METHODS OF MASS SPECTROMETRY AND SEPARATION OF ORGANIC MOLECULES

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.

Graduated in Experimental and Applied Biology- Curriculum  Cellular and Molecular Biology, with the teaching of METHODS OF MASS SPECTROMETRY  AND SEPARATION OF ORGANIC COMPOUNDS expands and deepens the basic knowledge in the field of integrative sector, acquired with the first degree. Moreover, hi/she matures an advanced scientific preparation on the aspects  of the chemical / biochemical methodologies of investigation, with particular focus on bioorganic compounds e compounds of biological interest.

In particular, the specific training objectives of this module are: to understand the principles on which the isolation of organic compounds from synthetic and natural matrices, including biological ones, is based, the methods and techniques used for this purpose, the principles underlying the characterization of organic compounds by mass spectrometry, the techniques used to obtain and interpret mass spectra and the principles of chromatography-mass spectrometry coupling.

 More specifically, with reference to the Dublin Descriptors, this teaching contributes to the acquisition of 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 by mass spectrometry is based. 

Ability to apply knowledge 

• ability to identify the most appropriate technique for the isolation and purification of organic compounds; 
• ability to identify an organic compound by studying its mass spectrum. 

Autonomy of judgment 

• critical reasoning ability;
• 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 orally, with proper language and terminological rigor, the result of a chromatographic procedure, mass spectrometry or chromatography/mass spectrometry analysis.

Learning skills

The students will have to develop learning skills that will enable them to continue studying in a self-directed or autonomous way.

These skills, as far as possible, will be stimulated by the teacher by proposing in-depth studies and carrying out exercises in the classroom during the course.

Information for students with disabilities and/or SLD

To guarantee equal opportunities and in compliance with the laws in force, interested students can request a personal interview in order to plan any compensatory measures, based on the educational objectives and specific needs.

In this case, it is advisable to contact the CInAP (Centre for Active and Participated Integration - Services for Disabilities and/or SLD) professor of the Department where the Degree Course is included.

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. Principle; 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. Operating principles. Construction and operation

Magnetic sector analyzer. Resolution. Double-focusing (magnetic- electrostatic) analyzer. High resolution mass spectrometry. Nominal mass, exact mass, monoisotopic 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 reactions of organic ions and interpretation of EI mass spectra. Quasi- equilibrium theory. Classification of the fragmentation reactions. Cleavage of sigma-bonds and rearrangements. Evenelectron 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  Reversed Phase-High Performance Liquid chromatography/MS (HP-HPLC/MS).

The exam of  "Methods of mass spectrometry and of separation of organic molecules" consists of an oral test carried out through an interview between the student and the examining commission aimed at ascertaining the degree of learning and understanding of the topics contained in the course program. In particular, the relevance of the answers to the questions asked, the quality of the contents, the ability to connect with other topics covered by the program, the ability to report examples, the technical language properties and the overall expressive ability of the student will be assessed.

The following parameters will be taken into account in assigning the final grade:

Grade 29-30 cum laude: the student has an in-depth knowledge of all the topics covered in class, is able to promptly and correctly integrate and critically analyze the situations presented, independently solving even highly complex problems; has excellent communication skills and property of language.

Grade 26-28: the student has a good knowledge of all the topics covered in class, is able to integrate and analyze the situations presented in a critical and linear way, is able to solve complex problems quite independently and exposes the topics clearly using appropriate language;

Grade 22-25: the student has a fair knowledge of the basic concepts of chromatography and mass spectrometry, even if limited to the main topics; is able to integrate and analyze the situations presented in a critical but not always linear way and exposes the topics quite clearly with a fair command of language;

Grade 18-21: the student has minimal knowledge of all the topics covered in class, has a modest ability to integrate and critically analyze the situations presented and presents the topics in a sufficiently clear way although the language skills are poorly developed;

Exam failed: the student does not possess the minimum required knowledge of the main contents of the course. The ability to use the specific language is very poor or non-existent and he/she is not able to independently apply the knowledge acquired.

Verification of learning can also be carried out on line, should the conditions require it.

The exam questions focus on all the topics covered in the lessons. Examples of questions are:

ESI source; magnetic and electrostatic sector analyzers; resolution in mass spectrometry; molecular ion and isotopic peaks; fragmentation of organic compounds; MALDI source; time-of-flight analyzers; quadrupole and ion trap analyzers; tandem mass spectrometry;

Van Deemter equation; resolution and selectivity; efficiency of chromatographic columns; high performance liquid chromatography; HPLC detectors; gas chromatogaphy; gas chromatography columns; gas chromatography detectors; molecular exclusion chromatography; ion exchange chromatography; affinity chromatography.

HPLC/MS and GC/MS interfacing.