1.0
INTRODUCTION
1.1
THEORY AND LITERATURE REVIEW
Vitamins are complex organic molecules required in small amounts by the body in
order to maintain health and well‐being. Generally, the daily requirements of the various
vitamins are very small quantities, but whenever these small quantities are not available, the
body cannot function properly. Vitamin C is important for the human body because it helps
the body to absorb iron, helps wounds to heal, helps red blood cell formation and helps to
fight infections. For example, a lack of vitamin C can cause a disease called scurvy, iron
deficiency and poor wound healing [1]. The healthy diet should include high amounts of
vitamin C because the human body cannot make it’s own vitamin C [2]. Oranges are an
excellent source of vitamin C [3,4]. We need to get vitamin C from the foods we eat [5,6].
Vitamin C is found in fruits such as oranges, limes, and grapefruit, and vegetables, including
tomatoes, green peppers, and potatoes. The recommended amount of vitamin C is 60 to 90
milligrams per day [7]. People who smoke need more vitamin C in their diet, because they
lose 25 mg. of vitamin C every time they smoke a cigarette. People who are stressed, have
infections, take antibiotics, drink lots of alcohol or have been injured need more vitamin C in
their diet [8]. Some people think they are receiving same concentration of vitamin C in any
type of fruits, even commercial fruit juices, or natural fruit juices. However, the commercial
fruit juice is typically designed to appeal to the taste preferences of the market, and will
therefore contain different flavour packs or chemicals depending on where it will eventually
end up. According to Hamilton, the juice created for the North American market tends to
contain high amounts of ethyl butyrate, which is one of the most commonly used chemicals
in both flavours and fragrances [9]. The method which Kabasakalis et al. (2000) used is to
determine concentration of vitamin C titration method where vitamin C in commercial fruit
juices was titrated against aqueous sodium dichlorophenolindophenol with starch as indicator.
1
The sodium dichlorophenolindophenol solution was standardised with sodium thiosulphate
will concentration of 0.01 N in a matrix of potassium iodide (50%) and HCI (1 N) using
starch as indicator. The amount of vitamin C determined in the samples were between 24 to
430 ppm of juice [10].
1.2
PROBLEM STATEMENTS
Fruit juices are liquid naturally contained in fruit or vegetable tissues. The labels of
fruit juice package may be misleading as the companies may underestimate or overestimate
the actual content. Thus, the problem is that the consumers do not know the actual amount of
vitamin C in the commercial fruit juices, unless the amount of vitamin C is stated on the label
of the package. Some people think they are receiving same concentration of vitamin C in any
type of fruits, even commercial fruit juices, or fresh fruit juices. However, the commercial
fruit juice is typically designed to appeal to the taste preferences of the market, and will
therefore contain different flavour packs or chemicals depending on where it will eventually
end up. Commercial fruit juice has already combined with oxygen, undergoes oxidation and
all of the nutrients have been destroyed. It also have artificial, including often a huge amount
of added sugar. However, fresh fruit juice has a shelf life of sometimes more than day, and
has hundreds of times more nutrients, enzymes, and phytochemicals. Thus, this study is
carried out to determine concentration of vitamin C content in three commercial fruit juices
chosen, that are apple, orange and lychee by using titration method. At the same time,
identifying the highest and the lowest vitamin C concentration in commercial fruit juices as
well as to differentiate the concentration of vitamin C concentration between commercial
fruit juices and fresh fruit juices.
2
1.3
OBJECTIVE
The objectives of the study are :
a) To determine the concentration of vitamin C in different commercial fruit juices,
(apple, orange and lychee) of same brand by using DCPIP titration.
b) To identify the highest and the lowest vitamin C concentration in commercial fruit
juices.
c) To differentiate the vitamin C concentration between commercial fruit juices and
fresh fruit juices (apple, orange and lychee).
2.0
METHODOLOGY
2.1
Titration Method
In this experiment, titration method is used to determine the concentration of Vitamin C
in freshly prepared and commercial fruit juice samples. Titration or called as volumetric
analysis is a common laboratory method of quantitative analysis that can be used to
determine the concentration of a known analyte. A titrant of known concentration is
used to react with a solution of the analyte of unknown concentration. Using a
calibrated burette, it is possible to determine the exact amount of titrant that has been
consumed when the endpoint is reached. The endpoint is the point at which the titration
is complete, as determined by the colour change of an indicator.
3
2.2
Materials
Natural Fruit Juices
Commercial Fruit Juices
Ascorbic Acid
0.5% Oxalic Acid
Distilled Water
2,6-dichlorophenolindophenol (DCPIP) solution
2.3
Apparatus
Titration Set (Burette, Stand, Clamp, Tile and funnel)
250ml Conical Flask
Buchner Funnel and Filter Paper
Pipetman 1000 and Pipette Tips
25 ml Measuring Cylinder
250 ml Beaker
Knife
2.4
Methods/ Procedure
Part A Preparation of Standard Ascorbic Acid Solution
1. 0.2 g of ascorbic acid is weighted out and make up to 1 L of distilled water.
2. The concentration of the ascorbic acid solution is calculated by using the formula
below:
Concentration of ascorbic acid =
4
Mole
Volume
=
Mass
Molar mass
Volume
PART B Preparation of DCPIP Solution
1. Approximately 0.24 g DCPIP is weighted out and make up to 1 L of distilled water.
2. The concentration of DCPIP solution is calculated by using the formula below:
Concentration of DCPIP solution =
Mole
Volume
=
Mass
Molar mass
Volume
PART C Standardization of DCPIP Solution
1. 25 ml of 0.5% oxalic acid is measured and transferred into a 250 ml conical flask.
2. 10 ml of standard ascorbic acid solution is added into the conical flask which
contains oxalic acid by using a pipetman.
3. A trial run of titration is carried out with a titration set. The ascorbic acid solution
is titrated rapidly with the DCPIP solution. The DCPIP solution is added through
the burette and the solution is vortex well. Colour change of DCPIP solution to
pink is observed when the solution contacts with the ascorbic acid solution and
then becomes colourless after shaking well.
4. After the trial run, another three actual titrations to the ascorbic acid standard
solution is conducted and the results are being average. Then, DCPIP solution is
added drop by drop carefully when the volume of DCPIP solution used is close to
the end point volume.
5. The volume of DCPIP solution used is recorded.
6. The concentration of the DCPIP solution is calculated by using the formula below:
CV (Ascorbic acid) = CV (DCPIP)
* C refer to concentration
* V refer to volume
5
Part D Determination of the Vitamin C Concentration in Fruit Juice
1. A fruit is cut in half with knife and the juice is squeeze out.
2. Fresh fruit juice is collected with the aid of a Buchner funnel and filter paper, the
flesh and seed is separated from the juice.
3. 10 ml of the fruit juice is pipetted into a 250 ml conical flask, which contains 25ml
of 0.5% oxalic acid, and 10 ml of distilled water is added.
4. The fruit juice solution is titrated with the DCPIP solution in the burette to a pink
end point.
5. The test is triplicated and average the results are being averaged.
6. The vitamin C concentration in the fruit juice is calculated by using the following
formula.
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
* Mr refer to molar mass
* C refer to concentration
* V refer to volume
6
3.0
OBSERVATION AND RESULTS
3.1
PART A : Standardization of DCPIP solution
Table 3.1 : Titration of ascorbic acid with DCPIP Solution (Standard solution)
Volume of DCPIP used to titrate 1 ml
Sample
Initial Reading
Final Reading
of Ascorbic Acid Standard Solution
Number
(ml)
(ml)
(ml)
Trial 1
0.00
21.30
21.30
Trial 2
21.30
42.50
21.20
Trial 3
0.00
21.20
21.20
Average
21.20
Concentration of Ascorbic Acid Solution (Standard)
Molecular formula of ascorbic acid is C6H8O6.
Molar mass of ascorbic acid = 176.12
Mass of ascorbic acid is 0.2 g.
Concentration of ascorbic acid =
=
=
Mole
Volume
Mass
Molar mass
Volume
0.20
176.12
1L
= 0.00114 mol/L
7
Concentration of DCPIP Solution
Molecular formula of DCPIP is C12H7NCl2O2.
Molecular formula of DCPIP = 268.10
Mass of DCPIP is 0.24 g.
Concentration of DCPIP solution =
=
=
Mole
Volume
Mass
Molar mass
Volume
0.24
268.10
1L
= 0.000895 mol/L
8
3.2
PART B : Vitamin C Concentration in Commercial Fruit Juices
Table 3.2 : Titration of commercial fruit juices with DCPIP solution
Sample number
Volume of
Average
Initial Reading
Final Reading
DCPIP solution
volume of
(ml)
(ml)
used (ml)
DCPIP solution
used (ml)
Trial 1
Apple
0.00
22.00
22.00
Trial 2
Apple
22.00
43.90
21.90
Trial 3
Apple
0.00
21.90
21.90
Trial 1
Orange
0.00
25.50
25.50
Trial 2
Orange
0.00
25.60
25.60
Trial 3
Orange
0.00
25.60
25.60
Trial 1
Lychee
0.00
16.80
16.80
Trial 2
Lychee
16.80
33.50
16.70
Trial 3
Lychee
0.00
16.80
16.80
9
21.90
25.60
16.80
Average Volume of DCPIP Solution used in the Titration of Commercial
Fruit Juices
Average volume of DCPIP solution used (ml)
30
25
20
15
10
5
0
Apple
Orange
Type of commercial fruit juice
Lychee
Graph 3.2 : Graph of average volume of DCPIP solution used in the titration of commercial
fruit juices
Mass of ascorbic acid (mg) in 100 ml of commercial apple juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0219
= 0.003452 g/10 ml
= 34.52 mg/100 ml
10
Mass of ascorbic acid (mg) in 100 ml of commercial orange juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0256
= 0.004035g/10 ml
= 40.35 mg/100 ml
Mass of ascorbic acid (mg) in 100 ml of commercial lychee juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0168
= 0.002648 g/10 ml
= 26.48 mg/100 ml
* Mr refer to molar mass
* C refer to concentration
* V refer to volume
11
3.3
PART C : Vitamin C Concentration in Fresh Fruit Juices
Table 3.3 : Titration of fresh fruit juices with DCPIP solution
Sample number
Volume of
Average
Initial Reading
Final Reading
DCPIP solution
volume of
(ml)
(ml)
used (ml)
DCPIP solution
used (ml)
Trial 1
Apple
0.00
24.80
24.80
Trial 2
Apple
24.80
49.50
24.70
Trial 3
Apple
0.00
24.80
24.80
Trial 1
Orange
0.00
27.50
27.50
Trial 2
Orange
0.00
27.60
27.60
Trial 3
Orange
0.00
27.60
27.60
Trial 1
Lychee
0.00
13.70
13.70
Trial 2
Lychee
13.70
27.30
13.60
Trial 3
Lychee
27.30
41.00
13.70
12
24.80
27.60
20.70
Titration of Fresh Fruit Juices with DCPIP Solution
Average volume of DCPIP solution used (ml)
30
25
20
15
10
5
0
Apple
Orange
Type of fresh fruit juice
Lychee
Graph 3.3 : Average volume of DCPIP solution used in titration of fresh fruit juices with
DCPIP solution
Mass of ascorbic acid (mg) in 100 ml of fresh apple juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0248
= 0.003909 g/10 ml
= 39.09 mg/100 ml
13
Mass of ascorbic acid (mg) in 100 ml of fresh orange juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0276
= 0.004351 g/10 ml
= 43.51mg/100 ml
Mass of ascorbic acid (mg) in 100 ml of fresh lychee juices
Mole (Vitamin C) = CV (DCPIP solution)
Mass
Molar mass
= CV
Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)
= 176.12 X 0.000895 X 0.0207
= 0.003263 g/10 ml
= 32.63 mg/100 ml
* Mr refer to molar mass
* C refer to concentration
* V refer to volume
14
3.4
PART D : Comparison of Vitamin C Concentration in Commercial and Fresh
Fruit Juices
Table 3.4 : Comparison of vitamin C concentration in commercial and fresh fruit juices
Average Vitamin C Concentration (mg/100 ml)
Types of Fruit Juices
Commercial Fruit Juices
Fresh Fruit Juices
Apple
34.52
39.09
Orange
40.35
43.51
Lychee
26.48
32.63
Comparison of Vitamin C Concentration in Commercial and Fresh Fruit Juices
Mass of Vitamin C (mg/100 ml)
50
45
40
35
30
25
Commercial Fruit Juice
20
Fresh Fruit Juice
15
10
5
0
Apple
Orange
Types of Fruit Juices
Lychee
Graph 3.4 : Comparison of vitamin C concentration in commercial and fresh fruit juices
15
4.0
DISCUSSION
Vitamin C can be determined by acid-base reaction or oxidation-reduction reaction.
2,6-dichlorophenolindophenol, DCPIP solution can be used as an indicator for vitamin C [11].
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:
DCPIP (blue)
+
H+
DCPIPH (pink) + Vitamin C
——→ DCPIPH (pink)
——→ DCPIPH2 (colourless)
Balanced equation :
C6H8O6
+
C12H7NCl2O2
——→
C6H6O6
+
C12H9NCl2O2
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.
For the estimation of ascorbic acid, dilutions of the volume of the juice samples used
were made. They were also acidified to remove ferric ions and protein components. Ascorbic
acid solutions are susceptible to air oxidation which can be counteracted by acidity
mechanism. It can be deduced from the results tabulated that both the orange juice and the
apple juice had different titre values to attaining their respective endpoints. Logically, the
16
apple juice would require a significantly greater amount than that of the orange juice simply
because of the ascorbic acid content present which was indeed reflected in the calculations.
From Table 3.4, concentration of vitamin C in commercial fruit juices is higher than
fresh fruit juices, such as apple, orange and lychee as analysed in the project. The highest
amount of vitamin C was orange about 44.14 mg/100 ml for commercial fruit juice and 47.59
mg/100 ml for fresh fruit juice respectively. Following by apple juice was about 42.76
mg/100 ml for commercial fruit juice and 37.75 mg/100 ml for fresh fruit juice. The lowest
concentration of vitamin C content was lychee about 28.97 mg/100 ml for commercial fruit
juice and 35.69 mg/100 ml for fresh fruit juice. It is a known faced that commercial orange
juice contains the highest amount of vitamin C and is highly concentrated causing 25.60 ml
DCPIP solution is used to titrate it. Both the apple and lychee juices contain vitamin C but
not as much as the orange juice. This was showed by the amount of vitamin C in three
different fruit juices between commercial fruit juices and fresh fruit juices, where orange
juices has the highest vitamin C concentration in it, following by apple juice and then lychee
juice which the lowest concentration of vitamin C.
It was known that fresh fruit juices normally contain more vitamin C compared to
commercial fruit juices. The observation and result prove that the commercial fruit juice is
typically designed to appeal to the taste preferences of the market, and will therefore contain
different flavour packs or chemicals depending on where it will eventually end up [12].
Although ascorbic acid is a stable solid that does not react with air, but commercial fruit juice
has already rapidly oxidised on exposure to air and light, and then undergoes oxidation when
in aqueous solution. The product of oxidation is dehydroascorbic acid. In this case, all of the
nutrients in fruit juice have been destroyed [13]. It also have artificial, including often a huge
amount of added sugar.
17
Another factor that affects the vitamin C content in fruit juices were type of storage.
According to the theory, the vitamin C content does not loss when the fruit juices are stored
at cool temperature, but vitamin C content will be lost at higher temperature. This is because
the vitamin C is more sensitive to temperature. Fruit juices must be stored at cool temperature.
However, it cannot be assured of there is no change in temperature if commercial fruit juices
during industrial process in the production of commercial fruit juices.
The result of this study was showed that all of the fresh fruit juices contains of higher
vitamin C concentration as compared to in commercial fruit juices, such as apple, orange and
lyvhee. In conclusion, fresh fruit juice is more suitable for drinking in daily life. Furthermore,
fresh fruit juice has a shelf life of sometimes more than day, and has hundreds of times more
nutrients, enzymes, and phytochemicals.
5.0
CONCLUSION
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 methods. 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 used iodometric titration method which involves iodine and
iodate solution. When iodine solution is a titrant, vitamin C is oxidised to form
18
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. Suntornsuk et al. (2002) determined vitamin C in
fresh and freeze dried herbal juices using direct titration method with iodine solution in acidic
potassium iodide. The iodine solution was standardised used primary standard arsenic
trioxide [14]. In future, research should be done to determine whether the temperature will
effect 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. All these research are beneficial to us in order to stay hale and hearty in our
daily life.
19
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