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

4. 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

5. 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

6. 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

7. Methods Day 1: Concentrations of Ascorbic acid we made
Ppm = mg/L ----> 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. 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

9. 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

10. 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

11. Results

12. Results

13. Results

14. 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
87.917
106560
93.522
40 ppm
1.508
92.306
153740
92.214
50 ppm
1.533
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

15. 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
98.501
148930
97.449
Grape
1.625
853583
67.686
107219
74.607
Cranberry
1.633
76.418
142170
81.828
Spiked Apple
1.600
99.821
824785
99.860
Spiked Orange
97.055
536584
99.110
Spiked Grape
1.883
91.225
999298
84.689
Spiked Cranberry
1.675
99.762
998171
99.545

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

17. 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

18. 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

19. 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