HOW DOES CONCENTRATION OF

ASCORBIC ACID/VITAMIN C IN

CITRUS FRUITS VARY OVER TIME?

SUBJECT: BIOLOGY

LEVEL: HL

CANDIDATE CODE: jyw118

SESSION: MAY 2022
Research Question: How does the concentration of Ascorbic acid/Vitamin C vary in a variety of

Citrus fruits over time?

Background information:

Having been in a homestead where horticulture is carried out on a small scale, have always

wondered which food nutrient is present in most citrus fruits. On doing research, I realized

Vitamin C and from this, I desired to investigate the amount of Vitamin C over a certain period

of time from the time of harvest to the time of consumption.

Vitamin C being readily available in most fruits, I decided to use Apple (Malus Domestica),

Orange (Citrus aurantium), Mango (Magnifera indica L.), Lemon (citrus lemon.) and

Pineapple (Ananas Comosus)

First of all, because they were in the season, at that time they are among the most consumed

fruits by the population in the tropical regions. I want to consider different genera since there

would be a significant difference in the vitamin C content putting in mind that these fruits have

been able to flourish despite the harsh weather conditions of the tropics.

Vitamin C also known as ascorbic acid is a water-soluble vitamin that is present in many fruits

and some vegetables. This is an essential anti-oxidant required by the body.

The human body does not synthesize vitamin C due to mutation of GLO gene which codes for

the production of the enzyme L-guluno-y-lactone oxidase, therefore our body has recommended

daily allowances of different age groups. In animals, collagen, protein contains proline in many

positions however there is a need to convert proline to hydroxyl-proline hence increasing

stability.

Vitamin C plays an important role in the synthesis of collagen as it is needed in the conversion of

proline to hydroxyl-proline, it also leads to abnormal production of collagen fibers that form
several tissues in the body such as ligaments, tendons, muscles, and skin, this gives them high

tensile strength.

Deficiency of Vitamin C causes scurvy. (Andrew Allot and David Mindorf).

The structure of ascorbic acid is as seen below;

Figure. 1

This being a reducing agent, vitamin C is prone to heat, oxidation and storage over time which

may alter the structure. During oxidation vitamin C is converted to hydro-ascorbic acid as seen

below;

Figure. 2

This is redox reaction where both reduction and oxidation occurs concurrently.

HYPOTHESIS
Null hypothesis: There is no significant difference in the vitamin C concentration between
different fruits. Any possibilities for variation in the vitamin C concentration will be due to
uncontrolled variables and not because of the independent variables.

Alternative Hypothesis: A significant difference will be observed in the Vitamin C concentration

of the different selected fruit over time.

Independent Variables: The fruits used

This is because different fruits have different genetic makeup as well as the conditions suitable
for their growth.

Dependent Variables: Vitamin C concentration

Ascorbic acid concentration would then be tested by using controlled volume of 4cm3 of each
fruit solution.

Table. 1 shows the controlled variables of this experiment

Variable How it was controlled Reason
1 Time A period of three days This is to allow room
was left between each for any variations of
experiment. significance

2 Freshness of the fruits All the fruits picked This was to reduce
from the same tree on the sources of error
the same day. that would come in
due to other factors.

Uncontrolled Variables

1. The temperature of the room could not be controlled but it affects the ascorbic acid
concentration due to its oxidation to hydroxyascorbic acid

2. The weather condition could also affects the concentration of Vitamin C to a small extent.
REAGENTS AND APPARATUS USED

o 0.1% dichlorophenol indole phenol

(DCPIP) o Distilled water o 2%
Standardized Vitamin C o Weighing
balance 1 o Blender 1 o Petri dish 4 o
Knife 1 o Dropper 4
o Measuring Cylinder (500 ml )
+_5cm3 o Beaker-glass 4 o Beakers –
plastic 4 o Test tube rack 1 o Burrette-
250cm (+_5cm3) o Test tubes- 5 and a
Clamp

Figure 3: Some of the apparatus that were used during my investigation are shown the figure
below.
SAFETY PRECAUTIONS

Use of hand gloves

Use of lab coat to protect from stains

Caution taken while using the knife Use

of goggles.

PROCEDURE

The fruits were washed with clean water and a knife was used to peel off their outer skin and

then cut into smaller pieces and the seeds removed from some of them.

For each fruit pieces, 80g was weighed and transferred into a blender to be crushed.

100 ml of distilled water was then added and then sieved into an empty clean plastic beaker and

the extract was then labelled using the fruit name.

The same procedure was repeated for all the remaining fruits and labeled.

Figure 4: Some of the Citrus fruits that were used are as seen below:
Standardization of DCPIP

The burette was filled with standard vitamin C solution to the mark.

4 cm3 of DCPIP solution was pipetted into a test tube and shaken.

Titration was carried out by opening the tap on the burette so that the ascorbic acid mixes with
the DCPIP solution. Upon mixing the two solutions, the solution turned pink and quickly to
colorless. This means the ascorbic acid was being reduced to dehydro-ascorbic acid.

The titration continued until faint pale color persisted for about 15 seconds.

This was repeated three times and the burette reading was recorded as seen in the table 2 below;

Table.2

DAY 1 DAY2
Titration No. 1 2 3 1 2 3
Final reading/cm3 1.50 3.20 4.80 1.00 2.00 2.90
Initial reading/cm3 0.00 1.50 3.20 0.00 1.20 2.00
Volume of 1.50 1.70 1.60 1.00 0.80 0.90
Ascorbic acid/cm3

Volume of DCPIP =y/2x

(A tenth volume is equivalent to 0.1mg of ascorbic acid)

Note

X – Concentration of standard ascorbic acid

For the extracts, same procedure was followed but instead of using standard ascorbic acid, I used
the extract of the different juices.
If the titre lies between 0.04 and 6 cm3, are calculated knowing the concentration of DCPIP in
milligram per cm3.

Table.3 below shows results of titration of DCPIP with a solution ascorbic acid of unknown
concentration.
Day 1 Day 2 Day 3
FRUIT Trials 1 2 3 1 2 3 1 2 3

ORANGES Final burette 0.50 1.10 1.70 0.70 1.40 2.20 0.90 1.80 2.70
reading(+_ 0.1cm3)

Initial burette 0.00 0.5 1.10 0.00 0.70 1.40 0.00 0.90 1.80

reading(+_0.1cm3)

Volume of Juice 0.50 0.60 0.60 0.70 0.70 0.80 0.90 0.90 0.90
used(+_0.1cm3)

PINEAPPLE Final burette 0.60 1.20 1.70 0.70 1.50 2.20 0.90 1.70 2.60
reading(+_ 0.1cm3)

Initial burette 0.00 0.60 1.20 0.00 0.70 1.50 0.00 0.90 1.70
reading(+_ 0.1cm3)

Volume of Juice 0.60 0.60 0.50 0.70 0.80 0.70 0.90 0.8 0.90
used(+_0.1cm3)

APPLE Final burette 1.00 1.80 2.90 1.10 2.30 3.50 1.30 2.60 4.00
reading(+_ 0.1cm3)

Initial burette 0.00 1.00 1.80 0.00 1.10 2.30 0.00 1.30 2.60
reading(+_ 0.1cm3)
 Volume of Juice 1.00 0.80 1.10 1.10 1.20 1.20 1.30 1.30 1.40
used(+_0.1cm3)

LEMON Final burette 0.20 0.41 0.63 0.24 0.49 0.76 0.27 0.55 0.83
reading(+_ 0.1cm3)

Initial burette 0.00 0.20 0.41 0.00 0.24 0.49 0.00 0.27 0.55
reading(+_ 0.1cm3)

Volume of Juice 0.20 0.21 0.22 0.24 0.25 0.25 0.27 0.28 0.28
used(+_0.1cm3)

MANGO Final burette 0.60 1.30 2.00 0.80 1.60 2.70 1.00 1.90 2.90
reading(+_ 0.1cm3)

Initial burette 0.00 0.60 1.30 0.00 0.80 1.70 0.00 1.00 1.90
reading(+_ 0.1cm3)

Volume of Juice 0.60 0.70 0.70 0.80 0.80 1.00 1.00 0.90 1.00
used(+_0.1cm3)

Table.4 shows average volume of ascorbic acid used in a day (cm3)

FRUITS DAY 1 DAY 2 DAY 3
Oranges 0.56 0.73 0.90
Pinepple 0.56 0.73 0.86
Apple 0.96 1.16 1.33
Lemon 0.21 0.24 0.27
Mango 0.66 0.86 0.96

DATA COLLECTION AND PROCESSING

Qualitative data

Color changes was observed whenever DCPIP was titrated with the vitamin C extracts of each
fruit.
The passion fruit and Orange extract turned pink on mixing.

For mango and Lemon, a pink layer is formed on mixing with DCPIP solution that disappeared
on shaking to colorless

Reaction between Ascorbic acid and DCPIP is as seen below in figure 5.

Quantitative

Volume of ascorbic acid = 1mg of DCPIP

= z/2x = 1.6/2(10)

=0.08 cm3

Calculation of DCPIP concentration:

0.08 cm3 of ascorbic acid was oxidized by 0.1 mg of DCPIP

0.4 Cm3 of vitamin C is oxidized by 0.1x0.4 = 5mg of ascorbic acid

0.08

2cm3--------5mg of ascorbic acid

100cm3 ------5x100 = 250 mg of ascorbic acid.

30cm3 0f Orange juice ----250mg of ascorbic acid

1cm3 of Orange Juice ------250 = 8.33mg 0f ascorbic acid

30
Table.5 showing ascorbic acid concentration in mg/cm3 for the different fruit juices

Ascorbic acid concentration( mg/cm3)

Oranges Pineapple Apple Lemon Mango

DAY 1 4.66 4.66 7.99 1.74 5.50
DAY 2 6.08 6.08 9.66 1.99 7.16
DAY 3 7.49 7.16 11.07 2.25 7.99

GRAPHICAL REPRESENTATION OF DATA

Graph 1 shows the ascorbic acid in the orange, Lemon, Apple, Mango pineapple.
ANALYSIS OF DATA USING STATISTICAL TEST

To analyze this data, I will use ANOVA variance (Analysis of variance):

Single factor test which is an analytical test in Microsoft excel instead of the test.

The values obtained are tabulated by software. ANOVA is most suitable considering the fact that
it is used to analyze data with more than three independent variables.

By using ANOVA test, will be using it to test the null hypothesis which is the different fruits
used will have no effect on the ascorbic acid concentration.

Table.6 shows ANOVA: single factor- vitamin C concentration for the selected fruits upon
testing over a given period of time

Anova: single factor

Groups Count Sum Average Variance

Orange 3 18.23 6.08
5.26 Pineapple 3 17.16 5.72
4.50 Apple 3 28.72 9.57
2.60
Lemon 3 5.98 1.99 4.04
Mango 3 20.65 6.88 1.40

CONCLUSION

From the ANOVA test results, the effective size of data was calculated using a notation N2 (sum

of squares in the first row divided by the total sum of squares in the final row). The sum of

squares used was the one between the groups as it is easier to use.
Based on the Cohen guidelines, the effect is used in determining the extent to which the

independent variables affect the dependent variables. The calculated value of n2 is 0.88 when

converted to a percentage, it is 88%.

The large effect size suggests that 80% of the variance in the ascorbic acid concentration is due

to the independent variables.

EVALUATION OF THE INVESTIGATION:

During my experiment, there were strengths and weaknesses which could also be considered as

sources of error.

My strength in this investigation was the control of freshness of the fruits where these fruits were

not purchased from the market but plucked directly from the tree and tested for ascorbic acid

concentration within a set time frame in my procedure.

However, the sources of error for example was the fact that the citrus fruits such as lemon and

orange contain citric acid which may interrupt the test for Vitamin C concentration.

Basing on research, certain fruits have higher citric acid levels during their early stages of growth

because, during fruit ripening, there is a downward trend in the concentration of organic acid due

to an increase in membrane permeability allowing the acid to be stored in respiring cells.

Reduction of the acid which has been translocated from the leaves reduces the ability of the fruit

to synthesize the organic acid. With maturity, the glucose levels increase in the fruit causing

change in the taste of sweetness.

The other source of inaccuracy in the data is the uncontrolled variables such as the room

temperature I couldn’t control by any means. The temperature must have had an effect on

ascorbic acid concentration whereby on exposure to high temperature, there would be a gradual

decline in the concentration of vitamin C.

The mass of the fruits was controlled by using the same mass of fruit samples.

This investigation could be improved using the fruits of the same species since similar species

have similar genetic makeup. This information obtained could be useful in the dietetics group as

many people could know when to take the fruits.

Bibliography

Y.H.Hui, Jozsef Bart, M. pilar Cano. Hand book of fruits and fruit processing.2008 Eddy,

Sam.Effective size of variance. 31 October 2010. 5 November 2018.

Rigoux, Bethany. Vitamin C .2 March 2013. 3 March 2019

George G, Y.H.Hui, Khachatourians.Food biotechnology.1996.

Chemistry, Oregon State. Vitamin C.2017.28 February 2019

Andrew Allott and David Mindorf.Biology course companion. London: Oxford University Press,
2014.