Hplc Lab Report

3745 WordsJan 3, 201415 Pages
Determination of food additives in unknown beverage by HPLC By Tallulah Day- 3016606 Aims 1. To determine the wavelength of maximum absorbance and A(1% 1cm) for each of the four food additives (tartrazine, saccharin, caffeine and vanillin). 2. To select a wavelength at which all 4 compounds absorb, with sensitivity suitable for the HPLC detector. 3. To quantify for each additive in a mixture using HPLC with UV/Vis detection and external calibration. 4. To assess the separating power and the efficiency of the HPLC column for each of the four additives. Introduction High performance liquid chromatography (HPLC) is used to separate compounds in a sample, identify compounds and can even be used to deduce the relative amounts of different compounds in a mixture. HPLC works under the same principle as thin layer chromatography using both a stationary and mobile phase. The mobile phase carries the mixture across the stationary phase which is used to separate the compounds. Although in HPLC mobile phase is tailor made to suit the polarity of the analytes. The mobile phase used in this particular experiment was Methanol and 0.1M sodium dihydrogen phosphate at a ratio of 30:70 and a pH of 4.5, slightly acidic. The stationary phase is the silica based particles packed within the column. The key characteristic of HPLC is the sheer pressure it works under. In fact HPLC is often referred to as high pressure liquid chromatography. The pressure at which the machine used in this investigation works is around 1900psi (pounds per square inch) which is the equivalent of 129.29 atmospheres! The pressure is maintained by pumps working within the system. The model of pump used in the experiment is the TSP 1500 isocratic pump set to a pressure of 1900psi. The term isocratic simply means that the mobile phase maintains a constant composition throughout the separation.
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