Background Information

Vitamin C is one of the essential vitamin needed for human. It is involved in many
processes in the human body including the production of collagen in the connective tissue; the
synthesis of neurotransmitter in the nervous system; and the synthesis of carnitine which is a
vital component in the transfer of energy to the cell mitochondria (Ajmil, 2013). Vitamin C is
also a water soluble vitamin (Bank, 1985).

There is also a research conducted on finding the Vitamin C content in human milk
which produce a result that shows the concentrations of Vitamin C in the milk samples (3.03-
3.52 mg/dl i.e.,30-35 mg/l) of the studied subjects was in agreement with Bangledeshi reference
value (National Research Council, 1989). A similar research is also done in Pakistan by Adeela
Yasmin (2011). Both sources uses titration method that I cannot use for my study because my
experiment requires different temperature from different storing condition which the heat can
lose out to the environment or increase the temperature of the sample if titration method is used.

Through my research, I have decided to use DCPIP solution. The method is taken from
research done by University of Sydney (2011) which uses fruit juices that is inserted into DCPIP
solution by using a syringe. This is because my research uses milk of different temperature and
can be done without releasing too much of its heat content. As vitamin C is a reducing agent, the
DCPIP is reduced forming a colourless compound of DCPIPH2 (VanderJagt, Garry, & Hunt,
1986).

A study done by Rita Mansour and Malak AlJoubbeh (2014) has given the idea to put
another independent variable, which is storage time. Therefore, I will examine the vitamin C
content of milk in two different temperature of storage temperature over a period of time after
the milk packaging has been opened.

Research Question

What is the rate of degradation of vitamin C in milk of two different storing condition after the
packaging has been opened?

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Problem Statement

To compare the vitamin Cs rate of degradation in milk at different temperature of storing

condition of different storage time after opening the milk carton.

Hypothesis

Vitamin C is a water-soluble vitamin, thus an increase in temperature lead the vitamin to be

leached in water. Thus high heat can reduce the amount of Vitamin C because it degrades the
vitamin (Ajmil, 2013). Heat alone will cause some reduction in vitamin C, but not as much as
when combined with loss of nutrients. (Sheehan, 2014).

This is because high heat kills the enzyme ascorbic acid oxidase which is mostly found in fruits
and vegetables (Vitaminable Staffs, 2013). Through the two condition of storage, room
temperature is higher than refrigerator temperature. Increasing exposure to room temperature
will result in the decrement of vitamin C content in milk (Mansour & AlJoubbeh, 2015).
Therefore, as the storage time of milk in room temperature increase, the concentration of
Vitamin C in milk decreases.

Variables

Variables Units Range

Room temperature and
Condition of storage -
INDEPENDENT refrigerator temperature
VARIABLE Hours ( 0.5
Storage time 0 to 96 hours
hours)
Percentage of Vitamin C
% -
DEPENDENT content in milk
VARIABLE Rate of vitamin C
- Gradient from the graph
degradation

Table 1: Independent and Dependent Variables

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 CONTROLLED
No. Units Method for control Possible effect(s) on results
VARIABLES
Only packaged cows milk
Each type of milk has different
1 Type of milk - are taken into account when
nutritional content
conducting the experiment
Different concentration of
A fixed concentration of
Concentration of ascorbic acid will have
2 0.1% ascorbic acid 0.1% is used
ascorbic acid different result on the
in this experiment
percentage of Vitamin C
A fixed volume of DCPIP Different volume may affect
Volume of DCPIP
3 - solution of 5 ml is used in the data of vitamin C
solution
the experiment concentration

Table 2: Controlled Variables

Materials and Apparatus

No. Materials Quantity Volume/Size

1 Milk 2 1 litre
2 Ascorbic acid - 0.1%
3 DCPIP solution - -
4 Syringe with needle 2 5 ml 0.1 ml
5 Specimen tubes 6 -
6 Beaker 1 100 ml 1 ml

Table 3: List of Materials and Apparatus

Method for Collecting Data

1. Measure and determine the volume of 0.1% ascorbic acid solution needed to decolourise
DCPIP solution.
2. Measure and determine the volume of milk needed to decolourise the same volume of
DCPIP solution for different temperature.
3. Calculate the percentage of vitamin C concentration in milk by comparing it with 0.1%
ascorbic acid solution.

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Procedure

1. Label two cartons of milk with A and B. Store both milk one day earlier before starting
the experiment, Milk A in room temperature and Milk B in the refrigerator.
2. The next day, 1 ml of DCPIP solution is inserted in a specimen tube using a syringe.
3. 5 ml of 0.1% ascorbic acid solution is filled into another syringe.
4. Ascorbic acid solution is added drop by drop and gently stirred with the needle of the
syringe. Do not shake the tube vigorously.
5. Ascorbic acid solution is added continuously until the DCPIP solution is decolourised.
6. The volume of ascorbic acid solution used is measured and recorded.
7. Take out Milk A and open it. Observe the time the milk is opened and record as 0 hours
after opened.
8. Repeat steps 1-7 for Milk B.
9. The percentage of Vitamin C content in milk of each storage condition is calculated by
using the following formula :
Volume of 0.1% ascorbic acid used
Percentage of Vitamin C concentration = 0.1
Volume of milk used

10. Repeat all the steps above after 24 hours for 10 days until 10 sets of data are collected.
11. After all the data is collected and calculated, the data is processed to find a regression
line.

Safety and Precaution

1. Carefully handle ascorbic acid solution by wearing gloves

2. Always take measurement at eye level to avoid parallax error
3. Conduct the experiment quickly for each sample as it is temperature sensitive
4. Dispose the sample properly after each repetition to avoid chemical pollution in water
sewage.
5. Use considerable amount of milk for each sample tested to avoid food wastage.

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Data Collection

Volume of ascorbic acid used ( 0.1 ml)

Trial 1 Trial 2 Trial 3 Mean
0.6 0.8 0.6 0.7

Table 4: Raw Data of Volume of Ascorbic Acid Used to Decolourise DCPIP Solution

Time since Volume of Milk A used ( 0.1 ml) Volume of Milk B used ( 0.1 ml)
package opened (
Trial 1 Trial 2 Trial 3 Mean Trial 1 Trial 2 Trial 3 Mean
0.5 hours)
0 3.8 4.0 3.8 3.9 3.4 3.6 3.6 3.5
24 4.0 3.8 4.2 4.0 3.4 3.6 3.8 3.6
48 3.8 3.8 4.0 3.9 3.6 3.8 3.8 3.7
72 4.0 4.4 4.4 4.3 3.8 3.6 3.6 3.7
96 4.6 4.4 4.6 4.5 3.6 3.8 3.8 3.7
120 4.8 4.6 5.0 4.8 3.8 4.0 3.6 3.8
144 5.8 5.4 5.4 5.5 3.8 4.0 3.8 3.9
168 6.0 5.8 6.0 6.0 4.2 3.8 4.0 4.0
192 6.4 6.6 6.6 6.5 4.2 4.2 4.0 4.1
216 7.2 7.0 7.0 7.1 4.2 4.4 4.2 4.3

Table 5: Raw Data of Volume of Milk A and Milk B to Decolourise DCPIP Solution

Observations

The initial colour of DCPIP solution is blue. After the addition of milk into the specimen tube,
the mixture turns into cloudy pale blue colour. The mixture changes completely into white
marking the endpoint of the milk titration into DCPIP solution.

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Data Processing

Time since Mean volume Percentage of Mean volume Percentage of

package of Milk A vitamin C of Milk B vitamin C
opened ( 0.5 used ( 0.1 concentration in Milk used ( 0.1 concentration in Milk
hours) ml) A (x 10-2 %) ml) B (x 10-2 %)
0 3.9 17.95 3.5 20.00
24 4.0 17.50 3.6 19.44
48 3.9 17.95 3.7 18.92
72 4.3 16.23 3.7 18.92
96 4.5 15.56 3.7 18.92
120 4.8 14.58 3.8 18.42
144 5.5 12.73 3.9 17.95
168 6.0 11.67 4.0 17.50
192 6.5 10.77 4.1 17.07
216 7.1 9.86 4.3 16.23

Table 6: Percentage of Vitamin C Concentration in Milk A and Milk B

Example of calculation (data taken for milk A for 0 hours since package opened):

Volume of 0.1% ascorbic acid used

Percentage of Vitamin C concentration = 0.1
Volume of milk used

0.7 ml
Percentage of Vitamin C concentration = 0.1
3.9 ml

Percentage of Vitamin C concentration = 17.95%

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 Graph of Percentage of Vitamin C Concentration in Milk Over Time (Error
Bar Represent Standard Error)
25
Percentage of vitamin C concentration (x 10-2 %)

20 y = -0.0154x + 20.01
r = 0.9738

Concentration
15 of vitamin C in
Milk A (%)

10 y = -0.0411x + 18.92 Concentration

r = 0.9807 of vitamin C in
Milk B (%)

0
0 50 100 150 200 250
Time since milk carton opened (hours)

Figure 1: Graph Shows Percentage of Vitamin C Concentration in Milk Over Time (Error Bar
Represents S.E.)

Figure 1 which is the graph of vitamin C concentration in milk over time is drawn by
using Microsoft Excel. In Graph 1, the Pearsons correlation coefficient, r value generated are
0.9738 for milk A and 0.9807 for milk B. As both value are close to 1, therefore the
concentration of vitamin C in both milks are strongly correlated with the time since the milk
carton is opened.

The rate of change of vitamin C concentration decrement in both milks can be

represented by the gradient of both best fit line. In milk A, the gradient is -0.0154 while in milk
B, the gradient is -0.0411 which is smaller.

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Conclusion

In conclusion, the experiment supported the hypothesis that as the storage time of milk in
room temperature increase, the concentration of Vitamin C in milk decreases.

Through the qualitative data observed, there is a change in colour of the sample from blue
to white. Thus, it indicates that DCPIP solution turns from blue to colourless. This is due to the
redox reaction that occurs between the vitamin C in the milk and the DCPIP molecule itself.
DCPIP acts as the oxidizing agent and vitamin C acts as the reducing agent (VanderJagt, Garry,
& Hunt, 1986). When the milk is added into the specimen tube, vitamin C in the milk reduced
the DCPIP, making it loses its colour. Therefore in this study, it can be said that the reducing
power of vitamin C is utilised to measure the vitamin C concentration in milk for each sample.

Besides, in Figure 1, it is analysed that the error bars generated in each trendline which
represents the standard error do overlaps each other. This representation shows that for each
sample taken between the 24 hours gap, the difference in percentage of vitamin C concentration
is not statistically significant. This is because the data collected are dependent samples. For each
24 hours of sample taken for each milk, the decrement of vitamin C concentration percentage is
dependent on the time taken since the milk cartons has been opened.

Furthermore, the rate of subsequent decrement of vitamin C concentration in milk A is

lower than milk B. This is in coherence with the data analysed by Rita Mansour and Malak
AlJoubbeh (2014) which concluded that increasing exposure to room temperature results in the
higher rate of decrement of vitamin C amount in infant milk. At the initial time since the milk
packaging has been opened of 0 hours, there is a slight difference in the concentration of vitamin
C in both milk. This is due to the different storing condition of 24 hours before the milk
packaging are opened.

According to Corleone (2015), vitamin C degrades over time because it is an unstable

acid compound. Therefore, vitamin C breaks down easily particularly in warm environments.
However by placing the milk in the refrigerator, it only slows down the degradation of vitamin
C.

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 By comparing the data in this study with the research done by Rita Mansour and Malak
AlJoubbeh (2014), the percentage of vitamin C loss in the carton milk of room temperature is
45.07% after approximately 216 hours, which is 10 days. Meanwhile the mean percentage of
vitamin C loss in baby milk powder at room temperature is 47.64% after three weeks. In terms of
processed milk, thus baby milk powder has a lower rate of vitamin C degradation than carton
milk eventhough the preparation of baby milk powder sample by them is done by using warm
water.

Evaluation

The trend of both best fit line drawn clearly shows the result as the correlation calculated
from Pearsons correlation coefficient for both lines are strong. Therefore, it is considerable to
say that there are no anomalies that may detract the data analysed and concluded. Besides, the
range of time taken since the milk carton opened is sufficient enough for it to represent the effect
of storage time on the concentration of vitamin C in milk.

The size of the uncertainty for each sample is 0.1 ml. Since there are three repeat sample
done each day for each type of milks storage condition, the uncertainty does not affect the
validity of the conclusion derived considering that the repetition made has increases the
accuracy. Nevertheless, the readings does not have a high precision due to the apparatus used for
the study which is a syringe.

By taking into account the qualitative data, there is a limitation regarding on to determine
the decolourisation of the DCPIP solution. This random error occurs because of human sight as
each person may view the endpoint decolourisation differently.

A few improvements can be made to this investigation by having a longer period of

storing time for the milk. This will show a more clear trendline of the vitamin C degradation.
Furthermore, a calibrated vessel with a higher precision of 0.01 ml can be used to titrate the
DCPIP solution. Besides, fresh cow milk may be used as the sample rather than carton milk as
they had been pasteurized and undergo food industrial processes, for example the addition of
preservatives which may affect the data collected in this study.

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 For further investigation, it is proposed to conduct a research on the change in pH of milk
over a period of time as pH also contributes to the factor of vitamin C degradation (Corleone,
2015). Furthermore, a study of vitamin C degradation can also be conducted on human milk as
nowadays, more moms prefer to store their milk by using Expressed Breastmilk (EBM)
technique for later use for their baby (NHS, 2014).

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References

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Serum of a Sample of Bangladeshi Mothers. Mal J Nutr 10(1), 1-4.

Ajmil, I. (2013). Effect Of Heating On Vitamin C Content Of Some. INTERNATIONAL

JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 2, ISSUE 11,
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Babycenter. (2013, February 1). Vitamin C in your child's diet. Retrieved June 7, 2015, from
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Bank, M. (1985). Effect of storage time and temperature on folacin and vitamin C levels in term
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Mansour, R., & AlJoubbeh, M. (2015). The effect of Storage Time and Humidity on Vitamin C
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Merriam-Webster. (n.d.). Milk. Retrieved June 8, 2015, from http://www.merriam-

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milk.aspx

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Ozrenk, E., & Inci, O. (2008). The effect of seasonal variation on the composition of cow milk in
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Sheehan, J. (2014, October 14). Does Cooking Food Reduce the Vitamin Content? Retrieved
June 8, 2015, from http://healthyeating.sfgate.com/cooking-food-reduce-vitamin-content-
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University of Sydney. (n.d.). Consumer Chemistry:Vitamin C in Fruit Juices. Retrieved June 10,
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VanderJagt, D. J., Garry, P. J., & Hunt, W. C. (1986, June). Ascorbate in plasma as measured by
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