Lab 7 Liquid Chromatography Analysis of Soft Drink Introduction 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 1 One 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 between two 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 1 Experimental Sample 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 The filtered 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 00 mL 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 L 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 mM samples 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 calculated value of 15 37 mg of caffeine When compared to the manufacturer s value of caffeine 46 mg this is lower and has a 66 6 error The concentration of each sample of