CROMATOGRAFIA E SPETTROMETRIA DI MASSA DI COMPOSTI ORGANICI

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.

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Lectures with classroom exercises.

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).

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 88-08-08959-21.