FACULTY OF SCIENCE

BACHELOR OF SCIENCE (HONOURS) DIETETICS

UDDN1304

NUTRITIONAL BIOCHEMISTRY

EXPERIMENT No: 6

EXPERIMENT TITLE: Determination of Vitamin C Using

Indophenol Method

STUDENT NAME &ID : Chan Hui Qi

LECTURER : Dr. Sinouvassane Djearamane

PRACTICAL GROUP : P6

DATE OF SUBMISSION : 17 April 2022

Experiment 6: Determination of Vitamin C Using Indophenol Method

1.0 Introduction & Objective

1.1 Introduction
Ascorbic acid or commonly known as vitamin C is a type of water-soluble vitamins that is easily
destroyed by heat and alkali. Vitamin C will be lost upon cooking. It is essential for the body to
maintain health. However, vitamin C cannot be produced by the body itself but it is taken from
diet. It plays a major role for the body to carry out metabolism and many body processes. For
example, vitamin C is very important for the collagen to be synthesised and provide tensile
strength to the fibres. Moreover, vitamin C is also helpful in haemoglobin metabolism, synthesis
of steroid hormones, formation of bile acids, and promotes the immunity system by stimulating
leukocytes and antibodies. Without vitamin C in diet, many health problems will occur such as
cataract, scurvy and internal haemorrhage. Scurvy is a health condition in which a patient has
deficiency of vitamin C. Scurvy will cause anaemia, edema, skin problem, gum disease, poor
wound healing and mental problems. In their diet, people could obtain vitamin C from fruits such
as strawberries, lemon, orange and vegetables such as spinach, potato and broccoli. Vitamin C
also can be gained through supplements. The recommended daily intake of vitamin C is 65 to 90
milligrams for adults and the maximum limit is up to 2000 mg per day. Too much vitamin C
intake or known as hypervitaminosis C will not lead to harmful situations but it has a chance to
cause nausea, diarrhea, abdominal pain and insomnia.

1.2 Objective
The objective of this experiment is to learn how to determine the vitamin C content in a sample
using the indophenol method.

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2.0 Materials and Method
2.1 Principle
There are many methods of analysis of vitamin C content in a sample. The most common
method is the indophenol method. It is the most rapid and controlled method for determining
vitamin C content (Nielsen, 2017). In this experiment, titration method with indophenol
colouring reagent or 2,6-dichlorophenolindophenol or commonly known as DCPIP is the main
principle. Indophenol indicator is to test the presence of vitamin C by oxidising reactions with its
colour-changing properties. Indophenol will reduce vitamin C to pink colour and oxidise to form
colourless solution (CH 112 Vitamin C Detective, n.d.). The end point of the titration is indicated
by the remaining colour of pink solution. Titration is a common laboratory test to determine the
concentration of a reactant. For this experiment, indophenol indicator is titrated to the sample
that contains an unknown amount of vitamin C. The sample is added with acids such as
metaphosphoric acid to react with the vitamin C contained in the sample. A standard vitamin C
solution is prepared and titrated to use as a reference. The amount of vitamin C present in a
sample can be calculated based on its quantitative relationship to the amount of standardised
indophenol indicator used.

2.2 Detailed procedure

The procedure is divided into three main parts:
A. Preparation of reagents
Indophenol colouring reagent
50g of 2,6 - dichlorophenolindophenol is dissolved in 80 ml of distilled water in a beaker. The
dissolved solution is then transferred to 100ml volumetric flask and made up to mark with
distilled water. The volumetric flask is shaked and the solution is filtered through filter paper.

20% Metaphosphoric acid solution

40g of metaphosphoric acid powder is measured in a beaker on a top-loading balance. 150ml of
distilled water is added to dissolve the acid using a magnetic stirrer. The solution is then
transferred and distilled water is added to reach 200ml in a measuring cylinder.

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5% Metaphosphoric acid solution
A 100 ml of 20% metaphosphoric acid solution is mixed with 300ml of distilled water in a
beaker.

B. Preparation for ascorbic acid standard

1. Add indophenol colouring reagent into a burette and mark the initial reading.
2. 5 ml of ascorbic acid is mixed with 5 ml of 5% metaphosphoric acid.
3. The solution is then titrated with an indophenol colouring agent.
4. The titration process is stopped immediately when pink colour appears.
5. The final reading of the burette is recorded.

C. Preparation of sample (Ascorbic acid content in orange juice)

1. An orange is peeled and juiced.
2. 5 ml of the orange juice is measured and transferred into a conical flask.
3. Another 5 ml of 5% metaphosphoric acid is added into the orange juice.
4. A burette is filled with indophenol colouring reagent and the initial volume is recorded.
5. The solution is titrated with indophenol colouring reagent from the burette.
6. The titration process is stopped immediately when pink colour appears.
7. The final reading of the burette is recorded.

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3.0 Results
500 × 𝑉2 × 100
Amount of ascorbic acid content (mg/100 ml) =
𝑉1 × 𝑌

500 = µg of standard ascorbic acid taken for titration

V1 = Volume of indophenol used to titrate 500µg of standard ascorbic acid
V2 = Volume of indophenol used to titrate 5ml of test sample
Y = Volume of the test sample taken for titration

V1 = 1.9 mg
V2 = 8.2 mg
Y = 5 ml

Amount of ascorbic acid content (mg/100ml)

500 × 8.2 × 100
=
1.9 × 5

410 000
=
9.5

= 43 157.9 mg/100ml

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4.0 Discussion
4.1 Physiological functions of vitamin C
Vitamin C is very important for maintaining body health. It is classified as a type of water
soluble vitamin and can be easily destroyed by heat and alkali. Vitamin C aids in the body
metabolism and many physiological processes. First, vitamin C is very essential for the
formation of protein for building up skin, tendons and ligaments as well as blood capillary. It
aids in formation of cross links in the collagen, thus it provides the tensile strength for the fibres
and for protein synthesis. In this way, vitamin C can also benefit in healthier skin, healing
wounds and forming scar tissue (Devaki and Raveendran, 2017). With vitamin C, it can prevent
formation of rough skin and coil-shaped body hair. Next, vitamin C enhances the iron absorption
from the intestine. It has the ability to reduce the ferric iron (Fe3+) to the ferrous iron (Fe2+) state.
In this way, the intestinal cell could absorb the iron more easily (Doseděl et al., 2021).
Absorption of iron is vital for haemoglobin production. Vitamin C is also important for
converting the methemoglobin which cannot transport oxygen to haemoglobin which transport
oxygen efficiently. Moreover, vitamin C can aid in maturation of red blood cells. Folic acid or
commonly known as vitamin B9 is important to prevent macrocytic anaemia. Vitamin C
stimulates the enzyme folate reductase to reduce folic acid to tetrahydrofolic acid (THF) which
could prevent the formation of abnormal red blood cells that might cause problems in the body.
Besides that, vitamin C also helps in the adrenal cortex found in the adrenal glands to synthesis
steroid hormones especially the corticosteroids. Next, vitamin C is commonly known to increase
immunity of the body as it can trigger the leukocytes to carry out phagocytosis to engulf the
bacterias that are not supposed to present in the body. Not only that, vitamin C also assists in the
formation of antibodies such as humoral immunity and antibody mediated immunity. With this,
vitamin C could prevent cough, flu and other infections. Lastly, vitamin C is an antioxidant
substance that could prevent cancer formation (Bürzle and Hediger, 2012). According to
research, vitamin C has much more function that associated with the properties stated above. For
example, a person that takes enough vitamin C could prevent joint pain, extensive bruising,
fatigue, and scurvy as well as preventing mental problems such as sudden horrible mood.

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4.2 Rich source of Vitamin C
Vitamin C is found abundantly in natural substances such as fruits and vegetables. The body does
not produce or store vitamin C itself, therefore, vitamin C is required from daily intake. An
orange or a cup of strawberries is enough to provide vitamin C for the day. There are other foods
that are rich in vitamin C including lemon, rose hip, chilli pepper, spinach, cranberry, broccoli,
cayenne pepper, potato, guava, pummelo, longan fruit, melon and papaya. Fortified cereals,
which are cereals that are mixed with minerals and vitamins are also a food that could provide
vitamin C (Devaki and Raveendran, 2017). Vitamin C can also be found in animal products such
as beef spleen as they could synthesise their own vitamin C. Kakadu plum from Australia and
camu-camu fruit from South America are found to have the richest vitamin C content (Doseděl et
al., 2021). People who might need more vitamin C will normally choose to take supplements.
Vitamin C supplements are very common these days. A single vitamin C tablet might contain
250 to 1000mg of vitamin C.

4.3 Deficiency syndromes of vitamin C

The Recommended Dietary Allowance (RDA) of vitamin C for a normal adult is between 65 to
90 milligrams daily. Those who are pregnant and lactation will need more vitamin C to maintain
their health. The recommended amount for this group of people is 85 to 120 mg per day (Carr
and Frei, 1999). In most developed countries, deficiency of vitamin C in normal people is
uncommon as it could be obtained from a healthy diet. However, those countries who are facing
a lack of food and starvation of their citizens might have a higher risk of developing diseases due
to insufficient nutrition. Not only that, smoking can also deplete vitamin C levels in the body.
Patients who are facing multiple diseases will also affect their absorption of vitamin C such as
people who are facing diarrhoea for a long period, high fever or inflammation, and surgery
(Johnson, 2020). The deficiency of vitamin C will lead to many health problems. The most
common syndrome is scurvy. The patients with scurvy will feel uncomfortable with their mouth
as it affects the gum from the teeth as the lack of collagen. The symptoms also include nostrils
bleeding, anaemia, myalgia, ulceration on legs, petechiae and skin problems (Maxfield and
Crane, 2021). When the intake of vitamin C is low, it might affect the formation of connective
tissues, bones and dentin which causes fragile blood vessels and might lead to haemorrhage and
defect in bone formation (Johnson, 2020). When collagen cannot form properly, the intercellular

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cement substance will become brittle. When the situation is dangerous, the conjunctiva and
retina might also bleed. Epistaxis is a syndrome that is caused by deficiency of vitamin C. The
symptoms are bleeding in the nasal cavity. Deficiency of vitamin C also leads to hematuria
which is the presence of red blood cells in the urine. Melena is defined as bleeding in the
intestinal region which results in dark black and tarry faeces and is associated with upper
gastrointestinal bleeding. Deficiency of vitamin C in children will cause bone tissue formation to
become abnormal which results in problems in growth and development. For pregnant women,
deficiency of vitamin C will affect the infant’s health and might lead to infantile scurvy. Infantile
scurvy or known as Barlow’s disease will occur in infants aged between 6 to 12 months in the
breastfeeding period. The lack of vitamin C in breast milk will affect the infant’s blood valves
(Melnitchouk et al., 2013).

4.4 Toxic effects of vitamin C in excess

The Tolerable Upper Intake Level (TUIL) of vitamin C is 2000 mg per day. Although too much
dietary intake of vitamin C is unlikely to be harmful and it is uncommon, but overdosing vitamin
C supplements or hypervitaminosis C might cause several effects. Taking vitamin C beyond
2000 mg will have higher chances of developing gastrointestinal distress and diarrhoea. It also
influences the balance of antioxidant-prooxidant in the body (Johnson, 2020). Excess vitamin C
in the body will strike hemolysis when the glucose-6-phosphate dehydrogenase (G6PD) is absent
or less in the body. Not only that, it will also cause renal colic, dental decalcification, occult
rectal bleeding and increase in estrogen levels (Rosenbloom, 2021). Too much vitamin C
supplementation will cause kidney stones and affect the excretion of oxalate and uric acid in
urine. Excess vitamin C could also cause problems in the ability of intaking other nutrients. For
instance, vitamin C may reduce the absorption of vitamin B12 or cobalamins and copper in the
body (Nall, 2019). As vitamin C could increase the absorption of iron, it may also cause
excessive levels of iron in the blood. According to research, the high level of vitamin C is likely
to cause bone spurs and face joint pain (Arthritis.org, 2016). Excessive intake of vitamin C will
cause adverse effects, therefore it is recommended that people should seek a doctor or pharmacist
for advice before taking the vitamin C supplement.

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5.0 References
‌Arthritis.org., 2016. [online] Available at:
https://www.arthritis.org/health-wellness/treatment/complementary-therapies/supplement
s-and-vitamins/vitamin-and-mineral-guide-for-arthritis [Accessed 16 April 2022].

‌Bürzle, M. and Hediger, M.A., 2012. Functional and Physiological Role of Vitamin C
Transporters. Co-Transport Systems, [online] pp.357–375. Available at:
https://pubmed.ncbi.nlm.nih.gov/23177992/ [Accessed 16 April 2022].

‌Carr, A.C. and Frei, B., 1999. Toward a new recommended dietary allowance for vitamin C
based on antioxidant and health effects in humans. The American Journal of Clinical
Nutrition, [online] 69(6), pp.1086–1107. Available at:
https://academic.oup.com/ajcn/article/69/6/1086/4714888 [Accessed 16 April 2022].

CH 112 Special Assignment #8 Vitamin C Detective. (n.d.). [online] Available at:

https://www.colby.edu/chemistry/CH112/Lab/Expt8.pdf.

Devaki, S.J. and Raveendran, R.L., 2017. Vitamin C: Sources, Functions, Sensing and Analysis.
Vitamin C. [online] Available at: https://www.intechopen.com/chapters/56440 [Accessed
16 April 2022].

‌Doseděl, M., Jirkovský, E., Macáková, K., Krčmová, L., Javorská, L., Pourová, J., Mercolini, L.,
Remião, F., Nováková, L. and Mladěnka, P., 2021. Vitamin C—Sources, Physiological
Role, Kinetics, Deficiency, Use, Toxicity, and Determination. Nutrients, [online] 13(2),
p.615. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918462/ [Accessed
16 April 2022].

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Johnson, L.E., 2020. Vitamin C Deficiency. [online] MSD Manual Professional Edition.
Available at:
https://www.msdmanuals.com/professional/nutritional-disorders/vitamin-deficiency,-depe
ndency,-and-toxicity/vitamin-c-deficiency [Accessed 16 April 2022].

‌Maxfield, L. and Crane, J.S., 2021. Vitamin C Deficiency. [online] Nih.gov. Available at:
https://www.ncbi.nlm.nih.gov/books/NBK493187/ [Accessed 16 April 2022].

‌Melnitchouk, S.I., Seeburger, J., Kaeding, A.F., Misfeld, M., Mohr, F.W. and Borger, M.A.,
2013. Barlow’s mitral valve disease: results of conventional and minimally invasive
repair approaches. Annals of cardiothoracic surgery, [online] 2(6), pp.768–73.
https://dx.doi.org/10.3978%2Fj.issn.2225-319X.2013.10.07‌

‌Nall, R., 2019. What happens when you take too much vitamin C? [online]
Medicalnewstoday.com. Available at:
https://www.medicalnewstoday.com/articles/326249#recommended-dosage [Accessed 16
April 2022].

Nielsen, S.S., 2017. Vitamin C Determination by Indophenol Method. Food Analysis Laboratory
Manual, [online] pp.143–146. https://doi.org/10.1007/978-3-319-44127-6_15‌

Rosenbloom, M., 2021. Vitamin Toxicity Clinical Presentation: History, Physical Examination.
[online] Medscape.com. Available at:
https://emedicine.medscape.com/article/819426-clinical#b1 [Accessed 16 April 2022].