Rachel Klein and Tasha Magloire Analysis of Vitamin C Content in Juice Drinks Using HPLC and FT-Raman Spectroscopy Rachel Klein and Tasha Magloire

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

Purpose 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.

Why use Raman Scattering? 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

Why use High Performance Liquid Chromatography (HPLC)? 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

Materials and Instruments Used 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

Methods 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

Methods continued… 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

Methods continued… 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

Methods continued… Day 4: Day 5: Day 6: 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

Results

Results

Results

Results Table 1. Standard calibration Table 2. Fruit juice conc. Ret. Time Area Area % Height Height % 10 ppm 1.408 307100 99.572 30690 99.662 20 ppm 1.458 745400 81.378 69914 90.042 30 ppm 1.483 1132929 87.917 106560 93.522 40 ppm 1.508 1606466 92.306 153740 92.214 50 ppm 1.533 2205540 99.994 217231 99.950 Table 2. Fruit juice conc. Concentration % Recovery1 % Recovery2 Orange 49 ppm 42.68% 120.4% Grape 23 ppm 26.73% --- Apple 12 ppm 0.42% 109.16% Cranberry 26 ppm 3.33% 500 mg spike 50 mg spike

Results Table 3. Summary table of information of fruit juices and fruit juices spiked with ascorbic acid Ret. Time Area Area % Height Height % Apple 1.567 344054 75.934 47568 91.205 Orange 1.683 2077012 98.501 148930 97.449 Grape 1.625 853583 67.686 107219 74.607 Cranberry 1.633 1021223 76.418 142170 81.828 Spiked Apple 1.600 6528139 99.821 824785 99.860 Spiked Orange 4365632 97.055 536584 99.110 Spiked Grape 1.883 27333634 91.225 999298 84.689 Spiked Cranberry 1.675 11326920 99.762 998171 99.545

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

Calculations continued… 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    

Errors 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

Conclusion 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