Instrumental Methods of Analysis

Code:

MI232112

Acronym:

MIA

Keywords
Classification	Keyword
OFICIAL	Physical Sciences

Instance: 2025/2026 - 1S

Active?	Yes
Responsible unit:	Applied Chemistry Laboratory
Course/CS Responsible:	MSc in Pharmaceutical Sciences

Cycles of Study/Courses

Acronym	No. of Students	Study Plan	Curricular Years	Credits UCN	Credits ECTS	Contact hours	Total Time
MICF	200	MICF - Transition Study Plan 2023/24 - 2024/25 - 2025/26	2	-	6	65	162

Teaching language

Portuguese and english
Obs.: apontamentos teóricos e lab. com tradução em inglês

Objectives

The natural, semi-synthetic and synthetic chemical / biological substances, as well as the instrumental devices that have potential benefit in protecting and promoting human health, constitute the axiom of specialization and pharmaceutical activity. The pharmaceutic profession is hence committed with the discover, evaluation of the effects, production, proposal and dissemination, dispensing and regulation of these substances use in a safe and effective manner. In accordance to such profile, this curricular unit intends to provide the trainee with basic knowledge and skills in common laboratory instrumentation use when the quality of chemical / biological substances and the respective effects is intended, whatever its context of the analysis: research, scale production with quality criteria, simple laboratory follow-up of its application in clinical trials, or in the service of public health. More broadly, this course aims to prepare pharmaceutical professionals with critical look over experimental and scientific information.

To achieve this end, two complementary objectives are established:

- Train the student for rational use of instrumental techniques and methods based on the interaction of light with matter, aiming biological and chemical characterization and quantification.

- Train the student in the use of statistical / computer tools which, according to the ICH Q2 and EURACHEM / CITAC guidelines, certify and measure the quality of the instrumental methodology and, therefore, the significance of the information made possible by it.

Learning outcomes and competences

The theoretical and theoretical-practical syllabus, together with laboratory work provide the trainee with:

- ability to decide on the most appropriate instrumental analytical techniques and methods, according to the particular nature, quantity and complexity of the sample under analysis;

- familiarization with laboratory analytical instrumentation use;

- familiarization with sample processing procedures in different contexts of drug control, from the identification tests to assay and up to contamination by elementary impurities (ICH Q3D);

- ability to develop and use spreadsheets for recurring calculation on laboratory data;

- ability to apply spreadsheets in the statistical measurement of uncertainty, namely the accuracy and precision of laboratory information.

Working method

Presencial

Pre-requirements (prior knowledge) and co-requirements (common knowledge)

Prerequesites:

Calculus, linear algebra, statistics – C. Unit of Mathematics and Biostatistics

Optics and electricity - C. Unit of Applied Physics

Nomenclature of organic compounds and functional identification by vibrational spectroscopy - C. Unit of de Organic Chemistry

Chemical, kinetic and thermodynamic equilibria - C. Units of Analytical Chemistry and Physical Chemistry

 

Co-requirements:

Enzymatic catalysis, proteins and nucleic acids - UC Biochemistry

Program

Syllabus:

Laboratorial - comprehends eight experiments:

Experiment 1: Colors and spectra. The trainee is committed with obtaining molecular absorption spectra in UV-vis, and with the association of a color, the spectral region determining it and with the observed complementary color. For this purpose, the solvatochromic effect on the Dimroth-Reichardt dye is used to determine the polarity of several solvents in the energy ET scale (30) and on a normalized scale.

Experiment 2: Conformity of the acetaminophen dose of a commercial drug formulation by UV-vis spectrophotometry. This work aims to familiarize the trainee in methodological validation (linear concentration range, sensitivity, detection limit and quantification, etc.) and use of colorimetric instrumental procedures in the quality control of medicines.

Experiment 3: Determination of stoichiometry and the binding constant of bilirubin to serum albumin by fluorimetry using the Job´s method. This work aims to familiarize the trainee in the use of qualitative instrumental procedures based on molecular photoluminescence, namely obtaining fundamental chemical information from the interaction of a chemical substance with its target.

Experiment 4: Determination of sodium and potassium concentration in bottled water by flame photometry; recovery assays. With this work it is intended to provide the contact with elementary analysis techniques and with the most common laboratory methodology in assessing the accuracy of results obtained by instrumental means.

Experiment 5: Use of the internal standard method in the determination of quinine in a formulation by HPLC with fluorimetric detection. With this work it is intended to provide the contact with one of the most common instrumental methodologies in pharmaceutical analysis and with individual use of chromatographic equipment.

Experiment 6: Conformity of the calcium dose of a commercial vitamin formulation by atomic absorption spectrophotometry. Comparison of results enabled by direct analysis after calibration with those provided by the method of successive standard additions. This work intends to familiarize the trainee with the use of the most common elementary analysis analytical technique in the evaluation of elementary impurities and with the importance played by the instrumental methodology in obtaining more accurate results in the face of matrix physical-chemical interferences.

Experiment 7: Potentiometric determination of fluoride in mothwash samples. This work intends to familiarize the trainee with the handling of electrochemical cells, reference electrodes and potentiometric sensors, its maintenance, relationship between ion activity vs- concentration and practical procedures for response validation. 

Experiment 8: Potentiometric titration of acetylsalicylic acid in granulat medicines. Conventional titration vs automatic titrators. This work intends to familiarize the trainee with instrumental titrimetic procedures, and the derivative tretament of potentiometric titration curves. The trainee is equally introduced to the combined glass electrode and its main features.

Theoretical-practical:

SUBJECT 1 (4 h): Calculations associated with procedures with instrumental measurement: resolution of type examples involving dissolution, dilution, preconcentration, extraction, doping.

SUBJECT  2 (2 h): Use of Excel spreadsheets for recurring calculations: general instructions for formatting and protection, renaming cells and cell ranges, using built-in and conditional functions, graphical formatting.

SUBJECT 3 (4 h): Use of Excel spreadsheets in instrumental analysis procedures. Identification of anomalous results by the Grubbs test, one-way ANOVA analysis. Errors associated with graphical interpolation, extrapolation and interception. Linear regression using the least squares method; interactive visual regression. Non-linear regression using optimization tools (Solver).

SUBJECT 4 (2 h): Use of Excel spreadsheet in methodological validation procedures. Repeatability, intermediate precision and reproducibility - population and sample standard deviation; normal distribution; z tables and Student's t parameters. Accuracy tests using reference materials and certified reference materials - hypothesis testing. Comparison of paired results with independent technique - correlation graphs; constant and proportional systematic deviations detection. Recovery tests: limitations. Accuracy and precision as components of uncertainty. Control charts.

SUBJECT 5 (1 h): Errors, failures, lapses in laboratory dynamics; classification and mitigation criteria; risk management. Automation of the flow of laboratory information.

Theoretical:

SUBJECT 1 (2 hours) - Introduction to Instrumental Methods of Analysis and its context in pharmaceutical activity. Chemical, physical and biological behaviour of atoms and molecules: examples with physiological relevance. The physical behaviour as the most sensitive way to inquiry atoms and molecules. Definition of instrument vs apparatus. Ways of obtaining chemical / biological information from physical responses: a) follow-up/screening. Medical devices as an example: lateral flow tests in pregnancy (aptamers vs antibodies as laboratory reagents, nanomaterials), glucometer in diabetes, microfluidic platforms in the identification of pathogenic strains of enterobacteria. b) samples transport and lab measurements: technique, method and instrumental analysis procedure. Examples: elementary analysis by ICP-MS in the forensic scope, leukocyte formula by flow cytometry. General advantages of instrumental analysis. Main instrumental techniques groups: spectral analytical, electroanalytical, separation, sample preparation and automated analysis;

SUBJECT 2 (2 hours) - General aspects of spectral techniques. Duality of light as an electromagnetic wave and a corpuscular vehicle of energy and moment: reflection, refraction, diffraction and photoelectric effect. Wave functions and the general wave equation. Principle of superposition; constructive and destructive interference. Iridiscence. Diffraction gratings, Michelson interferometer - Fourier transform. The electromagnetic spectrum; characterization quantities in space-time and energetics: multiples and submultiples. 

SUBJECT 3 (3 hours) - General aspects of the interaction of light with molecules and atoms. Spectral evidence of the discrete, discontinuous and energy quantized nature of electronic transitions in atoms. Electron, vibration and rotation transitions in molecules. Why atoms have color - energy patterns, spectroscopic notation and electronic selection rules. Why molecules have color - energy patterns and selection rules for electronic transition, vibration and rotation. Interpretation of typical atomic absorption and emission spectra and molecular absorption spectra in the UV-vis regions;

SUBJECT 4 (2 hours) - Molecular absorption spectrophotometry in the UV-vis regions. What is? What is it for? When it constitutes the first analytical option? Demonstration of the direct relationship between luminous energy absorption, concentration and the optical path (Bouguer-Beer-Lambert law); validity premises. Systematic instrumental and solution deviations to Beer-Lambert´s law; main causes for random errors. Relationship between absorbance-opacity and transmittance-transparency, reflected radiation. Instrumentation: constituent blocks; advantages and disadvantages of single-beam equipment vs. double beam; colorimeters, spectrophotometers, plate readers and densitometers. Common qualitative methods for determining drug pKas, stoichiometry of reactions, enzymatic activities; spectral derivatives. Common quantitative methods in the absence of sample interference, in the presence of matrix physical-chemical interference and in the presence of spectral interference. When a procedure fails; (examples); validation of procedures according to the EMA – ICH guidelines; validation parameters;

SUBJECT 5 (3 hours) - Atomic absorption spectrophotometry. What is? What is it for? When it constitutes the first analytical option? Importance of elemental determination in life sciences; why sodium is found in greater concentration in the extracellular environment; kosmotropic vs chaotropic ion. Elementary impurities in medicines - ICH Q3D standard. Need for sample atomization. Instrumentation based on discrete or continuous radiation sources of continuous radiation: constituent blocks; limits of detection, sensitivity its dependence on the atomization device: pneumatic, electrothermal and by hydride generation. Physical-chemical interferences; scattering and unspecific absorption (background correction systems). Instrument setup for analysis. Procedures comparison regarding the analysis of mercury, lead, cadmium and arsenic (class 1 risk impurities) at different concentration levels;

SUBJECT 6 (4 hours) - Techniques based on molecular luminescence. Molecular photoluminescence; radiant vs. non-radiant quenching, Stokes' law, late radiant emission phenomena: type E, P, recombinant and phosphorescence. Luminescence, half-life times for luminescence and their relationships with the molecular structure. Luminescence as a product of chemical and enzymatic reactions: direct vs indirect chemiluminescence. Fluorimetry and phosphorimetry. What are? What are worth for? When they constitute the first analytical option? Relationship between radiant absorption and emission - systematic instrumental and solution deviations. Instrumentation; constituent blocks. Spectrofluorimeters, luminometers and plate readers; comparative advantages and disadvantages. Common qualitative and quantitative methods used in pharmaceutical control; absorption, emission and synchronized spectra, indirect determination by quenching effects. Multiplexed analysis employing fluorescence. Techniques based on molecular radiant emission applied to biology. Fluorescent antibody and nucleic acid probes. Epifluorescence microscopy; FISH technique; Quantitative real time - PCR. Analytical and preparative flow cytometry. What is? What is it for? Instrumentation: constituent blocks. Interpretation of typical "dot plots".

SUBJECT 7 (1 hours) - Techniques based on the scattering of incident radiation: turbidimetry, nephelometry and Raman spectroscopy. What are? What are worth for? When they constitute the first analytical option? Why are the eyes sometimes blue; interaction of light with colloidal dispersions: Tyndall effect and Rayleigh scattering; dependence on polarizability and wavelength. Inelastic photon-particle collisions and anti-Stokes radiation: molecular thermometer. Absorption spectra in the mid IR region vs. Raman spectra. Instrumentation: constituent blocks; relationship with other generic equipment used in fluorimetry and UV-vis spectrophotometry. Analytical methods and applications in clinical biochemistry, microbiology and pharmaceutical analysis.

SUBJECT 8 (4 hours) -Introduction to electroanalysis, and its context in pharmaceutical activity; activity vs. concentration, ionic strength, importance of chemical speciation. Reversible and irreversible electrochemical cells; representation rules. Calculation of cell potentials from electrode potentials; relationship between potential and Gibbs free energy; Nernst equation. Electrode polarization; faradic and non-faradic currents in electrochemical cells. Electrical quantities applied to charge transport in electrolyte solutions. Equivalent conductance (or conductivity), ionic conductance (or conductivity) equivalent to infinite dilution. Static and dynamic electroanalytical techniques. Potentiometry as a static technique; modified Nernst equation; potentiometric selectivity coefficient. Reference electrodes; metallic and selective membrane indicator electrodes. Liquid junction potential. The glass electrode. Direct potentiometric determinations and potentiometric titrations. Dynamic electroanalytical techniques. Hydrodynamic voltammetry, differential impulse voltammetry, square wave voltammetry, cyclic voltammetry, anodic redissolution voltammetry, cathodic redissolution voltammetry and polarographic techniques. Application examples.

TOPIC 9 (4 hours) – Chromatography: introduction to analytical separation; chromatographic separations; main basic mechanisms of chromatographic separation processes; classification of chromatographic methods; HPLC and GS chromatographs; HPLC and GS chromatograms. General theoretical aspects: migration of mixture components; the widening of the bands (“peaks”) / column efficiency; variables that affect efficiency; obtaining the main chromatographic parameters from the chromatogram (resolution, number of theoretical plates, plate height, …). Comparison of HPLC, UHPLC and UPLC chromatography. The importance of chromatographic techniques in modern pharmaceutical and clinical laboratories and the food industry.

Mandatory literature

Skoog Douglas A.; Principles of instrumental analysis. ISBN: 0-03-001229-5
Rouessac Francis; Chemical analysis: modern instrumental methods and techniques. ISBN: 0-471-97261-4
Harris Daniel C.; Quantitative chemical analysis. ISBN: 0-7167-4464-3

Complementary Bibliography

James Miller e Jane C Miller; Statistics and Chemometrics for Analytical Chemistry, Pearson Education Limited, 2005. ISBN: 978-0-13-129192-8
E. Joseph Billo; Excel for Chemists:A comprehensive guide, 2001. ISBN: 978-0-471-39462-4

Teaching methods and learning activities

The contents of the discipline are given in 13 weeks. Fifety minutes theoretical classes are presented in DataShow, starting with the lesson summary and objectives. Practical lessons are concerned with discussion of procedures performance evaluation and statistical evaluation of analytical information mainly implemented in spreadsheets using personal laptops. Seven laboartorial experiments ilustrating the techniques and methods studied are provided. A report of each work with critical evaluation is required.

Evaluation Type

Distributed evaluation with final exam

Assessment Components

designation	Weight (%)
Exame	75,00
Trabalho laboratorial	25,00
Total:	100,00

Amount of time allocated to each course unit

designation	Time (hours)
Elaboração de relatório/dissertação/tese	50,00
Estudo autónomo	66,00
Frequência das aulas	46,00
Total:	162,00

Eligibility for exams

Studant attendance to the practical and laboratorial classes is mandatory. Those students whose attendance is lower than 3/4 of the classes effectively taught are considered without attendance.

Attendance to theoretical classes is not compulsory

Calculation formula of final grade

The 0 to 20 final classification corresponds to the final exam mark (75%) and to distribution assessment (25%).

Examinations or Special Assignments

not applied

Special assessment (TE, DA, ...)

Previous laboratorial evaluation is mandatory for students considered on the special regime (working students, etc)

Classification improvement

in accordance with "Normas de Avaliação"

Observations

Previous knowledge of analytical chemistry, organic chemistry, physics and statistics basic concepts are required for full comprehension of the different discipline issues. Classes are given in Portuguese language or if necessary in English.
The syllabus content is established for presencial teaching regime. It might be occasionally changed if extemporaneous measures to protect public health will determine closing facilities, holidays, etc.