Basic HPLC

Usually held Tuesday to Friday inclusive (may be in April or June/July each year).

Course content
(there may be small changes in the practicals dependent on instrument availability)

Day 1

    

Lecture 1

Introduction to HPLC

Basic HPLC components – mobile phase, pumps [isocratic, gradient], guard column, analytical column, detector, data system.
“Chromatogram”; retention time, capacity factor

Practical 1

Demonstrations

Making Connections
Mobile phase preparation
Introduction to instrumentation

Lecture 2

Columns and column packings

“Packings”
Reverse vs Normal phase
Separation mechanisms
Evaluation of column performance – retention time, van Deemter equation,
Care of columns

Practical 2

Setting up the instruments, and a simple separation

Obtaining a peak.
Varying mobile phase composition
Becoming familiar with operating the HPLC and data system

Day 2

    

Lecture 3

Mobile phases #1

Polarity
Separation mechanisms
Selectivity, Ion suppression, Ion pairing

Practical 3

Effects of mobile phase composition

Obtaining optimum separation of xanthine mix using binary mobile phases.  Comparison of several organic solvents

Lecture 4

Mobile phases #2

Resolution, Efficiency, Capacity
Solvent strengths

Practical 4

Effects of sample size and detector variables

Altering sample concentration, injection volume, detector wavelength, AUFS. calibration curves

Day 3

 

  

Lecture 5

Injectors and Detectors

Sample injection
Detectors – UV-vis, diode array, RI, others

Practical 5

Quantitative practicals

Optimisation
Calibration
A quantitative analysis

Lecture 6

Integration

Integration – what is this?
Area %
External standard
Internal standard
Quality – QC/QA, Method validation, Accreditation, etc

Practical 6

Application practicals

Caffeine in tea/coffee/coke
DI water quality
Fractions from red wine
Surfactants

Day 4

 

  

Lecture 7

Sample preparation and applications

Liquid-liquid extraction
Solid Phase Extraction (SPE)

Practical 7

Continue with previous samples

  

 

Wrap up discussion

Concludes approx. 1.30 pm