HPLC Determination of Caffeine and Benzoic Acid in Soft Drinks
1-To become familiar with the practice of high-performance liquid chromatography (HPLC).
2-To determine the caffeine and benzoic acid concentrations in various soft drinks.
Chromatographic techniques are widely used in all areas of science because they allow the analyst to separate and quantify the components of a mixture. In this experiment you will use high-performance liquid chromatography (HPLC) to determine the amount of caffeine and benzoic acid in a soft drink. The principle on which chromatographic methods are based is simple. Compounds in a mixture are separated from each other based on their preferences for one of two different solvents (or phases) in contact with each other. For example if a polar solvent and a nonpolar solvent are brought into contact, polar molecules will prefer to be in the polar solvent, while nonpolar molecules prefer to be in the nonpolar solvent. In a chromatography experiment one of these solvents is stationary (the stationary phase), while the other solvent (the mobile phase) flows…show more content… The column consists of small, porous silica particles that have hydrocarbon chains eighteen carbon atoms long (a C-18 column) chemically bonded to their surfaces. Mobile phases used in reversed-phase columns are polar. In this experiment you will use a mobile phase which is 75% 0.025 M phosphate buffer (pH 3.0) and 25% methanol. Separation of components in a sample relies on different substances having different preferences for the mobile and stationary phases. Molecules that are very polar will elute from the column early, since they prefer to stay in the mobile phase. Nonpolar molecules, preferring the environment of the stationary phase, will be retained on the column for longer periods of