magnetic- electrostatic) analyzer. High resolution mass spectrometry. Nominal mass, exact mass, nonoisotopic mass, relative molecular mass. Exact mass and the determination of molecular formula.

Molecular ion and isotopic peaks. Criteria for the recognition of the molecular ion peak. Information deducible from the molecular ion and isotopic pattern. Nitrogen rule.

Principles of fragmentation reaction of organic ions and interpretation of EI mass spectra. Quasi- equilibrium theory. Classification of the fragmentation reactions. Cleavage of sigma-bonds and rearrangements. Even-electron rule. Charge localization. Criteria for fragment ions intensity evaluation.

Sigma-bond cleavage in small non-functionalized molecules. Fragmentation of compounds containing double bonds or heteroatoms. Alpha-cleavage (radical site initiated process) . Inductive cleavage (Charge-site initiated process). Fragmentation of cyclic compounds. Rearrangements. Typical fragmentation of the most common classes of organic compounds.

The Matrix assisted laser desorption/ionization (MALDI) source. Operating principles. MALDI matrices. Sample preparation. Calibration in MALDI.

Time-of-Flight (TOF) analyzer. Basic principles. Improving resolving power: delayed extraction and ion reflector.

The Electrospray (ESI) source. Operating principles. Ions formation and ions transfer in ESI.

The Atmospheric Pressure Chemical Ionization (APCI). Operating principles.

Linear quadrupole analyzer. Operating principles.

Ion traps analyzer. Operating principles.

Tandem mass spectrometry. Tandem-in-space and tandem-in-time

Coupling gas-chromatography/MS (GC/MS) and High Performance Liquid chromatography/MS (HPLC/MS).
STRUCTURAL CHARACTERIZATION OF ORGANIC MOLECULES
Introduction

Importance of spectroscopic techniques in organic chemistry. Regions of the electromagnetic spectrum and corresponding spectroscopic techniques.

Ultraviolet and visible spectroscopy (UV-VIS)

Introduction and fundamental concepts. Analysis and interpretation of UV-VIS spectra: characteristic bands and absorption intensity. Main chromophores.

Exercises.

Infrared spectroscopy (IR)

Introduction and fundamental concepts. Analysis and interpretation of IR spectra. Characteristic absorptions of the main functional groups of organic molecules. Exercises.

Introduction to nuclear magnetic resonance spectrometry (NMR)

Magnetic properties of nuclei. Energy levels and population of nuclei immersed in a magnetic field. Downgrading processes. Pulse and Fourier transform NMR spectroscopy (PFT-NMR). NMR spectrometers.

¹H NMR spectra

Chemical shift. Screen constant and its components. Inductive effects, diamagnetic anisotropy, electric field; effects of intermolecular relationships on the chemical shift. Protons on oxygen and nitrogen atoms. Signal intensity. Spin-spin coupling.

Scalar and dipolar couplings. Zero-order and 1st order systems. Chemical equivalence and magnetic equivalence. 2nd order systems. Exercises.

¹³C NMR spectra

Theory. Spin decoupling (double resonance). Broadband heteronuclear decoupling. Overhauser nuclear effect (NOE). Heteronuclear NOE. Controlled decoupling (Gated decoupling). Exercises.

¹H NMR spectra and molecular structure

Equivalence, symmetry and chirality. Pairs of omotopic, enantiotopic and diasterotopic protons. Effects of a chiral centre. Virtual coupling. Coupling constants: structure and stereochemistry. Homonuclear couplings: geminal, vicinal and long-distance. Double homonuclear resonance ¹H-¹H. Overhauser effect difference spectrometry (NOE). Solvent effect and shift reagents. Exercises.

NMR in dynamic processes

Conformational equilibrium and chemical equilibrium. Fast exchange and slow exchange. NMR spectra at variable temperature. Eyring equation. NMR spectra of compounds containing "mobile" hydrogens.

Advanced experiments and 2D NMR

Advanced one-dimensional experiments: evolution time and mixing time. Evolution of magnetization in AX, AX2 and AX3 systems. J-modulation. DEPT Spectra. 1D-TOCSY Spectra. Two-dimensional NMR experiments (2D NMR). Homonuclear correlation spectra. Analysis of ¹H-¹H COSY and ¹H-¹H TOCSY spectra. 1H-1H NOESY and ROESY experiments and spectra analysis. Heteronuclear correlation spectra. Analysis of 1H-13C COZY spectra (HETCOR) and long-distance heteronuclear correlation ¹H-¹³C (HMBC). Exercises.

NMR spectra of heteronuclei

¹⁵N NMR spectra: characteristics, chemical shift region, constants ¹H-¹⁵N.

¹⁹F NMR spectra: characteristics, chemical shift region, constants ¹H-¹⁹F. ³¹P NMR spectra: characteristics, chemical shift region, constants ¹H-³¹P. Multinuclear couplings.

Protein-ligand interactions

Interactions with small ligands. Diffusion limited systems, KD, equations. Chemical exchange: slow and fast exchange. Time scales and chemical exchange. In vitro screening. Experiments for NMR characterization: WaterLOGSY, STD, DOSY.

Classroom exercises

Guided analysis of spectra (NMR, MS, UV, IR); integrated use of the studied spectroscopic techniques and development of problems of determination of the structure of organic molecules.

Laboratory

Identification of unknown molecules by NMR, MS, UV and IR analysis

Analysis of the metabolic profile of fruits and plants extracts by ¹H NMR

Textbook Information

CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI (Modulo 1)
1. Teaching material

2..R. Cozzi, P. Protti, T. Ruaro, ANALISI CHIMICA STRUMENTALE, Zanichelli, 2001
3. J. H. Gross, MASS SPECTROMETRY- A Textbook, Springer 2011
4. F.W. McLafferty, INTERPRETATION OF MASS SPECTRA University Science Books1980
5. K.A. Rubinson, J.F. Rubinson, Chimica analitica strumentale, 1a ed.,Bologna, Zanichelli, luglio 2002. ISBN 88-08-
08959-2
STRUCTURAL CHARACTERIZATION OF ORGANIC MOLECULES
1. R.M. Silverstein, F.X. Webster e D.J. Kiemle, Spectrometric Identification of Organic Compounds,” John Wiley & Sons
2. A. Randazzo, “Guida pratica alla interpretazione degli spettri NMR” Loghìa Publishing
3. L.D. Field, S. Sternhell, J.R. Kalman, “Organic Structures From Spectra” IV Edizione, John Wiley and Sons (Chichester New York Brisbane Toronto Singapore)
4. M. Hesse, H. Meier, B. Zeeh, Spectroscopic Methods in Organic Chemistry, Published by Thieme
5. Educational material made available on www.studium.unict.it

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