AIM:

To investigate the Vitamin C content in fruit juices based on standard

curve gained.

INTRODUCTION:

Vitamin C, also called ascorbic acid is a water-soluble vitamin that

is necessary for normal growth and development. Ascorbic acid, which
has the formula of C6H8O6, behaves as a vinylogous carboxylic acid,
where in the double bond transmits electron pairs between the hydroxyl
and the carbonyl. There are two resonance structures for the
deprotonated form, differing in the position of the double bond. Water-
soluble vitamins dissolve in water and the body cannot store them.
Leftover amounts of the vitamin leave the body through the urine.
Vitamin C is required for the growth and repair of tissues in all parts of
your body. It is necessary to form collagen, an important protein used to
make skin, scar tissue, tendons, ligaments, and blood vessels. Vitamin
C is essential for the healing of wounds, and for the repair and
maintenance of cartilage, bones, and teeth.

Vitamin C is one of many antioxidants. Antioxidants are nutrients

that block some of the damage caused by free radicals, which are by-
products that result when our bodies transform food into energy. The
build up of these by-products over time is largely responsible for the
aging process and can contribute to the development of various health
conditions such as cancer, heart disease, and a host of inflammatory
conditions like arthritis. Antioxidants also help reduce the damage to the
body caused by toxic chemicals and pollutants such as cigarette smoke.
The body does not manufacture vitamin C on its own, nor does it store it.
It is therefore important to include plenty of vitamin C-containing foods in
our daily diet.

All fruits and vegetables contain some amount of vitamin C. Foods

that tend to be the highest sources of vitamin C include green peppers,
citrus fruits and juices, strawberries, tomatoes, broccoli, turnip greens
and other leafy greens, sweet and white potatoes, and cantaloupe.
Other excellent sources include papaya, mango, watermelon, Brussels
sprouts, cauliflower, cabbage, winter squash, red peppers, raspberries,
blueberries, cranberries, and pineapples.

Vitamin C toxicity is very rare, because the body cannot store the
vitamin. However, amounts greater than 2,000 mg/day are not
recommended because such high doses can lead to stomach upset and
diarrhea. Too little vitamin C can lead to signs and symptoms of
deficiency, including dry and splitting hair, Gingivitis (inflammation of the
gums), bleeding gums, rough, dry, scaly skin, decreased wound-healing
rate, easy bruising, nosebleeds, weakened tooth enamel, swollen and
painful joints, anemia, decreased ability to fight infection and possible
weight gain because of slowed metabolism. A severe form of vitamin C
deficiency is known as scurvy, which mainly affects older, malnourished
adults.
 Vitamin C is highly sensitive to air, water, and temperature. About
25% of the vitamin C in vegetables can be lost simply by blanching
(boiling or steaming the food for a few minutes). This same degree of
loss occurs in the freezing and unthawing of vegetables and fruits.
Cooking of vegetables and fruits for longer periods of time (10-20
minutes) can result in a loss of over one half the total vitamin C content.
When fruits and vegetables are canned and then reheated, only 1/3 of
the original vitamin C content may be left. Consumption of vitamin C-rich
foods in their fresh, raw form is the best way to maximize vitamin C
intake.

DCPIP solution (2,6-dichlorophenolindophenol) is a blue chemical

compound which is used as a redox dye. Oxidised DCPIP is blue in
colour, but DCPIP is colourless when reduced. It is commonly used as a
monitor of the light reactions in photosynthesis because it is an electron
acceptor that is blue when oxidised and colourless when reduced. It is
part of the Hill reagents family. DCPIP is commonly used as a substitute
for NADP+ (nicotinamide adenine dinucleotide phosphate). The dye
changes colour when it is reduced, due to its chemical structure. The
nitrogen atom in the centre of the molecule is the atom that accepts
electrons, and it changes the double N-C bond to a single bond, which
forces bonds between carbons in the entire left ring to change. This
microscopic shift in the DCPIP structure causes the macroscopic
change in colour, from dark blue to colourless.
 DCPIP can also be used as an indicator for Vitamin C. If Vitamin
C, which is a good reducing agent, is present, the blue dye, which turns
pink in acid conditions, is reduced to a colourless compound by ascorbic
acid.

DCPIP (blue) + H+ ----------> DCPIPH (pink)

DCPIPH (pink) + VitC ----------> DCPIPH2 (colourless)

C6H8O6 + C12H7NCl2O2 ----------> C6H6O6 + C12H9NCl2O2

When all the ascorbic acid in the solution has been used up in a
titration, there will not be any electrons available to reduce the DCPIPH
and the solution will remain pink due to the DCPIPH. The end point is a
pink colour that persists for 10 seconds or more.

This experiment is carried out to study the Vitamin C content in

various fruit juices, be them the bottled or the fresh ones. The smaller
volume of fruit juice needed to decolourise the blue DCPIP solution, the
higher is the Vitamin C content in the fruit. Higher Vitamin C content may
be found in citrus fruits such as lemon and orange. Citrus fruits are
acidic; therefore this may explain why the Vitamin C content in citrus
fruits is higher compared to others. The smaller volume of fruit juice
needed to decolourise the DCPIP solution means that the DCPIP
solution is reduced at a faster rate; thus it can be assumed that the
concentration of H+ ions from the ascorbic acid in the fruit is higher.
PROBLEM STATEMENT:
Which type of fruit juice provides the most vitamin C?

HYPOTHESIS:
The smaller the volume of fruit juice needed to decolourise DCPIP
solution, the higher is the vitamin C content in the fruit juice. Lemon juice
has the highest content of vitamin C among the various fruit juices.

VARIABLES:
Manipulated variable : Type of fruit juices

Responding variable : Amount of vitamin C

Fixed variable : Volume of DCPIP solution, Concentration of

standard solution

APPARATUS:
Test tubes, beakers, spatula, syringes, mortar and pestle.

MATERIALS:
500mg vitamin C tablets, 1% dichorophenolinphenol (DCPIP), freshly
squeezed orange, lemon and lime juices, distilled water.
PROCEDURE:

Preparation of standard solution

1. A quarter of a vitamin C tablet is crushed into fine powder with
mortar and pestle.
2. The powder is dissolved in 100ml of distilled water to form 0.125%
ascorbic acid.
3. Steps 1 and 2 are repeated by using ½ tablet, 1 tablet, 1 ½ tablet,

1 ¾ tablet and 2 tablets of vitamin C to produce 0.25%, 0.5%,

0.75%, 0.875% and 1% of ascorbic acid respectively.

Preparation of standard curve

1. 1ml of DCPIP solution is measured and placed into a test tube.
2. Then, 1ml of 0.125% ascorbic acid is measured using a syringe.

3. The ascorbic acid is then titrated drop by drop into the test tube
containing DCPIP solution.
4. Ascorbic acid is added until the blue colour of DCPIP solution
turned colourless.
5. The volume of 0.125% of ascorbic acid is measured.
6. The experiment is repeated two times to get the average in order
to increase accuracy.
7. Steps 1-6 are repeated using ascorbic acid of concentration
0.25%, 0.5%, 0.75%, 0.875% and 1%.
8. A standard curve is plotted base on the result.
Testing the concentration of vitamin C in fruit juices
1. 1 ml of DCPIP solution is measured and placed into a test tube.
2. Then, 1ml of freshly squeezed orange juice is measured using a
syringe.
3. The juice is then added into the test tube containing DCPIP

solution.
4. Juice is continued being inserted into the test tube until DCPIP
solution decolourise.
5. The volume of orange juice needed to decolourised the DCPIP
solution is recorded.
6. The experiment using orange juice is repeated two more times to
get the average in order to increase accuracy of result obtained.
7. Steps 1 to 6 are repeated by replacing orange juice with freshly
squeezed lemon juice and lime juice.
8. The data obtained is tabulated.
9. The concentration of each fruit juices are figured out by using the
standard curve graph of vitamin C solution.
 RESULTS:

Volume of vitamin C needed to decolourise DCPIP solution (ml)

Amount of vitamin C 1st 2nd 3rd Average

Tablets Concentration (%) reading reading reading reading

¼ 0.125 3.20 3.30 3.20 3.23

(125mg)

½ 0.250 2.00 2.00 2.00 2.00

(250mg)

1 0.500 1.24 1.14 1.24 1.21

(500mg)

1½ 0.750 1.00 1.20 1.20 1.13

(750mg)

1¾ 0.875 0.98 0.98 1.10 1.02

(875mg)

2 1.00 1.00 1.00 1.00 1.00

(1000mg)

Table 1 shows the data obtained for volume of vitamin C needed to

decolourise DCPIP solution.
 Graph of volume of vitamin C against vitamin C
concentration
3.50

3.00
Volume of vitamin C (ml)

2.50

2.00

1.50

1.00

0.50

0.00
0.125 0.250 0.500 0.750 0.875 1.000
Concentration of vitamin C (%)
 Volume of juices needed to decolourise DCPIP solution (ml)

Type of 1st 2nd 3rd Average

juices reading reading reading reading

Orange 6.00 6.10 6.00 6.03

Lemon 3.90 3.70 3.90 3.83

Lime 4.00 4.00 4.00 4.00

Table 2 shows volume of various fruit juices needed to decolourise

DCPIP solution.

DISCUSSION:
Data interpretations:

Table 1 shows the vitamin C concentration and the volume of

vitamin C needed to decolourise blue DCPIP solution. Based on Table
1, there were six different vitamin C concentrations. The concentrations
of these standard solutions or ascorbic acid are 0.125%, 0.250%,
0.500%, 0.750%, 0.875% and 1.000%. The amount of ascorbic acid
used to decolourise blue DCPIP solution varied for each concentration.
The higher its concentration, the smaller the amount needed to
decolourise the blue DCPIP solution. By using the data obtained, I can
draw a standard a standard curve graph. The trend of the graph was
decreasing from 0.125% to 1.000% concentration of vitamin C.

Table 2 shows the concentration of vitamin C of different freshly

squeezed juices based on volume needed to decolourise DCPIP
solution. There were three types of fruit juices used in this experiment,
which are orange juice, lemon juice and lime juice. In this experiment,
three readings were taken and average readings were calculated. The
highest volume of juices needed was 6.03ml which was orange followed
by lime and smallest volume to decolourise DCPIP solution is lemon
juice which was 3.83ml.

The standard curve graph is supposed to be the guideline for us to

figure out the concentration in the fruit juices tested in this experiment.
However, due to some error which is small range in the graph plotted,
the value of vitamin C in fruit juices cannot be determined. This is the
limitation that leaves us without any definite result. We should have test
on more standard solutions with lower concentrations so we will have
bigger range to compare the value in the graph.

Nevertheless, we still can determine which fruit juice has the

highest amount of vitamin C although without any accurate figure or
value of concentration. This is by comparing the volume of juice needed
to decolourise DCPIP solution. By using comparison method, we know
that lemon juice has the highest amount of vitamin C followed by lime
juice. Orange juice tested has the lowest vitamin C concentration among
the fruit juices tested in this experiment.

Limitations:
 There are several limitations during this experiment. When fruit
juice is dropped into test tube containing DCPIP solution, a suspension
formed and its colour slowly change to black. The presence of the
suspension in DCPIP solution makes it harder to determine whether
DCPIP solution has been completely decolourise.

Next, the fruits used in this experiment- that is before they are
squeezed; were prepared earlier by the laboratory assistant and the
fruits had been exposed to air for a long time before experiment is
started. The air oxidized the fruits that already been cut into half
therefore reduce the amount of vitamin C in fruits.

Furthermore, being careless, we did not covered the beakers

containing freshly squeezed fruit juice which results in vitamin C amount
is further reduced because of oxidation. However, it is always better to
prepare only a little amount of freshly squeezed juice just before each
repeated experiment.

Another limitation is the presence of fruit residues in juices. Since

we did not sieve the fruit juices, some residues may have taken some
space in syringe and this reduce the reliability of this experiment. The
presence of air bubbles in syringe too can give less accurate reading for
the volume of fruit juices that is needed to decolourise the DCPIP
solution.

Therefore, repetitions of experiments are important so that we will

have several readings and average volume can be calculated in order to
obtain more accurate reading.
SAFETY PRECAUTION:
1. Although the chemical used in this experiment which is DCPIP
solution is not dangerous, it is advised to wear goggles during
experiment to prevent any of the chemical or juices get contact
with eyes.
2. During preparation of fresh fruit juices, cut the fruit carefully to
avoid any accident happened.
3. Do not consume any juices in experiment because they might be
contaminated.

CONCLUSION:

Every fruit has different concentration of vitamin C. The smaller the

volume of fruit juice needed to decolourise DCPIP solution, the higher is
the vitamin C content in the fruit juice. The volume of lemon juice
needed to decolourise blue DCPIP solution is the lowest. Thus, lemon
juice has the highest content of vitamin C among the various fruit juices.
REFERENCES:

1. http://www.nlm.nih.gov/medlineplus/ency/article/002404.htm
[medline plus]

2. http://www.3dchem.com/molecules.asp?ID=69 [3Dchem.com]

3. http://www.whfoods.com/genpage.php?tname=nutrient&dbid=109
[whfoods.org]

4. http://en.wikipedia.org/wiki/Vitamin_C [wikipedia.org]

5. Focus Super SPM Biology Reference book