HPLC Fundamentals

1 Day Course

This course introduces the fundamentally important concepts associated with High Performance Liquid Chromatography (HPLC) analysis including hardware basics, modes of analysis, basic troubleshooting, column chemistry, important parameters of the mobile phase such as amount and type of organic solvents, pH, buffer strength and type, and much more. Suitable as a refresher for the more experienced analytical chemist or as an invaluable introduction to the technique for those with limited experience, this course provides a priceless insight into HPLC principles and practice. This course is not instrument-specific.

We limit numbers to 20 per course so that each delegate gets the opportunity to ask questions and fully participate in tutorial exercises
When delivered on-site we can design the course material to suit your specific training needs
Customisable written assessments are available if required

Course Overview | Download course details »

"Chromatography is a physical method of separation, in which the components to be separated are distributed between two phases, one of which is stationary whilst the other moves in a definite direction".

This is the IUPAC definition of Chromatography, with which we begin a thorough investigation of modern Liquid Chromatography, from the underlying theory of analyte distribution to a detailed study of the chemistry of the column, via the importance of mobile phase composition to the instrumentation of the HPLC system. The different components of the chromatograph (pumping systems, injectors, detectors, etc) are fully explored, as well as the fundamentals of data analysis.

Who is this course for
Users of LC in any of its modes (NP, RP, HILIC, IEX, SEC…) or anyone interested in gaining a deeper understanding of the chromatographic separation of compounds amenable to analysis by LC.

Previous knowledge
No previous experience of chromatography is necessary, but a basic knowledge of chemistry is beneficial.

What you will learn

How the chromatographic process takes place
The essential theory behind chromatographic separations
The architecture and working principles of a modern HPLC system
The information contained in a chromatogram and the basic chromatographic parameters
The essential principles of sample preparation, mobile phases, buffers and eluent additives for HPLC

Course Outline

Basics of the Chromatographic Process

Main retention mechanisms in HPLC
Distribution constant
Retention theory
Model of the chromatographic process

Sample Preparation Protocols

Principles
Matrix elimination
Liquid and Solid Phase Extraction

Separation Mode/Retention Mechanisms

Adsorption (normal phase)
Reverse phase
Principles of ionisation (Ion Suppression Chromatography)
Ion pairing
Ion exchange

Quantitation

Integration parameters
System suitability testing

Injectors and Columns

Sample introduction
Rheodyne injectors/auto-samplers
Silica as a solid support
Column & packing geometry
Efficiency - the van Deemter & Knox equations

Detectors

Choosing the right detector
Operating principles
Optimisation
Typical operating conditions
UV (Diode Array)/RI/Fluorescence

Measuring and Optimising Chromatographic Parameters

Efficiency
Capacity factor
Selectivity
Resolution
Interdependence via the resolution equation