ANALYSIS OF VITAMIN C CONTENT IN
JUICE DRINKS USING HPLC AND
FT-RAMAN SPECTROSCOPY
Rachel Klein and Tasha Magloire
ASCORBIC ACID
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
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
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:
—
•
All standard concentrations of Ascorbic acid and non-spiked juice
samples were tested on the FT-Raman
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
10 ppm
20 ppm
30 ppm
40 ppm
50 ppm
Ret. Time
1.408
1.458
1.483
1.508
1.533
Area
307100
745400
1132929
1606466
2205540
Area %
99.572
81.378
87.917
92.306
99.994
Height
30690
69914
106560
153740
217231
Height %
99.662
90.042
93.522
92.214
99.950
Calibration Curve
2500000
y = 46579x - 197897
R² = 0.9931
2000000
Area
1500000
1000000
500000
0
Table 2. Fruit juice conc.
Orange
Grape
Apple
Cranberry
1.
2.
500 mg spike
50 mg spike
Concentration
49 ppm
23 ppm
12 ppm
26 ppm
0
% Recovery1
42.68%
26.73%
0.42%
3.33%
% Recovery2
120.4%
--109.16%
---
10
20
30
40
Concentration (ppm)
50
60
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
1.600
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)
Mg in Spiked Apple: ppm = mg/ L
mg= (114 ppm)(0.250L) = 28.5 mg
Example calc. of % Recovery (50mg)
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