THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF FOOD SCIENCE

Transcription

1 THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF FOOD SCIENCE DEVELOPMENT OF A PAPER ANALYTICAL DEVICE TO MEASURE VITAMIN C IN BEVERAGES JOHN J. DECARO III SPRING 2017 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Food Science with honors in Food Science Reviewed and approved* by the following: Dr. Joseph T. Keiser Senior Lecturer of Chemistry, Director of the General Chemistry Labs, and Assistant Head of Undergraduate Chemistry Thesis Supervisor Dr. John E. Hayes Associate Professor of Food Science Honors Adviser * Signatures are on file in the Schreyer Honors College.

2 i ABSTRACT In this project, the creation of a paper analytical device (PAD) with the capacity to measure vitamin C content in liquid food samples was explored. A paper analytical device (PAD) employs microfluidics by creating hydrophobic channel outlines through which samples and reagents can flow and interact in order to conduct a reaction. This project successfully developed a PAD that measures food samples for vitamin C content in the range of 2 mg/l 20 mg/l with a colorimetric assay using 1,10-phenanthroline, and in the range of 20 mg/l 300 mg/l utilizing a 2,6-dichloroindophenol titration.

3 ii TABLE OF CONTENTS LIST OF FIGURES... iii LIST OF TABLES... iv ACKNOWLEDGEMENTS... v Chapter 1 Introduction and Background Information... 1 Chapter 2 Previous Research... 7 Chapter 3 Methods and Materials... 8 Chapter 4 Results and Discussion for Analysis of Quantitative Methods Chapter 5 Results and Discussion for PAD Development Chapter 6 Conclusion BIBLIOGRAPHY... 26

4 iii LIST OF FIGURES Figure 1. Diagram of a PAD Figure 2. Average Absorbance Values of Gallic Acid Standards With Standard Deviation Error Bars...16 Figure 3. Average Absorbance Values of Vitamin C Standards With Standard Deviation Error Bars 20 Figure 4. Photo of 1,10-P Assay Standards...23 Figure 5a. Trial 1 of PAD Production with Standardized Sample Figure 5b. Trial 2 of PAD Production with Standardized Samples...27 Figure 6a. Trial 1 of PAD Production with White Grape Juice Sample Figure 6b. Trial 2 of PAD Production with White Grape Juice Sample... 28

5 LIST OF TABLES Table 1. Steps of the Folin-Ciocalteau Methods Procedure..17 iv Table 2. Absorbance Values at 729 nm of a 1500 mg/l Gallic Acid Standard With Varied Times Prior to Addition of Na2CO Table 3. Absorbance at 729 nm of Gallic Acid Standards With Varied Heating Times Table 4. Comparison of Average Experimental Results of Each Titration Method to Expected Results...21 Table 5. Summary of Time-Temperature Treatments of PADs 25

6 v ACKNOWLEDGEMENTS Foremost, I express my utmost gratitude to my thesis advisor, Dr. Joseph Keiser, for the continual academic and personal support. Further, I thank my honors advisor, Dr. John Hayes, for the helpful criticism and guidance throughout my writing process.

7 1 Chapter 1 Introduction and Background Information Antioxidants are molecules of interest for nutritionists and food scientists, due to the portprted health benefits they provide. One antioxidant in particular, vitamin C (also known as ascorbic acid) is quite important within the body, as it has the capacity to scavenge free radicals, which may play a role in disease prevention. 1 Further, consumption of another class of antioxidants, polyphenols, have been shown to be inversely related to diseases caused by oxidative stress. 2 For this reason, it is important to be able to quantify antioxidant levels in foods and beverages. The goal of this project was to create a paper analytical device (PAD) that could quantify vitamin C in liquid food samples. Initially, some efforts were made to also quantify polyphenol antioxidants. But, ultimately the focus became vitamin C quantification. PADs are platforms on which microscale chemistry can be performed, in which the reactions are run within the fibers of a piece of paper. Unlike other microscale analytical devices, PADs can be much simpler to fabricate, can be made more quickly, and are considerably cheaper. 3 The PADs created in this project utilize cellulose filter paper and heat-embedded wax channels as hydrophobic barriers that direct the flow of a sample into areas that contain dried reagents, in order to perform a quantitative analysis of the sample. Prior to PAD fabrication, it was necessary to assess the feasibility of applying various quantitative techniques for vitamin C and polyphenol analysis to a PAD. To quantify

8 polyphenols, the Folin-Ciocalteau method was assessed. Also, four vitamin C quantifications 2 were tested: a titration with an iodine solution, a titration with a 2,6-dichloroindophenol solution, a titration with N-bromosuccinimide, and a colorimetric assay with 1,10-phenanthroline. To determine which methods work best on a PAD, consideration must be taken regarding the accuracy of measurement, the safety of the reagents, and the ease of use of the methods. The Folin-Ciocalteau method is a spectrophotometric method in which a sample is reacted with the Folin-Ciocalteau reagent, composed primarily of phosphotungstate and phosphomolybdate, to yield a blue color. The absorbance of this mixture is measured and compared to a standard curve in order to assess polyphenol levels, measured as gallic acid equivalents. The mechanism of the Folin-Ciocalteau method reactions is unknown. 4 For vitamin C measurements utilizing an iodine solution, its concentration in a sample is quantified by an iodine titration with a reaction mixture that contains starch and excess iodide ions. In solution, potassium iodide dissociates to provide iodide anions that react with iodine to form the triiodide ion. Triiodide ions will oxidize vitamin C, or the excess iodine will complex with triiodide ions to yield polyiodide ions, I5 -. The oxidation of vitamin C occurs more quickly than polyiodide formation, therefore, the triiodide ions will react with all vitamin C molecules present prior to forming polyiodide ions. Upon oxidation of all vitamin C, excess iodine will complex with triiodide ions to yield I5 -. The polyiodide ions are linear molecules that react within the helix of starch molecules, yielding a complex molecule that has a blue-black color, which signifies the endpoint of the titration. The mechanism is shown in Scheme 1. 5

9 3 In solution: (excess I - is present) Oxidation of vitamin C: Once all vitamin C is oxidized: Scheme 1. Reactions of Iodine Titration For vitamin C measurements utilizing N-bromosuccinimide (NBS), its concentration is quantified by a NBS titration with a reaction mixture that contains starch and excess iodide ions. NBS will oxidize vitamin C, and will oxidize iodide in solution to yield iodine. However, the oxidization of vitamin C by NBS occurs more quickly than the oxidation of iodide, therefore NBS oxidizes all vitamin C prior to oxidizing iodide. Once all of the vitamin C has been oxidized, NBS will oxidize iodide ions to form iodine which will lead to the same series of

10 4 reactions shown above and the same blue-black end point. The mechanism is shown in Scheme 2. 6 Oxidation of vitamin C: Once all vitamin C is oxidized: Scheme 2. Reactions of NBS Titration For vitamin C measurements utilizing 2,6-dichloroindophenol (2,6-D), its concentration is quantified by a titration with 2,6-D. This reaction is unique, as 2,6-D is both is the titrant and indicator of the endpoint. Oxidized 2,6-D is blue, but upon reaction with vitamin C, the reduced 2,6-D yields a pink color. The mechanism is shown in Scheme 3. 7

11 5 Scheme 3. Reactions of 2,6-D Titration Lastly, vitamin C concentration can be quantified via a colorimetric analysis that utilizes a reaction with 1,10-phenanthroline (1,10-P), which is a component of a reagent solution referred to as the color reagent. In this reaction vitamin C from a sample reduces iron (III) from ammonium iron (III) sulfate dodecahydrate to iron (II), which in turn reacts with 1,10-P in solution to yield an iron-phenanthroline complex that has a reddish-orange color. The absorbance of this mixture is measured and compared to a standard curve in order to assess vitamin C concentration of the sample. The mechanism of the reactions that take place during this assay are shown in Scheme 4. 8

12 6 Scheme 4. Reactions of the 1,10-P Colorimetric Assay After determination of which analytical methods would be best used on PADs, it was necessary to determine the most effective time-temperature treatment necessary to achieve full wax embedment within the filter paper. Afterwards, application of the chosen methods were tested on a PAD.

13 7 Chapter 2 Previous Research Previously, I performed research that had results that are pertinent to this project. I performed experiments in which vitamin C content in cabbage, broccoli, teas, and fruit juices was quantified using titrations with an iodine solution. Those experiments demonstrated that accurate results are attainable when measuring relatively pure samples of vitamin C, such as prepared standards. However, results from the analyses of food samples were less accurate. It was determined that a complicating factor is the presence of other oxidizable substances, such as polyphenols. The complication occurs due to the relatively strong oxidizing capacity of I2, which has a standard reduction potential of V. 9 This discovery was the impetus for using the 2,6-D titration for vitamin C quantification, as the standard reduction potential for the reduction of 2,6-D is only V, meaning that it is a milder oxidizing agent than I2. Results from those experiments showed that indeed, titration of samples with 2,6-D gave more accurate results than titrations with iodine. 10 However, this project still assessed the use of iodine titrations on a PAD to determine if it would be a superior method on a PAD compared to 2,6-D.

14 8 Chapter 3 Methods and Materials Iodine Titration: 5 Three drops of g/l vitamin C containing 5.00 x 10-3 M EDTA, three drops of starch solution, and three drops of 0.1 M HCl were added to each well of a 12-well strip. A serial titration was performed with 2.84 x 10-4 M I2. 2,6-D Titration: 7 Three drops of g/l vitamin C with 5.00 x 10-3 M EDTA and three drops of an acetic acid mixture were added to each well of a 12-well strip. A serial titration was performed with 8.52 x 10-4 M 2,6-D. NBS Titration: 6 Three drops of 1.00 mm vitamin C with 5.00 x 10-3 EDTA, three drops of starch solution, two drops of 4% w/v potassium iodide solution, and two drops of M HCl were added to each well of a 12-well strip. A serial titration was performed with 1.00 mm NBS. FC Method Standard-Curve-Solution Preparation: g of gallic acid was dissolved in 10 ml of ethanol, then diluted with distilled water to 100 ml in a 100 ml flask. Then, 3, 5, 10, 20, and 25 ml of the gallic acid stock solution was added to 100-mL volumetric flasks respectively. Each was diluted to 100 ml with distilled water.

15 FC Method Reaction (for samples and standards): 4 9 First, 20 μl of sample was added to a cuvette. Then 1.58 ml of water and 100 μl of the FC reagent was added to the cuvette, mixed, and allowed to react for periods ranging from 30 seconds to 4 minutes. Next, 300 μl of saturated sodium carbonate solution was added to the cuvette, and mixed. Then, the cuvettes were placed in a 40 C water bath for 30 minutes. After incubation, the absorbance of the samples at 729 nm were analyzed with a SpectroVis spectrophotometer. 1,10-P Colorimetric Assay Color Reagent Preparation: 8 Added g 1,10-phenanthroline monohydrate, and 8.00 x 10-2 g of ammonium iron (III) sulfate dodecahydrate, to 49 ml of deionized water and 1 ml of 1M hydrochloric acid. 1,10-P Colorimetric Assay (for samples and standards): 8 Added 500 μl of sample and 500 μl of color reagent to a cuvette. Then, the absorbance values of the samples were analyzed with a SpectroVis spectrophotometer at 515 nm. PAD Production: PADs were created by drawing wax pathways onto 9-cm diameter cellulose filter paper. For the section of the PAD utilizing the 2,6-D titration, the center sample well was drawn with a 1-cm diameter, with four 0.7-cm wells extending 1.2 cm from the center sample well. For the section of the PAD utilizing the 1,10-P colorimetric assay, four rectangular wells were drawn with wax pencil, with 1-cm by 1-cm dimensions. The PAD was heated at 400 C on a hot plate for 3 minutes to cause the wax to soak through the paper. Then, for the section of PAD utilizing the 2,6-D titration, 0.5 μl of the 2,6-D solution was applied to each well, and then dried for 1 minute with nitrogen gas. The well that would test for 20 mg/l had g/l 2,6-D, the well for 100 mg/l vitamin C had g/l 2,6-D, the well

16 10 for 200 mg/l vitamin C had g/l 2,6-D, and the well for 300 mg/l vitamin C had g/l 2,6-D. Then, 0.5 μl of 0.1 M HCl was applied to each well and dried for 1 minute with nitrogen gas. For the section of the PAD utilizing the 1,10-P colorimetric assay, 5 μl of color reagent was added to each well, then dried with nitrogen gas. Lastly, 5 μl of a sample was applied to the 1,10-P portion of the PAD, and 7 μl of a sample was applied to the center of the sample well of the 2,6-D portion of the PAD. Results were read immediately after the solution fully traveled throughout the wells. A diagram of this PAD is shown in Figure 1. 2 mg/l vitamin C standard 1,10-P Portion 2,6-D Portion 10 mg/l vitamin C standard 300 mg/l vitamin C standard 20 mg/l vitamin C standard 20 mg/l vitamin C standard Sample well Sample well 200 mg/l vitamin C standard 100 mg/l vitamin C standard Figure 1. Diagram of Paper Analytical Device

17 Absorbance (at 729 nm) Chapter 4 11 Results and Discussion for of Possible Methods Folin-Ciocalteau Method: The Folin-Ciocalteau method requires the production of a standard curve from which the concentration of polyphenols, as gallic acid equivalents, can be determined. Numerous trials were performed to generate a standard curve and to assess reproducibility. Figure 2 below shows average values of the absorbance at 729 nm of gallic acid standards of 150 mg/l, 250 mg/l, 500 mg/l, 1000 mg/l, and 1500 mg/l for six trials y = x R² = Gallic Acid Concentration (mg/l) Figure 2. Average Absorbance Values of Gallic Acid Standards With Standard Deviation Error Bars These results are somewhat interesting, yet unexpected. First, Waterhouse has observed that the standard curve values were nearly linear, whereas this experiment yields values that appear to increase non-linearly. 4 However, the absorbance values of 150 mg/l, 250 mg/l, 500 mg/l, and 1500 mg/l gallic acid standards had quite low standard deviations, of 0.012, 0.021, , and , respectively, indicating that these values were very reproducible. The absorbance value of the 1000 mg/l gallic acid had a comparatively larger standard deviation of 0.106, but this was caused by one trial yielding an unexpectedly low absorbance value. Overall,

18 12 numerous trials yielded results that were quite similar, excluding the unexpected value for one trial of the 1000 mg/l gallic acid standard. Therefore, the accuracy and precision of the method may be adequate for use on a PAD. However, there are variations in the literature regarding the details of how the Folin- Ciocalteau Method should be carried out 4. Table 1 explains the procedure of method (a) in more detail. Table 1. Steps of Folin-Ciocalteau Method Procedure Step of Method Step 1 Procedure 20 μl of sample, 1.58 ml of water, and 100 μl of the FC reagent are added to a cuvette, then let react between 30 seconds and 4 minutes. Step μl of a saturated sodium carbonate solution is added to the cuvette, then mixed. The cuvettes are placed in a 40 C water bath for 30 minutes or in a 20 C water bath for 2 hours. Step 3 The samples are analyzed with a UV-Vis. spectrophotometer. Some of these variations were investigated. For example, Waterhouse wrote that the reaction time should be between 30 seconds and 4 minutes prior to Step 2. 4 The reaction time at the end of Step 1 was varied, then Step 2 was run at 40 C for 30 minutes, and Step 3 was performed as explained. The results of this investigation are shown in Table 2.

19 13 Table 2. Absorbance at 729 nm of a 1500 mg/l Gallic Acid Standard with Varied Times Prior to Addition of NaCO3 Time before addition of NaCO3 Absorbance at 729 nm 30 sec min mins mins mins Standard Deviation These results show minimal variance between absorbance values over the range of times, as evidence by small deviation about the average absorbance value. It seems that 30 seconds is enough time for completion of the reaction. Waterhouse wrote that the sample should either be heated at 40 C for 30 minutes or at 20 C for 2 hours. The heating step at the end of Step 2 was varied, then Step 3 was performed as explained. The results of this investigation are shown in Table 3.

20 Table 3. Absorbance at 729 nm of Gallic Acid Standards with Varied Heating Times 14 [gallic acid] Absorbance Absorbance Absorbance Standard Deviation (mg/l) after 30 min after 45 min after 60 min These results show minimal variance between absorbance values over the range of times, as evidence by small standard deviations throughout. Since it is desirable to limit reaction time for the FC method which is quite time intensive, a thirty second wait time and a thirty-minute heating time would be best. 1,10-P Colorimetric Assay for Vitamin C: The 1,10-P assay requires the production of a standard curve from which the concentration of vitamin C can be determined. Four trials were performed to generate the standard curve and to assess reproducibility. Figure 3 shows average absorbance values at 515 nm for vitamin C standards of 2 mg/l, 5 mg/l, 10 mg/l, 20 mg/l, 30 mg/l, and 40 mg/l.

21 515 nm y = 0.327ln(x) R² = mg/l vitamin C Figure 3. Average Absorbance Values of Vitamin C Standards With Standard Deviation Error Bars These results are encouraging, as they match the general trend shown in the literature. 8 Due to an acceptable shape of the trend line and rather small standard deviation values of mg/l, mg/l, mg/l, mg/l, mg/l, and mg/l vitamin C, respectively, this method seems to be acceptably accurate and reproducible. Vitamin C Titration Methods: The accuracy of the different vitamin C titrations was assessed. Table 4 summarizes the results from experiments, including the accuracy of each method.

22 16 Table 4. Comparison of Average Experimental Results of Each Titration Method to Expected Results Titration Method Expected Results Average Experimental Results Accuracy Iodine Titration g/l vitamin C g/l vitamin C 100 % (prepared standard) serial titration, 4 trials NBS Titration 1.00 mm vitamin C 1.04 mm vitamin C High by (prepared standard) serial titration, 8 trials 4.00% 2,6-D Titration g/l vitamin C g/l vitamin C High by (prepared standard) serial titration, 6 trials 2.60% These results show that the iodine method was the most accurate titration method. However, the deviation from the expected values for the 2,6-D method and the NBS method were also in an acceptable range. Ultimately, each method seems to be a sufficient method for analysis of vitamin C, yet other qualities of these methods must be considered. Safety of All Methods: Safety of the reagents must be considered. According to material safety data sheets, iodine, 2,6-D, Folin-Ciocalreau reagent, NBS, and the color reagent components are similar in terms of safety issues posed. Each compound is a skin and eye irritant, and each can create vapors that are harmful if inhaled. In terms of safety, the titrants are comparable, so other factors must be considered. 11,12,13,14,15,16

23 17 Ease of Use To assess the ease of use of each method, multiple factors were considered, including reaction time, number of reagents required, and endpoint color. Of the methods, the vitamin C titrations and the 1,10-P colorimetric assay had comparable reaction times, as each endpoint is reached instantaneously once the correct amount of titrant/color reagent is introduced into the reaction mixture. However, the FC method is quite time intensive, and application of heat is needed to allow the reaction to progress more quickly, which would pose a problem on a PAD. Further, of the titrations, the 2,6-D titration required the fewest reagents, as 2,6-D is both the titrant and the indicator, whereas the NBS titration and the iodine titration required additional compounds in order to indicate the endpoint of the reaction. The iodine titration required numerous compounds, with multiple separate reactions occurring in order to acquire the color change necessary to measure the vitamin C concentration. Of the spectrophotometric methods, the 1,10-P used only one reagent to achieve a color change, whereas the Folin-Ciocalteau method used many. In terms of simplicity of the methods, the 2,6-D was best titration, and 1,10-was the best spectrophotometric method due to the lack of a need for multiple reactions to occur, while the iodine titration method and Folin-Ciocalteau method were the most complicated, which would pose problems if transferred to a PAD. The endpoint color of each of the titrations was an important factor to consider, especially if colored beverages would need to be analyzed. Of the vitamin C analysis methods, both the NBS titration and iodine titration have endpoints with dark purple colors, while the 2,6- D titration has a pink endpoint. Depending on the color of the beverage being analyzed, one color of endpoint may be more easily recognized than others. Further, the 1,10-P assay gave

24 18 results that were easily distinguishable with the human eye, which is an important characteristic for application to a PAD, because the results need to be easily ascertained on the PAD. Figure 4 is a picture that demonstrates the clear differences in color between the vitamin C standards that have been titrated according to the 1,10-P method. Figure 4. Photo of 1,10-P Assay Standards (with units of mg vit. C/ L) Overall, it was decided that the 2,6-D titration method was the best vitamin C analysis for application on a PAD. It is accurate within 2.60% of the expected value, poses equivalent safety hazards as the other methods, and is much easier to use than the other vitamin C titration methods, as it requires the fewest reagents. Further, the 1,10-P colorimetric assay was also determined to be a good method for use on a PAD, as it used only one reagent, could be performed quickly, had comparable safety to the other methods, and gave easily distinguishable color changes. Lastly, it was concluded the FC method would pose serious difficulties when being applied to a PAD, specifically the long reaction time and numerous reagents required. By using two separate methods on the PAD, it can measure vitamin C concentrations within two separate ranges. By utilizing the 1,10-P colorimetric assay, the PAD will be able to measure vitamin C in the 2 mg/l to 20 mg/l range, whereas a section of the PAD using the 2,6- D titration will measure vitamin C in the mg/l range.

25 19 Chapter 5 Results and Discussion for PAD Development PAD Heating Analysis: In order to have a functional PAD, the wax channels must be fully embedded through the full depth of the filter paper. Embedment is achieved through adequate heating of the filter paper once the wax channels have been drawn on the surface using a wax pencil. PADs were heated for different times and at different temperatures, then a dye was applied to them in order to test for successful wax embedment. Table 5 shows the times and temperatures of different treatments, accompanied by a picture of the PAD after the dye had been applied.

26 Table 5. Summary of time-temperature treatments of PAD with Picture 20 Treatment Time Temperature Picture After Dye Application Result (min.) ( F) Channels leaked Channels leaked Channels leaked Channels did not leak

27 PAD Production and Tests: 21 The PADs produced in this project used that both 1,10-P method and 2,6-D titration to measure vitamin C. One half of the PAD was for the 1,10-P method, in which three standard reference wells received 2 mg/l, 10 mg/l, and 20 mg/l standards, and a fourth well that received the sample. These standards were chosen to be distinguishable from one another. The other half of the PAD used the 2,6-D titration with a design that allowed a sample solution to simultaneously travel to four wells that contained differing concentrations of 2,6-D. The different concentrations of 2,6-D in the wells are such that one could achieve a titration endpoint and color change at 20 mg/l vitamin C, 100 mg/l vitamin C, 200 mg/l vitamin C, or 300 mg/l vitamin C, depending on the concentration of vitamin C in the sample. Two trials with vitamin C standards were conducted, with a 15 mg/l vitamin C used to test the 1,10-P portion of the PAD, and a 300 mg/l vitamin C standard used to test the 2,6-D portion of the PAD. Pictures of both trial PAD are shown in Figure 5a and Figure 5b. Figure 5a. Trial 1 of PAD Production with Standardized Samples

28 22 Figure 5b. Trial 2 of PAD Production with Standardized Samples These trials show that, when using standardized vitamin C solutions, the PAD successfully measured samples for vitamin C. On the 1,10-P portion of the PAD, the sample well with a 15 mg/l vitamin C stock is noticeably darker in color than the 2 mg/l vitamin C and 10 mg/l vitamin C standard wells, but is lighter than the 20 mg/l vitamin C well. Further, on the 2,6-D portion of the PAD (right), a color change only occurred in the 300 mg/l well, which is expected outcome. This indicates the sample has a vitamin C concentration between 200 and 300 mg/l. Since the expected results were achieved, a sample beverage was analyzed to further show proof of the PADs functionality. White grape juice was chosen to be the sample, as the juice has a light, neutral color that would not interfere with the color changes that occur in the analyses. Pictures of both trial PADs with beverage are shown in Figure 6a and Figure 6b.

29 23 Figure 6a. Trial 1 of PAD Production with a White Grape Juice Sample Figure 6a. Trial 2 of PAD Production with a White Grape Juice Sample These results match the expected results. According to the white grape juice label, the juice had a concentration of 300 mg/l vitamin C. On the 1,10-P portion the sample well is dark and intensely colored, meaning that the concentration of vitamin C is either equal to or greater than 20 mg/l, therefore it must be tested on the 2,6-D portion. In the 2,6-D portion, both PADs showed a color change only in the 300 mg/l meaning that grape juice had a vitamin C

30 concentration between 200 mg/l and 300 mg/l. Therefore, this PAD was successful in 24 analyzing the white grape juice sample.

31 25 Chapter 6 Conclusion In conclusion, a PAD that measured vitamin C concentration in a white grape juice sample utilizing both 1,10-P colorimetric assay was successfully created. Further experiments should study the ability of a similar PAD to measure other beverage samples, especially samples that have a color that could interfere with the ability to accurately read the PAD. Also, it would be helpful to determine the minimum and maximum detectability limits using the quantitative experiments used in this experiment.

32 26 BIBLIOGRAPHY 1. National Institute of Health. Office of Dietary Supplements; Vitamin C, 2. Pandey, K. B., & Rizvi, S. I. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell. Longev, 2009, 2(5), Cai, L.; Wu, Y.; Xu, Xu, C.; Chen, Z. A Simple Paper-based Microfluidic Device for the Determination of the Total Amino Acid Content in a Tea Leaf Extract J. Chem. Educ , Waterhouse, Andrew. "Folin-Ciocalteau Micro Method for Total Phenol in Wine." Waterhouse Lab. University of California, Davis, 8 Aug Chemistry 113 FC Lab Manual, The Pennsylvania State University. 6. Baraket, M.Z., et. al. Anal. Chem. 1955, 27 (4), Chemistry 113 FC Lab Manual, The Pennsylvania State University. 8. Adem, S.M.; Leung, S. H.; Sharpe Elles, L.M.; Shaver L.A. A Laboratory Experiment for Rapid Determination of the Stability of Vitamin C. J. Chem. Educ , Standard Reduction Potentials (in Volts). California State University. 10. DeCaro, J. Laboratory Notebook National Center for Biotechnology Information. PubChem Compound Database; CID=67184,

33 12. National Center for Biotechnology Information. PubChem Compound Database; 27 CID=13726, National Center for Biotechnology Information. PubChem Compound Database; CID=807, Touro University. Material Safety Data Sheets; Folin-Ciocalteau Regeant National Center for Biotechnology Information. PubChem Compound Database; CID=1318, National Center for Biotechnology Information. PubChem Compound Database; CID=17907,

34 ACADEMIC VITA Academic Vita of John DeCaro 709 Plymouth Lane, Ellwood City, PA, Education Major(s) and Minor(s): Food Science Honors: Food Science Thesis Title: Development of a Paper Analytical Device to Measure Vitamin C in Beverages Thesis Supervisor: Dr. Joseph T. Keiser Work Experience May-August, 2016 Food Safety and Quality Assurance Intern Sheetz, Inc., Claysburg, PA Mrs. Chelsie Romberger Grants Received Awards: Frank S. and Nina Cobb Grant-in-Aid (Penn State University Department of Food Science), John Danhouse Martz, Jr. Scholarship (Penn State University College of Agriculture), Beliasov Family Scholarship (Penn State University College of Agriculture) Publications: Vitamin C, Penn State University Chemistry 113 FC Laboratory Manual, Community Service Involvement: Volunteer at Mt. Nittany Medical Center (2015-Present), Tutor at The Central Pennsylvania Institute of Science and Technology (Fall 2015), Language Proficiency: English (native language)