KYAMBOGO UNIVERSITY

FACULTY OF SCIENCE

DEPARTMENT OF CHEMISTRY

BACHELOR OF SCIENCE TECHNOLOGY CHEMISTRY

SEMESTER TWO, YEAR ONE

PRACTICAL REPORT

COURSE UNIT: SST 1203: INTRODUCTION TO BIOCHEMISTRY

LECTURER NAME: MR. OLADO SIMO PETER

STUDENT NAME: ODONGO TONNY

REG. NO: 18/U/CTD/269/GV

SIGNATURE…………

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ABSTRACT
The purpose of this experiment was to determine the amount of Vitamin C in orange fruit by using
a redox titration. The experiment was a success. The main objective of the experiment was to
determine the amount of vitamin C in an orange fruit, and the specific objectives were; to
determine the factors affecting the amount of vitamin C in a fruit, to determine the amount of
vitamin C required to be taken daily, to determine how many oranges one should take to meet the
daily requirement of vitamin C, and to investigate the effect of temperature on vitamin C.

The methodology used was by titration, where a burette was filled with orange juice orange and
DCPIP put in the conical flask. The average volume for the end point was calculated and used for
the calculations. The moles of vitamin C were calculated in order to determine the grams of vitamin
C. This value was then used to determine the of amount the vitamin C per 100ml of juice.

The amount of vitamin C per orange was found to be 54.34mg/100ml. The value obtained is small
compared to the standard value which is 85mg/100ml because the experiment was not conducted
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on a standard scale. Also, the number of fruits required for daily intake was found to be 12oranges.

The major factors that affect vitamin C concentration were found to be temperature and storage
time. Different methods such as temperatures and storage containers can influence oranges’
vitamin C concentration. It has been reported the citrus products lose vitamin C potency because
or those two major factors, temperature and storage time.

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TITLE

 EXPERIMENT TO DEMONSTRATE THE QUANTITATIVE DETERMINATION

OF VITAMIN C IN ORANGE FRUIT
Main objective
 To determine the amount of vitamin C present in orange fruit
Specific objectives

 To determine the amount of vitamin C in an orange fruit

 To determine the factors affecting the amount of vitamin C in a fruit
 To determine the amount of vitamin C required to be taken daily
 To determine how many oranges, one should take to meet the daily requirement of vitamin
C
 To investigate the effect of temperature on vitamin C

Introduction
Vitamin C (ascorbic acid, ascorbate, AA) is a water-soluble organic compound that participates in
many biological processes and the most common electroactive biological compound found in some
fruit species. It’s found in green vegetables, fruits, and potatoes. It is essential for a healthy diet. It
is the most widely taken nutritional supplement worldwide. One of its chemical properties is a
reducing agent. DCPIP is Dichlorophenol-indophenol, a blue dye which is decolourised by
ascorbic acid on account of its reducing properties. Methods for the detection of vitamin C involve
titrating it against a solution of an oxidizing agent like DCPIP.

There are many oxidizing agents that can be used and commonly used one is 2,6-dichlorophenol-
indophenol or DCPIP. This means finding out how much DCPIP reacts with a known amount of
vitamin C. the end point is usually a pink colour which persists for about 15 seconds or longer.
This experiment will majorly investigate the stability of ascorbic acid under different conditions
of temperature, and the main hypothesis for testing will; if heat destroys vitamin C, then heat
treated juice will have lower concentration of vitamin C.

Background
A vitamin is an organic molecule that is an essential micronutrient that an organism needs in
small quantities for the proper functioning of its metabolism. Essential nutrients cannot be
synthesized in the organism, either at all or not in sufficient quantities, and therefore must be
obtained through the diet

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Vitamins are essential nutrients that your body needs in small amounts to function. They fall into
two categories: water-soluble and fat-soluble. The water-soluble vitamins - C and the B-complex
vitamins - dissolve in water before your body can absorb them. Fat-soluble vitamins - A, D, E,
and K - dissolve in fat and can be stored in your liver and fat tissue until needed. Fat-soluble
vitamins have a multitude of functions from keeping your bones strong to helping your muscles
move.

Functions of Fat-Soluble Vitamins: Vitamin A helps your vision, immune, and reproductive
systems. It is essential to bone growth and tooth development. It also keeps your heart, lungs and
kidneys working properly. Vitamin D, along with calcium, keeps your bones strong by
preventing diseases such as rickets, a disorder that causes bones to become soft and weak in
children. It also helps prevent osteoporosis, which makes your bones weak and more likely to
break. Vitamin D also helps your muscles move, improves your immune function and helps
reduce inflammation. Vitamin E acts as an antioxidant that protects your body from free radicals,
which are molecules that damage your cells. It also boosts your immune system and keeps blood
moving through your blood vessels without clotting. Vitamin K, conversely, allows your blood
to clot. It also helps your body by making proteins for healthy bones and tissues.
Water-Soluble Vitamins: Nine of the 13 essential vitamins are classified as water-soluble
vitamins. That means they are found in great abundance in watery foods such as fruits and
vegetables and in the watery components of grains, nuts, seeds and animal products. The water-
soluble vitamins include A in the form of beta-carotene, B6, B12, riboflavin, thiamin, niacin,
folate, biotin, and C. Water-soluble vitamins are used immediately by your body or they are
excreted in your urine. Unlike fat-soluble vitamins, they cannot be stored in your body so they
should be replenished on a daily basis.

Citrus fruits are prominent for their high vitamin C content and nutritional values. Oranges in
particular have some of the highest concentrations of ascorbic acid (AA) also known as vitamin
C. Previous studies have revealed the nutritional values and health benefits from consuming
these citrus fruits. The results have shown that their benefits are excellent for human health.
Articles have also reported that scurvy, a disease that grows from a deficiency of vitamin C, can
be prevented by eating foods high in vitamin C contents. It has been mentioned that citrus fruits
have been used since early as 1564 to prevent scurvy disease (S. Nagy 1980). Back in the 1700’s
sailors also included citrus fruits in their diets to prevent this disease during their long voyages.
These fruits were used to supplement the essential vitamin C needed to maintain their bodies
healthy.
It has the formula: C6H8O6, IUPAC name: 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol,
Chemical name: Ascorbic acid (also L-ascorbic acid), and common name: Vitamin C

STRUCTURE:

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Oranges for example, are an excellent provider of this vitamin, although there do exist various
factors that affect their vitamin C contents. Experiments have been completed to study several of
those factors. One of those main factors studied are their storage methods. Different methods such
as temperatures and storage containers can influence oranges’ vitamin C concentration. It has been
reported the citrus products lose vitamin C potency because or those two major factors,
temperature and storage time (S. Nagy 1980).

Furthermore, it is important to study which ways oranges can be stored to preserve the fruits
vitamin C content for longer periods of time. In this particular experiment, different storage
methods along with temperature and aging factors were studied from different sets of oranges.
Vitamin C or ascorbic acid is essential for human life and is required for a range of
physiological functions in human body. It can be found either in fresh fruits and vegetables
naturally or in medical forms such as normal tablets, effervescent tablets and liquid vials. It is
the most widely taken supplement. Though daily requirements of vitamin C are changeable
according to the age, sex and conditions, it is around 75 to 90 mg per day for healthy adults and
no more than 2000mg per day is recommended. It is one of the most ubiquitous vitamins ever
discovered.

Adult men need about 90 milligrams of vitamin C each day, while women should have at least 75
milligrams daily. If you eat at least 2 1/2 cups of fresh fruits and vegetables each day, you should
easily reach this requirement. The Linus Pauling Institute recommends supplementing with two
250-milligram vitamin C doses per day, taking one in the morning and one in the evening, to give
you a daily intake of more than 500 milligrams a day.

Besides plays a paramount role as an antioxidant and free radical scavenger, it has been suggested
to be an effective antiviral agent. In addition, ascorbic acid has been widely used in the
pharmaceutical, chemical, cosmetic and food industry as antioxidant.

Therefore, there is a need to find an accurate, reliable, rapid, and easy-to implement method for
measuring the amount of ascorbic acid in a sample. However, there have been difficulties in
quantifying ascorbic acid due to its instability in aqueous
solution. The instability of ascorbic acid is due to its oxidation to dehydroascorbic acid, which is
a reversible reaction, and subsequently to 2,3-diketo-L-gulonic acid. The later reaction is
irreversible.

Ascorbic acid is a water-soluble vitamin with molecular weight of 176.12 g/mol and melting point
of 193˚C. World-wide accepted daily requirement of ascorbic acid is about 60–95 mg. Ascorbic
acid is a reducing agent which reverses the oxidation in aqueous solution. Increased amounts of
free radicals trigger the condition called oxidative stress which is kept under control by

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antioxidants. If there are not enough antioxidants some stress related diseases including
hypertension, atherosclerosis, chronic inflammatory diseases and diabetes might occur.

Everyone needs vitamin C for the growth and repair of tissues in all parts of your body. It is a
potent antioxidant. It helps the body make collagen, an important protein used to make skin,
cartilage, tendons, ligaments, and blood vessels. Inside our bodies, it functions as an essential
cofactor in numerous enzymatic reactions. Vitamin C is needed for healing wounds, and for
repairing and maintaining bones and teeth. It also helps the body absorb iron from non-heme
sources.
Vitamin C plays a role in protecting against the following: Heart Disease, High Blood Pressure,
Common Cold, Cancer, Osteoarthritis, and Age-related Macular Degeneration

The Institute of Medicine's Food and Nutrition Board has set the tolerable upper intake level, or
UL, of vitamin C for an adult over 19 years old at 2,000 milligrams per day. This means that unless
you regularly consume more than 2,000 milligrams of vitamin C each day including your intake
from both food and supplements you should not experience any potentially harmful side effects.

Postmenopausal women who have diabetes should avoid taking 1,000 milligrams of vitamin C
daily. A 2004 study published in the "American Journal of Clinical Nutrition" found that senior
women with diabetes who took more than 300 milligrams of supplemental vitamin C each day
were more likely to die from heart disease. However, different studies have yielded different
results, reports a 2016 review published in the International Journal of Molecular Science.

Vitamin C supplements can cause mild symptoms like an upset stomach, gas, nausea, vomiting,
headaches and diarrhea. Vitamin C supplements should be avoided if you're allergic to corn since
the majority of vitamin C preparations contain corn, if you've just had or are about to have a heart
procedure such as an angioplasty Vitamin C can interfere with the proper function of antacids,
statins, blood thinners like warfarin, estrogens, antibiotics such as tetracycline and fluphenazine.
In this study, manipulating a burette and carrying out a quantitative titration properly are essential
to improve the method to get a more accurate and better result in determining concentration of
vitamin C. Apart from titration with DCPIP solution, determination of vitamin C concentration
can also use iodometric titration method which involves iodine and iodate solution. When iodine
solution is a titrant, vitamin C is oxidised to form dehydroascorbic acid, while the iodine is reduced
to iodide ions. When all vitamin C has finished, the excess iodine solution will react will starch
solution to form blue-black colour as endpoint. The iodine solution needs to be standardised with
pure vitamin C or potassium thiosulphate because an iodine is unstable.

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REQUIREMENTS AND APPARATUS
Apparatus
 Volumetric flask(250ml)
 Two plastic Beakers
 Burette(50ml)
 Pipette
 Measuring cylinders(250ml)
 Analytical Balance
 Distilled water
 Heater
 Motor and pestle
 Conical flask(250ml)
 Glass beakers about 100mL
 Knife

Reagents
 DCPIP
 Vitamin C (reagent grade)
 Orange fruit

EXPERIMENTAL PROCEDURES
The actual experiment started with cleaning of the apparatus followed by the:

Part A: Preparing of 1% vitamin C standard solution

2.5g of vitamin was weighed, dissolved and transferred into 250ml volumetric flask, and water
added up to the mark.

Part B: Preparation of 1% DCPIP solution

2g of DCPIP was weighed, dissolved and transferred into 200ml volumetric flask, and water added
up to the mark.

Part C: Juicing two orange fruits

The orange juice was peeled and weighed.
It was then squeezed to get the juice and the remainder of the waste weighed again. The mass of
the juice was obtained by subtracting the mass of the waste from the mass of the juice plus the
mass off the peeling. The percentage yield of the orange fruit was calculated.

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Part D: Titration
10ml of DCPIP was pipetted and titrated with vitamin C solution from the burette. The end point
was indicated by the pink colour of the mixture in the conical flask, and the volume of the vitamin
C at the end point was read and recorded. The experiment was repeated two more times, and the
result obtained tabulated.
The above procedure was repeated, but this time with orange juice. The results obtained was also
tabulated.

Table of results
Volume of pipette used 10ml

Table 1: vitamin C titration

Experiment Final Reading Initial Difference

(ml) Reading (ml) (ml)
1 3.50 0.00 3.50
2 6.70 3.50 3.30
3 10.00 6.70 3.30
3.30+3.30+3.50
Average volume (titre) = = 3.37ml
3

Table 2: orange juice titration

Experiment Final Reading Initial Difference

(ml) Reading (ml) (ml)
1 12.00 0.00 12.00
2 23.80 12.00 11.80
3 11.80 0.00 11.80
11.80+11.80
Average volume (titre) = = 11.80ml
2

TREATMENT OF RESULTS
Reparation of orange juice

Mass of the orange + the peeling = 255.95g

Mass of the of peeling after squeezing out the juice = 77.65g

Mass of the juice = 178.26g

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 178.26
Percentage yield of the orange = 255.91 × 100% = 69.7%

Calculation on preparing ascorbic acid solution

𝑚𝑎𝑠𝑠, 𝑚
Concentration, C = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑚𝑎𝑠𝑠,𝑀 ; where m= 2.5g and M = 176.12g

2.5
∴ C= = 0.0141949M
176.12

Calculation on preparing DCPIP

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∴ C= = 0.0074599M
268.1

Standardizing the DCPIP solution

At the end point, the number of moles of the reactant are equal
Thus,
CV (ascorbic) = CV(DCPIP); where C and V are volume and concentration respectively.

0.0141949× 3.37 = C × 10

C = 0.0047837M
Calculation the mass of ascorbic acid in 100ml juice. Reaction ratio = 1:1
At the end point,
n(ascorbic) = n(DCPIP); where n is the number of moles
𝑚
= CV
𝑀

268.1 ×0.0047837×10
m = 1000

m = 0.0128250997g (in 11.8ml)

= 0.0128250997 ×100
m = 0.10868729g/100ml (or 108.68729mg/100ml)
3.67

= 108.68729
m = 54.34mg/100ml (for one orange, since two oranges were used to make juice)
2

The number of oranges required for daily intake

One orange has 54.34mg/100ml of ascorbic acid.

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But the standard value for the daily intake = 85mg/100ml of ascorbic acid.
85 1
According to this experiment, the daily intake would be = 54.34 = 1.56 ≈ 12 oranges

DISCUSSION OF RESULTS
Vitamin C recommendations vary depending on gender, certain life stages and whether or not you
smoke. Men, for example, need 90 milligrams of daily vitamin C from the age of 19 and all
throughout adulthood, according to the Office of Dietary Supplements. Women over age 19 require
75 milligrams per day.
However, during pregnancy, the recommendation is 85 milligrams and then increases further to
120 milligrams if you breast-feed. Smoking further ups your vitamin C needs by an additional 35
milligrams, since oxidative stress from free radicals increases when you smoke.
A medium-size orange is approximately 2 1/2 inches in diameter and weighs approximately 130
grams, or a little more than 4.5 ounces. A medium orange has nearly 70 milligrams of vitamin C.
If you find bigger oranges that are 3 inches in diameter and weigh approximately 185 grams or 6.5
ounces, has almost 100 milligrams of the vitamin C.

According to Lindsey Pine, M.S., R.D., owner of Tasty Balance Nutrition, Just one orange has
about 100 mg of vitamin C, which is 130 percent of that daily recommended intake. On getting
the average, an orange fruit contains 85mg/100ml of ascorbic acid.
Measurement of vitamin C in this experiment was done by titrating the juice against
dichlorophenolindophenol (DCPIP). It is a strong oxidizing agent with distinctive blue colour and
decolourised when being reduced by vitamin C.

First, the averages on the three different titration trials. The amount of vitamin C in 100ml of the
orange fruit was calculated using the average volume needed for each reaction to occur.

Using the two different methods of expressing vitamin C content (Vitamin C concentration and
total vitamin C content), and the concentration and volume of the DCPIP. The mass in mg/100ml
was calculated and was obtained to be 108.68mg/100ml, but this amount could be divided by two
as the juice was obtained from two oranges, and when this was done it was found to be
54.34mg/100ml. This means the one orange fruit has 54.34mg/100ml of ascorbic acid. This value
is less compared with the standard value. The number of oranges required for daily intake was also
1
calculated and was found to be 12oranges. The deviation could be due to experimental error as the
experiment was not conducted on a standard scale.

However, there do exist various factors that affect their vitamin C contents, which also might have
affected the results in this experiment. One of those main factors studied are their storage methods.
Different methods such as temperatures and storage containers can influence oranges’ vitamin C
concentration. It has been reported that citrus products lose vitamin C potency because or those
two major factors, temperature and storage time.

10
Titration with 2,6-dichlorophenolindophenol (DCPIP) solution is a suitable method to determine
vitamin C concentration in commercial or fresh fruit juices. This is because vitamin C can be
determined by oxidation-reduction reaction. Vitamin C is a good reducing agent. The DCPIP
solution is a strong oxidizing agent, so that it will not oxidize substances other than the ascorbic
acid in the sample of fruit juice. Besides that, titration method or called as volumetric analysis is
accurate and precision method compare another method.

DCPIP is used as a good indicator because ascorbic acid has two protons that can accept from
ascorbic acid and also ascorbic acid has two protons which can donate to DCPIP. The C=O from
DCPIP accepts to protons to eventually become C-OH and the C=O is a good proton acceptor
because the C=O is very reactive. If vitamin C, which is a good reducing agent. DCPIP solution is
a weak oxidizing agent, so that it will not oxidize substances other than the ascorbic acid in the
sample of fruit juice. In present, the blue dye, which turns pink in acid conditions, is reduced to a
colourless compound by ascorbic acid. The following reactions give a brief description of the
overall reaction:

In this titration, when all the ascorbic acid in the solution has been used up, 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

CONCLUSION:
The purpose of this experiment was to determine the amount of Vitamin C in orange fruit by using
a redox titration. The experiment was a success. A burette was filled with DCPIP solution, which
was used to titrate the orange juice orange juice was added in small increments until the flask
turned a pink color.
The final burette reading was calculated and used for the calculations. The moles of vitamin C
were calculated in order to determine the grams of vitamin C. This value was then used to
determine the of amount the vitamin C per 100ml of juice. The amount of vitamin C per orange
was found to be 54.34mg/100ml. The value obtained is small compared to the standard value which
1
is 85mg/100ml. Also, the number of fruits required for daily intake was found to be 12 oranges.

The major factors the affect vitamin C concentration were found to be temperature and storage
time. Different methods such as temperatures and storage containers can influence oranges’

11
vitamin C concentration. It has been reported the citrus products lose vitamin C potency because
or those two major factors, temperature and storage time

SOURCES OF ERRORS

When investigating the fruit, there was no focus on the varying sizes of fruits, as well as on the
thickness of their peel or their eatability.

The coloring of the fruit juice. Due to the already colored juice, it was hard to exactly determine
the endpoint of the titration because neither of us knew how the color change would look like. That
means it could be possible we were overshooting the end point and based on that, calculated a
slightly different concentration of vitamin c for the fruit juice.

Oxidation of DCPIP by oxygen in the atmosphere is one of the limitations of this experiment. This
affected the accuracy of the results.

The other limitation could be the side decomposition of vitamin C due to the presence of light and
air. Just like temperature, ultraviolet ray from incidence light causes the hydroxyl bond in ascorbic
acid to break, thus become oxidized.

The storage duration of the vitamin C might have also affected the results since the orange used in
this experiment was just bought from the market and therefore it was not a fresh orange fruit,
meaning that some of the vitamin C could have been lost from the fruit.

Recommendation

Several modifications can be made in the future to improve the accuracy and reliability of the
results of this experiment.

To reduce this limitation, the beakers containing orange juice to be stored at different temperatures
were neatly wrapped with aluminium paper which is a good reflector of light and heat. The
surfaced of the beakers were also sealed with Parafilm. This would prevent the entry of air and
significantly reduce unnecessary oxidation of vitamin C in the juice.

Hence, the test tubes were not shaken vigorously during titration in order to minimize this
limitation as vigorous moves can increase the rate of oxygen dissolving in a solution.

Iodine titration could be used as an alternative for DCPIP titration. Although this technique may
require the use of more reagents, the result from the trial experiment has shown that the end point
of titration is much easier to be identified.

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Besides, orange juice could be obtained by blending the peeled orange fruits instead of squeezing
them. This ensures maximum amount of juice obtained from each fruit as well as preventing
destruction of vitamin C by heat.

The orange juice to be used for the experiment should be fresh- just obtained from the orange tree
to obtained accurate result.

Finally, percentage of vitamin C degraded can be used as the response variable instead of
concentration. This would allow clearer illustration of the amount of vitamin C reduced for the
readers and also make the comparison process easier.

In future, research should be done to determine whether the temperature will affect the
concentration of vitamin C in fruit juices. Besides that, research also can be done to determine the
vitamin C concentration present at the time of consumption based on the expired date.

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1. References
2. Michels A, Frei B (2012). "Vitamin C" Biochemical, Physiological, and Molecular
Aspects of Human Nutrition (3rd ed.)
3. Gropper SS, Smith JL, Grodd JL (2005). Advanced nutrition and human metabolism. (4th
ed)
4. Gulati, K. B. (2014). senior practical physical chemistry (sixteenth edition).
5. S. Zumdal, S. (1998). Chemistry (Third edition).

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