Determination of Food Additives in a Beverage - Essay Example

Determination of food additives in a beverage Ultra violet or visible detector and high performance liquid chromatography (HPLC) was usedin this experiment which involved the use of the food four food additives Tartrazine, Saccharine, Caffeine, and Vanillin. For the first part of the experiment, the absorptiometry properties of the 4 analytes were measured using a maximum wavelength of 263.72nm, using it to the A (1%) of the 4 analytes. Using the same HPLC system, the second part of the experiment involved measuring the peak area of the 4 additives, to varying degrees containing all analytes using the resultant maximum wavelength of the first experiment with the inclusion of an unknown solution. The results of the experiment was used to plot a graph of varied peak areas against the concentration of specific food additives in the list. Then a calculation was performed to determine the resolution and efficiency of each food additive as part of the proof that each additive could be separated in content count within the food beverage. Introduction As an experiment, the main focus of this exercise was to create a determination of the suitable absorbance rate of Tartrazine, saccharin, vanillin, and caffeine using a precise calculation of the A (1% 1 cm.). In order to use a suitable HPLC detector, a maximum wavelength using all 4 analytes under ultraviolet radiation was selected. The quantified additives were composed of a mixture of HPLC and UV/vis detection along with external calibration. In order to properly assess the separation power and efficiency of the HPLC column per additive, the first part of the experiment used UV/vis detectors which is a wavelength that could determine the absorption of the 4 analytes. The second part of the experiment used a liquid chromatography separation method in order to determine the measurement of HPLC in the analytes. The experiment separated the non-volatile and unstable compounds for the identification of the analytes. Pharmaceutical drugs like aspirin and sodium chloride were used in the non-volatile compounds. The food additives Saccharine, Vanillin, Caffeine, and Tartrazine for the experiment was the obvious choice as these are the more commonly found additives in todays food. A conclusion that was reached through years of emphasis on the quality and importance of food and its additives in relation to its effect on the final product. The assessment needed to produce both quantitative and qualitative values related to the risks and effects of the use of these additives in food products. Food additives are required as a part of the commercial preparation of food in order to prevent the onset of spoilage, enhancing flavour, improving the texture and colour of the food products while on the shelf. Normally, food additives are composed of organic compounds such as sweeteners, flavouring agents, stimulant, and food colouring. Vanillin, Saccharine, Caffeine, and Tartrazine, all share the molecular structure C8H803, C7H5NO3S,C8H10N4O2,C16H9N4Na3O9S2 respectively. As such, these organic compounds have been known to possess chromophores which help in the absorptions of ultra violet radiation. The reason that ultraviolet radiation was used in this experiment stems from its ability to provide a high sensitivity, accuracy, and precision in measurement that makes it an ideal method of analyte quantification. Since Saccharine and Caffeine are known as polar compounds, the additives were easily separated using a reverse phase HPLC. The stationary phase is polar, therefore when separation occurs, the polar elements bond with non-polars, which leads to the mobile phase. Since the mobile phase is polar, it provides a good separation phase for the 2 aforementioned analytes. The compounds are then determined through retention time, which is the time that it takes for analyte movement from the HPLC column after injection. HPLC has been known to use 4 modes to separate the analytes namely the reverse phase, normal phase absorption and ion exchange, and size exclusion. For this experiment, we chose to use the reverse phase. The experiment utilized HPLC spectroscopic detection, refractive index, and florescence as the detection principle. Since there was more than one analyte in the experiment, the Beer Lambert Law was used in order to show the analyte concentration in proportion to their wavelength. This displays a linear relationship because when one analyte increases, so to the latter analytes. The following formula best represents the Beer Lambert Law: A=εbc A= absorbance ε=molar absorbability in l mol-1 cm-1 b=path length in cm c=concentration of sample being measured in mol l-1 A suggested wavelength was used to determine the component of the analytes in order to derive A (1% 1 cm) in order to derive maximum absorbance. In order to determine the concentration of unknown solutions, a graph of the variations using a specified wavelength composed of the 4 analytes were used. The combination of the results were used to calculate the resolution and efficiency of the 4 additives per column. Methodology METHODS Reagents used. Powdered solid samples of Tartrazine, Saccharin, Vanillin and Caffeine were provided. HPLC deionised water. HPLC mobile phase solvent consisting of, methanol 0.1mol dm-3 and sodium hydrogen phosphate (30:70, pH 4.5). Sample solution containing all four additives dissolved in the mobile phase. Procedures Step1: separate stock solutions for each beverage were prepared using a weighted sample of each beverage using a four scale balance transferred into a 100cm3 volumetric flask marking the HPLC deionised water. This method was also decided to be the best way with which to ensure the accurate knowledge of each concentrated additive of about 0.25mg/cm3 for tartrazine, vanillin and caffeine except saccharine which should be 1mg/cm3. The stock solutions were diluted yet again by transferring a calculated volume into a 10cm3 flask which was marked to get a concentration of 25µg cm⁻³ for tartrazine in a 10cm3, 25 µg cm⁻³for caffeine in 10cm3, 25 µg cm⁻³ for vanillin in 10cm3 and 100 µg cm⁻³ of saccharin in 10cm3. Scanning each diluted beverage solution for UV/Vis absorbance via UV/Vis absorbance double beam spectrophotometer and silica cuvettes was performed. A note was made of each solution with an estimated wavelength for each solution. These were then place in a drudge for further preservation and analysis over a weeks time. Step 2: After a week, the solutions were utilized in the preparation of multiple-standard solutions of each analyte. Each solution was prepared and labelled from 1-4, with each container holding a particular analyte. Solution 1 contained 400µl of each analyte, 600µl of each analyte in solution 2, 800µl of each analyte in solution 3, 1000µl of each analyte in solution 4. Each of the multi standard solutions and the solution from the previous week were aspirated separately onto the HPLC. The old individual solution was aspirated and its retention time marked. Each of the solution was aspirated into the HPLC in order to note the various peak areas produced coupled with the retention time that corresponded to the same retention time of a particular previous analyte. An unknown solution was also aspirated onto the HPLC, with its peak area and times also measured and noted. Discussion There was a linear relationship seen in the variation of peak area graphs of Tartrazine, Vanillin, Saccharine, and Caffeine. A note was made of the RS values of the four analytes as follows: 0.9999, 0.9999, 0.9995 and 0.9901 respectively. Rs values were always noted as being closer to one. The experiment followed the Beer Lambert Law to the letter as the concentration of analytes were increased with an increase in wavelength, which helped prove the linear relationship existing between concentration and wavelength. The basis of separation for the mixtures were the distribution of substances between the two phases of stationary and mobile phase, inclusive of its polarity, size, and / or shape. It appeared that it longer to leave the stationary phase when there was a greater relative affinity in an analyte in the stationary phase. This resulted in a longer retention time. Table 1 shows that Vanillin proved to have the highest retention time of 4. 819 with Tartrazine having the least retention time at 1.554. Therefore, Vanillin proved to have the greatest affinity during its stationary phase which produced its higher retention time. The opposite situation existed for Tartrazine. Such was the trend that continued into Table 2 which showed that the increased concentration of analytes produced decreased retention time in Tartrazine, Vanillin, Saccharine, and Caffeine with its peak areas increasing in the process. The existing polarization of the analyte resulted in a faster removal of the analyte. The experiment showed that Tartrazine was the most polar followed by Saccharine, Caffeine, and finally, Vanillin, as the least polar. The unknown solution marked as A,B,C,D in Table 3 were then identified using the retention time which resulted from Table 1 and 2. Identified as Tartrazine, Caffeine, Saccharine, and Vanillin, the retention time proved to be dependent on the pH and content of the mobile phase of the experiment. These 4 analytes are using across the world for various purposes. Caffeine is used in soft drinks, tea, and coffee. The USFDA has allowed the use of the aforementioned additives with a maximum amount of 6mg or 200ppm in beverages (Violeta Nour, 2010). Saccharine, which is used as a sweetener, had questions arise due to its link to tumor development in some animal experiments. So the USFDA decided to limit its use. Tartrazine is limited to dye use at the amount of 5mg (E 102). With the possibility of its link to cancer, the FAO.WHO decided that the maximum allowable amount in beverages was 7.5 mg. Vanillin on the other hand is allowed for beverage use up to a maximum of 10 mg. The concentrations of the unknown solutions of Tartrazine, Saccharine, Caffeine and vanillin when converted to mg were as follows 6.8×10-3,3.6×10-2,6.49 ×10-3 and 10.11×10-3 .the resulting values proved to be similar to the recommended amount of additives allowed by the FAO/WHO and the USFDA. Conclusion The main aim of the experiment was accomplished. In its efforts to determine the wavelength of maximum absorbance, it was determined that the value for Tartrazine, Vanillin, Saccharine, and Caffeine respectively was 263.72nm and A (1%1cm). 445.2,571.88,217.792 and 536.84 respectively. The wavelength for all 4 food additives was concluded to be 263.72nm. The separation power between the additives was 2.316, 3.174, and 3.726. A determined efficiency for the additives was found to be Saccharine - 2893.0055, Tartrazine - 3308.5504, Vanillin - 5910.5344, and Caffeine - 393.7536. Read More