ANALYSIS OF VITAMIN C IN COMMERCIAL FRUIT JUICES BY

IODOMETRIC TITRATION.

SHAMSUL AZRIN BIN MD. KANAFE

Final Year Project Report Submitted in Partial Fulfilment of the

Requirement for the Degree of Bachelor of Science (Hons.) Chemistry in the Faculty of Applied Sciences,

Universiti Teknologi MARA.

APRIL 2009

This Final Year Project Report entitled “ANALYSIS OF VITAMIN C IN COMMERCIAL FRUIT JUICES BY IODOMETRIC TITRATION” was submitted

by Shamsul Azrin Bin Md. Kanafe, in partial fulfillment of the requirements for the Degree of Bachelor of Science (Hons.) Chemistry, in Faculty of Applied Sciences, and

approved by

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ACKNOWLEDGEMENTS

First of all, I would like to ALLAH s.w.t. for giving the strength to face life as a

student in the Faculty of Applied Sciences in University Teknologi MARA. Thanks

to my supervisor, Pn Mashita Abdullah @ Mohd Noor, for her perpetual energy and

enthusiasm in suiding me to complete my project. My gratitude also goes to my

family for their unconditional love and support throughout my life. My friends who

have helped directly or indirectly during my student in the university.

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TABLE OF CONTENTS.

Page. ACKNOWLEDGEMENTS. iii TABLE OF CONTENTS. iv LIST OF TABLES. v LIST OF FIGURES. vi LIST OF ABBREVIATIONS vii ABSTRACT. viii ABSTRAK. ix

CHAPTER 1 INTRODUCTION. 1.1 Background. 1 1.2 Significant of study. 3 1.3 Objectives of study. 3

CHAPTER 2 LITERATURE REVIEW. 2.1 Vitamin C 4 2.2 Methods to determine vitamin C. 4 2.2.1 Direct titration. 4 2.2.2 High performance liquid chromatography (HPLC). 6 2.2.3 Enzymatic method. 6

CHAPTER 3 METHODOLOGY. 3.1 Method using iodometric titration 8 3.2 Materials and reagents. 8 3.3 Preparation of iodine solution. 8 3.4 Preparation of vitamin C standard solution. 9 3.5 Standardisation of iodine solution with the vitamin C standard solution 9 3.6 Titration of juice samples. 9

CHAPTER 4 RESULT AND DISCUSSION 10

CHAPTER 5 CONCLUSION AND RECOMMENDATION 15

CITED REFERENCES 16 APPENDICES 17 CURRICULUM VITAL 18

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LIST OF TABLE.

Table Caption Page

4.1 Table amount of vitamin C in fruit juice samples analysed. 11 4.2 Comparing amount of vitamin C in nature fruit juices and commercial fruit juices. 13

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LIST OF FIGURES.

Figure Caption Page 1.0 The structure of vitamin C or ascorbic acid 1 4.1 The amount of vitamin C for three commercial fruit juices analysed 12

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LIST OF ABBEREVIATIONS.

Ascorbic acid : Vitamin C D-IAA : D-isoascorbic acid HPLC : High pressure liquid chromatography. L-AA : L-ascorbic acid. Shelf life before expired : Period between experimental date and expired date UV-VIS : Ultraviolet-visible.

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ABSTRACT.

ANALYSIS VITAMIN C CONTENT IN COMMERCIAL FRUIT JUICE

SAMPLES SUCH AS APPLE JUICE, GUAVA JUICE AND MANGO JUICE.

The objective of study are determination vitamin C content in three commercial fruit juices (apple, guava and mango) using titration method. Beside that, comparing vitamin C content determined and to that stated on the label for each fruit juice sample. To relate the amount of vitamin C present at the time of consumption based on the expired date. Sample to determine vitamin C is apple juice, mango juice and guava juice in commercial fruit juice on different expiry date. From different expiry date can get different shelf life before expired days. Method to determine vitamin C in commercial fruit juice is titration method with iodine solution. Titration method is accurate and precision method compare another methods. From the result the higher amount of vitamin C was guava juice about 183 g/100mL for nature fruit and 20.25 g/100mL for commercial fruit juice. Following mango juice was about 28 g/100mL for nature fruit and 16.40 for commercial fruit juice. The lower amount of vitamin C was apple juice about 6 g/100 mL for nature fruit and 5.33 g/100mL for commercial fruit juice. However mango juice and guava juice were suitable to drinking compared apple juices because apple juice has lower vitamin C contained and the higher loss of vitamin C content. According from analysis, the longer shelf life before expiry, the higher amount of vitamin C in fruit juice. However, apple juice was lower amount of vitamin C because the range of vitamin C content was shorter compared mango juice and guava juice in commercial fruit juice.

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ABSTRAK.

ANALISIS KANDUNGAN VITAMIN C DI DALAM JUS SECARA KOMERSIL SEPERTI AIR EPAL, AIR JAMBU DAN AIR MANGGA. Matlamat di dalam analisis adalah mengetahui kandungan vitamin C di dalam tiga jenis air buah-buahan (epal, jambu dan mangga) menggunakan kaedah mentitrat. Di samping itu, membandingkan kandungan vitamin C secara analisis dengan kandungan vitamin C yang tertera di label setiap kotak minuman. Mengkaitkan kandungan vitamin C yang hadir dengan masa penggunaan berdasarkan tarikh tamat tempoh. Sampel yang digunakan didalam analisis adalah air epal, air jambu dan air mangga secara komersil yang menpunyai pembezaan tarikh tamat tempoh. Daripada tarikh tamat tempoh yang berbeza, akan dapat tempoh hayat yang berbeza. Kaedah yang digunakan untuk menentukan kandungan vitamin C adalah kaedah mentitrat dengan larutan iodine. Kaedah mentitrat merupakan kaedah yang jitu dan peka dibandingkan kaedah lain. Daripada keputusan mendapati bahawa kandungan vitamin C yang paling tinggi adalah air jambu sebanyak 183 g/100mL secara semulajadi dan 20.25 g/100mL secara komersil. Diikuti air mangga sebanyak 28 g/100mL secara semulajadi dan 16.40 g/100mL secara komercial. Air epal yang merupakan kandungan vitamin C yang rendah sebanyak 6 g/100 mL secara semulajadi dan 5.33 g/100 mL secara komersil. Disamping itu juga, air jambu dan air mangga sesuai di minum jika dibandingkan air epal kerana air epal mempunyai kandungan vitamin C yang paling rendah dan pengurangan vitamin C adalah tinggi. Menurut kajian semakin panjang tempoh hayat sebelum tarikh tamat tempoh, semakin banyak kandungan vitamin C didalam air buah-buahan . Tetapi air epal tetap mempunyai kandungan vitamin C yang sedikit kerana julat kandungan vitamin C adalah rendah jika dibandingkan air jambu dan air mangga secara komersil.

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CHAPTER 1

INTRODUCTION

1.1 Background and problem statement.

Vitamin C or ascorbic acid is important to the body for prevention of

scurvy. Scurvy is caused by lack of vitamin C in the body which can affect

muscles weakness, swollen and bleeding of gums, loss of teeth and

bleeding under the skin as well as tiredness and depression.

Figure 1.0 show that the structure of vitamin C or ascorbic acid.

Fruit juices are liquid naturally contained in fruit or vegetables tissue.

Juices are prepared by mechanically squeezing or macerating fresh fruits

or vegetables without the application of heat or solvents. The labels on

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 fruit

juices, unless the amount of vitamin C is stated on the label of the

package.

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Vitamin C can determine by acid-base reaction or oxidation-reduction

reaction. Vitamin C is a weak acid and a good reducing agent. Iodine is a

weak oxidizing agent, so that it will not oxidize substances other than the

ascorbic acid in the sample of fruit juice. As a strong reducing agent,

vitamin C will reduce I2 to I- very easily. The excess of iodine react the

starch as indicator to perform the amount of vitamin C is finishing in

redox reaction.

In this reaction, the ascorbic acid molecule gains oxygen (in the form of

OH groups). Each iodine atom in the I2 molecule accepts an electron and

become a negatively charge to form iodide ion. Thus that, the ascorbic

acid molecule was oxidized and the iodine molecule was reduced.

C6H8O6 + I2 + 2H2O C6H10O8 + 2I - + 2H+ …….equation 1

Excess iodine (I2) reacts with iodide ions (I-) to form triiodide ion (I3-)

which forms a very intense blue color when it reacts starch. This colour is

due to incorporation of the ions within the molecular structure of the

starch.

I2 + I- → I3- ………equation 2

To detect the end point, starch must be added at the beginning of the

titration in the conical flask. When all ascorbic acids have finished, the

excess of iodine solution will react the starch to form blue-black colour in

the solution.

I3- + starch → starch (I3

-) complex …….equation 3

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1.2 Significance of the study.

The result of the analysis of vitamin C in the commercial fruit juices, it

can be compared with the amount of vitamin C stated on the label. Thus,

the provide information the amount of vitamin C in commercial fruit

juices.

1.3 Objectives of study.

The objectives of the study are:

1. To determine the amount of vitamin C in three commercial fruit

juices (guava, apple and mango) using iodometric titration.

2. To compare the amount of vitamin C determined and to that

stated on the label for each fruit juice sample

3. To relate the amount of vitamin C present at the time of

consumption based on the expired date

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CHAPTER 2

LITURATURE REVIEW

2.1 Vitamin C.

Vitamin C or ascorbic acid can be obtained from fruits or vegetables. Vitamin C

also acts as an antioxidant, scavenging potential harmful molecules called free

radicals. Although not firmly established by clinic trials, this antioxidant

capacity may help boost immune function, protect against cancer, cataracts, age-

related macular degeneration of the retina and other chronic diseases. Vitamin C

intake may be particularly helpful to smokers, as they are more likely to suffer

from oxidative stress and cell damage that can deplete vitamin C.

2.2 Methods of determining vitamin C.

Methods which can be applied to determine vitamin C in fruits or vitamin

supplement can be summarised into five methods such as direct titration with

iodine solution, titration with dichlorolindophenol solution, using capillary

electrophoresis with UV-VIS and diode array detection in high performance

liquid chromatography [HPLC], using ion-pair reversed phase in HPLC and UV-

diode detection and enzymatic method.

2.2.1 Direct titration.

Determination vitamin C concentration involves iodine and iodate solution in a

redox titration method. When iodine solution is a titrant, vitamin C is oxidised

to form 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 of titration (ascorbic acid + I2→

2I- + dehydroascorbic acid). This method is a straight forward compared to the

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alternative method using iodate as the titrant Potassium iodate stable

compared to iodine and it does not have to be standardized as it is a primary

standard. The iodine solution needs to be standardised with pure vitamin C or

potassium thiosulphate because an iodine is unstable.

(www.outreach.canterbury.ac.nz)

In the alternative method, iodate solution (IO3-) is added into acidic solution

containing iodide ions (I-) where an oxidation-reduction reaction occur. The

iodate ions are reduced to form iodine (IO3- + 6H+ +5e- → ½ I2 + 3H2O ) while

the iodide ions are oxidised to form iodine ( 2I- → I2 + 2e-). The overall

reaction between iodate and iodide is 2IO3- +10 I- +12 H+ → 6 I2 +6 H2O. The

endpoint of sample was blue-black starch-iodine complex when excess iodine

reacts with the starch as indicator in the solution.

(www.outreach.canterbury.ac,nz)

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. Guava and emblic myrobolan have higher vitamin C content

compared to sweet pepper, passion fruit, lemon and G.schomburgkiana. The

limit of detection (LOD) and limit of quantitation (LOQ) was 2.2 and 7.26 mg.

After 4 weeks, vitamin C content has found to be reduced from 469 to 338 ppm

or 27.9 % for freeze-dried guava and from 1798 to 1727 ppm or 3.9 % for

freeze-dried emblic myrobolan. The vitamin C content in fresh juice is greater

than in freeze-dried sample while the stability of vitamin C in the freeze-dried

sample is greater than in freeze-dried juice after 4-week storage.

The method which Kabasakalis et al.,(2000) used is another titration method

where vitamin C in commercial fruit juices was titrated against aqueous

sodium dichlorophenolindophenol with starch as indicator. The sodium

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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.

2.2.2 High performance liquid chromatography [HPLC].

Versari et al., (2004) determined vitamin C in commercial fruit juices using

capillary electrophoresis with UV-VIS and diode array detection in high-

performance liquid chromatography (HPLC). The sample analysed were twenty-

six samples in three types of apricot commercial juices (5 organic, 5 inorganic

and 16 conventional). The limit of detection (LOD) was 1.6 and 1.1 mg/L for L-

AA and D-IAA. The best separation system to detect L-AA and D-IAA is by

capillary electrophoresis using capillary zone mode and micelle electrokinetic

capillary chromatography.

Both condition of analysis were tested, the best separation was obtained at 30ºC

using 50 mM tricine at pH 8.8 as running buffer. The maximum absorbances for

both samples were 265 nm, but a shift in the absorbance to shorter wavelength

was reported to occur with pH is lower to acidic condition.

2.2.3 Enzymatic methods.

Shekhovtsova et al., (2006) applied enzymatic method used to determine

vitamin C in juice sample, milk and sour-milk products for babies’ nutrition

from the signal of indicator reaction. The indicator reaction used were o-

dianisdini (OD) and 3,3’5,5’-tetramethylbenzidine (TMB).

Another method to determine vitamin C was using the pH-transistors. The pH-

transistor as the indicator reaction o-phenylenediamine-ascorbic acid (AsA)-

second substrate of horseradish (HRP), where vitamin C acts as a second

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substance of the enzyme.

The optimum pH value of indicator in the presence of vitamin C was found to

lie in the pH range of 4.5-6.5 and the solution was buffered with potassium

phthalate solution. The longest induction period appears in the presence of

vitamin C at pH 4.5 which is close to pKASA 4.25. The indicator reaction at pH

4.5 and application of the induction period duration as a analytical signal which

provide sufficiently sensitive determination of vitamin C in real sample or

foods in particulars.

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CHAPTER 3

METHODOLOGY

3.1 Method using iodometric titration

In the present research, vitamin C standard was used to standardise an

iodine solution prepared by mixing potassium iodate and potassium iodide.

Then vitamins C in fruit juice samples were determined by titration an

unknown aliquot against the prepared iodine solution. The method was

adoped from paws.wcu.edu/bacon/vitamin C.pdf.

3.2 Materials and reagents.

The list or reagents and materials used are :-

potassium iodide (KI) (HmbG).

potassium iodate (KIO3) (unilab).

3 M sulfuric acid.

ascorbic acid (R & M)

1 % starch solution (HmbG).

Commercial fruit juices sample obtained from Marigold peel fresh.

3.3 Preparation of iodine solution.

5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) were

dissolved into 500 mL beaker with 200 mL of distilled water. 30 mL of 3 M

sulfuric acid was added into the beaker and then diluted with distilled water

until 500 mL solution.

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3.4 Preparation of vitamin C standard solution.

0.250 g ascorbic acid was dissolved in the beaker with 100 mL distilled water.

The solution was transferred into 250 mL volumetric flask and diluted to the

mark will distilled water.

3.5 Standardisation of the iodine solution with the vitamin C standard

solution.

25 mL of vitamin C solution was pipetted into a 125 mL Erlenmeyer flask. 10

drops of 1% starch solution were added and then titrated against iodine

solution until blue-black colour was observed. Titrations were repeated four

times.

3.6 Titration of juice samples.

25 mL of juice samples were pipetted into a 125 mL Erlenmeyer flask.

Following by 10 drops of 1% starch solution and titrated against iodine

solution until blue-black colour was observed (Appendix D). Titrations were

repeated four times.

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CHAPTER 4

RESULT AND DISCUSSION

4.1 Introduction.

Suntornsuk et al., (2002) and Kabasakalis et al., (2000) determined vitamin C

in fruit juices by titration method. Titration method was accurate and precise

method to determine vitamin C compared other methods as high pressure

liquid chromatography [HPLC] method and enzymatic methods.

Using titration method, it was not suitable for detection of vitamin C in the

coloured sample because it was difficult to find blue-black colour at endpoint

after starch was added in titration method.

The molarity of iodine solution was determined by standardisation method.

The amount of vitamin C in each juice sample was calculated as shown in

Appendix A, B and C for apple, guava and mango juices respectively. Three

sets of titration were carried out for three different packages of different

expiry date each juice sample.

The result of the average value of vitamin C in each fruit juice samples are

tabulated in Table 4.1.

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4.2 Discussion on the result of vitamin C in the sample analysed.

Table 4.1 The amount of vitamin C in fruit juice samples analysed.

Vitamin C Average vitamin C *Shelf life days

Sample no. mg/ 100mL (mg/100 mL) before expired days

Set 1 Apple 4.75 17 Set 2 Apple 5.33 10 Set 3 Apple 1.18 3.75 ± 2.25 10 Set 1 Guava 20.25 46 Set 2 Guava 11.63 10 Set 3 Guava 15.00 15.63 ± 4.34 24 Set 1 Mango 15.40 46 Set 2 Mango 12.70 27 Set 3 Mango 16.40 14.83 ± 1.19 46

*Shelf life before expiry = period between experimental date and expired date.

In apple juice sample, set 2 has higher vitamin C content compared to set 3 and

the loss of vitamin C content in set 3 is higher compared to set 2 for the same

days before expiry date (10 days). Set 1 was higher vitamin C content and it

has half 2 weeks before expired period date compared set 3 (10 days before

expiry). In guava juice sample, set 1 has higher vitamin C content and longer

shelf life before expiry (46 days) compared set 2 (10 days). In mango juice, set

1 and set 2 have higher amount of vitamin C (15.40 and 16.40 mg/100 mL)

with longer period days before expiry (46 days) compared apple juice.

Mango juice can taking when shelf life before expiry was longer days. This is

because amount of vitamin C was higher in commercial fruit juices. In short

shelf life before expiry date (10 days), guava juice in set 2 (11.63 mg/100 mL)

and apple juice in set 2 (5.33 mg/100 mL) and set 3 (1.18 mg/100 mL). In

longer shelf life before expiry date (46 days), guava juice in set 1 (20.25

mg/100 mL) and mango juice in set 1 (15.40 mg/100 mL) and set 3 (16.40

mg/100 mL).

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However, guava juice has higher vitamin C content (11.63 mg/100 mL for 10

days and 20.25 mg/100 mL for 46 days) compared apple juice (1.18 mg/100

mL) and mango juice (16.40 mg/100 mL) for the same days before expiry (10

days for apple juice and 46 days for mango juice) respectively. Thus that guava

juice was suitable to customers and taken anytime, when the customers needed.

Comparing apple juice and mango juice in commercial fruit juices.

4.3 Comparing among of vitamin C in commercial fruit juices.

*Shelf life before expiry = period between experimental date and expired date.

Figure 4.1 The amount of vitamin C for three commercial fruit juices analysed

From Figure 4.1, if vitamin C versus true shelf life before expired period it

shows that. The longer shelf life before expired date, the higher amount of

vitamin C in commercial fruit juices. From the graph, guava juices (15.63

mg/100 mL) were higher amount of vitamin C, following mango juices (14.83

mg/100 mL) and the large shelf life before expiry ( 46 days)

The shelf life before expiry between 10 to 20 days for guava and apple juices

show that guava juices have higher amount of vitamin C compared apple

juices. So that, guava juices can maintain vitamin C content in short shelf life

before expire date compared apple juices in commercial fruit juices. A factor

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4.4 Comparing vitamin C content between nature fruit juices and

commercial fruit juices

The result of vitamin C in nature fruit juices is adopted as stated by

www.naturalhub.com/natural_food_guide_fruit_vitamin_c.htm

Type of Experimetal Fruit [ Vitamin C] * [Vitamin C] (mg/100 mL) (mg/100 mL) Apple 6 5.33 Guava 183 20.25 Mango 28 16.40

*The value of vitamin C obtained from

www.naturalhub.com/natural_food_guide_fruit_vitamin_c.htm

between guava juice and apple juice were type of fruit, guava juice has higher

vitamin C content than apple juice.

The shelf life before expiry between 20 to 30 days was guava and mango

juices in commercial fruit juices. Guava juices have higher vitamin C content

compared mango juices in this range. So that, guava maintained vitamin C

content in middle shelf life before expired date in commercial fruit juices. A

factor between guava juice and mango juice were type of fruit, guava juice has

higher vitamin C content than mango juice.

The shelf life before expiry between 40 to 50 days was mango juices and

guava juices. Guava juices have higher amount of vitamin C compared mango

juices in commercial fruit juices. Guava juices can maintain amount of vitamin

C in longer shelf life before expired date in commercial fruit juices. A factor

between guava juice and mango juice were type of fruit, guava juice has higher

vitamin C content than mango juice.

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Table 4.2 Amount of vitamin C in nature fruit juices and commercial fruit

juices as analysed in the project.

Amount of vitamin C in three fruit juices between commercial fruit juices and

nature fruit juices were guava juices are higher vitamin C content, following

mango juice and then apple juices.

Effect of vitamin C content in fruit juices were type of storage. Type of storage

is fruit juices must store at cool temperature. When the fruit juices are store at

cool temperature, the vitamin C content does not loss. But at higher

temperature, vitamin C content will be lost in fruit juices. This is because the

vitamin C is more sensitive to temperature.

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