PEPERIKSAAN SIJIL TINGGI PERSEKOLAHAN MALAYSIA

(MALAYSIA HIGHER SCHOOL CERTIFICATE EXAMINATION)

CHEMISTRY
PROJECT REPORT
STPM 2014
PAPER 4 (962/4)

TITLE:
THE INVESTIGATION OF THE CONCENTRATION OF
VITAMIN C IN ORANGE JUICE UNDER DIFFERENT
CONDITION
By

Lubna Binti Mohd Khalid (940728-10-5658)
Class of 6 Azam (Atas)

ABSTRACT


Orange is a good source of Vitamin C. Its Vitamin C content is about 50 mg per 100 g of
edible portion of the orange fruit. However, Vitamin C in orange juice is quietly degradable due
to oxidation. The main factors that affect Vitamin C loss is heat, light and condition of storage.
Therefore, this experiment is to determine under what condition will affect more the oxidation
process of Vitamin C. Sunkist is chosen because it has relatively high content of juices and
Vitamin C , thus will produce large amount of juices needed in this experiment. Acid-base
titration process by using Potassium Iodide , KI is chosen as it is more simpler and cheap rather
than by High Performance Liquid Chromatography [HLPC] process and by spectrophotometric
process which require more advanced technology and apparatus. The experiment is conducted
firstly by preparation of chemical properties such as 0.50mol of starch solution and diluting the
concentrated Potassium Iodide, of 1.00M into 0.05 M. Orange juice is peeled and filtered by
a clean cloth to remove pulps and seeds and then diluted by distilled water until 10.00ml. The
orange juice is then mixed with starch solution (as an indicator) then titrated with KI. The
mixture is left to be titrated until blue-black solution is observed. The end value of KI is recorded
and calculated in order to know the concentration of Vitamin C in various conditions. From the
experiment, it is found that freshly squeezed orange juice has the highest concentration of
Vitamin C and followed by heated, refrigerated and in room temperature juices. There are also
some other factors that needed to be considered as the results obtained is different from the pasts
research. In a nutshell, the highest concentration of Vitamin C is promoted by the freshly
squeezed orange juices as it is less time in oxidation to occur thus increase the diet intake of
Vitamin C in a human.

I NTRODUCTI ON

The orange is the fruit of the citrus species Citrus sinensis in the family Rutaceae. The
fruit of the Citrus sinensis is considered a sweet orange, whereas the fruit of the Citrus
aurantium is considered a bitter orange. The orange is a hybrid, possibly between pomelo (Citrus
maxima) and mandarin (Citrus reticulate ), which has been cultivated since ancient times.
As of 1987, orange trees were found to be the most cultivated fruit tree in the
world. Orange trees are widely grown in tropical and subtropical climates for their sweet fruit.
The fruit of the orange tree can be eaten fresh, or processed for its juice or fragrant peel. As of
2012, sweet oranges accounted for approximately 70% of citrus production. In 2010, 68.3
million metric tons of oranges were grown worldwide, production being particularly prevalent
in Brazil and the US states of California and Florida.
[1]

Vitamin C is an essential nutrient for humans and certain other animal species.

Vitamin C
refers to a number of vitamers that have Vitamin C activity in animals, including ascorbic acid
and its salts, and some oxidized forms of the molecule like dehydroascorbic acid. Ascorbate and
ascorbic acid are both naturally present in the body when either of these is introduced into cells,
since the forms interconvert according to pH. Vitamin C deficiency can lead to a disease called
scurvy, which is characterized by abnormalities in the bones and teeth. Many fruits and
vegetables contain Vitamin C, but cooking can destroys the vitamin.
Historically, Vitamin C was used for preventing and treating scurvy. Scurvy is now
relatively rare, but it was once common among sailors, pirates, and others who spent long
periods of time onboard ships. When the voyages lasted longer than the supply of fruits and
vegetables, the sailors began to suffer from vitamin C deficiency, which led to scurvy. It is then
discovered by Linus Pauling that Vitamin C has the antimicrobial and antioxidant that can
prevent scurvy hence giving its scientific name is ascorbic acid, which literally translated as
anti-scurvy acid.
[2]

Ascorbic acid is important as it is needed to make collagen, the glue that strengthens
many parts of the body, such as muscles and blood vessels. It also plays an important role in
wound healing and as natural antihistamine. Ascorbic acid also aids in formation of liver bile and
helps to fight viruses and to detoxify alcohol and others. Ascorbic acid is also an antioxidant that
is required for tissue growth and repair, adrenal gland function, and healthy gums. It enhances
immunity, protects against bruising and also aids in interferon production. Ascorbic acid works
well with Vitamin E.
One way to determine the amount of vitamin C in food is to use a redox titration. The
redox reaction is better than an acid-base titration since there are additional acids in a juice, but
few of them interfere with the oxidation of ascorbic acid by iodine. As the iodine is added during
titration, the ascorbic acid is oxidized to dehydroascorbic acid, while the iodine is reduced to
iodide ions.

Ascorbic acid +

+ Dehydroascorbic acid

Due to this reaction, the iodine formed is immedietly reduced to iodide as long as there is
any ascorbic acid present. Once all the ascorbic acid has been oxidized, the excess iodine is free
to react with the starch indicator, forming the blue-black starch-iodine solution. This is the
endpoint of the titration. The method is suitable used with Vitamin C tablet, fresh or packaged
fruit juices and solid juices and vegetables.
[7]

Figure 1: The structure of vitamin C.

LI TERATURE REVI EW

In recent research studies, the healing properties of oranges have been associated with a
wide variety of phytonutrient compounds. These phytonutrients include citrus flavones (types
of flavonoids that include the molecules hesperetin and naringenin), anthocyanins,
hydroxycinnamic acids and a variety of polyphenols. When these phytonutrients are studied in
combination with oranges-vitamin C, the significant antioxidant properties of this fruit are
understandable.
But it is yet another flavanone in oranges, the herperidin molecule, which has been
singled out in phytonutrient research on oranges. Arguably, the most important flavanone in
oranges, herperidin has been shown to lower high blood pressure as well as cholesterol in animal
studies, and to have strong anti-inflammatory properties. Importantly, most of this phytonutrient
is found in the peel and inner white pulp of the orange, rather than in its liquid orange center, so
this beneficial compound is too often removed by the processing of oranges into juice.
In Australia and New Zealand, the recommended daily intake (RDI) of Vitamin C is 60
mg. The Nobel-prize winning scientist, Linus Pauling (1901 1994), believed in regular mega
doses of vitamin C, but this is still regarded as unorthodox in conventional medicine. Vitamin C
is often used as an antimicrobial and antioxidant in foodstuffs. It was first isolated in 1928 and in
1932 it was proved to be the agent which prevented scurvy (hence its scientific name of
ascorbic acid, which literally translates as anti-scurvy acid). Its structure was determined in
1933 and confirmed by total synthesis soon after.
Ascorbic acid is needed to make collagen, the glue that strengthens many parts of the
body, such as muscles and blood vessels. Ascorbic acid also plays important roles in wound
healing and as a natural antihistamine. This ascorbic acid also aids in the formation of liver bile
and helps to fight viruses and to detoxify alcohol and other.
Ascorbic acid is an antioxidant that is required for tissue growth and repair, adrenal gland
function, and healthy gums. It enhances immunity, protects against bruising and promotes the
healing of wounds, also aid in interferon production. Ascorbic acid works well with vitamin E.
Antioxidants play an important role in manufacturing, packaging and storing fats and fatty
foods. Numerous compounds, both synthetic and natural in origin, have been developed as
antioxidants for food preservation, but only tert-butyl-4-hydroxyanisol (BHA) and tert-butyl-4-
hydroxytoluene (BHT) and tocopherol is natural antioxidants are practically used. However, the
most widely used antioxidants, BHA and BHT, are suspected of causing liver damage. On the
other hand, tocopherols are widely used as safe natural antioxidants, but they are not as effective
as synthetic antioxidants and the manufacturing cost is high. These circumstances stimulated the
isolation of an antioxidant from natural sources, especially from plant materials.














PROBLEM STATEMENT

Nowadays, people love to consume orange juice right after it is refrigerated. Some like to
warm up their drink in a microwave first and some are likely doing both. Is this act making the
concentration of Vitamin C remains the same when it is freshly squeezed? What is the condition
that will decrease the concentration of Vitamin C and how it can affect the humans health?


OBJ ECTI VE

To determine the concentration of vitamin C under different concentration.
To determine the effect of lack of vitamin C towards human.









METHODOLOGY

There are various method to determine the concentration of Vitamin C in an orange fruit,
which are:
1- Titration method
2- High Performance Liquid Chromatography [HPLC]
3- Spectrophotometric method
We are using the titration method to determine the concentration of Vitamin C because it is
cheaper and simpler than by using the HPLC and Spectrophotometric method.

APPARATUS

The list of apparatus used in these investigations are burette and stand with clamp, 250.00 ml
round bottom flask, 100.00 ml, 200.00 ml volumetric flask, oven, 10.00 ml pipette , filter funnel,
10.00 ml and 100.00 ml measuring cylinder, 250.00 ml and 500.00 ml beaker, dropper,
electronic balance, knife, plank.

MATERI ALS

The list of materials used in these investigation are 0.50mol of starch solution, diluted 0.05
moles of Potassium Iodide (KI), distilled water, orange juices (freshly squeezed, heated,
refrigerated, room temperature).


PROCEDURE
Sample Preparation
For fresh fruit juice: Squeeze the juice through a clean cloth to filter the pulp and seeds.
Solution Preparation
I odine solution: 1.00mol of iodine diluted with distilled water to 0.05mol concentration.
Starch solution: 0.50 gram of soluble starch is weighed on an electronic balance and added into
50.00 ml of near boiling water in a 100.00 ml conical flask. Stir the solution to dissolve and let it
cool.

1.) For freshly squeezed orange juice.
Fill the burette with 50.00 ml 0.05 moles of iodine solution. Then clamp the burette on
the retort stand. Put 10.00 ml of the sample in a 100.00 ml conical flask and add 75.00 ml of
distilled water. Add a few drops of starch solution into the conical flask. Place the conical flask
below the burette on top a white tile. Titrate the sample with 0.05 moles of iodine solution. The
end point of the titration is identified as the first permanent trace of a dark blue-black colour due
to the starch iodine complex. Repeat the titration until a concordant result obtained.

2.) For heated orange juice.
The orange juice sample is heated up in an oven for 5 minutes with 150
o
C. The
experiment is repeated the same as for freshly squeezed orange juice.

3.) For room temperature orange juice.
The ascorbic acid of orange juice was left at the room temperature 30 minutes. After that, the
procedure, the procedure was exactly the same as the experiment for freshly squeezed condition.
4.) For cold orange juice.
The ascorbic acid of orange juice was left in refrigerator to freeze for 15 minutes. After
that, the procedure is exactly the same as the experiment for freshly squeezed condition.




















OBSERVATI ON, RESULTS AND DI SCUSSI ON


Type of
condition
Reading of the volume if

solution,
( 0.10 )ml
Average
volume of

,
(0.10)ml
1 2 Average
Fresh 26.30 26.30 25.30 26.00
Heat 20.70 23.10 21.70 21.80
Cold 21.50 21.50 21.50 21.50
Room
Temperature
20.70 20.70 21.50 21.00
Table 1


CALCULATI ON

Reaction:
Ascorbic acid +

+ Dehydroascorbic acid
1.00mol of

react with 1.00mol of Ascorbic acid.

Molar mass of ascorbic acid (

) = 176.12 g

[1 gram per mole = 1000 milligram per mole]



Determining of the sample of freshly squeezed orange juice :

26.00cm
3
0.05M KI solution contains 0.05 x 0.026mol of KI.
Therefore, 10.00 ml of orange juice contains 0.05 x 0.026mol of Vitamin C.
Therefore, 10.00 ml of orange juice contains 0.05 x 0.026 x 176.12 g of Vitamin C .
Therefore, 10.00 ml of orange juice contains 0.228956 g = 228.956 mg of Vitamin C.
Therefore, content of Vitamin C in freshly squeezed orange juice =

= 22.8956 mg/ml.
= 22.90 mg/ml (2 d .p)

The calculation is then repeated to the sample of orange juice when heated, refrigerated and left
in room temperature. The calculation is then tabulated and analyzed.

Conditions
Content of Vitamin C in fresh
orange juice by experiment,
(mg/ml)
Content of Vitamin C in
fresh orange juice by
theoretically, (mg/ml)
Fresh 22.90 24.85
Heat 19.20 18.40
Refrigerated 18.93 21.20
Room temperature 18.49 23.22
Table 2



DI SCUSSI ON

The determination of Vitamin C by using titration method is used as it is simpler than
the chromatography and spectrophotometric process and easy to get the apparatus. From the
experiment, the highest concentration of Vitamin C is from the freshly squeezed by 22.90mg/ml
because it is less in time for the juice to be oxidized. The heated orange juice which is
19.20mg/ml is higher than refrigerated which is 18.93mg/ml and in room temperature which is
only by 18.49mg/ml.
In some of past research studies, the concentration of Vitamin C when freeze is higher
than the heated and left in room temperature. High temperature will speed up the oxidation
process. From the already conducted experiment, the lowest concentration of Vitamin C is from
the heated samples as the rate of decay is by 10% loss per day rather than the rate of decay of
refrigerated sample that only 3% per day.
[5]
The experiment we conducted is affected by some
external factors such as contamination by other food substances and microorganisms of the
environment inside of the refrigerator. From this experiment, we can analyzed that if someone is
practicing to cold the orange juice right after it is being squeezed, they actually consume less of
Vitamin C than orange juices that is being heated. If they practices this more often, they could
suffer from the symptoms of deficiency of Vitamin C such as fatigue, joint and muscles aches,
bruising, bad dental conditions, dry hair and skin and some inflammation. If the person
practicing frequently, they could have suffer from serious illness which is scurvy.
[4]
From this experiment, there are some of precautions need to be taken into
consideration. Firstly, the orange juice must be kept in a container with a lid to reduce oxidation.
After the juices is distributed into smaller portion, they need to be constantly closed with a finger
as to reduce further oxidation before put it into the conical flask the titration process. The eye
must be parallel to the reading of the burette while taking the measurement of the Potassium
Iodide to avoid error. There are some of the steps need to be improvise such as while conducting
the sample for cold orange juice, there is a need to close the conical flask with a tight lid in order
to prevent foreign substances inside the refrigerator contaminate the mixture.

CONCLUSI ON

Based on the results obtained, it is found that the freshly squeezed is higher in
concentration of Vitamin C compared to the conditions which heated in microwave, left cold in
refrigerator and left in room temperature. However, we found that from this experiment, external
factors such that exposing the juice with other foreign substances could accelerate the
oxidization of Vitamin C. Therefore, as a matter of suggestion, we should consume the orange
juice right after it is squeezed. If I were to conduct this experiment again, I will consider milk as
my sample experiment because milk is consumed daily as breakfast same as the orange juice.
Furthermore, milk also contain high vitamin and protein that can help in nutrition intake of a
human.












REFERENCES

[1] http://en.wikipedia.org/wiki/Orange_juice2
[2] http://www.orangejuicefacts.com/nutrition.html
[3] http://www.fitday.com/fitness-articles/nutrition/vitamins-minerals/8-symptoms-of-vitamin-c-
deficiency.html#b
[5] https://www.usc.edu/CSSF/History/2005/Projects/S0517.pdf
[6] Sullivan, Karen (1997) Vitamins and Minerals, A step-by-step Guide, Rockport, Massachussets
Element Books Limited (Page 30).
[7] Alpert, Howard (1997-2004), See For yourself Vitamin C Test-A Summary Retrieved January 10.2003.
[8] Silvester, Dr. Alvin, Robert(1992) Vitamin and Minerals, Brookfield, Connecticut The Millbrook
Press(Page 8).