Separation Methods


The overall objective is to provide the fundamental scientific background and practical training in Separation Methods included in the program, to know how to do the experimental work and solve the proposed problems.

General characterization





Responsible teacher

Jorge Manuel Pinto Lampreia Pereira, Maria Cristina Oliveira Costa


Weekly - 4

Total - 60

Teaching language



Available soon


Analytical Chemistry
Robert V. Dilts D. Van Nostrand, ISBN 0-442-22158-4

Physical Biochemistry: Principles and applications
1st Edition, David Sheehan, Wiley-VCH Verlag GmbH ISBN 0-471-98663-1

Biochemical Methods
A. Pingould, C. Urbanke, J. Hogget, A. Jeltsch, 1st Edition, Wiley-VCH Verlag GmbH ISBN 3-527-30299-9

Fundamentals of Analytical Chemistry (7th ed)
D. A. Skoog, D. M. West and F. J. Holler, Saunders College Publishing

Principles of Instrumental Analysis
Skoog, West, Holler, Nieman, Thomson Learning, ISBN – 0-03-002078-6

Quantitative Chemical Analysis
Daniel C. Harris, Freeman, New York, 5th Edition (1999) ISBN – 0-7167-2881-8

Practical High Performance Liquid Chromatography
Veronika Meyer, Wiley, ISBN0-471-941328.

Teaching method

Lectures  and problem-solving sessions, supported by data show projection of figures/plots/text included either in the documentation supplied directly by the teacher or in the recommended bibliography.

Evaluation method

Generically, the Separation Methods curricular unit (CU) happens” in Moodle. Thus, the documentation, instructions, planning, Calendar and delivery of Questionnaires is where it should be seen, done and corrected.

1. “Frequency”

Frequency for 1st enrollment students is obtained by:

i) Performing all Practical Works and delivery of all questionnaires.

Practical Assignments, as well as questionnaires, are carried out in groups of 2 or 3 students. The delivery periods will be defined in Moodle.

Students who are not aware of the protocols will be prevented from carrying out the work and, therefore, will not obtain attendance, “failing” the CU from the outset, in accordance with the FCT General Assessment Rules (Article 5);

ii) Attendance to at least 2 Theoretical-Practical classes

There are 4 Theoretical-Practical classes. 1st enrollment students or without previous attendance can only miss two of these classes. This rule does not apply to students with official Student Worker status.

iii) Students who “failed” the “Frequency”

In this case, in the next registration - for the next year - conditions i) and ii) will have to be fulfilled both or the one that is missing.

Also they cannot be evaluated this year in any way.

2. Theoretical Assessment

All students with frequency, and only those, will have two opportunities to carry out the theoretical component in one of the following ways:

i) By Continuous Assessment

Three quizzes, scheduled at CLIP, lasting approximately 1:30 hours each, with equal weight in the final grade of the theoretical component, in which the respective average must be equal to or greater than 9.5; there is no minimum score on any of the tests.

ii) or Final Exam

In the Final Exams Season there will be a Theoretical Exam with all the material and duration of 2 hours. The grade for this theoretical component will also have to be equal to or greater than 9.5, on a scale of 0-20. There will not be, in the exam, the possibility of doing it in parts.

Note.: About the Final Season

All students, with Frequency, who did not complete the CU in the Continuous Assessment Tests can access this Season.

3. Practical Assessment

All practical groups must take a laptop, or tablet, etc. to class, which will allow them to present graphs and/or the results obtained at the end of each shift. The practical component of the discipline, this academic year, will be carried out as follows:

1. Each Questionnaire delivered will be evaluated and will have one of 4 grades: 0, 2, 4, 5.

2. Being 5 Questionnaires, the practical component of the CU will have a normalized final grade that will vary between 0 and 2.5 values.

3. These scores for each Questionnaire and the respective final score for the practical component will be the same for each member of the Group.

4. There will be no other way or opportunity to earn a practice grade this year.

5. This grade will be counted for all editions of the CU made this academic year, with the exception of the ad-hoc Exam or Improvements referring to last year.

4. Grade Improvement

1. Registration for Grade Improvement is mandatory.

2. The Improvement will be made in the Final Exam Season of the respective academic year, or in the following year.

You can only apply for Improvement once in each CU.

About the Practical Assessment method

It is intended that students read and understand the protocol and answer the final questionnaire, as a group.

To answer and turn in the questionnaires:

1. use the Word file associated with the Practical Work Questionnaire link to answer the questions in the questionnaires, convert it to pdf and submit it, as well as an excel where you show the calculations and the values and results you used for your graphs, where applicable;

2. Files must be submitted within the deadlines indicated in Moodle. Failure to comply with this rule implies not having the maximum score in this questionnaire;

3. ONE submission is enough for each group; that is, just one element of each group to submit;

Please identify your files with your group, names, numbers and course.

4. Final Grade of the UC

Theoretical part normalized to 17.5 points + (0 to 2.5) of the practical part

Attention: It is possible to fail the CU even with a positive grade in the theoretical component, not yet standardized, if the “addition” of the practical component grade produces a final grade lower than 9.5.

Rounding of the Final Grade is done only once, in the last arithmetic operation.

5. General Provisions

1. All students must have a recognizable photograph in the CLIP.

2. Only your ID Card, two pens and a non-graphic calculator can be taken to the Assessments;

3. Cell phones or “smartphones”, “smartwatches”, nor draft sheets are not allowed;

4. In the Appeal Exam, the theoretical component is compulsorily done in a single test with the entirety of the material;

5. The final grade is rounded off only once, in the last arithmetic operation to calculate the final grade;

6. In accordance with the general rules of the FCT, registration for Grade Improvement is mandatory.

7. They must bear in mind the article on Plagiarism and Fraud of the Knowledge Assessment Regulation and the UNL Code of Ethics.

Subject matter


  1. Distribution equilibrium. Distribution (partition) coefficient (KD). Distribution ratio (D). Relation between D e KD in several cases.
  2. Solvent extraction: Batch extraction, Continuous extraction and Countercurrent extraction. Extraction of metal chelates. Extraction constant KE and pH1/2
  3. Chromatography. Principles of chromatography. Elution. Shape of the peaks. Partition coeficient. Retention time and peak widht. Factor capacity factor and selectivity. Velocity theory. Resolution. Number of theoretical plates.Partition chromatography. Liquid-liquid chromatography. Normal and reverse phase chromatography.Planar chromatography: Paper chromatography and Thin layer chromatography.Ion exchange chromatography.Size exclusion chromatography.Affinity chromatography.Gas chromatography.HPLC.
  4. Mass Sepctrometry.
  5. Electrophoresis.Principles of electrophoresis. Non-denaturing and denaturing electrophoresis. Isoelectric focusing.Two-dimensional electrophoresis. Proteomics. Electroblotting procedures. Capillary electrophoresis.
  6. Centrifugation and ultra-centrifugation.
  7. Hydrodynamics methods: sedimentation, ultrafiltration and dialysis.
  8. Purification of a membrane protein.


  1. Determination of caffeine in beverages- HPLC and GC
  2. Electrophoresis SDS PAGE


Programs where the course is taught: