Vitamin C (L-Ascorbic acid) is an important vitamin. Lack of ascorbic acid in diet causes scurvy,
a disease characterized by weakness, small hemorrhages throughout the body that causes gums
and skin to bleed, and loosening of the teeth. Scurvy was a serious problem for English sailors in
the 1600s and 1700s. James Lind, a British doctor. His test was conducted at sea in May 1747 and
consisted of two groups of men. One group was provided with lemon juice in addition to their
normal rations, the other was not. This test was considered to be the first example of a controlled
experiment comparing results on two populations of a factor applied to one group only with all
other factors the same. The results of the tests conclusively showed that lemons prevented the
disease. Around 1930 two scientists working independently isolated and published their findings
on vitamin C. The men found that vitamin C prevented and treated scurvy. The term ascorbic acid
was adopted to describe its ability to prevent scurvy.Since this discovery, the Royal Navy make
sure that all sailors had lemon juice to drink when they were at sea for longer than one month.
Figure 1: Structure of vitamin C (ascorbic acid).
Ascorbic acid (C6H8O6) is a water-soluble vitamin, whose structure is shown in Figure 1. Vitamin
C is easily oxidized, and the majority of its functions in vivo rely on this property. It plays a key
role in the body’s synthesis of collagen and norepinephrine by keeping the enzymes responsible
for these processes in their active reduced form. Vitamin C may also play a role in detoxifying
by-products of respiration.
The human body cannot produce ascorbic acid, and so it must be obtained entirely through one’s
diet. A very small daily intake of vitamin C (10-15 mg/day for an adult) is required to avoid
deficiency and stave off scurvy. The body requires extra vitamin C when fending infections.
Since ascorbic acid is a water-soluble vitamin, risk of getting an over dose is minimal and any
unused vitamin C will be excreted. The minimum daily requirement is 30 mg, and the
recommended daily allowance is 60-70 mg per person.
Fruits, vegetables, and organ meats (e.g., liver and kidney) are generally the best sources of
ascorbic acid; muscle meats and most seeds do not contain significant amounts of ascorbic acid.
The amount of ascorbic acid in plants varies greatly, depending on such factors as the variety,
weather, and maturity. But the most significant determinant of vitamin C content in foods is how
the food is stored and prepared. Since vitamin C is easily oxidized, storage and the cooking in air
leads to the eventual oxidation of vitamin C by oxygen in the atmosphere. In addition, ascorbic
acid’s water-solubility means that a significant amount of vitamin C present in a food can be lost
by boiling it and then discarding the cooking water.
Estimation of Vitamin C from Supplied Sample
Ascorbic acid as the name implies possesses the usual acidic property, (donation of H+ ion). The
acidity of vitamin C is not due to the carboxylic grouptied up in lactone from but to the ionization
of enol group. The method of estimation of vitamin C is based on the stoichiometric reduction of
dye 2,6-dichlorophenolindophenol to colourless compound by ascorbic acid. The titration is
conducted in presence of metaphosphoric acid mixture in order to inhibit the ascorbic acid
oxidation catalyzed by certain metallic ions (such as copper & silver ions present in distilled
water). In aqueous system, the vitamin is easily oxidized, the instability increases with increase in
pH. Metaphosphoric acid stabilizes the solution by lowering the pH. As a result of titration
vitamin C gets oxidized to dehydro ascorbic acid structurally as follows.
Vit. C + 2,6-dichlorophenolindophenol ↔ L-dehydroascorbic acid + 2,6-dichlorophenolindophenol
(Oxidized form. Blue) (Colourless)
(1) To examine the percentage of vitamin C content in supplied sample.
(2) To calculate the content of vitamin C of supplied sample based on content of vitamin C from
ascorbic acid powder as reference.
Slice of guava , 2,6-dichlorophenolindophenol , 3% metaphosphoric acid ,standard Vit.C solution.
Burette , conical flasks , retort stand , beakers , mortar.
Figure 2: Burette with retort stand Figure 3: Mortar Figure 4: Centrifuge
(1) 5.0 grams of guava is weighed. After that, it is grinded into guava juice with 20ml
3%metaphosporic acid. Guava juice is poured into two small tube (10ml each) and send to
(2) Standard vitamin C solution is prepared by taking 10 mg of vitamin C mixed with 100 ml of
3% metaphosphoric acid in 100 ml volumetric flask.
(3) 10 ml of standard vitamin C solution is taken in a conical flask and it is titrated with prepared
dye from a burette. The titration is terminated by the appearance of a permanent light pink
colour in the titration medium. The operation is repeated for two times and the burette
reading is recorded each time.
(4) The supplied sample is transferred in a 50 ml volumetric flask make up to the mark by 3%
metaphosphoric acid. 10 ml of this supplied solution is taken into a conical flask and it is
titrated with the dye. This operation is repeated for two times and the reading is taken each
Percentage error of burette: ±0.01 ml
Table1: Titration volume for standard Vit. C
Volume of standard
Vit. C (mg)
1 10 1 0.00 26.00
2 10 1 0.00 25.50
Table2: Titration volume for 5.0g of Guava
Average Reading (ml)
1 10 0.00 13.40
2 10 0.00 12.40
Content of Vitamin C in Guava
x=dye used to titrate ascorbic acid
y=dye used to titrate guava juice
10mg of standard vitamin C 100ml of solution
1mg of standard vitamin C 10ml of solution
mg of vitamin C × 5ml
= x 5ml
= 0.5010 mg
0.5010 mg x 20 = 50.10 mg
= 0.0501 g
*In every 100g of guava, there is 0.0501g of vitamin C in the content.
Vitamin C (ascorbic acid) gets oxidized to its dehydro form by air specially at alkaline pH.
However, it is suitable in an acidic solution. Therefore, vitamin C is extracted in metaphosphoric
acid or in a mixture of metaphosphoric and dilute acetic acid. Its estimation in the extract is
carried out by titrating it against 2.6-dichlorophenol indophenols solution. Oxidized form of this
dye is blue in colour in an alkaline medium and red in an acidic medium. Reduced form of the
dye, on the other hand, is colourless and is termed as its leuco form. The redox reaction occurring
during the titration process below:
From the results of experiment, I had found that there is varies between the result of vit. C
content come from the guava juice with the standard vitamin C solution. This variation may due
to several factors as below:
1) Production factors and climate conditions: High nitrogen fertilizer rates can lower vitamin
C levels in citrus fruits. Proper potassium levels are also needed for good vitamin C levels.
Additionally, climate, especially temperature -- total available heat -- affect vitamin C levels.
Areas with cool nights produce citrus fruits with higher vitamin C levels. Hot tropical areas
produce fruit with lower levels of vitamin C. Environmental conditions that increase the
acidity of citrus fruits also increase vitamin C levels.
2) Maturity state and position on the tree: Vitamin C decreases during the ripening process.
Immature fruit has the highest levels. The position on the tree also affects vitamin C levels.
Since sunlight exposure enhances vitamin C levels, fruit positioned on the outside of the tree
and on the south side have higher levels. Shaded inside fruit has the lowest.
3) Handling and storage: Oxygen is the most destructive ingredient in juice causing
degradation of vitamin C. However, one of the major sugars found in guava juice, fructose,
can also cause vitamin C breakdown. The higher the fructose content, the greater the loss of
vitamin C. Conversely, higher acid levels of citric and malic acids stabilize vitamin C.
Throughout this experiment, I had found that 100g of guava contain 0.0501g of Vitamin C.
3) Biochemistry Fourth Edition, Reginald H.Garret /Charles M.Grisham
4) Experimental Biochemistry A Student Companion