SCHOOL OF PHARMACEUTICAL SCIENCES

FAR 142/3

BASIC PHARMACOLOGY AND BIOCHEMISTRY

EXPERIMENT 3:

DETERMINATION OF ASCORBIC ACID IN VITAMIN C TABLETS USING

2,6-DICHOLOROPHENOLINDOPHENOL

NAMES : BEATRICE GILBERT (123864)

FATIN NASUHA BINTI MAHYUDDIN (123877)

KHO BOON KIE (123889)

LIM CHIN WEN (123901 )

LECTURER’S NAME : DR. SALIZAWATI MUHAMAD SALHIMI

DATE OF EXPERIMENT : 13th MARCH 2015

OBJECTIVES

1. To determine the ascorbic acid content in vitamin C tablets using titration with the

redox indicator DCPIP.

2. To determine the effect of ascorbate oxidase on the sample of vitamin C tablets.

3. To investigate the effect of standing at room temperature, boiling and hydrogen

peroxide on the stability of ascorbic acid.

OVERVIEW

Pure ascorbic acid is a white, crystalline solid with a molecular formula C6H8O6. It is water-soluble. Its main chemical property is that it oxidizes extremely easily in solution, thus reducing anything that can accept electrons. The oxidation process is impeded in acidic environment and at low temperatures. Ascorbic acid is a form of vitamin C which is easily reduced or destroyed by exposure to light, heat, oxygen, in basic solutions and in presence of metal ions such as Cu2+ or Fe3+. The ascorbic acid content can be determined by 2,6-dicholorophenolindophenol (DCPIP) titrimetric method for food production.DCPIP is used as the titrant because the titrant should only oxidized ascorbic acid and not other substances that may be present in the samples. It is also because DCPIP act as self-indicator where it only must be in one color in the presence of excess ascorbic acid and another color when all ascorbic acid has reacted. Ascorbic acid is determined by oxidizing it in acidic medium with DCPIP as indicator to dehydroascorbic acid.

Ascorbic acid reduces the DCPIP,indicator dye from oxidized form(red in acid) to a reduced form, colorless solution. At the end point of titration excess unreduced dye is a rose-pink color in the acid solution. The titer of the dye can be determined by using a standard ascorbic acid solution. Then the ascorbic acid in the food sample can be determined by the calculation using the volume of the titration.

Before DCPIP is used to determine the ascorbic acid, we must first know the concentration of ascorbic acid solution. In order to know that, we can use the reaction of DCPIP solution with a known concentration with a solution of ascorbic acid which is called as standardization. Then, the stability of ascorbic acid is studied by treating with air, boiling for 15 minutes and hydrogen peroxide. From this study, we can know the amount of ascorbic acid present before and after the treatment. Thus, it will shows the factors that influence the ascorbic acid reduction and how it affects the ascorbic acid.In part C where vitamin C is analysed, ascorbate oxidase solution is added to the second aliquot to be titrated with DCPIP to minimize the interference of other compounds that may be present in the samples which allows the determination of other reducing substances other than ascorbic acid. The first aliquot is titrated directly and total content of all reducing substances present is determined.

RESULTS

Experiment Volume of DCPIP used (mL)

1 2 3 Average

a. Preparation and standardization of ascorbic

acid standard solution15.20 14.20 13.90 14.43

b. Study on the stability of ascorbic acid

standard solution to the following treatments:

i. Air

ii. Boiling for about 15 minutes

iii. Hydrogen peroxide

13.10 13.40 - 13.25

13.10 12.40 - 12.75

15.20 14.90 - 15.05

c. Analysis of vitamin C or multivitamin tablets

i) Incubation of an hour with ascorbate

oxidase enzyme at 37°C

ii) No incubation, no ascorbate oxidase

enzyme added at room temperature

3.50 3.70 2.90 3.37

24.90 24.80 25.20 24.97

CALCULATIONS

A) Preparation and standardization of ascorbic acid standard solution

Weight of ascorbic acid = 0.02 g

Molecular weight of ascorbic acid, C6H8O6 = 176 g/mol

No of moles of ascorbic acid = 0.02g

176g /mol = 1.14x 10−4 mol

Volume of 0.5% oxalic acid = 100 mL

Concentration of ascorbic acid solution,M =nV

x 1000 = 1.14 x10−4

100x 1000

= 1.14x10−4 mol dm−3

Volume of DCPIP used for titration with 10 mL of solution = (15.20+14.20+13.90 )mL

3 = 14.43 mL

B) Study on the stability of ascorbic acid standard solution to the following

treatments

i) Air

Volume of DCPIP used

= (13.10+13.40 )mL

2

= 13.25 mL

Ascorbic acid content:

From part (a),

14.43 mL DCPIP is reduced by 2.00 mg ascorbic acid.

13.25 mL DCPIP is reduced by (13.25 mL ÷14.43 mL) × 2.00 mg

= 1.84 mg ascorbic acid

Reduction of ascorbic acid

= 2.00 mg –1.84 mg

= 0.16 mg

Percentage reduction of ascorbic acid

= (0.16 mg ÷ 2.00 mg)×100%

= 8.00%

ii) Boiling for 15 minutes

Volume of DCPIP used

= (13.10+12.40 )mL

2

= 12.75 mL

Ascorbic acid content:

From part (a),

14.43 mL DCPIP is reduced by 2.00 mg ascorbic acid.

12.75 mL DCPIP is reduced by (12.75 mL ÷ 14.43 mL) × 2.00 mg

= 1.77 mg ascorbic acid

Reduction of ascorbic acid

= 2.00 mg - 1.77 mg

= 0.23 mg

Percentage reduction of ascorbic acid

= (0.23 mg ÷ 2.00 mg)×100%

= 11.5%

iii) Hydrogen Peroxide

Volume of DCPIP used

= (15.20+14.90 )mL

2= 15.05 mL

Ascorbic acid content:

From part (a),

14.43 mL DCPIP is reduced by 2.00 mg ascorbic acid.

15.05 mL DCPIP is reduced by (15.05 mL ÷ 14.43 mL) × 2.00mg

= 2.09 mg ascorbic acid

Reduction of ascorbic acid

= 2.00 mg – 2.09 mg

= -0.09

Percentage reduction of ascorbic acid

= (-0.09÷ 2.00 mg)×100%

= -4.5% ?????

C) Analysis of vitamin C or multivitamin tablets

Weight of vitamin C tablets used:

i) 806.40 mg

ii) 805.50 mg

iii) 804.40 mg

Average weight of each vitamin C tablet = 805.43mg

Volume of DCPIP reduced by vitamin C without ascorbate oxidase enzyme

= 24.97 mL

Volume of DCPIP used reduced by vitamin C with ascorbate oxidase enzyme

= 3.37 mL

Volume of DCPIP reduced by vitamin C

= 24.97 mL – 3.37 mL

= 21.60 mL

From the results in part (a),

14.43 mL of DCPIP is used to oxidize 2.00 mg of standard ascorbic acid

21.60 mL of DCPIP is used to oxidize (21.60 ÷ 14.43) × 2.00

= 2.99 mg of ascorbic acid

Thus, 21.60 mL of DCPIP is used to oxidize 2.99mg of ascorbic acid.

For 10 mL of sample, there is 2.99 mg of ascorbic acid.

For 100 mL of sample, there is 29.90 mg of ascorbic acid.

Thus, 29.90 mg of ascorbic acid is present in 100.00 mg of vitamin C tablet.

Average weight of one vitamin C tablet = 805.43 mg

100.00 mg of vitamin C tablet contains 29.90 mg of ascorbic acid.

805.43 mg (1 tablet) of vitamin C contains 240.82 mg of ascorbic acid.

Percentage of ascorbic acid in 1 vitamin C tablet

=( 240.82 mg ÷ 805.43 mg) × 100% = 29.90%

Standard deviation of ascorbic acid content in vitamin C tablet

Volume of DCPIP used to reduce vitamin C on:

1st trial = 24.90 mL – 3.50 mL = 21.40 mL

2nd trial = 24.80 mL – 3.70 mL = 21.10 mL

3rd trial = 25.20 mL – 2.90 mL = 22.30 mL

The content of ascorbic acid in 10 mL sample:

First trial = 21.4 014.43

x 2 = 2.97 mg

Second trial = 21.1014.43

x 2 = 2.92 mg

Third trial = 22.3 014.43

x 2 = 3.09 mg

The content of ascorbic acid in 100 mL sample (100mg tablet):

1st trial = 29.70 mg

2nd trial = 29.20 mg

3rd trial = 30.90 mg

For 1 tablet (805.43 mg), the content of ascorbic acid:

First trial = 29.7 0

100 x 805.43 = 239.21 mg

Second trial = 29.20100

x 805.43 = 235.19 mg

Third trial = 30.90100

x 805.43 = 248.88 mg

The average value of DCPIP to oxidize ascorbic acid

= 239.21+235.19+248.88

3

=241.09mg

The standard deviation of ascorbic acid content in the tablet

= ± √ (239.21−241.09)2+(235.19−241.09)2+(248.88−241.09)2

3

= ± √ 99.033

= ± 5.75

DISCUSSIONS

A) Preparation and standardization of ascorbic acid standard solution

In part A, a standard ascorbic acid solution is prepared and standardized before determining the ascorbic acid in order to know its concentration. The concentration of ascorbic acid can be determined by titrating the 2,6-dichlorophenolindophenol or DCPIP (0.025%). Titration of the sample must give the same pink color as the pink color of titration product of ascorbic acid standard solution. Oxalic acid is used in this experiment to dissolve ascorbic acid and also as a stabilizer which helps to acidify ascorbic acid. This is because ascorbic acid is not stable in alkaline solution. Oxalic acid is also used to prevent further oxidation of dehydroascorbic acid which is the product of oxidation of ascorbic acid. From part A, we can see that the concentration of ascorbic acid is 1.14x10−4 mol dm−3 and the volume of DCPIP reduced by 0.02 g ascorbic acid is 14.43mL.

C) Analysis of vitamin C or multivitamin tablets

This part of the experiment is to analyze the content of vitamin C (ascorbic acid) with the presence and absence of ascorbate oxidase. Ascorbate oxidase is an enzyme that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. 

This enzyme belongs to the family of oxidoreductases, specifically those acting on diphenols and related substances as donor with oxygen as acceptor. This enzyme participates in ascorbate metabolism. It employs one cofactor, copper. It can eliminate ascorbic acid, which has high reducing power in clinical analyses, and detect levels of ascorbic acid.

Both solutions with and without the presence of ascorbate oxidase is then being titrated by using 2,6-dichlorophenolindophenol (DCPIP) solution that function as indicator in this experiment. The DCPIP will be decolourised when ascorbic acid particle comes into contact with the DCPIP particle while the solution will appear pink in colour if there is no ascorbic acid content in it. As the titration continues, we can find that the pink colour of the titrated solution will remain which mean that there is no more content of ascorbic acid in the solution.

By looking at the table, we can see that less amount of DCPIP solution is used in the ascorbic acid solution containing the ascorbate oxidase (3.37mL) when

Ascorbic Acid

Ascorbate oxidase

Dehydroascorbic Acid

DCPIP (blue) + H+ → DCPIPH (pink)

DCPIPH (pink) + Vitamin C → DCPIPH2 (colorless)

compared to the ascorbic acid solution without the addition of ascorbate oxidase (25.00mL). This shows that the there is less concentration of ascorbic acid in the solution that contain the ascorbate oxidase as the ascorbate oxidase have destroyed the ascorbic acid molecule in the solution.

According to British Pharmacopoeia (BP), ascorbic acid contained in one tablet of vitamin C is in the range of 237.5 - 268.75 mg.

Besides, British Pharmacopoeia (BP) states that the minimum tablets needed to determine the amount of active ingredient in tablets is 20 tablets. However, only 3 tablets of vitamin C provided, therefore it may lead to an inaccurate result and the vitamin C solution also may be oxidised during the opening and closing of the bottle. The other factor may be the tablets are not dissolve completely in distilled water before filtration is carried out. The undissolved vitamin C remains on the filter paper. As a result, the amount of ascorbic acid in the solution may become lesser from the standard value as stated in British Pharmacopoeia (BP).

PRECAUTION:

1. All the apparatus are required to be cleaned entirely to avoid contamination of chemicals from the last usage which can affect the results and observations obtained.

2. The readings should be taken approximately such as when titration to prevent parallax error.

3. Ascorbic acid prepared should be used immediately to avoid oxidation of the ascorbic acid by oxygen in the air.

4. Sufficient time of incubation of ascorbic acid sample with ascorbate oxidase is vital to ensure complete degradation of the ascorbic acid.

5. The conical flask should be swirled continuously and gently during the titration process.

6. The intensity of light pink colour, indicating the end point of titration should be consistent in every experiment done to ensure accuracy of results obtained. Thus, it is best if only one person doing all the titrations.

REFERENCES

1. http://chemlab.truman.edu/CHEM130Labs/VitaminC.asp 2. Voet, D., Voet J.D. and Pratt, C.W.: Fundamentals of Biochemistry(2nd ed),

John Wiley, 2006. 3. Practical Manual of FAR 142/3 Biochemistry and basic principles of

molecular biology (pg 14-18)4. David L.Nelson, Michael M.Cox. Lehninger-Principles of Biochemistry,4th

edition.5. http://www.rxlist.com/ascorbic_acid-drug.htm