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.

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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.

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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.

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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:
Mass
Mole Molar mass
Concentration of ascorbic acid = =
Volume Volume

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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:

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

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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
= CV
Molar mass

Mass = Mr (Vitamin C) X C (DCPIP) X V (DCPIP)

* Mr refer to molar mass

* C refer to concentration

* V refer to volume

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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.

Mole
Concentration of ascorbic acid = Volume

Mass
Molar mass
= Volume

0.20
176.12
= 1L

= 0.00114 mol/L

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Concentration of DCPIP Solution

Molecular formula of DCPIP is C12H7NCl2O2.

Molecular formula of DCPIP = 268.10

Mass of DCPIP is 0.24 g.

Mole
Concentration of DCPIP solution = Volume

Mass
Molar mass
= Volume

0.24
268.10
= 1L

= 0.000895 mol/L

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3.2 PART B : Vitamin C Concentration in Commercial Fruit Juices

Table 3.2 : Titration of commercial fruit juices with DCPIP solution

Volume of Average

Initial Reading Final Reading DCPIP solution volume of

Sample number (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 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 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 16.80

Trial 3 Lychee 0.00 16.80 16.80

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 Average Volume of DCPIP Solution used in the Titration of Commercial
Fruit Juices
30
Average volume of DCPIP solution used (ml)

25

20

15

10

0
Apple Orange Lychee
Type of commercial fruit juice

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
= CV
Molar mass

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

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Mass of ascorbic acid (mg) in 100 ml of commercial orange juices

Mole (Vitamin C) = CV (DCPIP solution)

Mass
= CV
Molar mass

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
= CV
Molar mass

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

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3.3 PART C : Vitamin C Concentration in Fresh Fruit Juices

Table 3.3 : Titration of fresh fruit juices with DCPIP solution

Volume of Average

Initial Reading Final Reading DCPIP solution volume of

Sample number (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 24.80

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 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 20.70

Trial 3 Lychee 27.30 41.00 13.70

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 Titration of Fresh Fruit Juices with DCPIP Solution
30
Average volume of DCPIP solution used (ml)

25

20

15

10

0
Apple Orange Lychee
Type of fresh fruit juice

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
= CV
Molar mass

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

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Mass of ascorbic acid (mg) in 100 ml of fresh orange juices

Mole (Vitamin C) = CV (DCPIP solution)

Mass
= CV
Molar mass

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
= CV
Molar mass

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

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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

45
40
35
30
25
Commercial Fruit Juice
20
Fresh Fruit Juice
15
10
5
0
Apple Orange Lychee
Types of Fruit Juices

Graph 3.4 : Comparison of vitamin C concentration in commercial and fresh fruit juices

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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)

DCPIPH (pink) + Vitamin C ——→ 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

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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.

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 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

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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.

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