1. Rachel Klein and Tasha Magloire

2. Chemical structure of Ascorbic acid Ascorbic acid (also known as Vitamin C): – Is a water soluble vitamin – An antioxidant and dietary supplement – Needed for the growth and repair of tissues in the body – Naturally present in some fruits and vegetables – Protects against Scurvy Heart disease Cancer Common cold High blood pressure – In some commercial juices

3.  To evaluate and analyze the Vitamin C content in four commercial fruit juices using Raman scattering and High Performance Liquid Chromatography (HPLC), and construct a calibration plot relating peak area for the ascorbic acid peak against concentration.

4.  Advantage of low sensitivity to water  Has narrow and highly resolved bands  Allows for nondestructive extraction of chemical compositions  Gives physical information about samples and aides in rapid on-line analysis

5.  An efficient and quick way to determine compositions  Simultaneously handles many analytes in a single test  HPLC is highly precise and accurate  Can be used to determine quantities of compositions

6. Materials: Ascorbic acid Ten 250 ml Volumetric flasks Five 100 ml Volumetric flasks Five 50 ml Volumetric flasks Pipettes Fruit juice(s): – Orange – Apple – Grape – Cranberry Instruments: FT-Raman HPLC

7.  Day 1:  Concentrations of Ascorbic acid we made  Ppm = mg/L ----> 1000 ppm = mg/0.250L = 250 mg of ascorbic acid  We made 5 standard concentrations of ascorbic acid  10 ppm – 0.5 ml  20 ppm – 1 ml  30 ppm – 1.5 ml  40 ppm – 2 ml  50 ppm – 2.5 ml

8. Day 2: – Made higher concentrations of our standards 50 ppm – 2.5 ml 100 ppm – 5 ml 150 ppm – 7.5 ml 200 ppm – 10 ml 250 ppm – 12.5 ml — Tested samples of cranberry, apple, grape, and orange juices

9.  Day 3:  Diluted juice samples to obtain clean peaks  10:1 dilutions of juice samples were made (10 ml juice to 100 ml DI water)  25:1 dilutions  We made spiked samples of each brand of juice, adding.5 g of Ascorbic acid to 10 ml of juice diluting with 250 ml of DI water  Lower concentrations of Ascorbic acid standards were made because the peaks weren’t under the calibration curve  10 ppm – 0.5 ml  20 ppm – 1 ml  30 ppm – 1.5 ml  40 ppm – 2 ml  50 ppm – 2.5 ml

10. Day 4: – All standard concentrations of Ascorbic acid and non-spiked juice samples were tested on the FT-Raman Day 5: — All spiked samples of cranberry, apple, orange, and grape juices were ran on the FT-Raman Day 6: — Juice concentrations were changed because the peaks had to be within the calibration curve. We retested two spiked samples of apple juice and orange juice on the HPLC, adding only.05 g of Ascorbic acid to obtain clean peaks that fit under the calibration curve

14. Ret. TimeAreaArea %HeightHeight % 10 ppm1.40830710099.5723069099.662 20 ppm1.45874540081.3786991490.042 30 ppm1.483113292987.91710656093.522 40 ppm1.508160646692.30615374092.214 50 ppm1.533220554099.99421723199.950 Table 1. Standard calibration Table 2. Fruit juice conc. Concentration% Recovery 1 % Recovery 2 Orange49 ppm42.68%120.4% Grape23 ppm26.73%--- Apple12 ppm0.42%109.16% Cranberry26 ppm3.33%--- 1.500 mg spike 2.50 mg spike

15. Ret. TimeAreaArea %HeightHeight % Apple1.56734405475.9344756891.205 Orange1.683207701298.50114893097.449 Grape1.62585358367.68610721974.607 Cranberry1.633102122376.41814217081.828 Spiked Apple1.600652813999.82182478599.860 Spiked Orange1.600436563297.05553658499.110 Spiked Grape1.8832733363491.22599929884.689 Spiked Cranberry1.6751132692099.76299817199.545 Table 3. Summary table of information of fruit juices and fruit juices spiked with ascorbic acid

16. Example calculation of fruit juice concentration Apple: y= 46579x-197897

17. Example calc. of % Recovery (500mg) Example calc. of % Recovery (50mg) Mg in Spiked Apple: ppm = mg/ L mg= (114 ppm)(0.250L) = 28.5 mg Mg in Spiked Apple: mg = (225 ppm)(0.250L)= 56.25 mg

18.  Errors that occurred:  Adding too much ascorbic acid to spiked juice samples created peaks that were outside of the calibration curve  Not diluting the regular juice samples right away created flat-top peaks

19.  Ascorbic Acid was successfully identified in all samples using Raman  Ascorbic Acid was successfully quantified in all samples using HPLC  The concentration of juices were as expected  Percent Recovery was exceptionally lower with 500 mg spiked juices  Percent Recovery was as expected with 50 mg spiked juices