AIM

To study the presence of vitamin C in various fruit juices by comparative analysis

OBJECTIVE
In this experiment we will determine the amount of vitamin C (ascorbic acid) in
different fruit juices by titration of the juice with a solution of iodine. The iodine
reacts rapidly with the vitamin C. If you have a juice you would like to analyze, bring
about 125 ml to the lab. Compare at least two juices.

Ascorbic acid
INTRODUCTION
Vitamin C is an essential supplement that is required for humans to keep their
overall health in a desired state. Humans (and other isolated species) do not have
the ability to synthesis Vitamin C vis-a-vis other species, making it an obligation for
them to intake it.  For many centuries, the condition scurvy was infamously known to
baffle humankind. Specifically for seafaring men and explorers, this mysterious
ailment inflicted aching pain and suffering, making each journey a gamble with
death. It was not until 1930, that scientists were able to determine the substance for
curing scurvy, and thus referring it to “Vitamin C. It is also known as L- ascorbate. In
living organisms ascorbate which is an ion of ascorbic acid acts as an antioxidant by
protecting the body against oxidative stress. It is also a co-factor in at least eight
enzymatic reactions . Vitamin C also aids in detoxification and in improving ferum
absorption. Other that that, vitamin C also ensure the maintenance of cartilage,
bone, denin and healthy blood vessel.. Humans and rodents cannot synthesis
vitamin C but most of other animals have the ability to synthesis vitamin C

Thus intake of vitamin C ensure healthy lifestyle. Since vitamin C is a water soluble
vitamin, since it cannot be stored in human body. The uses and recommended daily
intake of vitamin C are matters of ongoing debate, with RDI ranging from 45 to 95
mg/day. Vitamin C is mostly present in the liver and at least present in the muscles.
Since muscle provides the majority of meat consumed in the western human diet,
animal products are not a reliable source of the vitamins. Vitamin C is present in
mother's milk but, not present in raw cow's milk. All excess vitamin C is disposed of
through the urinary system.

Apricots, apples, banana, blackberries, cherries, kiwi, grapes, lemon, lime ,mango,
lychee, melon, orange, peach, pear, pineapple, plum, pumpkin, raspberries,
strawberry, tomato and watermelon are some of fruits that contain high content of
vitamin C. On the other hand, vegetables like artichoke, asparagus, avocado,
broccoli, cabbage, corn, paprika, mushroom and spinach have vast content of
vitamin C.
Benefits of Vitamin C include protection against immune system inadequacy,
prenatal health problems, eye disease, skin wrinkling and also cardiovascular
disease. Looking at the studies, one can derive that Vitamin C does indeed provide
numerous benefits to the body. For example, a contemporary study published in
Seminars in Preventive and Alternative Medicine looked and analyzed over 100
studies over 10 years and concluded that Vitamin C did indeed include many
benefits, such as the ones mentioned above.

Vitamin C is also required for the biosynthesis of collagen, certain neurotransmitters

and L-carnitine. In 2018, the University of Maryland conducted a study and
concluded that Vitamin C intake can reduce the pressure that’s built up on the bones
in a condition known as osteoarthritis. This could potentially prevent a person from
being diagnosed with arthritis. (University of Maryland Medical Center, 2018).

It can also assist in protein metabolism. (Y, 2019) Vitamin C also provides the
essential nutrients that help maintain the connective tissue and bones in our bodies.
It ensures the optimal functionality of several enzymes, by activating certain liver-
detoxifying systems. Vitamin C also acts as an antioxidant, as it reacts directly with
free radicals in the aqueous state. This is important as it protects cellular functions
and as a result, this function can aid in fighting bacterial infections and increase the
rate of regeneration of burns or wounds.

The chemical name for Vitamin C is ascorbic acid, and primarily exists in 2 forms- L-
ascorbic acid and D-ascorbic acid. The L variety can be found naturally, i.e in fruits or
vegetables but can also be found in its synthetic form (such as supplements), both
versions being interchangeable with their benefits. The D variety carries
indistinguishable antioxidant properties but not the vitamin C content of L-ascorbic
acid. In addition, the D form is not used in forms of supplement. Although they are
both chemically Vitamin C, the nutrient packed properties differ amongst them,
affecting their bio-availability.

Vitamin C (ascorbic acid) is an antioxidant that is essential for human nutrition.

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.  

Fruits have varying levels of vitamin C. In some fruits, the amount of vitamin C varies
between different varieties of the same species. The vitamin C content of many fruits
is higher when it is slightly unripe, and declines as the fruit becomes ripe. Vitamin C
content also decreases with storage. 

A deficiency in Vitamin C can ultimately be the leading factor to scurvy. Subclinical

deficiencies can lead to signs of inadequate wound healing and ulceration.  Early
signs of deficiencies are not too fatal, and may range from general weakness,
shortness of breath, lethargy, and possibly aching of the limbs. As time progresses,
other prominent conditions may become evident, such as petechiae after the
application of a sphygmomanometer (blood pressure monitor), perifollicular
haemorrhages, bleeding and swollen gums, pallor/anaemia (unhealthy pale
appearance after a result of prolonged bleeding). Unfortunately, groups that are at
high risk with these conditions include smokers, patients with diabetes and the
elderly.

Some people automatically assume that if a drink is advertised as ‘natural’ it will

contain as many nutrients and vitamins as the source it has been derived from.
However,  one must keep in mind companies will favour satisfaction of their target
more, and thus , means that commercial fruit juices (and sometimes the natural fruit
juices) will contain flavourings  and chemicals that will enhance the flavour and
preserve the liquids for longer. Also, commercial fruit juices react with oxygen, which
proves that most of the nutrients are lost due to oxidation. The method that will be
used is a titration method, where Vitamin C in fruit juices were titrated against
aqueous sodium dichlorophenolindophenol with starch as an indicator.

ACIDITY

Ascorbic acid behaves as a vinologous carboxylic acid, wherein double bond ("vinyl")
transmits electron pairs between the hydroxyl and carbonyl. There are two
resonance structures for the deprotonated form, differing in the position of double
bond.

Another way to look at ascorbic acid is to consider it an enol. The deprotonated form
is an enolate, which is usually strong basic. However, adjacent double bond stabilizes
the deprotonated form.

Ascorbic acid (vitamin C) is used as a dietary supplement when the amount of

ascorbic acid in the diet is not enough. People most at risk for ascorbic acid
deficiency are those with a limited variety of food in their diet, or who have
intestinal malabsorption problems from cancer or kidney disease. Ascorbic acid is
also used to prevent and treat scurvy (a disease that causes fatigue, gum swelling,
joint pain, and poor wound healing from a lack of vitamin C in the body). Ascorbic
acid is in a class of medications called antioxidants. It is needed by the body to help
wounds heal, to enhance the absorption of iron from plant foods, and to support the
immune system. It works as an antioxidant to protect your cells against free radicals,
which may play a role in heart disease, cancer and other diseases.

TAUTOMERISM

Ascorbic acid is rapidly in converts into two unstable diketone tautomers by proton
transfer, although it is the most stable in the enol form. The proton of the enol is
lost, reacquired by electrons in the form of double bond, to produce a diketone. It is
an enol reaction there are two possible forms: 1,2-diketone and 1,3-diketone.

DETERMINATION

The concentration of the solution of ascorbic acid can be determined in many ways;
the most common way involves titration with an oxidizing agent. 1.) DCPIP

A commonly-used oxidizing agent is the dye 2,6- dichlorophenol - indophenols, or

DCPIP for short. The blue dye is run into the ascorbic acid solution until a faint pink
colour persists for 15 seconds.

IODINE

Another method involving using iodine and a starch indicator, wherein iodine reacts
with ascorbic acid, and when all the ascorbic acid had reacted, the iodine is excess,
then forming a blue - black complex with starch indicator. This indicates the end
point of the titration. As an alternative, ascorbic acid can be reacted with iodine in
excess, followed by back titration with sodium thiosulfate while using starch
indicator.

IODATE AND IODINE

The above method involving iodine requires making up and standardizing the iodine
solution. One way around this is to generate the iodine in presence of ascorbic acid
by the reaction of iodate and iodide ion in acid solution.

N-BROMOSUCCINIMIDE A much-less-common oxidizing agent is N

bromosuccinimide, (NBS). In titration the NBS oxidizes the ascorbic acid 9 in

presence of potassium iodide and starch). When the NBS in excess (i.e., the reaction
is complete). The NBS liberates iodine from the potassium iodide, which then forms
the blue/black complex with starch, indicating the end-point of the titration.

USES AND PROPERTIES

Ascorbic acid is easily oxidized and so is used as a reductant in photographic

developer solutions amongst ethers and as a preservative.

Exposure to oxygen, metals, light, and heat destroys ascorbic acid, so it must be
stored in dark, cold and not metal container.

The L-enantiomer of ascorbic acid is known as vitamin C. The name "ascorbic" comes
from its property of preventing and curing scurvy.
Primates including humans and few other species of the same animal kingdom,
notably the guinea pig, have lost the ability to synthesize ascorbic acid, and must
obtain it in their food.

Ascorbic and its sodium, potassium, calcium salts are commonly used as antioxidant
food additives. These compounds are water-soluble and thus cannot protect facts
from oxidation. For this purpose, the fat soluble esters of ascorbic acid with long
chain fatty acid can be used as food antioxidants.

VITAMIN C CONTENT IN VARIOUS FRESH FRUITS

Determination of Vitamin-C content in fresh fruit juices

Samples of different fruits were prepared according to a previously written

procedure. Results of the total content of Ascorbic acid in the investigated samples
were calculated after the completion of titration. The highest content of total
ascorbic acid obtained by titration method was found in samples of fresh: orange
(41.93 mg/100 ml)>lemon (29.31 mg/100 ml))>apple (26.6 mg/100 ml)>grapes
(25.25 mg/100 ml) as shown in table 1.

Table 1:Vitamin-C content in fresh fruit juices

Volume of Iodine
Fresh Mean±S.D. Mass of vitamin Mass of vitamins
solution consumed
fruits a
C gm/20 ml mg/100 ml
(ml)

S1 S2 S3

Apple 5.9 5.8 5.9 5.9±0.05770.0053 26.60

Orange 9.4 9.3 9.3 9.3±0.05770.00838 41.93

Grapes 5.6 5.6 5.6 5.6±0.00 0.0050 25.25

Lemon 6.6 6.5 6.5 6.5±0.05770.0058 29.31

Determination of vitamin-C content in marketed fruit juices

Fruit juices with respective apple, orange, lemon, grapes flavoured were brought
from the market and samples were prepared according to a previously written
procedure on the due date. Result of the total content of Ascorbic acid in the
investigated samples obtained by titrimetric methods. The highest content of total
ascorbic acid obtained by titration was found in samples of marketed:

orange (46.44 mg/100 ml)>grapes (29.76 mg/100 ml)>lemon (24.8 mg/100

ml)>apple (24.35 mg/100 ml), as shown in table 2.

Table 2: Vitamin C content in various marketed fruits juices

Volume of titrant Mass of vitamin C Mass of vitamin C

Sample
consumed gm/20 ml mg/100 ml
S1 S2 S3 mean±S.D.
Real apple
5.3 5.5 5.5 5.4±0.1154 0.00487 24.35
juice
Real orange 10. 10. 10. 10.3±0.057
0.00929 46.44
juice 2 3 3 7
Real grapes
6.6 6.6 6.6 6.6±0.00 0.005953 29.76
juice
7up
3.6 3.5 3.5 5.5±0.0577 0.004961 24.8
nimbooz

Determination of vitamin-C content in marketed fruit juices

Thus received marketed fruit juices made up of apple, grapes, lemon, orange were
stored on the freeze at 0 °C and 10 °C and on the hot air oven at 20 °C, 30 °C, 40 °C,
50 °C for 72 h. After completion of storage, samples were prepared and analyzed for
determination of ascorbic acid using titrimetric as like fresh fruit juice. The content
of ascorbic acid in each fruit juice sample after storage at varying temperature is
shown in table 3.

Table 3: Vitamin-C content under varying temperature conditions in marketed fruit

juices (titrimetric analysis)

Vitamin C
Volume of Vitamin-C
Temperatur content Degradation
Sample titrant Mean±SDa content
e mg/100 in %
consumed µg/ml
ml
Trial Trial Trial
1 2 3
Real
24.56% at 50
apple 0 °C  5.3 5.3 5.4 5.3±0.0577 0.0047 23.90
°C
juice
10 °C  4.7 4.8 4.9 4.8±0.100 0.004 21.64
20 °C 4.8 4.7 4.7 4.7±0.0577 0.0042 21.19
30 °C 4.6 4.6 4.6 4.6±0.00 0.0041 20.74
40 °C 4.2 4.3 4.4 4.3±0.100 0.0038 19.39
50 °C 4.0 4.0 4.1 4±0.0577 0.0036 18.03

Real
10.1±0.057 10.89% at 50
orange 0 °C  10.1 10.2 10.1 0.0091 45.54
7 °C
Juice
10 °C  10.0 10.0 10.0 10.0±0.00 0.0090 45.09
20 °C  9.8 9.7 9.7 9.7±0.0577 0.0087 43.74
30 °C 9.4 9.5 9.3 9.4±0.100 0.0084 42.38
40 °C 9.4 9.4 9.5 9.4±0.0577 0.0084 42.38
50 °C  9.0 9.0 9.1 9±0.0577 0.0081 40.58

Real
12.70% at 50
grapes 0 °C  6.4 6.3 6.3 6.3±0.0577 0.0056 28.41
°C
juice
10 °C 6.3 6.3 6.2 6.3±0.0577 0.0056 28.41
20 °C  6.3 6.2 6.0  6.2±0.1527 0.0055 27.95
30 °C 6.0 6.0 6.0 6±0.00 0.0054 27.05
40 °C 6.0 6.0 6.1 6±0.0577 0.0054 27.05
50 °C 5.5 5.5 5.6 5.5±0.0577 0.0049 24.80

7up 0 °C  3.0 3.0 3.0 3±0.00 0.0027 13.52 50% at 50 °C

nimbooz
10 °C  2.8 2.9 2.8 2.8±0.0577 0.0025 12.62
20 °C  2.8 2.6 2.6 2.6±0.1154 0.0023 11.72
30 °C  2.3 2.2 2.2 2.2±0.0577 0.0019 9.92
2.2±0.0577 0.0019 9.92

HYPOTHESIS :
The higher the volume of fruit juices needed to decolourise DCPIP solution, the
lower the vitamin C content in the fruit juice. Fresh lime juice has the highest
content of Vitamin C among the fresh juices and orange cartoon juice has the highest
content of Vitamin C among carton juices.

Representation of variation of fruit Juices among various fruits

EXPERIMENT

APPARATUS REQUIRED
 Test tubes
 0.5ml syringe
 10ml syringe
 beaker
 mortar
 pestle

MATERIALS REQUIRED
 1000mg vitamin C tablets
 1% dichlorophenolindophenol solution (DCPIP)
 freshly squeezed lime
 Lemon and orange juices
 100ml distilled water
 Lime
 lemon
 orange carton juice.

PROCEDURE:
Preparation of standard solution
1. A full vitamin C weighing 1g was crushed into a fine powder with a mortar and
pestle.

2. The powder was then dissolved in 100ml of distilled water to form 1g/100ml
ascorbic acid.
3. Steps 1 and 2 were repeated thrice to get average volume of vitamin C needed to
decolourise DCPIP solution.

4. Steps 1 until 3 were repeated by using ¾ tablet, ½ tablet, ¼ tablet and ⅛ tablet of
vitamin C to produce 0.75g/100ml , 0.5g/100ml , 0.25g/100ml and 0.125g/100ml
respectively.

PREPARATION OF STANDARD CURVE

1. 0.5ml of 1.0% DCPIP solution was measured using 0.5ml syringe and placed into a
test tube.

2. Then, 1ml of 1g/100ml ascorbic acid was measured using a syringe.

3. The ascorbic acid was titrated drop by drop into test tube containing DCPIP
solution.

4. Ascorbic acid was added with extra care, drop by drop until the blue color of
DCPIP solution turned colourless.

5. The volume of 1g/100ml of ascorbic acid was measured and recorded.

6. Steps 1 to 5 were repeated using 0.75g/100ml , 0.5g/100ml , 0.25g/100ml and

0.125g/100ml respectively to obtain the respective measurement.

7. A standard curve was plotted based on the results.

Titration of ascorbic acid

TESTING THE CONCENTRATION OF VITAMIN C IN FRUIT JUICES

1. 0.5ml of 1.0%DCPIP solution was measured and placed into a test tube.

2. Then, 5ml of freshly squeezed orange juice was measured using a syringe.

3. The needle of the syringe was placed into the DCPIP solution.
4. The freshly squeezed orange juice was added drop by drop to the DCPIP solution.
The mixture is gently stirred with the needle of the syringe. The juice is continuously
added with extra care until the DCPIP solution is decolourised. The step is repeated
thrice.

5. The volume of fruit juice needed to decolourise the DCPIP solution was recorded.

6. If more than 5 cmᵌ of fruit juice needed to decolourise the DCPIP solution, then
one control colour was set up. With another test tube containing DCPIP solution,
fruit juice was added until the colour changed exactly the same. The volume of fruit
juices needed was recorded.

7. Steps 1 until 5 were repeated by replacing freshly squeezed orange juice with
freshly squeezed lemon juice and freshly squeezed lime juice. The data was
recorded.

8. Steps 1 until 5 were repeated by replacing freshly squeezed orange juice with
orange carton juice, lemon carton juice and lime carton juice. The data was
tabulated.

Titration of fruit juices

9. The concentration of each group of juice and each of the juice was calculated by
using the following formula :
V =K/C

10. The readings obtained were tabulated in a table.

CONCLUSION
Every fruit group has a different concentration of vitamin C. the smaller the volume
of fruit juice needed to decolourise DCPIP solution, the higher the content of vitamin
C in fruit juice. Fresh juice contains more vitamin C content than carton fruit juices.
Fresh lime juice has the highest vitamin C content and lime has the lowest vitamin C
content among fresh fruit juices, while for carton fruit juices, orange has the highest
and lime has the lowest content of vitamin C. The hypothesis is accepted.

BIBLIOGRAPHY
1. http://en.wikipedia.org/wiki/Vitamin_C
2. http://www.whfoods.com/genpage.php?tname=nutrient&dbid=109
3. http://www.lenntech.com/fruit-vegetable-vitamin-content.htm
4. http://www.google.com.my/search?q=vitamin+c