ANALYSIS OF TOXICOLOGICAL CHEMISTRY I

• Provide the student with the theoretical and practical knowledge of the toxicological chemical analysis and its applications
• Present the main sampling, handling and sample preparation methods for chemical analysis
• Acquired skills: theoretical and practical knowledge of sample treatment for chemical analysis; theoretical and practical knowledge of the main analytical techniques.

• Frontal lessons 28 hours
• Laboratory practice 36 hours

First module

Principles of Analysis of Toxicological Chemistry: general methodology. The analytical method. Definition of poison. General and specific analysis.

Sample preparation general concepts and main techniques: evaporation; appropriate choice of container; absorption of gas from the atmosphere; chemical alterations; conservation of unstable solids; sample concentration; sample purification.

General principles of the extraction process: vapor pressure, solubility and partition coefficient; electrolytic dissociation equilibrium, dissociation constant and ionization state, acid-base equilibrium; use of internal standards; sample pretreatment, protein homogenization and elimination, enzymatic and chemical digestion; Preparation of buffers at the desired pH value and their use in the extraction process, application of the Henderson-Hasselbalch equation; solutions, use and preparation; solvent types and solvent choice.

Extraction of semi-volatile substances from liquids:

• Liquid-liquid Extraction: extraction yield. Equilibrium distribution, Electrolytic dissociation equilibrium, phase ratio, number of extraction. Salting out. Type of solvent and selection. Extraction scheme of a basic compound. Extraction scheme of an acid and neutral compound. Separation of Acidic, Basic and Neutral Compounds. Aspirin extraction procedure. Caffeine extraction procedure.
• Solid-phase extraction (SPE): general considerations. Application fields. Types of interactions with the solid phase. Types of solid phases. Type of solvents and selection. Chemical mechanism of separation of acid/basic/neutral substances. Sample treatment. Stationary phase treatment. Sample application. Washing phases. Elimination and collection of contaminants. Drug recovery. SPE extraction of substances of toxicological interest.
• Solid phase microextraction (SPME): theory and principles of the SPME technique. Types of solid phase. Methodology and optimization. Applications.

Extraction of semivolatile substances from solids:

• Soxhlet extraction: general principles, methodology and optimization of the extraction process. Applications.
• Microwave assisted extraction (MAE): general principles, methodology and optimization of the extraction process. Applications.
• Extraction with supercritical fluids (SFE): general principles, methodology and optimization of the extraction process. Applications.

Extraction of volatile substances from solids and liquids:

• Headspace analysis: general principles. Equilibrium distribution, partition coefficient, phase ratio, salting out.
• Microdiffusion extraction: general principles and applications.

Sample preparation for metal analysis: main methods and applications.

Color tests: introduction and interpretation of color tests, color test types and methods. Color tests: ferric chloride, Marquis reagent, Mandelin reagent.

Second module

UV – Visible spectrophotometry: general principles, the electromagnetic radiation (amplitude, period, frequency, wavelength). Chromophore. Quantitative analysis, transmittance, absorbance, Beer-Lambert law, auxochrome. Solvent effect. Instrumentation: Generalities on spectrophotometers, the source, the monochromator, the cell, the cuvette, detectors. Spectrophotometers type: single and double beam spectrophotometers and diode array.

Chromatography methods: general principles. Chromatogram. Migration rate, retention and dead time, retention and dead volume. Column efficiency. The theoretical plates. The Van Deemter equation, turbulent diffusion, longitudinal molecular diffusion, mass transfer. Efficiency optimization. Selectivity. Resolution. Separation mechanism: adsorption, partition, ion exchange, exclusion

• Thin-Layer Chromatography: instrumentation and execution. Retention factor. TLC plate and stationary phases. Mobile phases and eluotropic series. TLC plate visualization. Derivatization, Dragendorff reagent. Selectivity, capacity, efficiency and resolution.
• High performance liquid chromatography (HPLC): general characteristics and advantages. Equivalent height to a theoretical plate. Direct and reverse phase chromatography. The HPLC column. The mobile phase and solvents in HPLC. Mobile phase modifiers, buffers, acidifiers, ion pair reagents. Solvents filtration and degassing. Isocratic and gradient elution. HPLC instrumentation: pumps, manual injector. Detectors. UV-Vis detector, the flow cell, diode array detector.

Mass spectrometer (MS): general principles. The mass spectrum. Ion mass and Dalton. Isotopic peaks and intensity. Exact and nominal mass. Resolution and resolving power. Instrumentation. Liquid chromatography-Mass spectrometry (LCMS): features and operative conditions.

Laboratory Experiences

• Preparation of solutions of acids and bases (HCl, NaOH, H₃PO₄, CH₃COOH, H₂SO₄).
• Preparation of buffers (phosphate buffers at different pH values).
• Color tests (Mandelin’s test, Sulfuric acid assay, Ferric chloride assay).
• Thin layer chromatography (TLC).
• Extraction Aspirin from urine (liquid-liquid extraction, color test and TLC).
• Imipramine extraction from blood (liquid-liquid extraction, color test and TLC).
• Diphenhydramine extraction from blood (solid phase extraction, color test and TLC).

• Somenath Mitra, Sample Preparation Techniques in Analytical Chemistry, John Wiley & Sons, Inc. (https://onlinelibrary.wiley.com/doi/pdf/10.1002/0471457817)
• Renato Cozzi, Pierpaolo Protti, Tarcisio Ruaro, Elementi di analisi chimica strumentale, seconda edizione, Zanichelli 2013
• Sabrina Moret, Giorgia Purcaro, Lanfranco S Conte, Il campione per l’analisi chimica, prima edizione, Springer 2014