PFT926 - Basic HP Liquid Chromatography

Outline info
Last revision date 2017-05-29 00:40:57.921
Last review date 2017-07-17 00:16:31.337

Subject Title
Basic HP Liquid Chromatography

Subject Description
This subject is an introduction to the theory and practical application of high performance liquid chromatography (HPLC). Students operate an automated HPLC system and learn HPLC theory through lectures and laboratory exercises. Topics cover both qualitative and quantitative aspects of HPLC. (Lectures and Labs)

Credit Status
One credit toward the Industrial Pharmaceutical Technology Certificate Program

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

1. Operate an automated HPLC system.

2. Manipulate HPLC mobile phases for the purpose of chromatographic separation.

3. Perform quantitative analysis.

4. Demonstrate experience in the analysis of a simple pharmaceutical formulation.

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
7 Week Delivery Mode

Class introductions; Laboratory safety procedures; WHMIS overview; Test and assignment due date; What is chromatography? HPLC equipment; Chromatographic process; USP formula of resolution; 3-Term formula of resolution; Column efficiency N; Factors affect on N; Peak dispersion: Mass transfer, eddy diffusion, longitudinal diffusion, column efficiency, extra column effects on N.
Laboratory #1: Efficiency of a chromatographic system and resolution between two peaks

Types of Chromatography; Normal phase, reverse phase; Comparison of parameters to describe peak retention t,v and k’; Factors affect k’;  Stationary phase polarity; Mobile phase strength/polarity; Column temperature; Mobile phase optimization techniques; RRM plot and log k’ vs %  mobile phase strength
Laboratory #2: Reversed phase HPLC effect of mobile phase strength on k’ in reversed phase system.

The selectivity (Alpha); Factors affect selectivity; Solvent chemistry; Column chemistry; Criteria of selection organic solvent; Commonly used solvents; Major interaction between molecules; Column chemistry ; Bonded phase column; Monomeric bonding and polymeric bonded reversed phase column; Column problems and remedies; Calculation of equal polarity of ACN mobile phase with Methanol mobile phase; Solvent selection triangle
Laboratory #3: Effect of solvent chemistry on separation

Mobile phase pH, ionic strength of buffer and ion-pair reagent on selectivity; Basic concept of acid/base, pH, pKa; Henderson Hasselbalch Equation;  How does pH affect ionization status of ionizable compounds; The correct way of preparation of mobile phase with buffer; Commonly used buffer and precaution; Ion-pair HPLC; application/mechanisim of ion-pair HPLC
Laboratory #4: The effects of pH on the retention of neutral, basic or acidic compounds in Reversed Phase System

Principle of quantitation by HPLC; Beer’s law; Methods of calibration; External standard calibration, internal standard calibration; System suitability, Data acceptability, precision; accuracy; Limit of detection  (LOD) and limit of quantitation (LOQ)
Laboratory #5: External Standard Method to determine the Acetaminophen and Caffeine in Excedrin Tablet

Review topics covered from laboratory #1 to 5; review method development process; mobile phase optimization using RRM plot
Laboratory #6: Project : Quantitiative analysis of the active ingredients in Excedrin tablet  by Internal Standard Method

14 Week Delivery Mode

Class Introductions; Test and Assignment Dates; Laboratory Safety Procedures; WHMIS Overview; What is Chromatography?  Chromatographic Process; Reversed Phase HPLC; Peak Retention; HPLC Equipment

Laboratory #1: Efficiency of a Chromatographic System and Resolution between two Peaks

Peak Dispersion:  Mass Transfer, Eddy Diffusion, Longitudinal Diffusion, Column Efficiency, Extra Column Effects on N

Laboratory #2:  Effect of Mobile Phase Strength on k' in Reversed Phase System

HPLC Solvent Properties; Stationary Phase Polarity; Mobile Phase Polarity; Resolution: two equations and the three parameters on which resolution depends

Laboratory #3:  The effects of solvent chemistry on retention and selectivity in a Reversed Phase System

pH and Buffers; Types of chromatography; Normal Phase, Reverse Phase, Ion Exchange, Size Exclusion

Laboratory #4:  The effects of pH on the retention of neutral, alkaline or acidic compounds in Reversed Phase System

Calibration, System Suitability, Data Acceptability, External Standard Curve, Internal Standard Curve

Laboratory #5: External Standard Method to determine the composition of a sample solution

Method Development, obtaining appropriate k' value, selectivity, analysis run time, Peak shape, pH, ion pair agent, special column chemistry

Laboratory #6:  Quantitiative analysis of the active ingredients in analgesic tablet by External Standard Method

Mode of Instruction
Classroom lectures detail the theory of HPLC. Laboratory experiments are performed weekly to provide practical application of the theory discussed. It is recommended that students expect to spend at least 3 to 6 hours per week on written assignments.

Prescribed Texts


Reference Material
Optional Reading:
High Performance Liquid Chromatography, 2nd Edition, Sadie Lindsay (1992), John Wiley & Son's Ltd. London. ISBN 13: 9780471931157

Introduction to Modern Liquid Chromatography, L.R. Snyder and J.J. Kirkland (1979), John Wiley & Sons Inc., New York. ISBN: 978-0470167540

Required Supplies
All students must wear a laboratory coat and safety glasses while in the lab.  This requirement is mandatory and consistent with safe practice.  Anyone not having the mandatory requirements will be asked to leave the lab.

Metric Ruler, Calculator, Graph Paper, Laboratory Coat, Safety Glasses

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:
Laboratory Assignments 25%
Laboratory Project 15%
Laboratory Skills 10%
Quizzes (3 X 5% each) 15%
Final Exam 35%


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