The main objective of this lab is to gain experience in using and reading results from the HPLC machine. 2) The first step is to inject a series of caffeine standards into the machine in order to get results/values of peak height and area. 3) The next step is to investigate the effects of a series of HPLC parameters on Retention time, peak height and peak area. 4) To use above data from peak height and area to construct two standard curves vs concentration.
Introduction: Caffeine is a common organic molecule found in many beverages such as coffee, tea, and cola.
It is a stimulant to the central nervous system. Which is why the majority of the population use it in one form or another to help stay alert. In general people drink caffeinated drinks like the ones mentioned above in order to obtain their “fix”. However with many recent studies in the area caffeine tablets are becoming a very popular way for athletes in lots of different sports to stay alert even when fatigue is setting in.
HPLC stands for High Performance Liquid Chromatography but is also referred to as High Pressure Liquid Chromatography.
It is used for separating mixtures either to analyse the mixture or to separate a required product from others in a reaction mixture. It can also be used to find the relative amounts of different components in a mixture. HPLC works along the same lines as paper chromatography. In paper chromatography a liquid (mobile phase) moves past a solid (stationary phase). In paper chromatography the stationary phase consists of water molecules bound to the cellulose in the paper, the mobile phase carries different components of the mixture along with it.
How fast each one moves depends on its relative affinity for the mobile and stationary phases. In HPLC the stationary phase is a solid packed into a column and the liquid is forced through the column by high pressure pumps. The pumps force the mixed solvents through the column the solvent emerging from the column carries the separated components of the mixture and is passed into the detector where a beam of ultraviolet light shines through it. There are many different types of detector depending on what you are
analysing. This light is at a wavelength that is absorbed by all the components to be separated. When the detector reading drops the component that is absorbing the UV light is coming out of the column and passing through the detector. The time it takes for each component to come off the column is called its retention time and can be used to help identify it. The more polar component comes off the column first followed by the less polar. Materials: As per laboratory manual.