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

In order to get frequency the student have to attend all 5 laboratory sessions (with previous preparation) and to submit the proposed questionnaire at the end of each practical session.

The evaluation has 2 components: theoretical (T) and laboratorial (L). The final classification is calculated according: 0.25 x L + 0.75 x T.

To get approval in the theoretical part it is necessary to obtain positive (higher or equal to 10) classification. It can be done by 2 mid-term tests or by final exam.

The laboratorial classification results from a test at the end of the term or by final exam. It is necessary to obtain classification equal or higher than 10.

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. Solvent extraction
  2. Thin layer chromatography and solvent extraction
  3. Determination of caffeine in beverages- HPLC and GC
  4. HPLC - Gel filtration
  5. Protein Purification (
  6. Electrophoresis SDS PAGE