Vitamin C Determination in
Various Fruit Juices Over Time
Andrew Mullenax
Sam Yatzkan
Instrumentation
• UV-Vis
• LC/MS
• HPLC
Procedure
• Samples diluted
– 1:10 (v/v)
• Room Temperature
• Half Opened, Half Closed
• Vacuum Filtered-Samples with visible solids
Samples
Simply
Lemonade
Brand
Dole Brand
Ascorbic Acid Standards
• 5, 10, 20, 30ppm
• Spiked Samples
• Characterized Wavelength: 262nm
• HPLC: Retention Time of ~1.6 minutes
– Solvent Mixture of .50mL/min degassed H2O and Methanol
• LC/MS: Retention Time of ~1.6 minutes
– Solvent Mixture of .75mL/min degassed Methanol and
.25mL/min degassed H2O
Data – UV-Vis
Day 2 Characterized
Wavelength (nm)
Day 3 Characterized
Wavelength (nm)
Day 4 Characterized
Wavelength (nm)
Cran-Apple Juice Closed
255
257
251
Pineapple Juice Closed
276
278
274
White Grapefruit Closed
301
316
298
Lemonade Closed
227
228
226
Cran-Apple Juice Opened
252
253
250
Pineapple Juice Opened
280
284
307
White Grapefruit Opened
200
331
Lemonade Opened
232
281
Samples
Day 1 Characterized
Wavelength
HPLC Calibration Curve
Concentration v. Area
1600000
1400000
y = 40879x + 78172
R² = 0.9867
1200000
Area
1000000
800000
600000
400000
200000
0
0
5
10
15
20
Concentration (ppm)
25
30
35
Data - HPLC
Samples
Day 1 Concentration (ppm)
Day 2 Concentration (ppm)
Day 3 Concentration (ppm)
Day 4 Concentration (ppm)
Cran-Apple Juice Closed
423.0
1094.0
1117.0
1249.0
Pineapple Juice Closed
1101.0
940.0
1171.0
1073.0
White Grapefruit Closed
353.0
354.0
67.0
99.0
Lemonade Closed
69.0
183.0
163.0
262.0
Cran-Apple Juice Opened
1160.0
873.0
781.0
1231.0
Pineapple Juice Opened
1296.0
922.0
563.0
460.0
White Grapefruit Opened
534.0
178.4
64.0
Lemonade Opened
35.0
230.0
36.0
LC/MS Calibration Curve
Concentration v. % Area
25
y = 0.7137x - 0.8725
R² = 0.9585
20
Area (%)
15
10
5
0
0
5
10
15
20
Concentration (ppm)
25
30
35
Data – LC/MS
Day 2 Concentration
(ppm)
Day 3 Concentration
(ppm)
Day 4 Concentration
(ppm)
Pineapple Juice Closed
264.0
220.2
30.7
Lemonade Closed
266.5
291.1
1080.2
Cran-Apple Closed
733.3
126.3
291.9
White Grapefruit Closed
699.5
640.3
246.0
Samples
Day 1 Concentration
(ppm)
Pineapple Juice Opened
542.2
30.7
299.5
Lemonade Opened
300.9
161.1
535.2
Cran-Apple Juice Opened
348.7
203.8
73.5
White Grapefruit Opened
924.4
1268.3
307.2
446.4
Conclusion
• Results not very reliable since we had varying
retention times
• Did not get very good separation in
chromatographs of the samples
• Too difficult to tell whether or not vitamin c
concentration actually decreased over time
Improvements To Be Made
• Better solvent system
• Better/Different Instrument Settings
• Dilute more
• Vacuum Filter All Samples Regardless
References
• Lee, H. S. HPLC Method for Separation and Determination
of Nonvolatile Organic Acids in Orange Juice. JAFC, 1993,
41(11), pp. 1991-1993.
• Nisperos-Carriedo, M. O.; Buslig, B. S.; Shaw, P. E.
Simultaneous Detection of Dehydroascorbic, Ascorbic, and
Some Organic Acids in Fruits and Vegetables by HPLC.
Chem. Anal. 1992, 40(7), pp 1127-1130.
• Silva, F. O.; Total ascorbic acid determination in fresh
squeezed orange juice by gas chromatography. Food
Control, 2005, 16(1), pp 55-58.
• V., K.; D., S.; E., M. Ascorbic acid content of commercial fruit
juices and its rate of loss upon storage. Food Chemistry,
2000, 70(3), pp 325-328.