Lab 7: Liquid Chromatography: Analysis of Soft DrinkIntroduction Most soft drinks contain carbonated water, sugar, colors, acids and caffeine. Specifically, the components include caffeine, aspartame, and sodium or benzoate. These components are readily available to be separated and quantified using analytical chemical techniques. 1One said technique for quantifying components of analyte is through the separation of compounds soluble in the solution, known as High Performance Liquid Chromatography (HPLC). This analytical technique involves the differential equilibria of the components betweentwo phases: the stationary and mobile phases. The stationary phase is packed in a tube of known length, called the column. When the mixture is introduced to the column, the components can be separated by their affinity to either the stationary or mobile phase. The result of this technique is the various components separated into bands as they pass through the stationary phase. The constituent parts of the sample separate due to the difference in the relative affinities of the different molecules for the mobile and stationary phases used in the separation. 1 Common techniques for HPLC differentiate the mobile and stationary phases by polarity. As the sample passes through the column, the more polar components will have a greater affinity to the polar phase, and the same goes for the nonpolar components with the nonpolar phase. Thus, one must decide whether it would be beneficial to have a polar stationary phase, or normal phase chromatography, or a nonpolar stationary phase, or reverse phase chromatography. In one technique of Liquid chromatography, known as Ultra Performance Liquid Chromatography (UHPLC) the stationary phase is based various aspects, including adsorption, partition, ion exchange, size exclusion, and specific affinity. The stationary phase includes nonpolar octadecyl groups (C18H37, or C18), suggesting that the interactions will be reverse-phased, so the components will elute in order of decreasing polarity. 2 Once the components elute, detection of LC can be based on various properties, including refractive index, electrical conductivity, oxidation-reduction behavior, or absorption of UV or visible radiation. The UV or visible radiation method is a very common type of quantifying the separation of HPLC. When a solution elutes, the absorbance increases and is quantifiable by a UV/vis spectrophotometer, as a peak is observed. 1ExperimentalSample Preparation: The lab was split into two groups, one for the analyzation of Diet Coca Cola and the other for Diet Pepsi. The sample was filtered prior to examination with a 3 mL syringe with a filter attached to the end. 1 mL of sample was filtered and collected in a small, graduated cylinder. Thefiltered soda was diluted by half, by adding 1 mL of water. Next, seven 10 mL volumetric flasks were obtained, in order to create five standards for caffeine, one standard for aspartame, and one standard for benzoic acid. In the five volumetric flasks for caffeine, 0.5 mL of 1000 ppm standard of caffeine was added to the first flask, 0.75 mL of the standard to the second flask, 1.00mL to the third flask, 1.50 mL to the fourth flask, and 2.00 mL to the fifth. These five samples were then diluted to the 10 mL mark. For the benzoic acid flask, 1.50 mL of the 1000 ppm standard of benzoic acid was added to a 10 mL volumetric flask and diluted to the 10 mL mark. For the aspartame flask, 1.00 mL of the 2000 ppm aspartame standard was added to a 10 mL volumetric flask and diluted to the 10 mL mark. Finally, the samples were placed in small screw top autosampler vials, along with a sample of the soda and one filled with DI water to serve as a blank.Instrument Preparation: Mass Lynx v4.1 on the computer connected to the instrument was set up for the 2489 UV/vis and the lamp was turned on for 30 minutes before use. Solvent A (0.1% Acetic Acid in Water) was set to flow at 82.5% at 2 ml/min. Solvent B (0.1% Acetic Acid in Acetonitrile) was automatically set to 17.5%. The switch on the inside of the instrument was switched to 2 and the samples were placed in the sample holder. The placement of each sample was recorded. The machine was set to run at a solvent composition of Column A at 82.5 and Column B 17.5 for 2 minutes. The UV detector was set to scan at 215 nm. The injection volume was set to 20 µL of sample. This procedure was repeated for all the samples, after changing the vial number for each.The machine was left to run for about 25 minutes. The UV/Vis detector data was collected and saved for each molecule.Data Analysis: The Data was used in order to determine the retention times and retention factors for caffeine,aspartame, and benzoic acid. This data was used to prepare calibration tables and plots of peak area versus concentration for caffeine. The line of best fit was used to determine the concentration of the caffeine in the beverage. This value was then compared to the manufacturers’ stated values and these results were summarized in a table. The retention time of each species and the separation of the soda were entered into plots.Results and Discussion The elution times (Table 2) for each compound can be used to compare to those in the soda sample. Peaks were observed at 0.37, 1.38, and 0.45 minutes for caffeine, benzoic acid, and aspartame, respectively. In the soda sample, peaks were observed at 0.37, 0.46, and 1.38 minutes, which directly corresponds to the compounds that had measured standards. This shows that caffeine, benzoic acid, and aspartame are all found in Diet Coke, as they had the same, or very similar elution times and interactions with the mobile and stationary phases. The order of elution times would show that caffeine is the most polar and benzoic acid is the leas polar, as a longer retention time shows a greater affinity for the non-polar stationary phase. A calibration curve (Figure 1) was generated from the peak areas, or peak integrations for the caffeine samples. This curve does not include the integration from the 1.0300 mM or 1.2283 mMsamples, as these were identified as outliers and would have led to a negative caffeine concentration for the soda sample. Additionally, these data points deviated from the general trend of the line. Using the trendline, the Diet Coke sample was found to have a concentration of 0.2230 mM. In a typical serving of Diet Coke, 12 fl oz, there is a