This course has been designed for novice HPLC users and those who have some experience of running HPLC methods. Although the main focus will be on the fundamental theory of HPLC we will discuss slightly more advanced topics in order to allow you to progress easily to the next, more advanced stage in your HPLC training.
This course is approved by the Royal Society of Chemistry & American Chemical Society - Division of Analytical Chemistry.
The fundamental principles of high performance liquid chromatography (HPLC) will be covered to begin your training. The fundamental chromatographic parameters which govern resolution within chromatography will be detailed.
We will then move on to discuss reversed phase HPLC, the most widely used mode of chromatography. The fundamental separation mechanism will be detailed along with discussions on the effect of mobile phase components and how to select the most appropriate eluent for a separation. Working with ionizable analytes requires specific considerations when designing an HPLC protocol such as eluent pH, appropriate buffer choice, and the possibility of unwanted secondary interactions within the HPLC column. All of these topics will be covered in order to give you a full understanding of this ubiquitous separation mode.
Selecting the correct HPLC column for your application is absolutely crucial. This session will examine the available column stationary phases and support materials (including conventional fully porous and core-shell/superficially porous materials), column dimensions, and the effect that they can have on your chromatography.
Following this, the use of gradient elution in HPLC will be considered. We will cover the fundamental principles of this technique which lead to the characteristic sharp chromatographic peaks produced by this elution mode. We will demonstrate how to optimize essential gradient parameters using scouting gradients and simple calculations in order to allow you to quickly achieve suitable separation of analytes.
Each part of the liquid chromatograph will be considered starting, in this session, by considering any precautions which must be taken when preparing a mobile phase - this includes selecting the correct purity of solvent, the appropriate buffer, and the essential steps included in mobile phase preparation such as filtering and degassing.
Next we will consider the pumping system and autosampler. The components which make up these parts of the instrument will be detailed along with some basic troubleshooting for when problems arise.
The final component within the liquid chromatographic instrument is the detector. There are several common detectors which are commonly used within modern laboratories including UV-visible, refractive index, fluorescence, and evaporative light scattering detectors. We will look at the working principles, application, and advantages and disadvantages of each detector.
Some basic column and system care and maintenance will be covered in order to allow you to utilize your instrument to its full potential and keep it in good working order.
Finally, we will discuss quantitative and qualitative HPLC and the information which can be gained from the chromatographic data produced from an HPLC experiment.