PFT503 - Separation Chromatography / Spectrophotometry

Outline info
Last revision date 2018-07-20 11:49:13.302
Last review date 2018-07-20 11:49:22.934

Subject Title
Separation Chromatography / Spectrophotometry

Subject Description
This subject provides a theoretical and practical study of separation chromatography used in the testing and development of pharmaceutical products as well as evaluation of chromatographic results by Spectrophotometry. The subject concludes with the study of accelerated testing of pharmaceutical shelf-life stability. (Lectures and Labs)

Credit Status
One credit towards the Pharmaceutical Instrumentation Analyst (PIA) Certificate Program

Learning Outcomes
Upon successful completion of this subject the student will be able to:

1. Demonstrate proficiency in the use of different chromatographic techniques (paper chromatography, thin layer column chromatography, affinity chromatography and ion-exchange chromatography)

2. Comprehend principles of chromatographic separation.

3. Perform analysis of pharmaceuticals using paper chromatography, TLC and open column chromatography.

4. Complete identification of compounds by Rf - factor, visualization and detection procedures.

5. Perform two dimensional and preparative TLC.

6. Apply TLC to assess stability of pharmaceutical compounds.

7. Show competency in accelerated stability testing of pharmaceutical products.

8. Implement trouble shooting TLC techniques

9. Demonstrate proficiency in the use of spectrophotometry for evaluation of chromatographic analysis.

Cheating and Plagiarism
Each student should be aware of the College's policy regarding Cheating and Plagiarism. Seneca's Academic Policy will be strictly enforced.

To support academic honesty at Seneca College, all work submitted by students may be reviewed for authenticity and originality, utilizing software tools and third party services. Please visit the Academic Honesty site on for further information regarding cheating and plagiarism policies and procedures.

All students and employees have the right to study and work in an environment that is free from discrimination and/or harassment. Language or activities that defeat this objective violate the College Policy on Discrimination/Harassment and shall not be tolerated. Information and assistance are available from the Student Conduct Office at

Accommodation for Students with Disabilities
The College will provide reasonable accommodation to students with disabilities in order to promote academic success. If you require accommodation, contact the Counselling and Disabilities Services Office at ext. 22900 to initiate the process for documenting, assessing and implementing your individual accommodation needs.


Topic Outline

Course Content
Mode of evaluation
Laboratory Safety Rules and WHMIS overview, GLP
Chromatographic Principles of Separations
Different Types Chromatographic Methods
Definitions of Thin Layer Chromatography (TLC)
Sample Application and Plate Development
Laboratory Work
Analysis of the Components of Analgesic Drugs (Caffeine, Acetaminophen, Ibuprofen, and Acetyl Salicylic Acid) by Thin Layer Chromatography.

Demonstration of TLC Equipment
Different Type of Plates
Procedures for Activation of the Plates
Development of the Plates and Visualization
Laboratory Work
Determination of Free Salicylic Acid in Aspiring Tablets by TCL

Different Types of Chromatographic Materials Used as Solid Support
Modification of the Solid Support, Selection of Coatings
Selection of Mobile Phase
Detection or Visualization Procedure, Calculating Rf Factor
Use of Spectroscopy for Quantification, Standards Unifying TLC
Two-dimensional Electrophoresis
Application of TLC, Method Accuracy
Laboratory Work
Identification of vitamin A by TLC

Open Column Chromatography
Hydrophobic Interaction Chromatography (HIC); Affinity chromatography (Protein A as a Ligand to Binf IgG) Optimization of Chromatographic Separation
Laboratory Work
Column Chromatography - Separation of Pigments

Spectrophotometry - Basic Concepts
Quantitative Determination of the Concentration of the Sample in a Solvent by Spectroscopic Method Application in Pharmaceutical Industry and Biotechnology
Laboratory Work
Determination of cortisone tablets potency by TLC and Calorimetric Methods

Stability of Pharmaceuticals: Relevant Terms and Definitions
Physical Factors Affecting Stability
Chemical Factors Effecting Stability
Real Tim Accelerated Stability Testing
Calculation of the Shelf Life Time by Accelerated Stability
Laboratory Work

Final Exam

Mode of Instruction
Lecture sessions combined with practical demonstrations and experiments

Prescribed Texts
None - Handouts

Reference Material

  • Laboratory Techniques in Biochemistry and Molecular Biology, Editor: T.S. Work and E. Work; Elseview Science ISBN: 072-044-2214
  • An Introduction to Gel Chromatography, by L. Fishe, 1969, North Holland Publishing ASBN: B000Q9TDC6
  • Quantitative Pharmaceutical Chemistry, by, Adelbert M. Knevel, Frank E. Digangi and Stephen R. Bryn, 1982, Waveland Press ISBN: 088-133-0019 

Promotion Policy

Grading Policy
A+ 90%  to  100%
A 80%  to  89%
B+ 75%  to  79%
B 70%  to  74%
C+ 65%  to  69%
C 60%  to  64%
D+ 55%  to  59%
D 50%  to  54%
F 0%    to  49% (Not a Pass)
EXC Excellent
SAT Satisfactory
UNSAT Unsatisfactory

For further information, see a copy of the Academic Policy, available online ( or at Seneca's Registrar's Offices.

Modes of Evaluation

  • Assignments are due at the beginning of the class on which they are due.
  • A late penalty of 10% per day is assessed for late assignments, including those not handed in at the beginning of class when due.
  • Material will not be accepted after one week following the due date and/or when the marked material is returned to students, whichever comes first.
  • Assignments are to be prepared by computer.

Absenteeism and Exams
  • Students should be aware that absenteeism almost guarantees an inability to achieve satisfactory grades.
  • Students who are absent for an examination due to an emergency (e.g., motor vehicle accident, hospitalization or death in the family) may provide official documentation within five days of the missed exam and be provided a deferred exam at a later date.  Official documentation includes a death notice or an original doctor’s certificate identifying the date, length of time expected absence and the specific reason for the absence.  Examinations missed without official documentation and approval result in a grade of zero.
  • There are no deferred options for missed tests.  The value of missed tests, at the discretion of the Faculty, will be added to other evaluation components

English Proficiency
  • All written work should demonstrate the following characteristics for clarity and conciseness:
-writing is consistent with the rules of English grammar
-spelling and punctuation are correct
-sentences are structured correctly
-main ideas are supported with specific, relevant examples and reasons
-work flows logically through supporting statements/paragraphs
-work is arranged in correct format (e.g., as a report, essay)
-up to 10% of the final grade may be deducted on all work if the above English competencies are not met.

Format for Assignments
  • Students must use the standard, APA style for quoting sources.   Help is available at:

Laboratory Attendance

The laboratory component is essential and therefore it is strongly recommended  that you attend all labs.  Any missed labs must be supported with a legal document with three days of the lab.  Any student who fails to attend 2 scheduled laboratory classes for a 7 week subject and more than 3 laboratory classes for a 14 week subject will not pass the subject.    

Laboratory Safety
Students are required to review and understand the safety procedures and guidelines outlined on the first class and sign the sheet to this effect before beginning work in the laboratory.  Students must also wear a lab coat and safety glasses when conducting experiments.
A student who arrives without the proper safety equipment will not be permitted to participant in the lab but will be asked to leave the class.  The student will receive no grade for the lab missed.

Grading is based on the following marking scheme:
Lab Assignment (3) 40%
Mid Term Test or Quiz  20%
Final Exam 40%


Other Evaluation Considerations
The student is expected to comply with the Safety Rules for working in the laboratory, sign the safety contract, and WILL NOT UNDER ANY CIRCUMSTANCES WEAR CONTACT LENSES in the laboratory.  The student will know where all safety equipment is located in the laboratory and will be familiar with WHMIS concepts and signage.


Approved by: Denis Gravelle