Sarah Gao
4/8/12
Checkpoint 6
Period 5
The Effect of Storage Time on Vitamin C Content in Oranges and
Orange Juice
Sarah Gao
Takoma Park Middle School 2011-2012
Abstract
It is known that Vitamin C is essential to health and oranges have abundant Vitamin C. My testable
question was: Does Vitamin C stay longer in oranges or orange juice during storage? My hypothesis was: If
oranges and orange juice are stored for 0, 2, 4, and 6 weeks, then the oranges would have more Vitamin C
throughout the time intervals, because oranges have coverings around them that protect Vitamin C from being
degraded. To test Vitamin C, I squeezed juice from oranges, and stored the juice and un-squeezed oranges in a
refrigerator for the above time intervals. Vitamin C was measured by titrating the orange juice and a Vitamin C
standard using the iodine titration and starch indicator solutions. The results showed that for the stored orange
juice, Vitamin C mean concentration was 0.57 mg/mL at week 0, which continuously reduced to 0.53 mg/mL at
week 2, 0.50 mg/mL at week 4, and 0.47 mg/mL at week 6. For oranges, Vitamin C mean concentration was
0.57 mg/mL at week 0 and 0.56 mg/mL at week 2, which increased to 0.59 mg/mL at week 4 and 0.65 mg/mL
at week 6. My hypothesis was supported because throughout the time points, the oranges had more Vitamin C
than the orange juice. Some implications are to drink freshly squeezed juice, while not to drink stored juice after
storing for 4 weeks. Key terms for my study are: iodine solution, starch solution, titration, oxidation, reduction,
Vitamin C, oranges, and orange juice.
Introduction and Review of Literature
Does Vitamin C stay longer in oranges or
orange juice during storage? I am interested in this
topic because Vitamin C is essential to our health [6]
(our body needs it but cannot make it) and it is
important to know how much Vitamin C is left in
the oranges and orange juice that we consume.
From previous experiences, I have learned that
Vitamin C is not very stable, and I know that orange
juice gets contaminated more quickly than oranges.
I have also done some experiments before that
involve chemical reactions which helped me with
this study.
My levels of Independent Variable are 0
(control level), 2, 4, and 6 weeks of storage in the
refrigerator, with 0 weeks being freshly squeezed
oranges. I chose these levels because 0-6 weeks is
how long people would usually keep oranges and
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orange juice before they are consumed. Also, these
levels provide a time window to observe how much
Vitamin C is lost between each time interval. The
Dependent Variable is the concentration of Vitamin
C in oranges and orange juice at each time interval
(0, 2, 4, and 6 weeks). The units I measured in are
milliliters. To measure the Dependent Variable, I
added starch solution to the orange juice in a flask,
and filled a pipette with the iodine titrating solution
using a ball filler dispenser. I added the iodine
solution drop by drop into the flask containing
orange juice and starch solution, and swirled the
flask every time. When the solution turned greenish
brown, I knew that all of the Vitamin C had been
oxidized and the iodine started to react with the
starch. The volume of the iodine solution used to
The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
titrate the Vitamin C was the measure of the
One previous study was performed by
Dependent Variable.
Christopher W. Allen on April 2, 2005, entitled
One scientific principle that I used to
“Cooking Away the Vitamins.” [1] This experiment
measure Vitamin C was oxidation-reduction
was testing Vitamin C content in broccoli under
[3]
reaction (redox reaction) . Redox reaction
different cooking conditions. An iodine (a bluish
happens when an electron or electrons transfer from
black element of the halogen group that transfers
one atom to another in a chemical reaction. When
into a violet irritating gas) and starch-based Vitamin
iodine receives electrons from Vitamin C, iodine is
C indicator solution was made, which turned from a
reduced to iodide ions, and Vitamin C is oxidized.
dark blue to a lighter blue in the presence of more
Iodine can react with starch, and turn it a blue color,
Vitamin C. Using different concentrations of
but in its reduced form, iodide ions, it cannot.
Vitamin C supplement powder in water, and adding
However, when all of the
a constant amount of each to
Vitamin C is oxidized, the
tubes of indicator solution,
Filler Button
iodine is free to react with
five different colors from
Dispenser Button
the starch, turning it to a
lightest to darkest blue were
blue color. The amount of
made. The amount of Vitamin
Vitamin C is measured by
C in each was: 2.4 mg/ml
the milliliters of iodine
water, 2.2 mg/ml, 2.1 mg/ml,
added to turn the solution
1.9 mg/ml, and 1.7 mg/ml.
blue. Another scientific
Four ounces of broccoli were
principle is the titration
then boiled, drained,
Pipette
method [5]. Titration is the
processed with 100-ml water,
process of chemical analysis
and the juice was strained.
when the quantity of an
The juice was added drop by
unknown sample is
drop to indicator solution until
determined by adding to it a
it matched the color produced
measureable amount of
by the 2.2 mg/ml water
another substance that will
concentration, using the other
react with the sample. In my
concentrations to aid in the
experiment, I titrated
matching. A formula was used
Vitamin C to determine its
to find the mg/oz of Vitamin
amount by adding
C in the sample of broccoli.
measureable amounts of
Data was recorded. This
iodine. An additional
process was repeated with the
scientific principle is that
other cooking methods and
bacteria can decompose
the control of raw broccoli,
Vitamin C. This principle is
ten trials each. Results show
supported by the classical
that all of the cooking
Figure 1: Ball filler dispenser attached to a
study performed in 1943. [7]
conditions caused Vitamin C
pipette
In my experiment, over the
reduction. Boiling caused
storage time, the Vitamin C
Vitamin C to reduce the most.
content reduced in the orange juice, which might be
The next was microwaving, and steaming reduced
caused by bacteria decomposition.
Vitamin C content the least. Factors that cause this
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The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
are the water-solubility (capability of being
dissolved) and high reactive properties of Vitamin
C. This experiment relates to my study because I
was also using iodine solution and starch indicator
solution to test the amount of Vitamin C.
Another previous study was conducted by
Irfan S. Habib on April 2, 2009, entitled “Vitamin C
Content: Analysis of Food by Titration.” [2] This
experiment was testing the Vitamin C content in
different fruits and vegetables of different
conditions. A standard vitamin C solution was
titrated (the process of chemical analysis when the
quantity of an unknown sample is determined by
adding to it a measureable amount of another
substance that will react with the sample) with
iodine (starch used as indicator) until endpoint.
Titrations were done to determine concentration of
vitamin C in various fruits and vegetables.
Temperature effect was investigated by titrating
boiled, then frozen orange juice. The following
equation was used to find the content of vitamin C
in all solutions tested: ?mg of vitamin C / # of
iodine drops = 25mg of Vitamin C / # of drops of
iodine. The results show that temperature does
affect vitamin C content. Boiling almost destroyed
it, but freezing had little effect. Also, strawberry
juice had most Vitamin C. Cauliflower, orange and
green pepper juice had close amounts. Pear and
plum juice had the least amount of Vitamin C. My
experiment relates to this study because I was also
using iodine titrating solution to titrate and using
starch indicator solution as an indicator. Also, I was
testing Vitamin C in oranges and orange juice, and
determined which had more Vitamin C.
The last previous study was done by Sarah
L. Pilegard, on April 2, 2008, entitled “Which One
Has More Vitamin C, Freshly Picked and Squeezed
or Store Bought Orange Juice?” [4] This experiment
was testing the Vitamin C contents in freshly picked
and squeezed orange juice and store bought orange
juice. An indicator solution was used which gave a
color variation when orange juice was added. A
color scale was developed to judge each test. The
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indicator solution consisted of cornstarch, water and
iodine. That was put in a test tube and orange juice
was added. If the resulting color stayed dark, that
indicated there was less Vitamin C in that juice, but
if the resulting color was lighter than the indicator
solution, then there was more Vitamin C in that
juice. The juice was sampled from 18 separate
oranges from the same tree and 18 separate bottles
of orange juice from the same brand of juice. The
results were that the fresh orange juice had more
Vitamin C than the store bought orange juice. The
fresh orange juice averaged lighter on the color
scale than the store bought, which shows that the
fresh orange juice has more Vitamin C. My
experiment relates to the previous study because I
was also using iodine and starch, and testing which
kind of orange juice, freshly squeezed or stored
juice, had more Vitamin C.
I designed my investigation based on the
background information and previous studies in a
few ways. From previous studies, I know that
Vitamin C is not stable. Also, previous studies
helped me develop levels of Independent Variable
and Dependent Variables. From background
information, I know that a protective covering
around an orange helps Vitamin C to degrade more
slowly. Also, I know that oranges and orange juice
are usually kept for 0-6 weeks before consuming, so
it is important to know how much Vitamin C is lost
in those weeks.
The hypothesis of my study is: If oranges
and orange juice are stored in the refrigerator for 0,
2, 4, and 6 weeks, then the oranges would have
more Vitamin C throughout the time intervals,
because the oranges have a covering around them
that would protect Vitamin C from being degraded.
From previous experiences, I know that orange
juice gets contaminated with germs fast, but oranges
can be stored for a longer time. Since bacteria can
decompose Vitamin C, I think that Vitamin C is
likely going to degrade faster in orange juice than in
oranges.
The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
Materials and Methods
color in the solution that lasted for longer than 20
seconds. I wrote down the final volume of the
iodine solution in the pipette in a notebook with a
pencil. I repeated the titration of the Vitamin C
standard solution five times at each time that I made
Vitamin C measurements of the juice samples.
I bought 20 California Seedless Oranges and
stored them in a refrigerator. To make orange juice,
I peeled off the skin from the oranges and put the
skinless oranges in a blender to blend for 10
seconds. I poured everything in the blender onto
cheesecloth already laid out on a bowl. I brought the
ends of the cheesecloth together to make a pouch
and I squeezed out the juice into the bowl. I made
sure that no pulps were left in the juice. At the time
interval of 0 weeks, I squeezed out juice from ten
oranges (500 mL). I poured out 100 mL for testing
Vitamin C content as a starting point for both
testing groups, oranges and orange juice. The rest of
the orange juice (400 mL) was stored in an empty
store-bought orange juice container and put in the
refrigerator for further testing Vitamin C content in
orange juice stored for 2, 4, and 6 weeks. To test the
Vitamin C content in stored oranges at the time
intervals 2, 4, and 6 weeks, I squeezed out juice
from three oranges (100 mL) into a measuring cup
at each time interval.
One of my controlled variables was the
same brand and the same box of oranges (California
Seedless Oranges), assuming that the oranges were
picked and stored roughly at a similar time, and
have a similar content of Vitamin C. Another
controlled variable was keeping the oranges and
orange juice at the same temperature, so that the
variation of Vitamin C among the samples was not
a result of temperature. The same brand of iodine,
Iodine Tincture USP First Aid Antiseptic, and the
same preparation procedure were used to make the
iodine titration solution. To keep the experimental
system consistent, I also used the same amount of
juice and the same type and amount of starch
indicator solution for each testing.
I prepared an iodine titration solution, a
starch indicator solution, and a Vitamin C standard
solution by the following procedures. For the iodine
titration solution, I diluted the Iodine Tincture USP
First Aid Antiseptic iodine in a 1 to 10 ratio, by
pouring 30 mL of the iodine into a 500 mL glass
bottle and adding 270 mL of distilled water using a
500 mL graduated cylinder to bring the total volume
to 300 mL. I tightly covered the bottle, wrote
“Iodine Titration Solution” on the bottle with a
marker, and stored it in a dark location. I made this
solution every time interval that I made Vitamin C
measurements. To make the 0.5% starch indicator
solution, I added 1 g of Cock Brand Tapioca starch
to 200 mL of distilled water in a 500 mL flask. I
boiled it, stirred it, and allowed it to cool. When
cooled, I poured the solution into a 500 mL glass
bottle, labeled it “Starch Indicator Solution” with a
marker, and stored it in an undisturbed location. I
also made this solution every time interval. For the
Vitamin C standard solution, I dissolved a 500 mg
Vitamin C tablet in 500 mL of distilled water in a
500 mL graduated cylinder. I made this standard
solution each time that I made Vitamin C
measurements at 0, 2, 4, and 6 weeks.
To titrate the Vitamin C standard solution, I
measured 20 mL of Vitamin C standard solution
into a clean 50 mL Erlenmeyer flask using a 10 mL
pipette attached to a ball filler/dispenser. I used a 1
mL plastic transfer pipette to add 10 drops of starch
indicator solution into the flask. I filled another 10
mL pipette with the iodine titration solution to the 0
mark on the pipette using a ball filler/dispenser. I
placed the Erlenmeyer flask (containing the Vitamin
C standard and starch indicator solutions) under the
pipette and added the iodine titration solution drop
by drop into the flask by pressing on the dispenser
button (see Figure 1). I swirled the flask to mix the
iodine solution and the Vitamin C standard and
starch solutions together after each addition of the
iodine titration solution. The titration was complete
when the iodine titration solution created a blue
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Page 4 of 8 The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
To measure the Vitamin C concentration in
the mean concentration to 0.50 mg/mL and at week
both groups, oranges and orange juice, I used a 10
6, reducing the mean concentration to 0.47 mg/mL.
mL pipette attached to a ball filler dispenser to
However, the trend was different for the oranges. At
measure 20 mL of the orange juice from either
week 0, the mean concentration was 0.57 mg/mL.
group into a 50 mL Erlenmeyer flask. I added 10
At week 2, the mean concentration was 0.56
drops of starch indicator solution with a 1 mL
mg/mL. Considering the standard deviation (Figure
plastic transfer pipette to the Erlenmeyer flask. I
2), this mean concentration is not significantly
filled another 10 mL pipette with iodine titration
different from the mean concentration at week zero.
solution to the 0 mark on the pipette using the ball
Surprisingly, the mean concentration of Vitamin C
filler dispenser. I placed the Erlenmeyer flask
increased at week 4 to 0.59 mg/mL and at week 6 to
(containing the orange juice and starch indicator
0.65 mg/mL.
solution) under the pipette and added the iodine
titration solution drop by drop. I
DV: Amount of Vitamin C (mg/mL)
IV:
swirled the flask to mix in the iodine
Sample
Mean ±
Time
Trial Trial Trial Trial Trial
titration solution after each addition.
Type
Standard
Intervals
1
2
3
4
5
The titration was complete when the
Deviation
iodine titration solution created a
0 weeks 0.57 0.56 0.57 0.56 0.57 0.57 ± 0.01
greenish brown color change in the
2 weeks 0.57 0.56 0.56 0.56 0.56 0.56 ± 0.00
orange juice/starch indicator solution. Orange 4 weeks 0.59 0.59 0.59 0.59 0.59 0.59 ± 0.00
I recorded the final volume of the
6 weeks 0.65 0.65 0.65 0.65 0.65 0.65 ± 0.00
iodine titration solution in the pipette
0 weeks 0.57 0.56 0.57 0.56 0.57 0.57 ± 0.01
in a notebook with a pencil. The
Orange 2 weeks 0.54 0.54 0.52 0.52 0.52 0.53 ± 0.01
difference between the initial and the
Juice
4 weeks 0.50 0.50 0.50 0.49 0.49 0.50 ± 0.01
final volumes was the volume used to
6 weeks 0.47 0.47 0.47 0.47 0.47 0.47 ± 0.00
titrate the sample. I repeated the
titration of the orange juice five
Figure 2: The Effect of Storage Time on the Vitamin C Content in
times for each time interval, 0, 2, 4,
Oranges and Orange Juice
and 6 weeks for each group,
oranges or orange juice. The Vitamin C
concentration was calculated using the formula: X=
Discussion and Analysis
Vsample (mL) / Vstandard (mL). Vsample was the volume
My data showed different trends for the
of iodine titration solution used to titrate the orange
change of Vitamin C concentration in orange juice
or orange juice samples. Vstandard was the volume of
and oranges. For the orange juice, the Vitamin C
the iodine titration solution used to titrate the
concentration decreased continuously at each time
Vitamin C standard solution. I used the GraphPad
point, weeks two, four, and six. For the oranges, the
Prism Software to calculate the standard deviation.
Vitamin C concentration remained almost the same
[8]
Results
at week two compared to week zero, and then
increased continuously at weeks four and six. This
increase of Vitamin C concentration in oranges
most likely happened because of loss of juice
volume in the oranges. When doing the experiment,
oranges in weeks four and six showed clear signs of
The results are shown in Figures 2 and 3.
For the orange juice, the mean concentration of
Vitamin C was 0.57 mg/mL at week 0. At week 2,
the Vitamin C mean concentration reduced to 0.53
mg/mL. This trend continued at week 4, reducing
Name of Journal
Page 5 of 8 The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
Mean Vitamin C Concentration (mg/mL)
dehydration. The Vitamin C content
might have remained the same in the
oranges, but the dehydration might
have caused Vitamin C concentration
to increase. This didn’t happen to the
orange juice, however, because the
0.70
orange juice volume did not decrease,
but the Vitamin C content decreased.
0.60
Throughout the whole
experiment, my hypothesis was
0.50
supported by my data, which showed
that the oranges had more Vitamin C
0.40
than orange juice. At week zero, the
oranges and orange juice both started at
0.30
a Vitamin C mean concentration of
Orange
0.57 mg/mL. At 2 weeks, the mean
Orange Juice
0.20
concentration of Vitamin C in oranges
was 0.56 mg/mL and the mean
0.10
concentration for orange juice was 0.53
mg/mL. At 4 weeks, the mean
0.00
concentration of Vitamin C in oranges
Week 0
Week 2
Week 4
Week 6
was 0.59 mg/mL and the orange juice
had a mean concentration of 0.50
Storage Time
mg/mL. At 6 weeks, the mean
concentration of Vitamin C in oranges
Figure 3: The Effect of Storage Time on Vitamin C Content in
was 0.65 mg/mL and the mean
Oranges and Orange Juice
concentration of orange juice was
0.47 mg/mL. At weeks 2, 4, and 6, the
degrade Vitamin C. I observed that the orange juice
oranges always had a greater Vitamin C mean
had a gloppy substance at the bottom of the
concentration than orange juice.
container at weeks four and six, most likely due to
My results relate to my original purpose,
bacteria contamination. This happened during the
which was to test whether there would be a higher
time that Vitamin C was reduced, which suggested
Vitamin C concentration in oranges or orange juice
that the degrading of Vitamin C in orange juice was
during storage. From this experiment, I found out
due to bacteria contamination. My results also
that oranges had a higher concentration of Vitamin
related to the previous studies. My results and other
C than orange juice during storage.
scientists’ results all agreed that Vitamin C is not
My results from this experiment relate to my
stable. Also, my results showed that the redox
previous main scientific principles. One way that
reactions and titration process are reproducible in
my results related to my main scientific principles is
different experiments.
that I applied the redox reaction and the titration
There were a few sources of error in my
principles to my experiment, and they helped me to
experiment. One error was rounding the mean
achieve expected results. Also, the orange juice
concentration for each time point. For example, for
results related to the principle that bacteria can
Name of Journal
Page 6 of 8 The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
the oranges week zero actually had 0.567 mg/mL,
but was rounded up to 0.57 mg/mL. The second
week actually had 0.562 mg/mL, but was rounded
down to 0.56. However, when considering the
standard deviation, these two numbers are not
significantly different. Another source of error was
the difficulty to control the titration process to
produce the exact same color in each trial. In some
trials, the green-brown color was a little lighter or
darker than the others. If the color was lighter, it
would seem like the oranges or orange juice had
less Vitamin C, and if the color was darker, it would
seem like the oranges or orange juice had more
Vitamin C.
A real world ramification would be not to
store orange juice for more than up to four weeks
because the Vitamin C content is reduced by about
12% from week zero and the potentially
contaminating bacteria may cause health problems.
From this experiment, it can be applied that Vitamin
C in oranges is much more stable than in orange
juice, and oranges usually do not get bacteria
contamination, so squeezing juice from stored
oranges is much better for health than stored orange
juice. Finally, a future research related to these
results would be experimenting whether oranges
really lose Vitamin C, by testing whether the
percent of dehydration is equal to or different from
the percent of increased Vitamin C concentration.
There are a few further related experiments
that I could perform. One experiment could be
testing which fruit has the most Vitamin C, and
another testing which vegetable has the most
Vitamin C. Also, I could test which storage
container would keep the oranges to maintain the
most Vitamin C.
References
1. Allen, C. W. (2005, April 2). Cooking Away
the Vitamins. In California State Science
Fair 2005 Project Summary. Retrieved from
http://www.usc.edu/CSSF/History/2005/
Projects/J0501.pdf
2. Habib, I. S. (2009, April 2). Vitamin C
Content: Analysis of Food by Titration. In
California State Science Fair 2009 Project
Summary. Retrieved from
http://www.usc.edu/CSSF/History/2009/
Projects/J0410.pdf
3. Oxidation-reduction Reaction. (2011).
Retrieved November, 2011, from
http://www.britannica.com/EBchecked/
topic/436636/oxidation-reduction-reaction
4. Pilegard, S. L. (2008, April 2). Which One
Has More Vitamin C, Freshly Picked and
Squeezed or Store Bought Orange Juice? In
California State Science Fair 2008 Project
Summary. Retrieved from
http://www.usc.edu/CSSF/History/2008/
Projects/J1818.pdf
5. Titration. (2011). Retrieved November 16,
2011, from Encyclopedia Britannica
website: http://www.britannica.com/
EBchecked/topic/597325/titration
6. Vitamin C. (2011). Retrieved November,
2011, from Encyclopedia Britannica
website: http://www.britannica.com/
EBchecked/topic/631079/vitamin-C.
Acknowledgements
I would like to thank Ms. Epling, who
helped me do this project by correcting and grading
each of my checkpoints. Also, I would like to thank
my family for helping and encouraging me
throughout the experiment.
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Page 7 of 8 The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice
Sarah Gao
4/8/12
Checkpoint 6
Period 5
7. Young, R. M., & Rettger, L. F. (1943,
October). Decomposition of Vitamin C by
Bacteria [Scientific Research Paper].
Retrieved from
http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC373828/?page=1
8. http://www.graphpad.com/prism/prism.htm
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The Effect of Storage Time on Vitamin C Content in Oranges and Orange
Juice